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Methods in
Molecular Biology 2303
Kuberan Balagurunathan
Hiroshi Nakato
Umesh Desai
Yukio Saijoh Editors
Glycosamino-
glycans
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
Glycosaminoglycans
Methods and Protocols
Second Edition
Edited by
Kuberan Balagurunathan
Departments of Biology, Bioengineering, and Medicinal Chemistry, University of Utah,
Salt Lake City, UT, USA
Hiroshi Nakato
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis,
MN, USA
Umesh Desai
Department of Medicinal Chemistry and Institute for Structural Biology, Drug Discovery
and Development, Virginia Commonwealth University, Richmond, VA, USA
Yukio Saijoh
Department of Neurobiology & Department of Nutrition & Integrative Physiology,
University of Utah, Salt Lake City, UT, USA
Editors
Kuberan Balagurunathan Hiroshi Nakato
Departments of Biology Department of Genetics, Cell Biology and Development
Bioengineering, and Medicinal University of Minnesota
Chemistry Minneapolis, MN, USA
University of Utah
Salt Lake City, UT, USA
Umesh Desai Yukio Saijoh

Department of Medicinal Department of Neurobiology & Department
Chemistry and Institute for Structural of Nutrition & Integrative Physiology
Biology University of Utah
Drug Discovery and Development Salt Lake City, UT, USA
Virginia Commonwealth University
Richmond, VA, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1397-9 ISBN 978-1-0716-1398-6 (eBook)
https://doi.org/10.1007/978-1-0716-1398-6
© Springer Science+Business Media, LLC, part of Springer Nature 2022
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
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Cover Caption: Drosophila ovary, one of the best model systems for the study of the stem cell niche where
glycosaminoglycans play major roles. Green color shows expression of Dally, a Drosophila glypican.
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Dedication
Dedicate this special volume on glycosaminoglycans to the memory of Professor Robert

D. Rosenberg who made pioneering contributions to our understanding of anticoagulant
actions and biosynthesis of heparin
v
Preface
It is fast becoming clear that glycosaminoglycans (GAGs) are nature’s “go-to tools.” Nature
tends to use GAGs to bring protein ligands and receptors together, alter protein conforma-
tion, effect internalization of ligands into cells, stabilize proteins and signaling complexes,
prevent protein aggregation, and, quite simply, sequester cations and water. Interestingly,
each of the six types of GAGs, e.g., heparan sulfate, heparin, chondroitin sulfate, dermatan
sulfate, keratan sulfate, and hyaluronic acid, play these fundamental roles to different
extents. Thus, distinct GAGs have been found to play critical roles in numerous physiologic
or pathologic responses. For example, heparin is most associated with modulation of
hemostasis, whereas heparan sulfate, in its proteoglycan form, is associated with growth
and morphogenesis. Likewise, chondroitin sulfate is most associated with osteoarthritis. Yet,
GAGs contribute to many specific biologic responses such as neurogenesis, stem cell
differentiation, left-right axis induction, synaptic plasticity, and bacterial/viral infections
among others. Much remains to be understood about the role of GAGs in these processes;
specifically, the molecular and structural mechanisms at play, the selectivity of recognition,
and the therapeutic and diagnostic potential.
The first edition of this volume in 2015 brought together a range of scientific disci-
plines, including developmental biology, chemical biology, organic synthesis, structural
biology, biochemistry, cell signaling, drug discovery, stem cell biology, tissue engineering,
bioinformatics, and computational glycobiology, to understand and disseminate the value of
GAGs in biology. The volume proved to be very useful as judged by the number of down-
loads and citations. In this volume, we capitalize on the gains made earlier and offer more
than 30 new and significantly revised chapters that update the content as we understand
today. We continue to emphasize and expand upon prior knowledge on the structural
analysis of GAGs; chemical and enzymatic synthesis of GAGs; biophysical, biochemical,
and computational analysis of GAG–protein interactions; molecular, genetic, and imaging
approaches for understanding and manipulating GAG cell biology; biomedical applications
of GAGs; and molecular and genetic approaches involving animal models and organoids.
This volume also retains the primary goal of offering insight and guidance to
non-glycoscience researchers undertaking advanced experiments on GAGs. Considering
the difficulty experienced by many researchers with tools and techniques of the glycoscience
trade, we have made it a point to incorporate suggestions and technical notes/tips on
successful performance of experiments—one of our high priority areas. This should enhance
the value of this volume even more to the practicing researchers including graduate students
and postdoctoral fellows. At the same time, the sheer breadth of the field should also help
practicing GAGologist learn tricks of the trade in a related area, e.g., a synthetic chemist
learning biophysical analysis.
We also dedicate this volume to Professor Robert D. Rosenberg, a mentor to one of the
editors (KB) at the Massachusetts Institute of Technology, who passed away on April
01, 2020. Professor Rosenberg was a stalwart in GAG studies, especially the antithrom-
bin–heparin system. Starting from purifying human antithrombin from plasma, his group
elucidated the mechanism of anticoagulation by heparin, derived the critical heparin
sequence that binds to antithrombin, led the isolation and characterization of GAG biosyn-
thetic enzymes, and elucidated the biosynthetic pathway of heparin anticoagulants. He was
vii
viii Preface
truly an exemplary mentor and researcher who trained numerous postdoctoral fellows and
enabled many of his trainees including physicians choose the field of GAGs as a primary
research topic in their career worldwide.
Overall, this volume should serve as a very useful experimental manual for cutting-edge
methodologies and practical tips to overcome barriers in understanding the chemistry and
biology of GAGs.
Salt Lake City, UT, USA Kuberan Balagurunathan

Minneapolis, MN, USA Hiroshi Nakato
Richmond, VA, USA Umesh Desai
Salt Lake City, UT, USA Yukio Saijoh
Contents
Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
PART I GAG STRUCTURES
1 One-Pot Enzymatic Synthesis of Heparin from N-Sulfoheparosan . . . . . . . . . . . . 3

Li Fu and Robert J. Linhardt
2 Solution NMR Spectroscopy for Characterizing
Protein–Glycosaminoglycan Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Prem Raj B. Joseph, Krishna Mohan Sepuru, Krishna Mohan Poluri,
and Krishna Rajarathnam
3 Metabolic Labeling of Proteoglycans and Analysis of Their Synthesis
and Sorting in Filter-Grown and Polarized Epithelial Cells . . . . . . . . . . . . . . . . . . . 25
Kristian Prydz and Ravi Adusumalli
4 Fluorous-Tag-Assisted Synthesis of GAG-Like Oligosaccharides . . . . . . . . . . . . . . 37
José L. de Paz and Pedro M. Nieto
5 Aqueous Molecular Dynamics for Understanding Glycosaminoglycan
Recognition by Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Balaji Nagarajan and Umesh Desai
6 A Nonradioactive Method to Measure Hyaluronan Synthase Activity . . . . . . . . . . 63
Davide Vigetti, Evgenia Karousou, Manuela Viola, and Alberto Passi
7 A Glycoproteomic Approach to Identify Novel Proteoglycans . . . . . . . . . . . . . . . . 71
Fredrik Noborn, Mahnaz Nikpour, Andrea Persson, Carina Sihlbom,
Jonas Nilsson, and Göran Larson
8 Analysis of Protein–Glycosaminoglycan Interactions Using Traveling
Wave Ion-Mobility Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Robert V. Williams and I. Jonathan Amster
9 Ascidian (Chordata-Tunicata) Glycosaminoglycans: Extraction,
Purification, Biochemical, and Spectroscopic Analysis. . . . . . . . . . . . . . . . . . . . . . . . 93
Felipe C. O. B. Teixeira and Mauro S. G. Pavão
10 Quantitative Disaccharide Profiling of Glycosaminoglycans
from Two Different Preparations by PMP and Deuterated
PMP Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Meng Zhang, Yi An, and Lijuan Zhang
11 Preparation and Characterization of Heparan Sulfate-Derived
Oligosaccharides to Investigate Protein–GAG Interaction
and HS Biosynthesis Enzyme Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Cédric Laguri, Rabia Sadir, Evelyne Gout, Romain R. Vivès,
and Hugues Lortat-Jacob
ix
x Contents
12 Heparan Sulfate Structure: Methods to Study N-Sulfation

and NDST Action. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Anders Dag€ a lv, Anders Lundequist, Beata Filipek-Gorniok,
Tabea Dierker, Inger Eriksson, and Lena Kjellén
13 Methods for the Production of Recombinant Heparosan,
a Critical Heparin Precursor, from Nonpathogenic E. coli Strains . . . . . . . . . . . . . 151
Anindita Roy, Varun Garg, Sai K. Mehta, Alessandro Rossi,
and Kuberan Balagurunathan
14 De Novo Sequencing of Heparin/Heparan Sulfate Oligosaccharides
by Chemical Derivatization and LC-MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Quntao Liang and Joshua S. Sharp
15 Production and HPLC-Based Disaccharide Analysis of Xyloside-Primed
Glycosaminoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Andrea Persson, Emil Tykesson, Ulf Ellervik, and Katrin Mani
16 Nuclear Magnetic Resonance Methods in Structural Characterization
of Glycosaminoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Vitor H. Pomin and Barbara Mulloy
17 Aggrecan: Approaches to Study Biophysical and Biomechanical Properties . . . . . 209
Hadi Tavakoli Nia, Christine Ortiz, and Alan Grodzinsky
18 Chromatographic Molecular Weight Measurements for Heparin,
Its Fragments and Fractions, and Other Glycosaminoglycans . . . . . . . . . . . . . . . . . 227
Barbara Mulloy and John Hogwood
19 Mass Spectrometric Methods for the Analysis of Heparin
and Heparan Sulfate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Miroslaw Lech, Ishan Capila, and Ganesh V. Kaundinya
20 Affinity Purification of Glycosylphosphatidylinositol-anchored
Proteins by Alpha-Toxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Kevin Huang and Sungjin Park
21 Thermodynamic Affinity and Nature of Forces Defining
Glycosaminoglycan-Protein Systems Using Fluorescence Spectroscopy . . . . . . . . 259
Rio S. Boothello and Umesh Desai
22 Isolation and Characterization of Heparan Sulfate Containing
Amyloid Precursor Protein Degradation Products . . . . . . . . . . . . . . . . . . . . . . . . . . 279
Katrin Mani
23 Chemical Approaches to Prepare Modified Heparin
and Heparosan Polymers for Biological Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Karthik Raman and Sailaja Arungundram
24 Glycosaminoglycan Quality Control by Monosaccharide Analysis . . . . . . . . . . . . . 297
Yiran Zhang, Xuexiao Ma, and Lijuan Zhang
25 Characterizing Thermodynamics of Protein-Glycosaminoglycan
Interactions Using Isothermal Titration Calorimetry . . . . . . . . . . . . . . . . . . . . . . . . 307
Amit K. Dutta, Krishna Mohan Sepuru, Jörg Rösgen,
Contents xi
26 Automated Synthesis of Chondroitin Sulfate Oligosaccharides. . . . . . . . . . . . . . . . 319

Chien-Fu Liang, Heung Sik Hahm, Narayana Murthy Sabbavarapu,
and Peter H. Seeberger
27 Capillary Electrophoretic Analysis of Isolated Sulfated Polysaccharides
to Characterize Pharmaceutical Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Zachary Shriver and Ram Sasisekharan
28 Preparation of Isotope-Enriched Heparan Sulfate Precursors
for Structural Biology Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Victor V. Xylophone, Vy My Tran, Kuberan Balagurunathan,
and Thao Kim Nu Nguyen
29 Methods for Measuring Exchangeable Protons in Glycosaminoglycans . . . . . . . . 349
Consuelo N. Beecher and Cynthia K. Larive
30 Advances in Studying Glycosaminoglycan–Protein Interactions
Using Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
Aiye Liang and Umesh Desai
31 anexVis: A Transcriptome Tool to Visualize Organ/Tissue-Specific
Glycosaminoglycan Biosynthetic and Catabolic Pathways
in Human Health and Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Kishan Thambu and Kuberan Balagurunathan
PART II GAG FUNCTIONS

32 Molecular Genetic Techniques for the Proteoglycan Functions
in Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
Nanako Bowden, Masahiko Takemura, and Hiroshi Nakato
33 Measuring Sulfatase Expression and Invasion in Glioblastoma . . . . . . . . . . . . . . . . 415
Anna Wade, Jane R. Engler, Vy My Tran, and Joanna J. Phillips
34 Analyzing the Role of Heparan Sulfate Proteoglycans
in Axon Guidance In Vivo in Zebrafish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427
Fabienne E. Poulain
35 Regulation of 3-O-Sulfation of Heparan Sulfate During Transition
from the Naı̈ve to the Primed State in Mouse Embryonic Stem Cells. . . . . . . . . . 443
Hayato Ota and Shoko Nishihara
36 Analysis of Human Hyaluronan Synthase Gene Transcriptional
Regulation and Downstream Hyaluronan Cell Surface Receptor
Mobility in Myofibroblast Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
Adam C. Midgley and Timothy Bowen
37 Methods to Analyze the Effect of Diet-Derived Metabolites
on Endothelial Inflammation and Cell Surface Glycosaminoglycans . . . . . . . . . . . 469
Brett Ronald Cutler, Jie Shi Chua, Kuberan Balagurunathan,
and Pon Velayutham Anandh Babu
38 Assays for Evaluation of Substrates for and Inhibitors
of β-1,4-Galactosyltransferase 7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477
Emil Tykesson, Andrea Persson, Katrin Mani, and Ulf Ellervik
xii Contents
39 Enzymatic Alteration of ECM to Explore Muscle Function

and Motor Control of a Learned Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487
Caitlin Mencio, Kuberan Balagurunathan, and Franz Goller
40 Procedures to Evaluate the Role of Heparan Sulfate on the Reactivity
of Resistance and Conductance Arteries Ex Vivo. . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
Jae Min Cho, Kellsey Ly, Steven Ly, Seul-Ki Park,
Pon Velayutham Anandh Babu, Kuberan Balagurunathan,
and J. David Symons
41 Computerized Molecular Modeling for Discovering Promising
Glycosaminoglycan Oligosaccharides that Modulate Protein Function. . . . . . . . . 513
Nehru Viji Sankaranarayanan and Umesh Desai
42 Imaging Glycosaminoglycan Modification Patterns In Vivo . . . . . . . . . . . . . . . . . . 539
Hannes E. Bülow
43 Isolation and Purification of Versican and Analysis of Versican Proteolysis. . . . . . 559
Simon J. Foulcer, Anthony J. Day, and Suneel S. Apte
44 Identification of Cell Autonomous and Non-Cell Autonomous
Functions of Heparan Sulfate Glycosaminoglycan Chains by Creating
Chimeric Mouse Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Isao Matsuo and Chiharu Kimura-Yoshida
45 Methods for Assessing the Effects of Xylosides on Angiogenesis . . . . . . . . . . . . . . 595
Jie Shi Chua, Geethu Muruganandam, Yukio Saijoh,
46 Role of HSPGs in Systemic Bacterial Infections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605
Rafael S. Aquino, Kazutaka Hayashida, Atsuko Hayashida,
and Pyong Woo Park
47 Generation of Drosophila Heparan Sulfate Mutant Cell Lines
from Existing Fly Strains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627
Eriko Nakato, Nanako Bowden, and Hiroshi Nakato
48 Application of a Mutant Cell Library to Determine the Structure–Function
Relationship of Heparan Sulfate in Facilitating FGF2-FGFR1 Signaling . . . . . . . 637
John Faulkner, Xuehong Song, and Lianchun Wang
49 Manipulation of Glycosaminoglycans Using Synthetic Xylosides
to Study Their Roles in Lung Branching Morphogenesis in Ex Vivo
Lung Bud Culture System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645
Jie Shi Chua, Kuberan Balagurunathan, and Yukio Saijoh
50 Informatics Ecosystems to Advance the Biology of Glycans . . . . . . . . . . . . . . . . . . 655
Lewis J. Frey
51 Analysis of 30 -Phosphoadenosine 50 -Phosphosulfate Transporters:
Transporter Activity Assay, Real-Time Reverse Transcription Polymerase
Chain Reaction, and Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 675
Hideo Egawa and Shoko Nishihara
52 Preparation of Nanosensors for Detecting the Activity
of Glycosaminoglycan Cleaving Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687
Mausam Kalita, April Joice, Khoi Dang Le, Yiling Bi,
Gurusankar Ramamoorthy, Orlando Antelope, Anindita Roy,
Contents xiii
53 Detection of Glycosaminoglycans in Pancreatic Islets

