Received: 6 April 2023 Revised: 15 May 2023 Accepted: 23 May 2023

DOI: 10.1002/JPER.23-0224

ORIGINAL ARTICLE

Altered expression of MZB1 in periodontitis: A possible link

to disease pathogenesis

Deniz Sunnetci-Akkoyunlu1 Esra Guzeldemir-Akcakanat2 Begum Alkan3

Busra Gurel4 V. Merve Balta-Uysal2 Emel Akgun4 Ahmet Tarik Baykal4
Vakur Olgac5

1 Department of Medical Genetics, Faculty

of Medicine, Kocaeli University, Kocaeli,
Turkey
2 Department of Periodontology, Faculty
of Dentistry, Kocaeli University, Kocaeli,
Turkey
3 Private Practice, Istanbul, Turkey
4 Department of Medical Biochemistry,
School of Medicine, Acibadem Mehmet
Ali Aydınlar University, Istanbul, Turkey
5 Department of Oral Pathology, Faculty of
Dentistry, Istanbul University Turkey,
Istanbul, Turkey
Correspondence
Esra Guzeldemir-Akcakanat, Kocaeli
University, Faculty of Dentistry,
Department of Periodontology, 41190
Basiskele, Kocaeli, Turkey.
Email: esragd@yahoo.com
Funding information
Technological Research Council of Turkey
(TUBITAK), Grant/Award Number:
214S008; Ankara, Turkey, and Kocaeli
University the Scientific Research Projects
(BAP), Grant/Award Number: KOU
2013/5
Abstract
Background: Our previous study explored the molecular signatures of gen-
eralized aggressive periodontitis (GAgP) using gingival tissues through omics-
based-whole-genome transcriptomic analysis. This continuation study aimed to
investigate the whole protein profiling of these gingival samples through liquid
chromatography-mass spectroscopy/mass spectroscopy (LC-MS/MS) analysis
and to validate the identified proteins through immunohistochemistry to provide
further evidence for the quality of the results.
Methods: In previous study, gene expression patterns were identified in gingival
tissues from 23 GAgP and 25 control individuals. In the current study, compara-
tive proteomic analysis was performed on isolated proteins from the same study
groups using LC-MS/MS analysis. The data from the transcriptomics study pub-
lished before and the proteomics data were integrated to reveal any common
genes and proteins. Additionally, immunohistochemical analysis was conducted
to further investigate the findings.
Results: The most upregulated proteins in patients compared to controls were
ITGAM, AZU1, MMP9, BPI, UGGG1, MZB1, TRFL, PDIA6, PRDX4, and PLG.
The top six pathways associated with these proteins were involved in innate
immune system, post-translational protein phosphorylation, interleukin-4 and
-13 signaling, toll-like receptors cascades, and extracellular matrix organization.
Based on the integration and validation analysis of transcriptomics and pro-
teomics data, as well as immunohistochemical analysis, MZB1 was identified as
a shared gene and protein that were upregulated in the patients.
Conclusions: MZB1 is a protein that is involved in the development of B cells and
the production of antibodies. Its upregulation in periodontitis suggests that there
may be a dysregulation of the immune response in this condition, and MZB1 may
be a potent biomarker for periodontitis.

KEYWORDS
genomics, immunohistochemistry, inflammation, MZB1, periodontitis, proteomics

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium,
provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2023 The Authors. Journal of Periodontology published by Wiley Periodicals LLC on behalf of American Academy of Periodontology.