and Lymphoid Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 695
Marika Bogdani, Charmaine Simeonovic, Nadine Nagy,
Pamela Y. Johnson, Christina K. Chan, and Thomas N. Wight
54 Histochemical Analysis of Heparan Sulfate 3-O-sulfotransferase
Expression in Mouse Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 719
Tomio Yabe and Nobuaki Maeda
55 Mapping Proteoglycan Function Using Novel Genetic Strategies . . . . . . . . . . . . . 731
Mauricio Cortes, Leslie K. Cortes, and Nancy B. Schwartz
56 Xyloside Derivatives as Molecular Tools to Selectively Inhibit
Heparan Sulfate and Chondroitin Sulfate Proteoglycan Biosynthesis . . . . . . . . . . 753
Caitlin Mencio, Kuberan Balagurunathan, and Mamoru Koketsu
57 A Strategic Approach to Identification of Selective Inhibitors
of Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
Nirmita Patel, Somesh Baranwal, and Bhaumik B. Patel
58 Applications of Xylosides in the Manipulation of Stem Cell Niche
to Regulate Human Neural Stem Cell Differentiation and Neurite
Outgrowth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779
Jie Shi Chua, Geethu Muruganandam, Daeun Sung,
Yukio Saijoh, and Kuberan Balagurunathan
59 Murine Models in the Evaluation of Heparan Sulfate-Based
Anticoagulants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789
Bassem M. Mohammed, Qiufang Cheng, Ivan S. Ivanov,
and David Gailani
60 Investigating the Roles of Heparan Sulfate Structures
in Alpha-Synuclein Aggregation in Cell Culture Models . . . . . . . . . . . . . . . . . . . . . 807
Anindita Roy, Akila V. Chalapathi, and Kuberan Balagurunathan
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 821
Contributors
RAVI ADUSUMALLI • Department of Biosciences, University of Oslo, Oslo, Norway

I. JONATHAN AMSTER • Department of Chemistry, University of Georgia, Athens, GA, USA
YI AN • Systems Biology and Medicine Center for Complex Diseases, Center for Clinical
Research, The Affiliated Hospital of Qingdao University, Qingdao, China
PON VELAYUTHAM ANANDH BABU • Department of Nutrition and Integrative Physiology,
College of Health, University of Utah, Salt Lake City, UT, USA
ORLANDO ANTELOPE • Departments of Biology, Bioengineering, and Medicinal Chemistry,
SUNEEL S. APTE • Department of Biomedical Engineering, Lerner Research Institute,
Cleveland Clinic, Cleveland, OH, USA
RAFAEL S. AQUINO • Division of Respiratory Diseases, Boston Children’s Hospital, Harvard
Medical School, Boston, MA, USA
SAILAJA ARUNGUNDRAM • Chemistry Department, Pacific Lutheran University, Tacoma, WA,
USA
KUBERAN BALAGURUNATHAN • Departments of Biology, Bioengineering, and Medicinal
Chemistry, University of Utah, Salt Lake City, UT, USA
SOMESH BARANWAL • Division of Hematology and Oncology, Department of Medicine,
Virginia Commonwealth University, Richmond, VA, USA
CONSUELO N. BEECHER • Department of Chemistry and Biochemistry, College of the
Canyons, Santa Clarita, CA, USA
YILING BI • Departments of Biology, Bioengineering, and Medicinal Chemistry, University of
Utah, Salt Lake City, UT, USA
MARIKA BOGDANI • Matrix Biology Program, Benaroya Research Institute at Virginia
Mason, Seattle, WA, USA
RIO S. BOOTHELLO • Division of Hematology and Oncology, Department of Internal
Medicine, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA,
USA
NANAKO BOWDEN • Department of Genetics, Cell Biology and Development, University of
Minnesota, Minneapolis, MN, USA
TIMOTHY BOWEN • Wales Kidney Research Unit, Division of Infection and Immunity, School
of Medicine, College of Biomedical and Life Sciences, Cardiff University, Cardiff, UK
HANNES E. BÜLOW • Department of Genetics, Albert Einstein College of Medicine, Bronx,
NY, USA; Dominick P. Purpura Department of Neuroscience, Albert Einstein College of
Medicine, Bronx, NY, USA
ISHAN CAPILA • Momenta Pharmaceuticals, Cambridge, MA, USA
AKILA V. CHALAPATHI • Departments of Biology, Bioengineering, and Medicinal Chemistry,
CHRISTINA K. CHAN • Matrix Biology Program, Benaroya Research Institute at Virginia
QIUFANG CHENG • Department of Pathology, Microbiology and Immunology, Vanderbilt
University, Nashville, TN, USA
JAE MIN CHO • Department of Nutrition and Integrative Physiology, Molecular Medicine
Program, University of Utah, Salt Lake City, UT, USA
xv
xvi Contributors
JIE SHI CHUA • Departments of Biology, Bioengineering, & Medicinal Chemistry, University
of Utah, Salt Lake City, UT, USA
LESLIE K. CORTES • Department of Pediatrics, University of Chicago, Chicago, IL, USA;
Scholar Rock, Cambridge, MA, USA
MAURICIO CORTES • Department of Pediatrics, University of Chicago, Chicago, IL, USA;
Cellarity, Cambridge, MA, USA
BRETT RONALD CUTLER • Department of Nutrition and Integrative Physiology, College of
Health, University of Utah, Salt Lake City, UT, USA
ANDERS DAGA€ LV • Department of Medical Biochemistry and Microbiology, Science for Life
Laboratory, Uppsala University, Uppsala, Sweden
ANTHONY J. DAY • Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences,
University of Manchester, Manchester, UK
JOSÉ L. DE PAZ • Glycosystems Laboratory, Instituto de Investigaciones Quı́micas (IIQ),
cicCartuja, CSIC and Universidad de Sevilla, Sevilla, Spain
UMESH DESAI • Department of Medicinal Chemistry and Institute for Structural Biology,
Drug Discovery and Development, Virginia Commonwealth University, Richmond, VA,
USA
TABEA DIERKER • Department of Medical Biochemistry and Microbiology, Science for Life
AMIT K. DUTTA • Department of Biochemistry and Molecular Biology, The University of
Texas Medical Branch, Galveston, TX, USA
HIDEO EGAWA • Department of Bioinformatics, Graduate School of Engineering, Soka
University, Hachioji, Tokyo, Japan
ULF ELLERVIK • Department of Chemistry, Lund University, Lund, Sweden
JANE R. ENGLER • Department of Neurological Surgery, Brain Tumor Research Center,
Department of Pathology, University of California, San Francisco, San Francisco, CA,
USA
INGER ERIKSSON • Department of Medical Biochemistry and Microbiology, Science for Life
JOHN FAULKNER • Department of Molecular Pharmacology & Physiology, Morsani College of
Medicine, University of South Florida, Tampa, FL, USA
BEATA FILIPEK-GÓRNIOK • Department of Medical Biochemistry and Microbiology, Science for
Life Laboratory, Uppsala University, Uppsala, Sweden
SIMON J. FOULCER • Department of Biomedical Engineering, Lerner Research Institute,
Cleveland Clinic, Cleveland, OH, USA
LEWIS J. FREY • Department of Public Health Sciences, College of Medicine, Medical
University of South Carolina, Charleston, SC, USA
LI FU • Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic
Institute, Troy, NY, USA
DAVID GAILANI • Department of Pathology, Microbiology and Immunology, Vanderbilt
VARUN GARG • Department of Biology, University of Utah, Salt Lake City, UT, USA;
Department of Bioengineering, University of Utah, Salt Lake City, UT, USA
FRANZ GOLLER • School of Biological Sciences, University of Utah, Salt Lake City, UT, USA;
Institute of Zoophysiology, University of Münster, Münster, Germany
EVELYNE GOUT • Université Grenoble Alpes, Centre National de la Recherche Scientifique,
and Commissariat à l’Énergie Atomique et aux Énergies Alternatives, Institut de Biologie
Structurale, UMR 5075, Grenoble, France
Contributors xvii
ALAN GRODZINSKY • Department of Biological Engineering, Electrical Engineering and

Computer Science, and Mechanical Engineering, Massachusetts Institute of Technology,
Cambridge, MA, USA
HEUNG SIK HAHM • Department of Biomolecular Systems, Max Planck Institute of Colloids
and Interfaces, Potsdam, Germany; Department of Chemistry and Biochemistry, Freie
Universit€
at Berlin, Berlin, Germany
ATSUKO HAYASHIDA • Division of Respiratory Diseases, Boston Children’s Hospital, Harvard
Medical School, Boston, MA, USA
KAZUTAKA HAYASHIDA • Division of Respiratory Diseases, Boston Children’s Hospital,
Harvard Medical School, Boston, MA, USA
JOHN HOGWOOD • National Institute for Biological Standards and Control, Hertfordshire,
UK
KEVIN HUANG • Department of Neurobiology, University of Utah, Salt Lake City, UT, USA
IVAN S. IVANOV • Department of Pathology, Microbiology and Immunology, Vanderbilt
PAMELA Y. JOHNSON • Matrix Biology Program, Benaroya Research Institute at Virginia
APRIL JOICE • Departments of Biology, Bioengineering, and Medicinal Chemistry, University
of Utah, Salt Lake City, UT, USA
PREM RAJ B. JOSEPH • Departments of Biochemistry and Molecular Biology, Sealy Center for
Structural Biology and Molecular Biophysics, The University of Texas Medical Branch,
Galveston, TX, USA
MAUSAM KALITA • Departments of Biology, Bioengineering, and Medicinal Chemistry,
EVGENIA KAROUSOU • Department of Surgical and Morphological Sciences, University of
Insubria, Varese, Italy
GANESH V. KAUNDINYA • Momenta Pharmaceuticals, Cambridge, MA, USA
CHIHARU KIMURA-YOSHIDA • Department of Molecular Embryology, Research Institute,
Osaka Women’s and Children’s Hospital, Osaka Prefectural Hospital Organization,
Osaka, Japan
LENA KJELLÉN • Department of Medical Biochemistry and Microbiology, Science for Life
MAMORU KOKETSU • Department of Chemistry and Biomolecular Science, Faculty of
Engineering, Gifu University, Gifu, Japan
CÉDRIC LAGURI • Université Grenoble Alpes, Centre National de la Recherche Scientifique,
CYNTHIA K. LARIVE • Department of Chemistry and Biochemistry, University of California
Santa Cruz, Santa Cruz, CA, USA
GÖRAN LARSON • Department of Laboratory Medicine, Institute of Biomedicine, Sahlgrenska
Academy at the University of Gothenburg, Gothenburg, Sweden; Laboratory of Clinical
Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden
KHOI DANG LE • Departments of Biology, Bioengineering, and Medicinal Chemistry,
MIROSLAW LECH • Momenta Pharmaceuticals, Cambridge, MA, USA
AIYE LIANG • Department of Chemistry, Charleston Southern University, North Charleston,
SC, USA
xviii Contributors
CHIEN-FU LIANG • Department of Biomolecular Systems, Max Planck Institute of Colloids