J Periodontol. 2023;94:1285–1294. wileyonlinelibrary.com/journal/jper 1285


1286
1 INTRODUCTION
Periodontitis is a bacterially induced multifactorial inflam-
matory disease characterized by progressive destruction
of the periodontal attachment apparatus. Although peri-
odontal diseases are non-communicable diseases, they are
still a significant public health burden and the most preva-
lent chronic inflammatory non-communicable disease.
Periodontitis is initiated by the dysbiotic plaque biofilm
and its release of substances such as lipopolysaccharides
and toxins, which leads to the dysregulation of immune-
inflammatory processes.1 Hence, the destruction of tissue
and bone by stimulating various cells and producing pro-
inflammatory mediators such as chemokines, cytokines,
matrix metalloproteinases, and prostaglandins, resulting
in the local immune response.
Several studies have examined gene expression in the
gingival tissue of patients with periodontitis using microar-
rays, revealing differences in gene expression between
periodontitis and healthy gingival tissues, and gene expres-
sions related to inflammation, apoptosis, and cell death are
found to be particularly prevalent in periodontitis.2–9
The interaction and collaboration of genes and proteins
are crucial for the proper functioning of biological sys-
tems. With the accumulation of an enormous amount of
genomic data and advancements in computational tech-
niques, researchers now have the opportunity to better
comprehend these complex systems. Researchers have
started to explore not only steady-state systems but also
perturbed systems through advanced high-throughput
data analysis approaches.10 To accurately capture the com-
plexity of biological systems, it is necessary to consider
biological interactions as networks. Consequently, the
integration of systems biology approaches and biologi-
cal network analysis has become increasingly important
for identifying gene-protein networks and unraveling the
underlying mechanisms of these systems.11
Despite the effectiveness of genomics in producing com-
prehensive genome-level data on the disease, and, the
valuable insights gained from our previous studies,2,3 that
have enhanced our understanding of the subject, it is
important to recognize that the biochemical processes
involved in periodontitis are dynamic and that the patho-
genesis of the disease may not solely be determined by
numerous genes but also by proteins.12
Proteins are synthesized based on the genetic code of
a specific gene. Proteomics, which involves the large-
scale analysis of proteomes, can offer valuable biological
insights into diseases by examining factors such as expres-
sion levels and post-translational modifications.13 With
advances in mass spectrometry (MS) technology, it is now
possible not only to obtain quantitative data but also
to compare differentially labeled peptides from samples
using a mass spectrometer.10
19433670, 2023, 11, Downloaded from https://aap.onlinelibrary.wiley.com/doi/10.1002/JPER.23-0224, Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SUNNETCI-AKKOYUNLU et al.
Evidence suggests that protein abundances tend to be
more conserved compared to mRNA levels due to regu-
latory processes such as post-transcriptional, translation,
and protein degradational regulation. These regulatory
mechanisms help to maintain protein abundances at evo-
lutional preferred levels. However, mRNA levels can differ
over time.10,14 Therefore, while the gene itself is stable and
ubiquitous, the proteome is dynamic and represents the
final product of a gene, which is closer to function than
the gene itself.
Through our analysis, we have identified gene expres-
sion patterns and proteomics in gingival tissues from
patients with chronic periodontitis.3 The data obtained
from transcriptomics and proteomics were integrated,
validated, and revealed two common shared genes and pro-
teins for chronic periodontitis, namely, MZB1 and ECH1.
Based on our findings, it was concluded that MZB1 may be
a promising candidate for chronic periodontitis.
In a previous study conducted by our group, we utilized
whole-genome approaches to identify molecular markers
and potential candidate genes for aggressive periodontitis.2
Gene expression profiling revealed 125 downregulated and
389 upregulated genes, as well as four gene networks.
The most upregulated genes included MZB1, TNFRSF17,
PNOC, FCRL5, LAX1, BMS1P20, IGLL5, MMP7, SPAG4, and
MEI1, while the most downregulated genes were LOR,
LAMB4, AADACL2, MAPT, ARG1, NPR3, AADAC, DSC1,
LRRC4, and CHP2. The study concluded that TNFRSF17,
FCRL5, MZB1, and DSC1 were important candidate genes
in characterizing aggressive periodontitis, and reported
gene networks were closely related to humoral immune
response and organismal injury/abnormalities. However,
further evaluation in terms of proteomics and immuno-
histopathology was not conducted at that time.
These two studies were conducted prior to the pub-
lication of “The 2017 Classification of Periodontal and
Peri-implant Diseases and Conditions”.15 The 2017 World
Workshop acknowledged that while localized early-onset
periodontitis has a distinct presentation, the underly-
ing or pathological factors responsible for this presenta-
tion remain undefined, as is the mechanism responsi-
ble for the development of generalized periodontitis in
young individuals.16 Therefore, to address the differences
between generalized aggressive periodontitis and chronic
periodontitis, we subjected the same aggressive periodon-
titis group from our previous whole-genome transcrip-
tomics study2 to proteomics and immunohistopathological
evaluations.
2 MATERIALS AND METHODS
The Ethics Committee of the Medical Faculty of Kocaeli
University (KOU KAEK 2013/15) approved this study

SUNNETCI-AKKOYUNLU et al.
protocol, which was conducted in compliance with the
2000 revised version of the Declaration of Helsinki of 1975.
This clinical trial has been registered with ClinicalTri-
als.gov under the identifier NCT02327533.
This study is a follow-up study to a previously pub-
lished study,2 which provided a detailed description of the
study materials. The study recruited patients with gener-
alized aggressive periodontitis and healthy controls from
individuals seeking periodontal and/or dental treatment
at Kocaeli University, Faculty of Dentistry, Department of
Periodontology. A comprehensive medical, dental, peri-
odontal, and radiographic examination was conducted,
and the periodontal diagnosis of individuals with gener-
alized aggressive periodontitis was determined based on
the 1999 International World Workshop for a Classification
of Periodontal Diseases and Conditions17 using clinical
and radiographic criteria. The study protocol was thor-
oughly explained to the patients, and those who agreed to
participate provided written consent.
Gingival tissue samples were collected from patients
with generalized aggressive periodontitis during periodon-
tal surgery and extractions, without any prior supra- or
subgingival instrumentation.17 Biopsy sites showed persis-
tent bleeding on probing and clinical attachment loss at
these sites was greater than 4 mm, with corresponding
alveolar bone loss observed on radiographic evaluation.
Control specimens were obtained from crown lengthen-
ing procedures, gingivectomies, and extractions prior to
orthodontic treatment.
Under local anesthesia* , gingival biopsies were taken
using an inverse bevel incision to obtain tissue from the
underside of the papilla. The samples consisted of the sul-
cular and junctional epithelium, underlying connective
tissue, and granulation tissue, and were thoroughly rinsed
with a sterile saline solution following dissection.
Tissue specimens were divided into three pieces, and
two of them were promptly snap-frozen in liquid nitrogen
and stored at −80◦ for later analysis. The remaining tissue
specimen was preserved in a 10% formalin fixation solu-
tion. One of the pieces from these samples was utilized for
transcriptomic evaluations in the previous analysis.2
2.1 Proteomic analysis
2.1.1 Sample preparation for LC-MS/MS
analysis
The tissue samples that had been homogenized were lysed
by incubating them at 100◦ C in UPX Buffer† . After protein
* UltracainDS 40mg/mL+6mcg/mL, Sanofi, Istanbul, Turkiye.
† UniversalProtein Extraction (UPX) Buffer, Integrated DNA Technolo-
gies, Expedeon, UK.
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1287
isolation, the total protein concentration was determined.
The Filter Aided Sample Preparation protocol (FASP)‡ was
used to obtain tryptic peptides. In brief, 50 μg protein was
initially washed with 6 M urea on a 30 kDa cut-off spin col-
umn and then alkylated with 10 mM iodoacetamide in the
dark at room temperature for 20 min. The tryptic peptides
were eluted from the spin column, and their concentration
was measured with a fluorometer§ . Prior to injection into
the liquid chromatography-MS/MS (LC-MS/MS) system,
the peptides were diluted to 100 ng/ul using 0.1% formic
acid.
2.1.2 LC-MS/MS analysis
Tryptic peptides were separated from the trap** col-
umn and transferred to the analytic columns†† using a
mass spectrometry instrument‡‡ with 3%–40% acetoni-
trile gradient for 90 min. After the ESI ionization, they
were examined using quaternary-based ultraperformance
instrumentation§§ . Peptide signal data between 50 and
1900 m/z values were collected without any precursor ion
preselection. MS and MS/MS functions were used over
0.7-s intervals, and 100 fmol/ul Glu-1-fibrinopeptide B was
used as lockmass calibrant.
2.2 Immunohistochemical (IHC)
analysis
To verify the identified proteins in gingival tissues,
immunohistochemistry was performed, which provided
evidence for the quality of the results. The tissue sam-
ples were fixed in a 10% formalin solution and rinsed in
dH2 O, dehydrated in a graded series of ethanol, cleared,
infiltrated with paraffin, and embedded in paraffin. The
paraffin blocks were cut into 6 μm sections and the sections
were stained with hematoxylin and eosin*** . However,
immunohistochemistry was delayed until the comple-
tion of whole-genome transcriptomic and whole-protein
profiling.
Once the transcriptomic and proteomics data were
analyzed together, specific proteins were selected for
‡ Filter Aided Sample Preparation (FASP) kit, Integrated DNA Technolo-
gies, Expedeon, UK.
§ Qubit 2.0 Fluorometer, Thermo Fisher Scientific, Waltham, MA, USA.
** ACQUITY UPLC M-Class Symmetry C18 Trap Column, 5um, 180 um
i.d. x 20 mm, Waters Corporation, Milford, MA, USA.
†† ACQUITY UPLC M-Class Peptide CSH C18 Column, 1.7 um, 75 um i.d.
x 250 mm, Waters Corporation, Milford, MA, USA.
‡‡ ACQUITY UPLC- M-Class System, Waters Corporation, Milford, MA,
USA.
§§ ACQUITY UPLC- ESI- SYNAPT G2Si QTOF, Waters Corporation,
Milford, MA, USA.
*** H&E, Mayer haematoxylin solution, Italy.