and Interfaces, Potsdam, Germany
QUNTAO LIANG • College of Biological Science and Engineering, Fu Zhou University, Fu
Zhou, P. R. China
ROBERT J. LINHARDT • Center for Biotechnology and Interdisciplinary Studies, Rensselaer
Polytechnic Institute, Troy, NY, USA
HUGUES LORTAT-JACOB • Université Grenoble Alpes, Centre National de la Recherche
Scientifique, and Commissariat `a l’Énergie Atomique et aux Énergies Alternatives,
Institut de Biologie Structurale, UMR 5075, Grenoble, France
ANDERS LUNDEQUIST • Department of Medical Biochemistry and Microbiology, Science for
Life Laboratory, Uppsala University, Uppsala, Sweden
KELLSEY LY • Department of Nutrition and Integrative Physiology, Molecular Medicine
STEVEN LY • Department of Nutrition and Integrative Physiology, Molecular Medicine
XUEXIAO MA • Systems Biology and Medicine Center for Complex Diseases, Affiliated
Hospital of Qingdao University, Qingdao, China
NOBUAKI MAEDA • Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
KATRIN MANI • Department of Experimental Medical Science, Division of Neuroscience,
Glycobiology Group, Lund University, Lund, Sweden
ISAO MATSUO • Department of Molecular Embryology, Research Institute, Osaka Women’s
and Children’s Hospital, Osaka Prefectural Hospital Organization, Osaka, Japan
SAI K. MEHTA • Department of Biology, University of Utah, Salt Lake City, UT, USA;
CAITLIN MENCIO • Departments of Biology, Bioengineering, and Medicinal Chemistry,
University of Utah, Salt Lake City, UT, USA; Interdepartmental Program in
Neuroscience, University of Utah, Salt Lake City, UT, USA
ADAM C. MIDGLEY • Key Laboratory of Bioactive Materials for the Ministry of Education,
Rongxiang Xu Center for Regenerative Life Science, College of Life Sciences, Nankai
University, Tianjin, China
BASSEM M. MOHAMMED • Department of Biochemistry and Molecular Biology, Saint Louis
University School of Medicine, Doisy Research Center, St. Louis, MO, USA
BARBARA MULLOY • Glycosciences Laboratory, Department of Medicine, Imperial College,
London, UK; Institute of Pharmaceutical Science, King’s College London, London, UK;
National Institute for Biological Standards and Control, Hertfordshire, UK
GEETHU MURUGANANDAM • Departments of Biology, Bioengineering and Medicinal
BALAJI NAGARAJAN • Department of Medicinal Chemistry and Institute for Structural
Biology, Drug Discovery and Development, Virginia Commonwealth University,
Richmond, VA, USA
NADINE NAGY • Matrix Biology Program, Benaroya Research Institute at Virginia Mason,
Seattle, WA, USA; Division of Infectious Diseases and Geographic Medicine, Department of
Medicine, Stanford University School of Medicine, Stanford, CA, USA
ERIKO NAKATO • Department of Genetics, Cell Biology and Development, University of
HIROSHI NAKATO • Department of Genetics, Cell Biology and Development, University of
Contributors xix
THAO KIM NU NGUYEN • Faculty of Biology, VNU University of Science, Vietnam National
University, Hanoi, Vietnam
HADI TAVAKOLI NIA • Department of Biomedical Engineering, Boston University, Boston,
MA, USA
PEDRO M. NIETO • Glycosystems Laboratory, Instituto de Investigaciones Quı́micas (IIQ),
cicCartuja, CSIC and Universidad de Sevilla, Sevilla, Spain
MAHNAZ NIKPOUR • Department of Laboratory Medicine, Institute of Biomedicine,
Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden
JONAS NILSSON • Department of Laboratory Medicine, Institute of Biomedicine, Sahlgrenska
Academy at the University of Gothenburg, Gothenburg, Sweden; Laboratory of Clinical
Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden
SHOKO NISHIHARA • Department of Bioinformatics, Graduate School of Engineering, Soka
University, Hachioji, Tokyo, Japan; Glycan & Life System Integration Center (GaLSIC),
Soka University, Hachioji, Tokyo, Japan
FREDRIK NOBORN • Department of Laboratory Medicine, Institute of Biomedicine,
CHRISTINE ORTIZ • Department of Materials Science and Engineering, Massachusetts
Institute of Technology, Cambridge, MA, USA
HAYATO OTA • Department of Bioinformatics, Graduate School of Engineering, Soka
University, Hachioji, Tokyo, Japan
PYONG WOO PARK • Division of Respiratory Diseases, Boston Children’s Hospital, Harvard
Medical School, Boston, MA, USA; Department of Pediatrics, Harvard Medical School,
Boston, MA, USA
SEUL-KI PARK • Department of Nutrition and Integrative Physiology, Molecular Medicine
SUNGJIN PARK • Department of Neurobiology, University of Utah, Salt Lake City, UT, USA
ALBERTO PASSI • Department of Surgical and Morphological Sciences, University of Insubria,
Varese, Italy
BHAUMIK B. PATEL • Division of Hematology and Oncology, Department of Medicine,
Virginia Commonwealth University, Richmond, VA, USA
NIRMITA PATEL • Hunter Holmes McGuire VA Medical Center, Richmond, VA, USA
MAURO S. G. PAVÃO • Laboratorio de Bioquı́mica e Biologia Celular de Glicoconjugados,
Programa de Glicobiologia – Instituto de Bioquı́mica Médica Leopoldo de Meis, Hospital
Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, Brazil; Rua Professor Rodolpho Paulo Rocco, Cidade Universitária, Ilha do
Fundão, Rio de Janeiro, Brazil
ANDREA PERSSON • Department of Laboratory Medicine, Institute of Biomedicine,
JOANNA J. PHILLIPS • Department of Neurological Surgery, Brain Tumor Research Center,
USA
KRISHNA MOHAN POLURI • Department of Biotechnology, Indian Institute of Technology
Roorkee, Roorkee, Uttarakhand, India
VITOR H. POMIN • Department of Biomolecular Sciences, Research Institute of
Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, Oxford, MS, USA
FABIENNE E. POULAIN • Department of Biological Sciences, University of South Carolina,
Columbia, SC, USA
KRISTIAN PRYDZ • Department of Biosciences, University of Oslo, Oslo, Norway
xx Contributors
KRISHNA RAJARATHNAM • Department of Biochemistry and Molecular Biology, The University

of Texas Medical Branch, Galveston, TX, USA; Sealy Center for Structural Biology and
Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX, USA
GURUSANKAR RAMAMOORTHY • Departments of Biology, Bioengineering, and Medicinal
KARTHIK RAMAN • Department of Bioengineering, University of Utah, Salt Lake City, UT,
USA
JÖRG RÖSGEN • Department of Biochemistry and Molecular Biology, Penn State College of
Medicine, Hershey, PA, USA
ALESSANDRO ROSSI • Department of Biology, University of Utah, Salt Lake City, UT, USA;
ANINDITA ROY • Departments of Biology, Bioengineering, and Medicinal Chemistry,
NARAYANA MURTHY SABBAVARAPU • Department of Biomolecular Systems, Max Planck
Institute of Colloids and Interfaces, Potsdam, Germany
RABIA SADIR • Université Grenoble Alpes, Centre National de la Recherche Scientifique, and
Commissariat `a l’Énergie Atomique et aux Énergies Alternatives, Institut de Biologie
YUKIO SAIJOH • Department of Neurobiology, & Department of Nutrition & Integrative
Physiology, University of Utah, Salt Lake City, UT, USA
NEHRU VIJI SANKARANARAYANAN • Department of Medicinal Chemistry & Institute for
Structural Biology, Drug Discovery and Development, Virginia Commonwealth
University, Richmond, VA, USA
RAM SASISEKHARAN • Department of Biological Engineering, Koch Institute of Integrative
Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
NANCY B. SCHWARTZ • Department of Pediatrics, University of Chicago, Chicago, IL, USA;
Cellarity, Cambridge, MA, USA
PETER H. SEEBERGER • Department of Biomolecular Systems, Max Planck Institute of Colloids
and Interfaces, Potsdam, Germany; Department of Chemistry and Biochemistry, Freie
Universit€at Berlin, Berlin, Germany
KRISHNA MOHAN SEPURU • Departments of Biochemistry and Molecular Biology, Sealy Center
for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch,
Galveston, TX, USA
JOSHUA S. SHARP • Department of BioMolecular Sciences, University of Mississippi, Oxford,
MS, USA; Department of Chemistry and Biochemistry, University of Mississippi, Oxford,
MS, USA
ZACHARY SHRIVER • Department of Biological Engineering, Koch Institute of Integrative
Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
CARINA SIHLBOM • Proteomics Core Facility, Sahlgrenska Academy, University of
Gothenburg, Gothenburg, Sweden
CHARMAINE SIMEONOVIC • Diabetes/Transplantation Immunobiology Laboratory,
Department of Immunology and Infectious Disease, The John Curtin School of Medical
Research, The Australian National University, Canberra, ACT, Australia
XUEHONG SONG • Department of Molecular Pharmacology & Physiology, Morsani College of
DAEUN SUNG • Departments of Biology, Bioengineering and Medicinal Chemistry,
Contributors xxi
J. DAVID SYMONS • Department of Nutrition and Integrative Physiology, Molecular Medicine

Program, University of Utah, Salt Lake City, UT, USA; Division of Endocrinology,
Metabolism, and Diabetes, University of Utah, Salt Lake City, UT, USA
MASAHIKO TAKEMURA • Department of Genetics, Cell Biology and Development, University of
FELIPE C. O. B. TEIXEIRA • Laboratorio de Bioquı́mica e Biologia Celular de
Glicoconjugados, Programa de Glicobiologia – Instituto de Bioquı́mica Médica Leopoldo de
Meis, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, Brazil
KISHAN THAMBU • Departments of Biology, Bioengineering, and Medicinal Chemistry,
VY MY TRAN • Department of Neurological Surgery, Brain Tumor Research Center,
USA
EMIL TYKESSON • Department of Experimental Medical Science, Division of Neuroscience,
Glycobiology Group, Lund University, Lund, Sweden
DAVIDE VIGETTI • Department of Surgical and Morphological Sciences, University of
MANUELA VIOLA • Department of Surgical and Morphological Sciences, University of
ROMAIN R. VIVÈS • Université Grenoble Alpes, Centre National de la Recherche Scientifique,
ANNA WADE • Department of Neurological Surgery, Brain Tumor Research Center,
USA
LIANCHUN WANG • Department of Molecular Pharmacology & Physiology, Morsani College of
THOMAS N. WIGHT • Matrix Biology Program, Benaroya Research Institute at Virginia
ROBERT V. WILLIAMS • Department of Chemistry, University of Georgia, Athens, GA, USA
VICTOR V. XYLOPHONE • Department of Medicinal Chemistry, University of Utah, Salt Lake
City, UT, USA
TOMIO YABE • Department of Applied Life Science, Faculty of Applied Biological Sciences,
Gifu University, Gifu, Japan; Center for Highly Advanced Integration of Nano and Life
Sciences (G-CHAIN), Gifu University, Gifu, Gifu, Japan
LIJUAN ZHANG • Systems Biology and Medicine Center for Complex Diseases, Center for
Clinical Research, The Affiliated Hospital of Qingdao University, Qingdao, China
MENG ZHANG • Systems Biology and Medicine Center for Complex Diseases, Center for
Clinical Research, The Affiliated Hospital of Qingdao University, Qingdao, China
YIRAN ZHANG • Systems Biology and Medicine Center for Complex Diseases, Affiliated
Hospital of Qingdao University, Qingdao, China
Part I
GAG Structures
Chapter 1
One-Pot Enzymatic Synthesis of Heparin

from N-Sulfoheparosan
Li Fu and Robert J. Linhardt
Abstract
Heparin, a glycosaminoglycan-based anticoagulant drug, is prepared as an extract of animal tissues.
Heparosan, an Escherichia coli (E. coli) K5 capsular polysaccharide with the structure !4)-β-D-glucuronic
acid (1 ! 4)-β-D-N-acetylglucosamine (1!, corresponds to the precursor backbone in the Golgi-based
biosynthesis of heparin. Anticoagulant heparin is prepared in a one-pot synthesis using a chemically
prepared derivative of heparosan called N-sulfoheparosan (NSH), recombinant Golgi enzymes expressed
in E. coli, and the 3-phosphoadenosine-5-phosphosulfate (PAPS) cofactor.
Key words Heparin, Heparan sulfate, Heparosan, Enzymes, 3-Phosphoadenosine-5-phosphosulfate,

Biosynthesis
1 Introduction
Heparin (Fig. 1), a glycosaminoglycan-based anticoagulant drug, is

prepared as an extract of animal tissues, including beef lung and pig,
beef, and sheep intestine [1]. Heparin, discovered over 100 years
ago [2], is a widely used essential drug for the practice of modern
medicine [3]. Since heparin is prepared from the tissues of food
animals, its production involves both the food and drug chains and
thus is difficult to control and regulate [4]. In 2007–2008, as a
result of a regulatory failure, pharmaceutical heparin products were
contaminated with an inexpensive adulterant, oversulfated chon-
droitin sulfate (OSCS), leading to a number of patient deaths
[5]. While the control and regulation of heparin production from
animal tissues have been improved by upgraded pharmacopoeial
monographs, regular inspections of manufacturing facilities, and
improved record keeping [6], there remain concerns over the safety
and availability of these critically important drugs [4].
The biosynthesis of heparin in the Golgi of animal cells has
been extensively studied over the past 50 years [7]. A number of
laboratories have cloned and expressed many of the Golgi enzymes
Kuberan Balagurunathan et al. (eds.), Glycosaminoglycans: Methods and Protocols, Methods in Molecular Biology, vol. 2303,
https://doi.org/10.1007/978-1-0716-1398-6_1, © Springer Science+Business Media, LLC, part of Springer Nature 2022
3
4 Li Fu and Robert J. Linhardt
Fig. 1 One-pot synthesis of heparin. (a) Conversion of heparosan to NSH. (b) One-pot conversion of NSH to
anticoagulant heparin with cofactor recycling
responsible for heparin biosynthesis [8–10]. These enzymes, which

are commonly expressed in Escherichia coli (E. coli) as truncated
forms and/or fusion proteins to improve their solubility, stability,
and catalytic properties [11], have been used as useful tools in
understanding Golgi function and more recently in heparin and
heparin oligosaccharide synthesis [12]. There are multiple
approaches at different stages of development for using recombi-
nant heparin biosynthetic enzymes to prepare heparins and heparin
oligosaccharides. Two such approaches, metabolic engineering,
involving the cell-based synthesis of heparin [13], and iterative
chemoenzymatic synthesis [14], involving the stepwise assembly
of heparin oligosaccharides using glycosyltransferases and activated
sugar donors along with polysaccharide modifying enzymes (i.e.,
sulfotransferases and epimerases), are not described in this methods
chapter.
This method chapter describes in detail a simple one-pot
approach for the enzymatic conversion of a polysaccharide sub-
strate, N-sulfoheparosan (NSH), to anticoagulant heparin (Fig. 1)
[15]. NSH is prepared from heparosan (with the structure
!4)-β-D-glucuronic acid (1 ! 4)-β-D-N-acetylglucosamine
(1!, which has an average molecular weight of 50–150 kDa) and
the capsular polysaccharide of E. coli K5 obtained from the super-
natant of its fermentation on either glucose or glycerol (Fig. 1)
[16]. Treatment of heparosan with sodium hydroxide and
One-Pot Enzymatic Synthesis of Heparin from N-Sulfoheparosan 5
Table 1
Enzymes used in the one-pot synthesis of heparin
Enzyme Source species Truncation Fusion protein or tag Reference

C5Epi Human – Transmembrane domain Mannose binding protein [18]
2OST Chinese hamster – Transmembrane domain Mannose binding protein [18]
6OST-1 Mouse – Transmembrane domain Mannose binding protein [19]
6OST-3 Mouse – Transmembrane domain Mannose binding protein [20]
3-OST-1 Mouse – Transmembrane domain His-tag [21]
AST-IV Rat – None His-tag Unpublished
N-sulfonation with triethylamine-sulfur trioxide complex affords

NSH with the structure !4)-β-D-glucuronic acid (1 ! 4)-β-D-N-
sulfoglucosamine (1!, which contains some residual N-acetylglu-
cosamine residues (2–15%) and an average molecular weight of
10–20 kDa (Fig. 1) [17].
In this one-pot synthesis, NSH is treated with recombinant
Golgi enzymes, 2-O-sulfotransferase (2OST), C5-epimerase
(C5epi), 6-O-sulfotransferase-1 and -3 (6OST-1 and 6OST-3),
and 3-O-sulfotransferase (3-OST-1), which are all expressed in
E. coli (Table 1), as well as a sub-stoichiometric amount of 3-phos-
phoadenosine-5-phosphosulfate (PAPS) together with a cofactor
regenerating system consisting of p-nitrophenylsulfate (PNPS) and
arylsulfotransferase-IV (AST-IV) (Fig. 1) [15]. The resulting hepa-
rin product has a specific activity of >100 units/mg based on either
anticoagulation assay or amidolytic assays.
2 Materials
2.1 Combinational 1. 500 mM MES buffer: 500 mM MES, pH 7.0. Weigh 0.39 g
One-Pot Enzymatic 2-(N-morpholino)ethanesulfonic acid (MES hydrate) and
Reaction transfer into a 4 mL centrifuge tube. Add 3 mL DI water to
dissolve completely. Adjust pH to 7.0 using 2% NaOH and
bring the volume up to 4 mL.
2. 50 mM MES buffer: 50 mM MES, pH 7.0. Mix 1 mL of
500 mM MES buffer into 9 mL DI water. Check pH and adjust
pH to 7.0 if necessary, using HCl or NaOH. Store at 4 C.
3. Solution S: Weigh 2 mg of purified NSH (see Note 1) and
dissolve into 2 mL of 50 mM of MES buffer in a 50 mL
Eppendorf centrifuge tube.
4. 3 mM PAPS: 3 mM PAPS. Weigh 1.5 mg of PAPS and dissolve
into 1 mL DI water completely. Store at 4 C.
5. 50 mM PNPS: 50 mM PNPS. Weigh 51.5 mg of PNPS and

dissolve it into 4 mL DI water completely. Store at 4 C.
6. Purified C5 epimerase (C5-epi), aryl sulfotransferase (AST) IV,
and sulfotransferases (2-OST, 6-OST-1, 6-OST-3, and
3-OST-1): All enzymes (Table 1) are concentrated to at least
5 mg/mL before use. All enzymes are at 5 mg/mL unless
otherwise specified (see Note 2).
2.2 Strong Anion 1. 3 kDa molecular weight cut-off (MWCO) centrifugal unit
Exchange (SAX) (Amicon centrifugal filter units, Millipore).
Purification 2. Strong ion exchange spin column: 20 mL Vivapure Ion
of Bioengineered Exchange Q-Column (Sartorius).
Heparin 3. SAX calibration buffer: 50 mM NaCl. Dilute 2 mL of SAX
washing buffer in 18 mL of DI water.
4. SAX washing buffer: 500 mM NaCl. Dilute 10 mL of SAX
elution buffer in 30 mL of DI water.
5. SAX elution buffer: 2 M NaCl. Weigh 11.69 g NaCl and
dissolve into 100 mL of DI water.
2.3 Enzymatic 1. Ammonium acetate buffer: 50 mM ammonium acetate, pH