1288
further analysis using immunohistochemistry. The tis-
sue sections were incubated with primer antibodies for
selected proteins, followed by the addition of horseradish-
peroxidase††† . The slides were then mounted‡‡‡ and exam-
ined under a light microscope using 200× and 400×
magnifications in five different areas. In each area, 100
cells (a total of 500 cells) were counted, and percentages
of positively stained cells were calculated, with a focus on
cytoplasmic images.
Based on the comparison and evaluation of the pro-
teomic and transcriptomic data, MZB1, ECH1, PPB1, and
ZBP1 were selected for further analysis using immunohis-
tochemistry. To confirm the findings from the proteomic
analysis, tissue specimens were stained with related anti-
bodies.
2.3 Statistical analysis
2.3.1 Proteomic analysis
Progenesis QI-P software (Non-linear Dynamics) was used
for the identification and statistical analysis of protein
expression. All proteins were identified by at least three
unique peptide sequences. All p values < 0.05 were con-
sidered significant throughout the study and a minimum
1.4-fold expression difference between groups was used as
a filter. The pathway and co-expression analysis were done
by online pathway analysis tools§§§,**** .
2.3.2 Immunohistochemical analysis
The results of the immunohistochemical evaluation were
analyzed using image analysis software†††† , and statisti-
cal analysis was carried out using a software program.
A p-value of less than 0.05 was considered statistically
significant.
3 RESULTS
3.1 Proteomic analysis
In this study, the protein expression changes in the gingival
tissue biopsies from healthy individuals and patients with
††† ScyTek, SensiTek HRP Anti-Polyvalent Lab Pack, Logan, UT, USA.
‡‡‡ DDK, dDMount Mounting Media (Entellan), Italy.
§§§ ELIXIR, STRING (version 10.5, online), Cambridgeshire, UK. (https://
string-db.org)
**** Reactome version 3.5 (https://string-db.org)
†††† Olympus AnalySIS 5 image analysis software, Tokyo, JAPAN.
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SUNNETCI-AKKOYUNLU et al.
generalized aggressive periodontitis were using nanoLC-
ESI- qTOF system. Progenesis QI-P software was used for
data acquisition, deisotoping, and charge state deconvo-
lution. A total of 715 proteins were identified by at least
three peptides in all identified proteins, and 147 proteins
showed significant alterations in expression (p < 0.05
and fold change > 1.4). Among these proteins, 85 were
upregulated and 62 were downregulated in generalized
aggressive periodontitis compared to controls (See Table
S1 in online Journal of Periodontology), and a list of
38 proteins was obtained by enrichment of the results
(>2 fold) (Table 1). The most upregulated proteins were
ITGAM, CAP37, MMP9, BPI, UGGG1, MZB1, TRFL,
PDIA6, PRDX4, PLMN and downregulated proteins were
CRNN, GAPD1, MAP4, ACTBM, DNMBP, THIO, K1C16,
ZN407, CBR3, SPB13, CBR3, K2C3, NDUS1, LEG7, AK1C2,
AMY1, A2ML1, K1C23, GARS, KCRB, CALML5, K1C25,
K2C1, K1C10 (Table 1).
The top six pathways (p < 0.05) were determined by
an overrepresentation test analysis of these 38 proteins
(Figure 1). The proteins related to these pathways were
also analyzed using the STRING database to reveal any
relationships based on protein networks or co-expression.
Figure 1 shows some of the common proteins shared by
these pathways.
Figure 2 represents the network of these proteins
and related pathways. The biological process of differen-
tially upregulated proteins was related to protein folding,
response to stress, single-organism catabolic process, reg-
ulation of peptidase activity, and negative regulation of cell
death. The molecular function was related to structural
molecule activity, anion binding, poly(A) RNA binding,
peptidase regulatory activity, and protease binding. The
protein network and co-expression graph are shown in
Figures 2 and 3.
3.2 Integration of the transcriptomics
and proteomics data for common genes
To clarify the network results, some nodes in the net-
work were eliminated until the common proteins were
identified in both genomic and proteomic analysis. Fol-
lowing the integration of whole-genome transcriptomic
and whole-protein profiling data, identified genes were
reviewed for their relevance to the pathogenesis of the peri-
odontal disease, and MZB1, ECH1, EPHA7, and PPB1 were
thought to contribute to the pathogenesis of generalized
aggressive periodontitis. However, IHC results provided
evidence of the presence of MZB1 (p < 0.001) (Figure 4)
but ECH1, EPHA7, and PPB1.
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SUNNETCI-AKKOYUNLU et al. 1289

TA B L E 1 The list of up and downregulated proteins.