Digestion 7.4.
2. Heparin lyases I, II, and III [22]: 2 mU/μL separately prepared
in ammonium acetate buffer (see Note 3).
3. YM-10 micro-concentrator (Millipore).
2.4 LC-MS 1. Agilent 1200 LC/MSD instrument (Agilent Technologies,

Inc. Wilmington, DE) equipped with a 6300 ion trap and a
binary pump followed by a UV detector equipped with a high-
pressure cell.
2. Poroshell 120 C18 column (2.1 100 mm, 2.7 m,
Agilent, USA).
3. LC-MS Eluent A: 12 mM tributylamine (TBA), 38 mM
ammonium acetate (NH4Ac) in water/acetonitrile (85:15,
v/v), pH 8.5. Dissolve 2.22 g TBA and 2.93 g NH4Ac
completely in 800 mL DI water. Adjust pH to 8.5 using acetic
acid. Bring up volume to 850 mL. Add 150 mL acetonitrile.
Adjust pH back to 8.5 if necessary. Store at 4 C.
4. Disaccharide and tetrasaccharide standards for LC-MS system
are prepared to concentrations of 0.1, 0.5, 1, 5, 10, 20, 50, and
100 ng/μL.
5. LC-MS Eluent B: 12 mM TBA, 38 mM NH4Ac in water/
acetonitrile (35:65, v/v), pH 8.5. Dissolve 2.22 g TBA and
2.93 NH4Ac completely in 300 mL DI water. Adjust pH to 8.5
using acetic acid. Bring up volume to 350 mL. Add 650 mL
acetonitrile. Adjust pH back to 8.5 if necessary. Store at 4 C.
2.5 NMR 1. D2O: 99.996% D2O (Sigma).

Spectroscopy 2. NMR micro tubes, OD 5 mm (Norell).
3. Bruker Advance II 600 MHz spectrometer (Bruker BioSpin).
4. Topsin software (Bruker Topspin).
2.6 Molecular Weight 1. HPLC system consisting of an LC-10Ai pump, a CBM-20A

Determination controller, and an RID-10A refractive index detector
(Shimadzu).
2. GPC mobile phase: 0.1 M NaNO3. Dissolve 17 g NaNO3 in
2 L of DI water. Store at 4 C.
3. TSK-GEL G3000PWxl size exclusion column (Tosoh
Bioscience).
4. Column heater (Eppendorf).
5. LC solution Version 1.25 software (Shimadzu).
6. USP heparin sodium Mw calibrant and system suitability solu-
tion (US Pharmacopeia).
2.7 In Vitro 1. BIOPHEN ANTI-IIa and ANTI-Xa (Two-Stage Heparin

Anticoagulant Activity Assay) kits (Aniara).
Measurement
3 Methods
3.1 Combinational 1. Add 4 mL of purified 2OST-1, C5-epi, and AST-IV each into
One-Pot Enzymatic solution S.
Reaction 2. Add 0.4 mL of purified 6OST-1, 6OST-3, and 3OST-1 into
solution S.
3. Add 2 mL of 3 mM PAPS and 50 mM PNPS each into
solution S.
4. Bring up the final volume to 20 mL, gently mix, transfer the
tube into 37 C immediately, and incubate overnight.
3.2 SAX Purification 1. After an overnight one-pot enzymatic reaction, boil the sample
of Bioengineered for 10 min, cooldown to room temperature, and filter through
Heparin 0.22 μm membrane.
2. Wash a 20 mL 3 kDa MWCO centrifugal unit with DI water to
remove glycerol.
3. Desalt the clarified permeate by loading into washed centrifu-
gal unit and centrifuge at 5000 g for 20 min, bring up the
volume back to 20 mL with DI water. Repeat the centrifuga-
tion steps 3–5 times to remove the salt and other low molecu-
lar weight substances.
4. Wash a strong ion exchange spin column with SAX calibration

buffer by centrifuging at 500 g for 3–5 min (see Note 4) to
remove glycerol and equilibrate the column.
5. Load the desalted polysaccharide solution into the washed
column and centrifuge at 500 g for 3–5 min until the
solution flows through completely. Repeat the centrifugation
step with 10 mL of SAX washing buffer to remove protein or
peptide.
6. Elute bioengineered heparin with 5 mL of SAX elution buffer.
Collect the elution and desalt using 3 kDa MWCO spin col-
umn as step 2 in Subheading 3.2.
7. Weigh a 2 mL Eppendorf centrifuge tube and determine its tare
weight in mg. Collect the purified bioengineered heparin using
the weighed tube and freeze-dry.
8. After the sample is completely dried, weigh the tube containing
the dried sample and subtract the tare weight to determine the
mass of recovered heparin.
9. Add DI water to dissolve heparin to make 10 μg/μL solution as
a test sample for further analysis.
3.3 Enzymatic 1. Add 1 μL of the test sample into a 1.5 mL tube, add 5 μL
Digestion (10 mU) each of heparin lyase I, II, and III into the test sample,
bring up the volume to 100 μL using ammonium acetate
3.3.1 Disaccharide
buffer, and incubate at 35 C for 10 h to degrade heparin
Analysis
sample completely [23].
2. Load the resulting disaccharide solution into a YM-10 micro-
concentrator and centrifuge at 10,000 g for 10 min. Collect
the permeation, which contains disaccharide, and freeze-dry.
3. Dissolve the digested heparin disaccharides in DI water to a
concentration of 25–50 ng/μL for LC-MS analysis.
3.3.2 Tetrasaccharide 1. Add 5–10 μL of the test sample and 20 μL (40 mU) of heparin
Mapping lyase II into a 1.5 mL tube, bring up the volume to 100 μL with
ammonium acetate buffer, and incubate at 35 C for 10 h to
degrade heparin sample completely [23].
2. Freeze-dry the resulting tetrasaccharide for further LC-MS
analysis [24].
3.4 Analysis 1. Set the electrospray interface for LC-MS system in negative
Using LC-MS ionization mode with a skimmer potential of 40.0 V, a capil-
lary exit of 40.0 V, and a source temperature of 350 C, to
3.4.1 Disaccharide
obtain the maximum abundance of the ions in a full-scan
Analysis
spectrum (200–1500 Da). Use nitrogen (8 L/min, 40 psi) as
a drying and nebulizing gas.
2. Calibrate the LC-MS system with Eluent A. For sample testing,

set the gradient as solution A for 5 min, followed by 0–40%
linear gradient Eluent B from 5–15 min. Set flow rate as
150 μL/min.
3. Inject disaccharide standards first to make quantification cali-
bration curves. Linearity was assessed based on the amount of
di- or tetrasaccharide and the peak intensity in extracted ion
chromatogram (EIC).
4. Inject the digested test sample (disaccharides) and quantify
based on the established calibration curves.
3.4.2 Tetrasaccharide 1. Use the same LC-MS system as disaccharide analysis (step 1 in
Mapping Subheading 3.5) for tetrasaccharide analysis.
2. Calibrate the LC-MS system with Eluent A. For sample testing,
the gradient is set as solution A for 2 min, followed by 0–30%
linear gradient Eluent B from 2 to 40 min. Set flow rate as
150 μL/min.
3. Inject tetrasaccharide standards first to make quantification
calibration curves. Linearity was assessed based on the amount
of tetrasaccharide and the peak intensity in extracted ion chro-
matogram (EIC).
4. Inject the digested test sample (tetrasaccharides) and quantify
based on the established calibration curves.
3.5 NMR 1. Take about 100 μL (~1 mg) of a test sample and freeze-dry.
Spectroscopy 2. Redissolve the dried test sample in 100–200 μL D2O.
3. Repeat above deuterium exchange (steps 1 and 2) twice to
remove most H2O from the test sample.
4. Dissolve deuterium exchanged test samples into 450 μL of
D2O and transfer the solution to NMR micro tubes for NMR
analysis.
5. Tune and shim the Bruker Advance II 600 MHz spectrometer
to have optimal conditions.
(a) 1D 1H-NMR experiment conditions: wobble sweep
width of 12.3 kHz, acquisition time of 2.66 s, and relaxa-
tion delay of 8 s at 298 K.
(b) 2D 1H-13C HSQC experiment conditions: 32 scans,
sweep width of 6.15 kHz, acquisition time of 0.33 s, and
relaxation delay of 0.90 s.
6. Process the NMR data using Topsin software.
3.6 Molecular Weight 1. Set a sample injection volume of 20 μL and a flow rate of
Determination 0.6 mL/min in the HPLC system.
2. Maintain TSK-GEL G3000PWxl size exclusion column at
40 C with a column heater.
3. Record the SEC chromatograms with the LC solution Version

1.25 software and determine molecular weight properties
using the “GPC Postrun” function.
4. Inject and analyze USP heparin sodium Mw calibrant and
system suitability solution to confirm the system suitability.
5. Inject the test sample and calculate the molecular weight based
on the calibration of the USP calibrant.
3.7 In Vitro 1. Analyze the anti-IIa and anti-Xa activities using BIOPHEN
Anticoagulant Activity ANTI-IIa and ANTI-Xa (Two-Stage Heparin Assay) kits fol-
Measurement lowing the instruction provided.
4 Notes
1. Purified NSH [17] is freeze-dried before use.

2. The sulfotransferases should be assayed prior to use with a
chomogenic assay using NSH and PAPS (coupled with PNPS
and AST-IV) [25]. The C5-epimerase should be assayed using
chomogenic assay using NSH, 2OST, and PAPS (coupled with
PNPS and AST-IV).
3. The heparin lyases should be assayed prior to use with heparin
or heparan sulfate as a substrate by measuring a change in
absorbance at 232 nm [26].
4. Usually, 10 mL of solution can be all flown through the strong
ion exchange spin column. Extend the centrifugation time if
there is still a solution on top of the resin.
Acknowledgments
The authors are grateful for funding from the National Institutes of
Health in the form of grants # DK111958 and CA231074.
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Chapter 2
Solution NMR Spectroscopy for Characterizing

Protein–Glycosaminoglycan Interactions
Prem Raj B. Joseph, Krishna Mohan Sepuru, Krishna Mohan Poluri,
Abstract
Solution nuclear magnetic resonance (NMR) spectroscopy and, in particular, chemical shift perturbation
(CSP) titration experiments are ideally suited for mapping and characterizing the binding interface of
macromolecular complexes. 1H-15N-HSQC-based CSP studies have become the method of choice due to
their simplicity, short-time requirements, and minimal working knowledge of NMR. CSP studies for
characterizing protein–glycosaminoglycan (GAG) interactions can be challenging due to binding-induced
aggregation/precipitation and/or poor quality data. In this chapter, we discuss how optimizing experi-
mental conditions such as protein concentration, choice of buffer pH, ionic strength, and GAG size, as well
as sensitivity of NMR instrumentation can overcome these roadblocks to obtain meaningful structural
insights into protein–GAG interactions.
Key words Nuclear magnetic resonance (NMR), Chemical shift perturbation, Protein–ligand inter-
actions, Glycosaminoglycan, Dissociation constant, Heparan sulfate, Heparin
1 Introduction
Crosstalk between macromolecules both in the intracellular and

extracellular milieu orchestrate all cellular processes and under-
standing the structural basis and molecular mechanisms that dictate
the specificity and affinity requires a detailed atomistic description
of the complexes. Nuclear magnetic resonance (NMR) spectros-
copy and X-ray crystallography are both routinely used for structure
determination of macromolecular complexes. However, NMR is
the only alternative available when complexes fail to crystallize,
which could be the case if one or both of the partners are dynamic
and/or if the interactions are weak in nature.
Glycosaminoglycans, such as heparin and heparan sulfate (HS),
chondroitin sulfate (CS), and dermatan sulfate (DS), are highly
negatively charged linear polysaccharides that are widely expressed
by most cell types and are also present in the extracellular milieu.
13
14 Prem Raj B. Joseph et al.
They mediate a wide variety of crucial functions due to their ability

to bind and regulate the activities of large classes of proteins
[1, 2]. We refer the reader to other chapters for a more detailed
description of GAG structures and properties. Remarkably very
little is known regarding the structural basis of how GAGs interact
with proteins and mediate function. The challenges that have
plagued both NMR and X-ray studies can be attributed to multiple
interrelated factors that include binding-induced protein aggrega-
tion/precipitation, especially at the high concentrations used in
structural studies, the flexibility and high negative charge of
GAGs, intrinsic heterogeneity and GAG size, and the dynamic
nature of the protein–GAG interface.
In this chapter, we outline how NMR chemical shift perturba-
tion (CSP) methods can be effectively used to study protein–GAG
interactions. The popularity of this method is due to the high
sensitivity and robustness of 2D 1H-15N HSQC experiments.
Chemical shifts are exquisitely sensitive to binding-induced
changes in the local electronic environment and can provide
residue-level details of the binding interface, binding affinities,
and timescales of protein–ligand interactions. The CSP information
together with molecular docking tools like HADDOCK can also
provide structural models of the protein–ligand complexes [3].
CSP-based NMR experiments have been routinely used for
studying protein binding to other proteins, peptides, DNA, and
small molecule ligands, but have been challenging for characteriz-
ing protein–GAG interactions. In addition to the other challenges
described above, other reasons could be nonoptimal sample and
experimental conditions. In this chapter, we discuss different fac-
tors that must be taken into consideration in the experimental
design and, in particular, the importance of protein concentration,
choice of buffer, pH, ionic strength, GAG size, sensitivity of NMR
instruments, and do’s and don’ts for obtaining quality NMR
data (see Notes 1–6). Our guidelines are based on our experience
of characterizing heparin and, more recently, HS, CS, and DS
oligosaccharides binding to neutrophil-activating chemokines [4–
12]. Structure function and animal model studies have shown that
gradients formed by chemokines bound to the cell surface, and
soluble GAGs direct and regulate neutrophil trafficking from the
blood to the tissue [13].
An HSQC spectrum provides a fingerprint of the backbone
amides of a protein. Each backbone amide is represented by a
peak corresponding to the chemical shifts of the 1H (x-axis) and
15
N (y-axis) nuclei. The total number of peaks corresponds to the
number of amino acids in the protein chain excluding prolines
(which do not have a backbone amide NH) plus those
corresponding to side chain NH2 of asparagine and glutamine
residues. In general, chemical shifts are available from literature or
the BMRB data bank (http://www.bmrb.wisc.edu); if not, the
Solution NMR Spectroscopy for Characterizing Protein–Glycosaminoglycan. . . 15
chemical shifts must be assigned using established NMR

procedures.
An overlay of a series of HSQC spectra of a heparin oligosac-
charide titration to a 15N-labeled chemokine, and the 2D histo-
gram plot of the weighted average of 1H and 15N chemical shift
changes ((Δδ ¼ [ΔδH2 + (ΔδN/5)2]1/2) as a function of individual
residues, are shown in Fig. 1a, c, respectively. The data show
selective perturbation of a subset of protein peaks indicating specific
binding. GAGs are acidic, and GAG-binding proteins are basic or
contain clusters enriched in basic residues. Not surprisingly, NMR
studies show CSP for lysines (Lys), arginines (Arg), and, occasion-
ally, histidines (His). It is generally assumed that residues showing
the largest CSP are involved in the binding process. However, CSP
changes are correlative and not necessarily causative of the binding
process. Therefore, it is possible that not all of the interfacial
residues will show significant CSP upon binding. Considering
that binding is mediated via lysine NH2 and arginine guanidinium
groups, chemical shifts of some of the binding-interface Lys/Arg
amides may not be sensitive enough due to the long intervening
side chain. Similarly, changes in chemical shift may also occur due to
indirect binding, arising from structural rearrangements or changes
in packing interactions remote from the binding interface. There-
fore, it is important that the CSP data be interpreted cautiously. If
the structure of the protein is known, a residue level description of
the binding site can be obtained (Fig. 1d). Analysis of the protein
structure also helps in teasing out direct interactions from indirect
interactions. Binding affinities can be obtained from the CSP of the
individual residues as described previously (Fig. 1b) [14, 15].
We have recently shown that the chemical shifts of Lys, Arg,
and His side chains are sensitive probes for identifying binding
interface residues [16–18] (Fig. 2). Assigning Lys and His side
chain chemical shifts require additional experiments, and especially
challenging for lysines because of their rapid exchange with the
bulk solvent. However, we have shown such experiments are feasi-
ble and can unambiguously identify binding interface residues and
provide novel insights that are not possible from any other techni-
ques [16–18].
2 Materials
2.1 Protein NMR experimental conditions that enable titrations at low protein
Concentration concentrations will substantially increase the probability of acquir-
and NMR Titrations ing quality data, considering high concentration can lead to aggre-
gation/precipitation. Availability of high-field NMR instruments
equipped with cryoprobes and gradient accessories has significantly
improved the scope and complexity of experiments including the
ability to work at low protein concentrations. For instance, we have
Fig. 1 Mapping of chemokine-heparin oligosaccharide binding interface using NMR chemical shift pertubation
data. (a) A section of a 1H-15N HSQC spectrum showing heparin oligosaccharide binding-induced chemical
shift changes in a chemokine. The unbound and final bound peaks are in black and red and the intermediate
NH – CαH – CβH2 – CγH2 – CδH2 – NεH – Cζ = NηH2+

A NηH2
WT R26 K64R R26 C

84.5
R60 R64 160
Nε2-Hδ2
Nε1-Hε1
R6B R60
85.5 R6B
R6 R6 Nε1-Hε1 Nε2-Hδ2
(ppm)
R47
180
R47
86.5
15Nε
K20R/K64R R26 K64RK67R R26

84.5
R64 R64 200
R20
R60
(ppm)
R6B H18
R6B R60 R67
85.5
R6 R6
15N
R47 R47 220
86.5
H33 δ1N δ2
8.5 8.1 7.7 7.3 6.9 8.5 8.1 7.7 7.3 6.9
ΝΗ
1Hε (ppm) ε1 ε2
N2 tautomer
240 Nδ1-Hε1
B
NH – CαH – CβH2 – CγH2 – CδH2 – CεH2 – NζH3+
Nδ1-Hε1
32.0
K21 260
K21 8.0 7.5 7.0
1H (ppm)
(ppm)
32.5 K65
K45
K49
15N
K61 K60
33.0
K29
33.5
7.5 7.0 7.5 7.0
1H (ppm)
Fig. 2 (a) Overlay of the Nε-Hε of selective 1H-15N HISQC spectra of Arg side chains in the free (black) and
heparin-bound (red) forms in different CXCL8 mutants. Arrows represent the connection between free and
bound peaks. A schematic of Arg side chain and NεH that is detected in the spectra is encircled. (b) The 1H-15N
HISQC spectra of CXCL1 Lys side chains in the free (left) and heparin-bound (right) forms. A schematic of Lys
side chain and NH3+ that is detected in the spectra is encircled. (c) Overlay of 1H-15N HMQC spectra of His side
chains in the free (black) and heparin-bound (red) forms. The Nε2 tautamer of His side chain is shown in the
inset.The spectra were adapted from refs. 16–18
Fig. 1 (continued) peaks are shown in cyan, blue, and green. Note the selective perturbation of a subset of
amino acids indicated by an arrow. (b) A representative plot of CSP vs. GAG/chemokine concentration (in molar
ratio), which allows calculation of the dissociation constant (Kd). (c) A histogram of the CSP as a function of
chemokine sequence. Dotted line indicates the cutoff for residues to be considered perturbed. (d) A surface
plot of the chemokine showing residues (blue) that are significantly perturbed on GAG binding
acquired an HSQC spectrum of a ~100 μM chemokine sample

(MW ~16 kDa) in 10 min on an 800 MHz spectrometer equipped
with cryoprobe and field gradient accessories. Time required can be
reduced by a factor of ~2 when using higher field instruments
(800 vs. 600 or 500 MHz) and by a factor ~8 when using an
NMR instrument with and without a cryoprobe. However, the
sensitivity of a cryoprobe is dependent on ionic strength, with low
salt conditions providing the best sensitivity. In terms of protein
concentration, reducing the concentration by a factor 2 will
increase the time requirement by factor of 4. Therefore, time
requirement for a 50 μM compared to a 200 μM sample will be
16 times higher to achieve a similar signal to noise (s/n).
For some proteins, binding studies may be feasible only at
concentrations as low as ~10 μM. It is still possible to collect an
HSQC spectrum of a ~10 μM sample in less than 12 h on an
800 MHz spectrometer with a cryoprobe. As several titrations
may not be practical and cost effective, we suggest collecting the
spectrum of the unbound protein at higher concentrations and a
few spectra of the GAG-bound protein at lower concentrations.
Ideally, these data can identify which residues mediate binding and
allow describing the geometry of the binding interface. However,
chemical shift assignments of all the residues in the GAG-bound
form may not be possible, especially if the interface residues
undergo significant binding-induced chemical shift changes. In
this case, additional titration experiments must be performed to
better define the binding-interface residues. We also suggest, if
possible, characterizing the protein–GAG complexes using other
biophysical techniques such as dynamic light scattering (DLS) and
sedimentation velocity under similar sample conditions to gain
insights into the relationship between protein concentration and
aggregation state of the complexes before carrying out the NMR
experiments.
2.2 Choice of GAG NMR studies are generally carried out using commercially available
for NMR Titrations size-defined heparin oligosaccharides. Therefore, we discuss our
strategy on the basis of our experience using heparin oligosacchar-
ides. Our discussion is applicable to heparan sulfate and all other
GAGs. Most biophysical and structural studies reported in litera-
ture have also used unfractionated or size-fractionated heparin
oligosaccharides. Heparin oligosaccharides are available in various
sizes from a disaccharide to a 26mer from specialized vendors like
Iduron.
2.3 NMR Sample The protein must be isotopically labeled with 15N for characterizing
Preparation binding interactions using 1H-15N HSQC titrations. Isotopically
labeled proteins are overexpressed in E. coli grown in minimal
media using 15NH4Cl as the sole nitrogen source. Since the growth
characteristics of E. coli in minimal media is somewhat
compromised compared to growth in rich media (LB media),

growing larger cultures is necessary to produce sufficient quantities
of protein. In recent years, adding labeled growth supplements
(available from vendors such as Cambridge Isotopes and Spectra
Isotopes) circumvents this problem by increasing cell growth rates
and overall protein expression. Once the protein is purified, the
purity and molecular weight are confirmed using mass spectrome-
try. Sample purity is important because contaminations could com-
plicate interpreting real signals from spurious noise peaks.
Protein samples for NMR studies are typically ~500 μl in vol-
ume, containing ~5–10% D2O for spectrometer frequency lock,
1 mM 2,2-dimethyl-2-silapentanesulfonic acid (DSS) for spectral
referencing, and 1 mM sodium azide (NaN3) to prevent microbial
growth. Sample temperature for NMR data collection typically is
between 20 and 40 C and can vary depending on the protein.
The choice of buffer can influence the quality of the
GAG-bound spectra (see Note 1). If the initial choice of buffer
results in poor quality, we suggest collecting spectra at different
buffers, pH, and ionic strength. In our experience, significant line
broadening could occur under nonoptimal pH conditions.
It is also important to ensure that the protein and GAG samples
are prepared in the same buffer to minimize chemical shift changes
due to pH changes which could complicate and, in worst case
scenario, lead to wrong interpretation of the binding interactions.
If necessary, dialyze the protein and ligand in the same buffer.
Alternatively, the lyophilized powders can be dissolved in the
NMR buffer of interest, but the pH of the samples needs to be
checked and adjusted before proceeding with the experiments.
3 Methods
3.1 Experimental Prior knowledge of the protein, including behavior of the protein in
Design solution, dimerization and oligomerization properties (including
Kd), its GAG-binding properties including binding-induced oligo-
merization and precipitation issues would be useful. Using the right
protein concentration is a critical parameter for successful titration.
A major problem is binding-induced precipitation, especially at
high protein concentrations typically used in NMR studies. We
propose an initial concentration of ~200 μM and, in the event of
precipitation, alter pH/salt conditions and/or reduce the protein
concentration. For chemokines, we carried out titrations on sam-
ples anywhere between 50 and 150 μM, and occasionally at lower
concentrations. In principle, lower concentrations will require lon-
ger data acquisition times, and therefore, whenever possible, we
strongly recommend using spectrometers with cryoprobes.
We propose that the initial experiments are carried out in low
ionic strength buffers, which could lead to stronger binding,
resulting in lower GAG requirements. If the binding data suggest

nonspecific interactions, spectra collected at varying ionic strengths
could allow teasing out specific vs. nonspecific interactions. If start-
ing protein concentrations is dependent on the oligosaccharide
length, then titrations have to be carried out with shorter oligosac-
charides or the protein concentration must be reduced sufficiently
where the complex does not aggregate or precipitate.
3.2 NMR Titrations CSP experiments involve collecting a series of HSQC spectra by
and Data Analysis adding GAG aliquots until essentially there are no binding-induced
changes in protein chemical shifts. We suggest collecting a mini-
mum of 6–8 spectra, which includes those of the free protein,
around 50% fraction bound population, and at saturation. More
data points around 50% bound population helps in better defining
the binding isotherms for accurate calculation of the dissociation
constant. Using stock solutions of ~10 mM heparin oligosacchar-
ides minimizes errors in protein concentration due to dilution.
Prior knowledge of an estimate of binding affinity can be useful in
selecting the starting protein concentration and amount of GAG to
be added.
NMR data processing and analysis can be carried out using
NMRpipe, NMRview, Sparky, or instrument-specific Bruker and
Varian software [19–21]. Most processing and analysis script and
programs for data fitting are available in the respective software
websites. Binding affinity of protein–GAG interactions can vary by
orders of magnitude (nM to mM) and, accordingly, kinetics (espe-
cially off rate, koff) can vary by many orders. The kinetics of binding
are classified as slow, intermediate, and fast exchange on the NMR
timescale, which can influence the nature and quality of the spectra
[14]. If binding occurs in the slow exchange regime, spectra will
contain separate peaks at the chemical shifts of the free (δP) and
GAG-bound (δPL) protein. As the ligand is titrated into the protein,
the peak intensity at δP will decrease and of the peak at δPL will
increase. If binding occurs in the fast exchange regime, spectra will
contain a single set of peaks at the population-weighted average
chemical shifts. As the ligand is titrated into the protein, the peak
position will move from δP to δPL. If binding is in the intermediate
exchange regime, peaks are exchange-broadened resulting in poor
quality spectra.
The CSP experiments are best performed under the fast
exchange regime as assigning chemical shifts of the GAG-bound
form is straightforward as shown in Fig. 1a, b. However, in the
intermediate and slow exchange regimes, assigning chemical shifts
of all the residues in the GAG-bound form may not be possible as
the information on the direction and magnitude of the individual
peak movements are missing. In particular, this will be the case if
the binding residues are in the crowded region of the spectrum
and/or undergo large chemical shift changes. Therefore, it may be
necessary to change experimental conditions so that the binding

occurs in the fast exchange regime by using smaller GAGs, altering
ionic strength, and/or other experimental parameters.
In the fast exchange regime, the CSP follows a hyperbolic
dependence as a function of ligand concentration and the Kd can
be fitted using nonlinear least squares analyses using two indepen-
dent variables (ligand and protein concentrations) and two depen-
dent parameters (Kd and maximum CSP) as shown in Fig. 1b
[6]. Proper choice of protein and ligand molar ratio can increase
the accuracy of the measured dissociation constant.
4 Notes
1. Initial experiments must be carried out in low ionic strength

buffers. We observed no significant differences in the perturba-
tion profile or the binding affinities between low and high salt
conditions for the binding of heparin oligosaccharides to che-
mokines, though the overall extent of perturbation was higher
in low salt buffers. Using low ionic strength buffers can result
in non-native interactions leading to aggregation and precipi-
tation. On the other hand, use of high salt buffers can lead to
screening of electrostatic interactions and weak binding.
2. Binding-induced oligomerization and precipitation effects are
highly sensitive to heparin oligosaccharide chain length and the
protein of interest. We suggest starting with shorter oligosac-
charides such as a disaccharide and then progressing to higher
oligosaccharides. For some proteins, titrations even with a
tetrasaccharide lead to precipitation.
3. Shorter oligosaccharides will be a compromise but nonetheless
can provide useful information on binding. Some limitations
include smaller chemical shift perturbations, lower binding
affinity, multiple binding modes, and nonspecific interactions.
Therefore, it may not be possible to come up with a unique
binding model, and one needs to be cautious and not over
interpret the data. On the other hand, selective perturbations
would suggest specific binding and can provide the binding
geometry. Further, studying shorter oligosaccharides can also
be exploited for designing GAG decoys that could function as
therapeutics in a clinical setting.
4. Line broadening can occur due to aggregation or due to inter-
mediate exchange binding kinetics. In the latter case, the peaks
will reappear on excess GAG titration (fractional bound popu-
lation >90%). Therefore, we suggest not to abort the experi-
ment, but to collect a few additional spectra in the presence of
excess GAG.
5. Choice of pH can be a critical factor in obtaining quality NMR

CSP data. One can collect trial HSQC spectra of a protein–
GAG sample by varying pH conditions (say pH 5.0–8.0). This
can help in arriving at the right pH condition for the titration
experiments. For some chemokines, we observe severe line
broadening below pH 7.0 for some heparin oligosaccharides
but not all.
6. When performing titration experiments, it is important that the
sample is thoroughly mixed. After collecting the first HSQC
spectrum of the free protein, the sample is transferred back to
an Eppendorf tube using a glass pipette before addition of the
ligand. After adding an aliquot of GAG (~2–10 μl), the sample
is mixed well by pipetting the solution a few times. Look for
any cloudiness or precipitation. Precipitate sticking to the sides
of the NMR tube will interfere with the shimming of the
sample. Switch sample to a new NMR tube if necessary. Shim-
ming the sample again may be necessary before starting the
next experiment. It is not uncommon for the initial cloudiness
to disappear on subsequent GAG addition. We advise not to
add GAG directly into the NMR tube, which can cause uneven
mixing leading to errors in the estimation of the protein–GAG
molar ratios.
References
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6. Sepuru KM, Nagarajan B, Desai UR, Rajarath- nam K (2018) Structural basis, stoichiometry,
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293:17817–17828 17. Sepuru KM, Iwahara J, Rajarathnam K (2018)
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sulfate, chondroitin sulfate, and dermatan sul- using NMR spectroscopy. Analyst
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Chapter 3
Metabolic Labeling of Proteoglycans and Analysis of Their

Synthesis and Sorting in Filter-Grown and Polarized
Epithelial Cells
Kristian Prydz and Ravi Adusumalli
Abstract
Studies of synthesis, turnover, and secretion of macromolecules in cell culture are carried out to address
mechanisms of cellular and physiological importance. Culture systems have been developed to mimic the
in vivo situation as much as possible. In line with this aim, epithelial and endothelial cells have been grown
on filters for more than three decades. Growing such cells on permeable support allows for nutrient uptake
via the basolateral membrane of tight epithelial monolayers, from a medium reservoir underneath the filter.
While this basolateral medium reservoir resembles the blood supply, the apical medium reservoir resembles
the organ lumen. Growing the cells in a polarized manner allows for studies of differential transport and
localization of apical and basolateral proteins and of endocytic and secretory transport at both sides of the
epithelium. Here we describe how metabolic labeling of proteoglycans (PGs) with 35S-labeled sulfate
enables analysis of synthesis of different types of PGs, with respect to size, glycosaminoglycan (GAG)
chain length, and charge. We also describe protocols for studies of intracellular PG sorting, in the apical and
basolateral direction in polarized epithelial cells, in the absence and presence of inhibitors of synthesis and
transport.
Key words Glycosaminoglycans (GAGs), Proteoglycans (PGs), Madin-Darby canine kidney (MDCK)
epithelial cells, Golgi apparatus
1 Introduction
Proteoglycans (PGs) consist of protein cores that are inserted

co-translationally into the endoplasmic reticulum (ER) lumen.
During anterograde transport through the secretory pathway,
they acquire long, linear glycosaminoglycan (GAG) chains. GAG
chains attached to PGs are classified by the nature of repeating
disaccharide units that they have in common. These disaccharide
units are modified by sulfation at various positions and to a variable
extent [1]. For example, 35S-sulfate added to the culture medium
will be incorporated into keratan sulfate (KS), chondroitin sulfate
(CS), dermatan sulfate (DS), heparan sulfate (HS), and heparin
25
26 Kristian Prydz and Ravi Adusumalli
GAG chains. The polymerization and sulfation mechanisms take

place in the Golgi apparatus, where the responsible enzymes are
localized [2].
PGs are important components of the extracellular matrix of
epithelial and endothelial cells, but some are also important as
membrane anchored molecules and as secretory products [1]. A
number of studies have been carried out to address bulk synthesis
and secretion of PGs in epithelial Madin-Darby canine kidney
(MDCK) cells [3, 4] and CaCo-2 [5] cells. Other studies have
addressed the synthesis and intracellular transport through the
secretory pathway of individual PGs, normally after stable cellular
expression of recombinant PG protein cores [6–8]. Yet other stud-
ies address xyloside-based GAG synthesis in polarized epithelia
[9, 10], while even other studies employ PGs as markers of the
apical and basolateral secretory pathways when the cells are
challenged with perturbants of cellular pathways and functions
[11–14].
2 Materials
2.1 Filters, Cell Filters of various materials and pore sizes for cell culture are com-
Lines, and Equipment mercially available. For the experiments described here are mostly
for Polarized Culture polycarbonate filters with an area of 4.7 cm2 and a pore size of
0.4 μm used (Costar Corning Transwell 3412).
The cell lines used in the experiments referred to are MDCK I,
MDCK II, and CaCo-2. The culture conditions used: MDCK I and
MDCK II: DMEM, supplemented with 5% FCS, 1% penicillin/
streptomycin (P/S); CaCo-2: DMEM, supplemented with 20%
FCS, 1% P/S, 10 μg/ml insulin, 1% nonessential amino acids.
The method of filter growth would apply to most epithelial cell
lines.
Before seeding of cells, the filters are transferred to holders in
autoclaved glass Petri dishes (Duroplan, diameter 14 cm, height
2.5–3.0 cm). When confluent monolayers have formed, the filters
are transferred back to the original six-well plates.
2.2 Metabolic 35S-sulfate (Hartmann Analytic) is added to the labeling medium

Labeling, Harvesting, at 0.2 mCi/ml. The labeling medium is a special order sulfate-free
and Cell Lysis RPMI-1640 medium (Gibco 041-90985M).
Buffers for cell lysis:
Lysis buffer A: for complete solubilization of both cell membrane
and matrix components: 4 M guanidine, 2% Triton X-100,
0.05 sodium acetate, pH 6.0.
Lysis buffer B: compatible with subsequent use of antibodies, for
instance, in immune precipitation: 1% Nonidet P-40, 50 mM
Tris, pH 7.5, 2 mM EDTA, 150 mM NaCl, and 35 μg/ml
phenylmethylsulfonyl fluoride.
Metabolic Labeling of Proteoglycans and Analysis of Their Synthesis and. . . 27
2.3 Reagents Heparitinases I, II, and III purchased from Grampian Enzymes,
for Enzymatic Aberdeen, Scotland. Stock solutions of 2 mU/μl of each enzyme
and Chemical are diluted to a final concentration of 0.01 mU/μl in a 5 mM Hepes
Degradation buffer, pH 7.0 with 1 mM CaCl2, and 50 mM NaCl, with addition
of Glycosaminoglycan of bovine serum albumin (BSA) to 0.05% before use.
(GAG) Chains Chondroitinase ABC (cABC) purchased from Amsbio, Abing-
don, UK, is diluted to a final concentration (indicated in Methods -
Subheading 3) in cABC buffer: 33 mM Tris–HCl and 33 mM
sodium acetate, pH 8.0.
Nitrous acid (HNO2) reagent for HS degradation is the super-
natant made from mixing of 0.5 M H2SO4 and 0.5 M Ba(NO2)2
followed by centrifugation.
2.4 Gel Filtration For removal of unincorporated 35S-sulfate: 4 ml columns of Sepha-

Columns dex G-50 fine (Pharmacia) made in 10 ml disposable pipettes.
Elution with water or phosphate-buffered saline (PBS).
For determination of GAG chain length: Sepharose CL-6B
(Pharmacia) or Sephadex G25 (Pharmacia), 1 40 cm columns.
Sample dissolved in 200–500 μl of elution buffer: 50 mM Tris,
0.15 M NaCl, 0.1% Triton X-100, pH 8.0 including markers of Vo
(Blue Dextran (Mw 2,800,000; 2–5 mg/ml) and Vt (potassium
chromate, 2–3 mg/ml) in each sample.
2.5 Scintillation 3 ml of Ultima Gold XR scintillation cocktail (PerkinElmer) which

Counting is compatible with high salt solutions is added to each sample in a
scintillation vial.
3 Methods
Several types of filter support for cell culture are available. Early
experiments use “homemade” equipment, where nitrocellulose
filters are autoclaved and placed in holders made for the purpose
[15]. The studies referred to in the Introduction are mostly carried
out with Costar Corning Transwell polycarbonate, fitting in
six-well plates, but other dimensions are also available. MDCK
cells require 4–6 days on filter, depending on the cell seeding
density, to establish a confluent and tight epithelial monolayer
(Fig. 1). The growth conditions chosen must allow for optimal
growth of the epithelial cells seeded onto the filters [16] (see
Note 1).
3.1 Metabolic Proteoglycans may be labeled radioactively in the protein core by

Labeling adding radioactive amino acids to the medium, in the GAG chains
of Proteoglycans (PGs) by adding radioactive sugars to the medium, or in the sulfate
groups modifying their GAG chains by adding 35S-sulfate to the
medium. The latter is the most specific, although other glycan
structures (attached to proteins and lipids) than GAGs may be
28 Kristian Prydz and Ravi Adusumalli
Tight junctions
Fig. 1 Growth of polarized epithelial cells on filter supports. A schematic representation of a confluent
epithelial cell monolayer grown on a filter in the well of a well-plate. The cells on the filter are joined by
tight junctions, which limit intercellular passage. This allows for separate harvest of apical and basolateral
media at the end of labeling experiments
sulfated, and sulfation also occurs on tyrosine residues. These sulfa-

tion reactions are catalyzed by sulfotransferases in the Golgi appa-
ratus, but also cytoplasmic molecules like the bile acid lithocholic
acid may be sulfated in tissues like liver, kidney, and intestinal
cells [17].
3.1.1 Protocol for Growth 1. (a) Plating of epithelial cell type of interest (for instance,
and Continuous Metabolic MDCK) on filter support. For Transwell filters fitting in
Labeling with 35S-Sulfate six-well plates, transfer the filters to holders in an autoclaved
glass Petri dish (diameter 14 cm, height 2.5–3.0 cm) with
90 ml DMEM, supplemented with 5% FCS and 1%
Pen-Strept. Then add 1.5–2.0 million cells in 1.6 ml of the
same medium onto each filter. Leave in incubator at 37 C with
5% CO2 until confluent (3–4 days for MDCK cells).
(b) Add 2.6 ml DMEM (as in a) to each well of the six-well
plate. Then add 1.5–2.0 million cells in 1.6 ml of the same
medium onto each filter. Change medium every day until cells
are confluent (3–4 days for MDCK cells).
2. Add 2 ml RPMI 1640 without sulfate, supplemented with 2%
fetal calf serum (FCS) and 0.3 mCi/ml 35S-sulfate to each well
of a suitable six-well plate. Transfer the filters with cells to be
labeled to the wells of the six-well plate with a forceps. Remove
the apical growth medium and replace this with 1.2 ml of the
sulfate-depleted medium before placing the filter onto the rim
of the well to avoid pressure from below. Such pressure could
lead to cell detachment. Incubate at 37 C and 5% CO2 for the
desired incubation time. Typical incubation times are 16–24 h
(see Note 2).
3. Harvest apical and basolateral media into separate tubes.
4. Centrifuge down eventual loose cells in the media (particularly
the apical media often contain some loose cells). Transfer
medium supernatants to new tubes.
Another random document with
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help it. Yet she wronged you not. She always spoke of you with
true respect And said you were my wife, she but a slave. Then you
went forward. After that she bowed To Natural law and called
herself my wife. But her proud spirit would not brook restraint,
Nor act the puppet part of Consort Queen. When I and
Sanctimonious sought to force This part undignified upon her, she
Left me and sought the refuge of her home. I claimed her back,
but Bernia’s Prince refused To yield his sister up; and so our
Church And State divorced her, made her an outcast And left, of
course, the child to be my care. Merani, you so kind, with heart so
large, Will understand and will forgive the King. Oh! sorry fate.
How long must I sham on? How long must I approve what I
detest, And be a slave? What! sign my son’s death warrant? Never!
I will not murder my own child. Thank goodness he escaped, and
yet, alas! If they should catch young Fortunatus and Arrest
Vulnar, the law will hang these men As murderers of the
policeman Grett; And I shall sign approving warrant, I, The father
of Vergli whose life they saved. Was ever man more sorely tried
than I? Oh! sorry, sorry fate to be a King.”
Enter Larrar: “Sire, there is most important news arrived.” (Reads)
“‘Three masterly arrests have just been made— Of Vulnar, Scrutus
and young Fortunatus. One of their followers turned traitor and
Betrayed the hiding place where Vergli lurked. Young Fortunatus,
though entrapped himself, Managed to send a warning to Vergli;
He and Vulnar and Scrutus stood their ground And held the
entrance to their chief’s retreat. Fearing that Vergli would refuse
to fly And leave the others to their fate, the youth Resorted to a
subterfuge, saying Vergli must meet them on the Bawn co Pagh,
Whither they were retiring. He knew well That once on Bawn co
Pagh, the citadel And fortress of Vulnar, Vergli was safe And
midst a band of men true to his cause; But for this cunning
message here detailed, Vergli would have returned to aid his
friends And been entrapped and made a prisoner. E’en as it was
the others might have fled, But had they done so would have
doomed Vergli; And so they fought it out and thus gained time,
But were at length o’ercome and captive made.’ The name of him
who worked this clever ruse By which this mountain hiding place
was found, Is Judath, who feigned fealty to their cause But turned
informer and betrayed them all.”
King Hector (aside): “Curses upon him. Black-souled son of Hell,
Monster of foul and base iniquity.” (To Larrar) “So, so, they’ve
caught the three who murdered Grett; Now will the law avenge
itself, the mob Wreak its all-fathomless resentment on The men
whom Judath has so deftly nailed. And I, yes I, must bow with
smothered love Crying within my bosom to my soul, And sign the
rights of these men to fair life Away into the black abyss of wrong.
Larrar, what piteous fate e’er made me King?”
Larrar. “Not fate, Sire. You can cast the title off And just become an
ordinary man. Children like dolls, the grown-up child likewise
Makes you its doll and pays you for your trouble. What are you,
Sire, but the paid servant of A government of nondescript
creation? You do its work and call yourself a King. I am your
servant, but you in your turn Are mine, because I am part of that
state Which pays the piper to pipe forth its tune. Vergli would
have the King part of the State, The chairman, so to say, one with
real pow’r. Paid, but a real King, not a mere cypher To whom men
bow, although but to a slave. Were you a real King you could
speak your mind And guide your peers and people to be fair, Or
influence them to espouse the right. I say not Kings should be all
absolute, But they should be Chairmen of the State. At least this is
the creed preached by Vergli And long ago his words converted
me, I am at heart an Evolutionist.”
King Hector (aside): “And I, too. Who would be the farce I am?” (To
Larrar) “Larrar, you are presuming. Have a care, Kings’ waiting-
men are servants, too, you know; A waiting groom and waiting
lord are paid. If I’m a puppet, all who wait on me Are puppets,
too! What shall we call the thing Which this queer puppet-mixture
has evolved? Merry-go-round or Humbug spinning round? I think
the latter, ’tis more suitable; For Humbug is in the ascendant now
And Sham the Idol of Society, And over all King Hector spreads
his wings; Shall they be free wings or their pinioned stumps?”
[He walks towards the Castle, musing.
SCENE II.
On the ramparts of Bawn co Pagh Castle. Vergli and Verita, the
latter in male attire, are pacing up and down conversing earnestly.
Vergli (passionately): “And they are captives, while I stand here
free! Alas! ’tis terrible. What can I do? Isola, you a captive and
condemned, Vulnar likewise and faithful Scrutus, too?
Condemned to die for giving me my life! Shades of Iniquity!
Horrible fact! Isola, whom I love, condemned to death, Vulnar,
whose home protects this wanderer, Scrutus, who was the first to
stand by me, All doomed to die, all doomed to die for me.”
[He sobs.
Verita. “Not so, Vergli. Fret not. They fight for Truth, Of which you
are the representative; They die that you may live to win that prize
And give it, from them, to posterity. Vergli, live to reward their
sacrifice, Live to see triumph—that for which they’ll die I know I
echo dear Isola’s thoughts, Do you not feel them hov’ring round
you now?”
Vergli. “Yes, they steal round me, gently kissing me, Bidding me be
a hero not a cur. Dearest Isola, I shall work for you And win the
Right we both desire so much. To go to you, to die by your dear
side, That is the wish of Vergli’s yearning heart; To live for you, to
win the Truth you love, Shall be the duty done for you and Right.”
Verita. “Spoken as heroes speak, noble Vergli, Your answ’ring
words will cheer Isola’s heart; They’ll flood with light her prison’s
lonely cell And bring her happiness and restful peace. Now will I
start for Infantlonia. There! The sun is sinking, all is red and gold,
The colours flood the far off western sky. Red is Blood’s sign, but
Gold’s the sign of Truth, And Martyr’s blood shall win Truth’s
victory.”
[She bids Vergli farewell and goes.
Vergli (solus): “Mine be the task to wake a sleeping world And force
it to espouse the cause of Truth. Merani, Mother! Dost thou hear
thy son? Thy dear lips taught him Truth. Thy noble words Live all
unfading in his Memory. Thou art not dead. Thy life is with me
now. I am thyself, I am thy property. What I do that thou doest,
Mother mine, My voice is but the echo of thy own. And you, Isola,
your thought hovers near Mixing with ours, making mine doubly
strong. Oh! Thought amalgamate with subtle force, Flood me with
pow’r to think and to express And to enforce it on Humanity.
Thought, mighty Thought, essence of God Divine, Wax great and
multiply. Attain the Truth.”
[He enters the Castle of Bawn co Pagh.
SCENE III.
In the exercising ground of the Prison of Holdfast. Vulnar,
Fortunatus and Scrutus are at exercise. The first two have halted
and are engaged in conversation. It is the day before their trial.
Vulnar. “This I assume is the last chance I’ll have To speak to you,
Isola. Without doubt The verdict will be Guilty, sentence Death.
My lawyer tells me that the angry wave Of that most fickle Judge,
Public Opinion, Is rabid for our instant execution! We are, in fact,
condemned before being tried; A wave of anger has possessed the
land, Fostered, encouraged by the powers that be. Ah! well, t’will
soon be o’er. I fear not death, To die beside you is enough for me.
Vulnar asks not a better fate, indeed, But to be faithful to the very
end— To Love, to Justice and to mighty Truth, All three the
seraphs of a perfect Life. Forgive me, Isola, for breathing love, But
I have loved you faithfully and well. To feel you feel this and
forgive Vulnar, Would make his last days peaceful and content.
He could not help his love, it came on him Long long ago when he
was yet a boy; He loved this love and hugged it very tight, And
nurtured it, until it grew so strong He knew no mortal pow’r could
sever it; The sapling had become, in fact, an oak— An oak
impervious to ev’ry storm. Kind Isola, I know that you forgive And
do not blame Vulnar for loving you.”
Isola. “Why do men love me thus? What is the spell Which makes
them love with such unselfish love? Oh! Vulnar, could I blame you
for such love? Rather, I thank you for your brave devotion. Kind
Vulnar, loving friend of Escanior, ’Tis good, indeed, to have so
true a friend; If it to you is joy to have loved me, Believe me, ’tis a
joy to me, Vulnar. I would not sell your love for all the world; I
would not barter it for Life itself. Such love in man is so
uncommon, rare, To own a gem so rare is wealth, indeed. Yes,
Death is nigh, that death men fear so much. Why do they fear it, if
their God is good? Why fear to go to what is loving, kind? If God is
as a father, they should laugh And clap with joy their hands at
sight of Death. This they do not, but fear it fearfully. Why?
Because they have made an untrue heav’n; A cruel hell, a hydra-
headed God Whom they call Good and yet fear to approach,
Whom they adore and yet seek to evade! Small wonder seeing
they are human and This God is most inhuman. Oh! fair Truth
Prevail, prevail, come quickly and prevail. Well, Vulnar, Life is fair
and Life is Life— To us who know that Thought can never die And
is the soul of Life, we fear not Death; Because we feel ’tis but an
open door Where Life rejoins the Thought which cannot die, And
starts afresh upon Life’s pilgrimage. I will not say farewell, we’ll
meet again, You and my fair-haired, blue-eyed Escanior; We’ll
meet, our forceful thought attracting us To be together. Yes, to be,
to be.”
Warder (approaching): “Time’s up for exercise. Back to your cells.
Silence. No further speaking is allowed.”
[All re-enter the prison.
SCENE IV.
In the Palace of Sham, the Infantlonian residence of the Ardrigh.
Sanctimonious and Conception sit together in the study of His
Graciousness.
Conception. “Your Graciousness, I’ve thought of everything. None
but the Prince of Bernia and that jade Whom they call Verita,
possess the fact That Isola is Fortunatus, too. Charged with
conspiracy, both are in gaol; There they shall stop till Isola is
dead. His Majesty has no suspicion, has he?”
Sanctimonious. “No, none, Conception. We’ll take care of that, I and
Sirocco, the Prime Minister. Now that Vulnar and his accomplices
— Scrutus and Fortunatus—are condemned, The danger of
detection is quite nil. I trust to you, of course, to keep the truth
Barred in the prison till they are no more. ’Tis fortunate they led
their own defence, And that Isola scorned to plead her sex And so
secure a respite for herself. Yes, Fortunatus, you shall hang,
indeed, And I’m revenged on Lady Isola!”
Conception (starting): “Your Graciousness, the Prince of Scota’s
there Staring at you with all his might and main, Where did he
spring from? Is’t a shadow wraith? God! how his features mirror
Isola’s.”
Sanctimonious (testily): “’Tis but a child. He often stays with me,
Comes for instruction. Plays in the Garden. Nothing to fear from
him. A mere, mere child. How now, my son, what stops you in
your play?”
Prince Bernis. “A voice called me. I thought it was Mamma’s.
‘Bernis,’ it said, ‘Come, darling, come here quick!’ I ran so fast. I
thought it was Mamma.”
[Enter Prince Bernis’s nurse by same window as he had entered.
Nurse. “Fie, Bernis! Fie! I’ve called you sev’ral times.”
Prince Bernis. “I thought it was Mamma and ran in here.”
Nurse. “Hush! Do not speak of Lady Isola. Make salutation to His
Graciousness, Then come with me, we must be going home.”
Sanctimonious. “My blessing on you, Prince. Be a good boy. Come
again soon and have a game of play.” [Exit Prince Bernis and
Nurse. (To Conception) “’Tis fortunate he is a little child And
would not understand what I was saying.”
Conception (uneasily): “I hope he did not, but his eyes were wide,
They seemed to me to be Isola’s eyes.”
Sanctimonious. “Tut! tut! you are a fool, Conception. The Prince of
Scota is a baby still.”
Conception. “Some babies are too sharp, your Graciousness.
However, you know best. I am a fool.”
Sanctimonious. “To-morrow they will die, I wish ’twas o’er. I shall
not freely breathe till their breath’s gone.”
Conception (rising): “Sharp on the stroke of eight they’ll die to-
morrow. Your Graciousness may eat in peace at nine.”
Sanctimonious. “Well spoken, man. Unparalleled Conception.”
[Exit Conception.
SCENE V.
In a condemned cell in the Prison of Holdfast. Fortunatus is seated
at a small wooden table writing. Close to him a warder sits
reading.
Fortunatus (writes): “When these words reach you, Hector, o’er the
tide Which leads from Death to Life I shall be moving. This
Thought, which now inhabiting my brain Sends forth to you this
message, will have sped Forward to mingle with Escanior’s. Yet
e’er it leaves its human canopy, It wafts you the last words of
Isola. These are they ‘Be you just and merciful, Become a king in
deed rather than name, Work with your people and for them,
Hector; Let King mean brother, treat all men as such. Sweep from
the statute book all useless law, All law which harrows progress,
or degrades. See to it that the young shall learn the Truth, Learn
to be useful, moral, just and kind— To give to every living thing
that breathes The right which Nature gives it, Happiness. Train up
the youth to say “Thou shalt not kill,” To say it and to practise it
as well. Abolish War and raise up Arbitration. See that each child
is taught a trade, or shewn How to use hands given for work and
use. See that all men have opportunity To work and win the fruits
of honest toil. Let all work be Co-operative and Give unto woman
what you give to man. Let principles of Fair Play animate All laws
and regulations of the State; Let Reason guide their framing, not
the lust Of gold, or greed, or selfishness. Be fair. Let it be ordered
“Privilege shall die, Just laws alone rule o’er the Destinies Of
Man and beast.” Crush Cruelty to earth. See to it that the base,
ignoble crime Cursed Vivisection, be swept clean away— Totally
abolished, treated as a crime, And stains no more the fame of our
dear land.’ One last word, Hector. Watch o’er our Bernis, Make
him a hero not a bauble prince; Let him be what Isola bore him
for, To be an honest and an upright man. And with this last word
let me bid you rise And call unto your side your first-born son,
Give him the right to be that which he is— The Prince of Scota and
your rightful heir. Farewell, Hector! For Right and Truth I die, See
to it that I do not die in vain.”
Warder. “Will you not take some rest? The hour grows late. I
counsel you, young Fortunatus, sleep.”
Fortunatus, rising, lies down on his bed. Then he turns on his back,
puts his hands behind his head and looks up at the ceiling,
mentally saying: “Bernis, my darling, be Isola’s child. Good-bye,
my little man. Be kind. Be true. Use thought to think right things,
be just, be brave; Be mother’s child, reflection of Isola.”
[Sleeps
SCENE VI.
The Palace of Dreaming in Infantlonia. King Hector tosses restlessly
in his bed and mutters to himself: “Grey dawn is coming, bringing
in its hand Death for the three who saved my son from death,
And I have signed the warrant for their deaths— I, the lone King
of poor Saxscoberland. Oh! Isola, had you been by my side, Had
you been reigning jointly with me now As you declared you had
the right to reign, Such foul injustice never had been done. Isola,
noble Isola, divorced, Driven from Hector’s side by unjust law,
Come to me, drive away the imp Remorse Which grinning sits
before me, mocking me.”
Enter Prince Bernis (in his nightdress, peeping in): “Papa, mamma
is calling. I heard her. Who is Isola? Is it mamma, papa?”
Hector (springing up): “What brings you here, my child? Bernis,
what is’t? By all the Gods! What is it, Bernis boy?”
Prince Bernis. “Mamma called me to come here. I have come.
Where is mamma? Is mamma Isola? Nurse calls her Lady Isola,
papa; But, yesterday, I heard His Gaysiousness Say ‘Isola was
Fortunatus.’ Who? Papa say, who is Fortunatus, and How can he
be my dear mamma, Isola?”
Hector (seizing the boy and staring at him): “He said that
Fortunatus was Isola? Speak, Bernis, did His Graciousness say
that?”
Prince Bernis. “Oh! yes, papa. Conception said it, too. I heard the
Ardrigh and Conception say it. Tell me, papa, where is mamma
and why Is she called Fortunatus by them both, And nurse calls
her the Lady Isola?”
King Hector (dressing hastily): “Oh! God Almighty, I shall be too
late. ’Tis twenty-nine miles to the prison gates. They die at eight.
’Tis now far after six. Almighty God! How reach Holdfast in time?
Oh! for the pow’r to flash the word ‘Reprieved’ Into the hands of
Holdfast’s Governor. Surely the Universe holds property Able to
send forth silent messages.” (To the child) “Run back to bed, my
darling, run, Bernis; Papa is going to try to save Mamma. No. I
can’t take you, run to bed, Bernis. Almighty God! can I get there
in time?”
[He rushes from his room.
End of Act V.
ACT SIXTH.
SCENE I.
Early morning in the condemned cell where Isola lies sleeping. In
one corner of it a warder sits, with his head sunk on his chest,
asleep. The first sign of day dawn is stealing through the barred
window.
Isola (gradually awaking, says dreamily): “’Tis somewhat hard my
rugged, earthy couch, Yet the brown heather nurtures Liberty. I’d
rather nestle in its arms, than lie Cushioned and canopied on regal
couch.” [Wakes more fully, and starts up into a sitting posture,
as consciousness and remembrance return. “’Tis neither, though.
Memory has returned. Morning is breaking on my last one here.
In a few hours my deathless Counterpart Will meet once more my
loved Escanior. Escanior! I am coming, Escanior! They sought to
part us. We shall meet again.” (She looks at the dim light in the
cell, and says): “’Tis a lone scene. A dreary aspect. Cold.” [Shivers.
“Bare walls, grey dawn, a flick’ring light at play A drowsy gaoler,
with his sleeping head, Nodding upon his almost soulless breast.
What is he but a thing mechanical, The tool of icy and unfeeling
law? Law, sacred law! No matter how unjust. An idol to be viewed
with veneration! Yes, Death is nigh, nigh unto Isola. It has no
terror for her, still she fain Would turn aside its grip from dear
Vulnar, And faithful Scrutus, too, if possible. Why should they die
for saving Hector’s son? Hector, awake! Save them, preserve their
lives. What is their crime? Did they not save Vergli, Half-brother
of our little Bernis? Hark! Far off I hear a clock tower tolling six.
Just two hours more. Bernis, awake? My child. Bernis, arouse
your father, bid him save, Bid him give Scrutus and Vulnar their
lives. It matters not for me, but for these two, Bernis awake him,
bid him think of them. My little boy, make haste. Time glides
along; It waits for no one, peasant, peer, or king.”
[Enter another gaoler, the drowsy one starts up.
Gaoler. “The pastor’s here. Would you converse with him? And let
him shrift your soul from coal black sin? What will you have to
eat? Name your desire, And I will see it is attended to. You must
be hungry, aye, and thirsty too, For two whole days food has not
passed your lips, Nor water either. Are you not famishing?”
Fortunatus. “Ask the wild bird, deprived of Liberty, And caged
inside a narrow prison cell, Either to eat of seed or drink of water!
I am not hungry friend, I need no food, Nor do I need the pastor’s
aid to shrive My soul of some imaginary sins. Let me be left in
peace. ’Tis all I ask, And when the hour arrives for me to die, I’ll
leave this cage ever so joyfully.”
Gaoler. “You’re a queer lot, you evolutionists. I would not like to
die, at all, at all, And without eating, or a steadying dram To keep
the nerves together. Think of it! It is to me incomprehensible.
Queer fish indeed these evolutionists.”
Isola (musingly to herself): “Hector might wake. My voice may have
reached him, Those thoughts of mine might possibly strike home!
Somehow I feel he’ll wake and send reprieve. Send it, yes, but will
it arrive in time? I’ll claim the privilege of dying first. Each
moment saved is precious. Dear Vulnar, Your staunch fidelity to
me and Truth, Merits not death, but Honour, Liberty. And you,
too, Scrutus, you so faithful. No, You do not merit such a
punishment. Hector! Art coming? Give these men their lives.”
SCENE II.
On the scaffold. An immense crowd is assembled outside the prison
of Holdfast. The three prisoners have been pinioned, and have
reached the spot of execution.
Fortunatus (to the hangman): “I claim the privilege of dying first,
Being the youngest of us three condemned, So man, make me
your first experiment, And take your time, don’t hurry, be
composed. Tut man, don’t tremble! What is there to fear? Learn
from young Fortunatus how to die. Adjust the rope. There! Steady.
Hark! I hear. [Listens. ’Tis the far echo of a horse’s feet, Surely,
yes surely, both will now be saved, I feel it, bless thee Hector, Vic
——”
A tremendous roar is heard outside. The words “Reprieve,
Reprieve, the King himself! The King!” suddenly penetrate to the
scaffold. A minute later and the King hurries thereon.
King Hector. “Reprieved! Governor hear! They are reprieved!”
[Staring at the group. “Two only here? Where is young
Fortunatus?”
The Hangman. “Dead, Sire! The word ‘reprieve’ reached me too
late, The sound arrived just as I pulled the bolt. His last words
were ‘Bless thee Hector, Victory!’ I heard them uttered as he fell
below, His death was speedy, instantaneous.”
Hector, laying both hands on Vulnar’s shoulder and bowing his
head on them, sobs out: “Isola! Isola! too late! too late! Oh! Isola
forgive. I rode my best. I rode not as a King, but as a man Whose
heart was bursting to reach you in time. I rode the horse you used
to love so well. The chestnut Saladin. He cleft the air, He seemed
to fly like arrow from the bow. He did his utmost. I did mine. Alas!
Fate was against us. Fate inexorable.”
The Governor of Holdfast prison exclaiming to himself: “Isola!
Fortunatus, Isola? By all the gods! This is a pretty pass. [To a
Warder. Haste man! Cut down young Fortunatus. See. Quick!
bear the body to my private rooms. Explain the situation to my
wife. Tell her to lay Isola on the bed. Apprise her that the King is
here. The King! A pretty pass! A tragedy indeed!”
Vulnar (to the hangman): “Unpinion me and Scrutus. Do it sharp,
man.” [A pause. Turning to the King and taking his hand: “Oh!
sire, grieve not, you did your very best. Would I had died first, and
saved Isola. I never dreamed of a reprieve. Brave heart! She died
to give me life. She died for Truth. Sire, see to it she did not die in
vain. Her last words, ‘Bless thee Hector. Victory!’ Shall ring into
your soul and make you just, Oh! yes, they shall. Her name will
gain the day, Isola dead, shall win bright Victory.”
King Hector (still sobbing): “Take me to Isola. Isola! I tried to save
thee, but I came too late. I strove with human might to be in time,
The human heart was beating in my breast. All royal mummery
had left my side, It was the man and not the King that strove,
Though Kings can feel, they are just human beings, Albeit
barbaric customs make them dolls. And I, I loved thee Isola. I did.
Who could help loving one so kind, so true?” (To Vulnar) “Vulnar,
where is she? Take me to her side, I tried to save her, but I came
too late.”
[Sobs.
Vulnar (linking the King’s arm in his and signing to the Governor,
standing close by, to lead forward): “Come, sire, I’ll take your
Majesty to her. Take comfort thinking how she blest you, sire;
Mourn not for her, she died as she had lived, With valiant heart
beating for others’ woes. Death had no terrors for her, sire,
indeed, It cannot claim the soul of Isola, Her deathless Thought,
that which made her a pow’r, Lives on and will live on eternally.
Doubtless ’tis roving with Escanior’s, She loved him, loved no
other all her life, I, his old Comrade, testify to this, I who e’er
worshipped where her feet have trod. And yet she’ll hover round
you sire again, And influence your heart to make the Cause, For
which she died, triumphant everywhere. She claimed to reign with
you, see to it Sire That her loved voice shall wake this world
again.”
They follow the Governor to his private apartments, and this latter
and Vulnar silently stand aside as the King enters the one in
which Isola has been laid.
King Hector (solus): “Yes, she is dead. Isola, thou art gone, That
which o’ertakes all men has come to thee. Vulnar spoke rightly,
when he said that thou, Dead should ne’erless obtain the Victory.
Yes, thou hast won it. Here, I swear to thee, All thou did’st die for
shall be realised, Right shall prevail, and Men shall own their
own, There shall be no more disinherited. Saxscober’s
Constitution shall become The constitution of a people free, And I
will be their real, not dummy King, Their brother worker, their
companion. While Life is left to me to work, I’ll work, I’ll make
Saxscoberland a dreamland scene, It shall reflect thy dream dear
Isola, Its face shall be the mirror of thy soul. Vergli shall aid me.
My first act shall be To do him justice and proclaim him heir; Our
little Bernis shall not act the thief, He shall be what thou sought’st
to keep the child, A human being, not a puppet slave. He shall be
his brave mother’s substitute, In him already shines thy deathless
soul. Isola, thou hast won, I swear it, Love. Thy death has won
Saxscober’s Liberty,”
He bends over and kisses her forehead. Then leaves the room.
Meeting Vulnar outside, he says: “Vulnar, I leave her body in your
care. Treat her as you would treat a reigning Queen. She shall
reign over fair Saxscoberland In deed, in fact, in true reality. Unto
the other nations of our Erth Her message shall be borne and shall
prevail, The bright example of Saxscoberland Shall move the
smaller fry to imitate, A bright example has its magnetism, And
draws men to solicit its embrace. Hector is clasping Isola’s. No
force Shall ever tear it from his grasp. No fear! I leave you, Vulnar.
Do your part. I go. My share in Evolution has begun. With Vergli I
will lead its sacred cause, With him will realize Isola’s dream.”
[He wrings Vulnar’s hand, and calling the Governor to him walks
away by his side.
Vulnar. “Is it a dream or Truth’s reality? Can it be fact or is it only

fancy? Isola dead, I living, Scrutus free, Vergli no longer outlawed,
but our Prince? It seems a dream, and yet ’tis not a dream, ’Tis
true, and Isola has triumphed. Sure! My love! my love! Who died
to save Vulnar, Who died for noble Truth, which he upheld, And
dying, won Saxscober’s liberty. Yes, it is won. Though Opposition
strong Will struggle to retain the law of Might, Right shall prevail,
and noble Truth prevail, That Right and Truth for which Isola
died.”
[He beckons Scrutus, who is standing near, to follow him, and goes
out. In the streets around the prison loud cheers can be heard.
They are given to King Hector, who is driving away in the
Governor’s carriage. So far, the death of Fortunatus and the fact
that Fortunatus is Isola, has not transpired. Vulnar interviews the
Governor, and makes every preparation for the removal of Isola’s
body to the residence of her brother, The Prince of Bernia.
SCENE III.
The fortress Castle of Bawn co Pagh. A voice sings: “Where Liberty
with Love entwines its arms, Its Life possesses vast, magnetic
charms; Cold, lifeless Licence is not liberty, To be a King means
not that you are free. Laws docked of Nature are not Freedom’s
joys, But just mechanical and puppet toys, Laughed at by men,
who scorn their puny sway, And treat them as just made to
disobey. ’Tis Love whose occult Pow’r alone conceives What
properties makes freedom. She receives Into her gentle bosom
Truth’s mandate And guided by it learns how to create Those laws
which fashion Liberty divine, And which alone from Love’s soft
eyes can shine. Oh! Love, thou child of the Almighty Pow’r,
Seductive as the sweetest scented flow’r, Thy influence is
paramount to save, Teaching men to be just, be fair, be brave, To
be the sons of Liberty and thee, True mates who can alone
produce the free, Those free, whose eyes are fixed on Love’s bright
Star, Speaking to them in flashes from afar. Be thou my guide all
through my mortal Life, Holding thy hand let me destroy the strife
Which Cruelty creates and scatters round, Sowing its poisoned
grain in fertile ground. I will, by aid of thee, uproot this grain,
Upon it Fire’s consuming powers rain, Burn it to ashes, sow
instead thy seed Which shall Love’s golden luscious harvest breed,
Whose sustenance shall nourish and inspire Kindness to triumph
over Selfish ire.”
Vergli (coming to the ramparts and looking over them): “Do my
ears mock me? Sure, ’tis Vulnar’s voice, None other owns such
subtle melody. Is it your Spirit serenading me, Comrade in arms,
friend of my boyhood, too? Vulnar, sure voice like yours is quite
unique, You have no rival, so it must be you. You have no equal,
whose melodious touch Sends through the being thrills of ecstacy.
Vulnar, where are you? Is your presence nigh, In body or in spirit
calling me? It seems to me as though Isola’s voice Whispers unto
me, ‘Vergli, Victory,’ And now I hear song rippling from your lips,
Song such as Vulnar’s lips alone can frame, Song in whose
melody, immortal Truth Mingles with mortal utterance in tune.”
Enter Vulnar: “Hail, Prince of Scota. Welcome to my home.
Welcome, Prince Vergli, to our Bawn co Pagh.”
Vergli (seizing his hand): “Vulnar alive! Vulnar not dead? Not gone?
Are my eyes clear, or am I dreaming dreams? Vulnar saluting me
as Hector’s heir, Calling me Prince of Scota? Hark! I hear.
Whispers are whispering within my brain, I hear Isola’s voice
addressing me. It comes from Vulnar, yet it is her voice. ‘Vergli,’ it
says, ‘Hail Vict’ry? You are free.’”
Vulnar. “Yes, Vergli, it is Victory indeed. From Isola, whom both of
us adore, I bear you the last word her dear lips framed, She died
while utt’ring it. ’Twas ‘Victory.’”
Vergli. “Isola dead! And you alive, Vulnar? Can it be possible?
Speak man. Explain.”
Vulnar recounts events to Vergli. The latter listens in silence, then
exclaims: “Isola dead. Happy Escanior. You revel in a being we
have lost. Lost, yet not lost, for Isola is nigh. Around me is her
presence. Ev’rywhere! Her Thought permeates my soul,
entrancing it, The breath of Memory is on my brow, Within my
brain her voice is speaking Love, Love, velvet Love, to Vergli and
Vulnar. Yes, Vulnar, love to you, and love to me, For Isola is Love
itself. Her Life Was one long act of love. Cold Cruelty Was the sole
thing she hated on our Erth.”
Vulnar. “Sir, Diamond Truth falls from inspired lips, Your words
are echoes of that attribute. There was no hate or fear in Isola,
Save of the awful demon Cruelty, And him she feared and hated
cordially. Her words through Hector, my dear lord, The King, I
bear you now. ‘Come, take your own, Vergli, You are The Prince of
Scota, true born son Of Noble Merani. Saxscober’s heir.’ Hail Sir,
as such, no courtly homage mine. But just acknowledgment of
brotherhood, There is but one nobility, one claim, Which I
acknowledge as nobility, And that is Merit, child of Perfect
Thought, That perfect thought which love alone can frame. Lo!
sinks the sun behind the Bawn co Pagh. Amidst a perfect sea of
yellow gold, Whence shoots aloft a fan of brilliant rays, Blue, opal,

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Glycosaminoglycans Kuberan

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Pharmacology and Toxicology of Cytochrome P450 - 60th

For further volumes:

Methods and Protocols

Umesh Desai Yukio Saijoh

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Dedicate this special volume on glycosaminoglycans to the memory of Professor Robert

Salt Lake City, UT, USA Kuberan Balagurunathan

PART I GAG STRUCTURES

1 One-Pot Enzymatic Synthesis of Heparin from N-Sulfoheparosan . . . . . . . . . . . . 3

12 Heparan Sulfate Structure: Methods to Study N-Sulfation

26 Automated Synthesis of Chondroitin Sulfate Oligosaccharides. . . . . . . . . . . . . . . . 319

PART II GAG FUNCTIONS

39 Enzymatic Alteration of ECM to Explore Muscle Function

53 Detection of Glycosaminoglycans in Pancreatic Islets

RAVI ADUSUMALLI • Department of Biosciences, University of Oslo, Oslo, Norway

ALAN GRODZINSKY • Department of Biological Engineering, Electrical Engineering and

CHIEN-FU LIANG • Department of Biomolecular Systems, Max Planck Institute of Colloids

KRISHNA RAJARATHNAM • Department of Biochemistry and Molecular Biology, The University

J. DAVID SYMONS • Department of Nutrition and Integrative Physiology, Molecular Medicine

One-Pot Enzymatic Synthesis of Heparin

Key words Heparin, Heparan sulfate, Heparosan, Enzymes, 3-Phosphoadenosine-5-phosphosulfate,

Heparin (Fig. 1), a glycosaminoglycan-based anticoagulant drug, is

responsible for heparin biosynthesis [8–10]. These enzymes, which

Enzyme Source species Truncation Fusion protein or tag Reference

N-sulfonation with triethylamine-sulfur trioxide complex affords

5. 50 mM PNPS: 50 mM PNPS. Weigh 51.5 mg of PNPS and

2.3 Enzymatic 1. Ammonium acetate buffer: 50 mM ammonium acetate, pH

2.4 LC-MS 1. Agilent 1200 LC/MSD instrument (Agilent Technologies,

2.5 NMR 1. D2O: 99.996% D2O (Sigma).

2.6 Molecular Weight 1. HPLC system consisting of an LC-10Ai pump, a CBM-20A

2.7 In Vitro 1. BIOPHEN ANTI-IIa and ANTI-Xa (Two-Stage Heparin

4. Wash a strong ion exchange spin column with SAX calibration

2. Calibrate the LC-MS system with Eluent A. For sample testing,

3. Record the SEC chromatograms with the LC solution Version

1. Purified NSH [17] is freeze-dried before use.

Solution NMR Spectroscopy for Characterizing

Crosstalk between macromolecules both in the intracellular and

They mediate a wide variety of crucial functions due to their ability

chemical shifts must be assigned using established NMR

NH – CαH – CβH2 – CγH2 – CδH2 – NεH – Cζ = NηH2+

WT R26 K64R R26 C

K20R/K64R R26 K64RK67R R26

acquired an HSQC spectrum of a ~100 μM chemokine sample

compromised compared to growth in rich media (LB media),

resulting in lower GAG requirements. If the binding data suggest

necessary to change experimental conditions so that the binding

1. Initial experiments must be carried out in low ionic strength

5. Choice of pH can be a critical factor in obtaining quality NMR

1. Karamanos NK, Piperigkou Z, Theocharis AD, CXCL5-glycosaminoglycan interactions. J Biol

KC/mCXCL1 and MIP2/mCXCL2 binding mediating chemokine-glycosaminoglycan

Metabolic Labeling of Proteoglycans and Analysis of Their

Proteoglycans (PGs) consist of protein cores that are inserted

GAG chains. The polymerization and sulfation mechanisms take

2.2 Metabolic 35S-sulfate (Hartmann Analytic) is added to the labeling medium