Accession Symbol Protein name Fold-change p-Value
P11215 ITGAM Integrin alpha-M 10.37 2.70E−04
P20160 AZU1 Azurocidin 10.26 2.21E−03
P14780 MMP9 Matrix metalloproteinase-9 4.32 3.85E−05
P17213 BPI Bactericidal permeability-increasing protein 4.11 1.11E−04
Q9NYU2 UGGG1 UDP-glucose: glycoprotein glucosyltransferase 1 3.28 3.54E−07
Q8WU39 MZB1 Marginal zone B- and B1-cell-specific protein 3.22 1.58E−07
P02788 TRFL Lactotransferrin 2.73 2.09E−03
Q15084 PDIA6 Protein disulfide-isomerase A6 2.55 1.19E−08
Q13162 PRDX4 Peroxiredoxin-4 2.20 1.80E−05
P00747 PLG Plasminogen 2.20 1.37E−04
P14625 HSP90B1 Endoplasmin 2.19 8.29E−08
P02671 FGA Fibrinogen alpha chain 2.12 3.61E−03
P01861 IGHG4 Ig gamma-4 chain C region 2.04 2.44E−03
Q15154 PCM1 Pericentriolar material 1 protein 2.04 2.35E−02
Q9UBG3 CRNN Cornulin 0.49 1.11E−06
Q14C86 GAPD1 GTPase-activating protein and VPS9 domain-containing 0.49 4.96E−02
protein 1
P27816 MAP4 Microtubule-associated protein 4 0.49 1.05E−02
Q9BYX7 ACTBM Putative beta-actin-like protein 3 0.46 1.24E−02
Q6XZF7 DNMBP Dynamin-binding protein 0.46 4.68E−02
P10599 THIO Thioredoxin 0.46 2.04E−05
P08779 K1C16 Keratin, type I cytoskeletal 16 0.45 2.55E−03
Q9C0G0 ZN407 Zinc finger protein 407 0.45 3.58E−02
O75828 CBR3 Carbonyl reductase [NADPH] 3 0.44 4.97E−06
Q9UIV8 SPB13 Serpin B13 0.43 1.53E−04
P01008 CBR3 Antithrombin-III 0.43 3.47E−06
P12035 K2C3 Keratin, type II cytoskeletal 3 0.42 3.29E−04
P28331 NDUS1 NADH-ubiquinone oxidoreductase 75 kDa subunit, 0.41 7.84E−03
mitochondrial
P47929 LEG7 Galectin-7 0.40 2.72E−08
P52895 AK1C2 Aldo-keto reductase family 1 member C2 0.39 1.20E−04
P04745 AMY1 Alpha-amylase 1 0.38 4.73E−02
A8K2U0 A2ML1 Alpha-2-macroglobulin-like protein 1 0.37 9.56E−04
Q9C075 K1C23 Keratin, type I cytoskeletal 23 0.35 1.25E−03
P41250 GARS Glycine–tRNA ligase 0.33 3.63E−03
P12277 KCRB Creatine kinase B-type 0.31 3.56E−06
Q9NZT1 CALML5 Calmodulin-like protein 5 0.31 7.08E−04
Q7Z3Z0 K1C25 Keratin, type I cytoskeletal 25 0.28 4.45E−03
P04264 K2C1 Keratin, type II cytoskeletal 1 0.24 2.29E−07
P13645 K1C10 Keratin, type I cytoskeletal 10 0.16 6.46E−06
Note: Fold-change values represents the protein expression ratio of P to controls; values > 1 represent the upregulation of the protein in P while the downregulation
in P is represented when the fold-change is between 0 and 1.

4 DISCUSSION results in loss of periodontal attachment and radiographi-

cally assessed alveolar bone loss.16,18
Periodontitis is described as a multifactorial inflamma- Humans have been suffering from gingival and peri-
tory disease characterized by dysbiotic plaque biofilm that odontal diseases since the beginning of recorded history.19
 19433670, 2023, 11, Downloaded from https://aap.onlinelibrary.wiley.com/doi/10.1002/JPER.23-0224, Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1290 SUNNETCI-AKKOYUNLU et al.

Pathway name p value

1 Innate Immune System 5.52E-04

Regulation of Insulin-like Growth Factor (IGF)
transport and uptake by Insulin-like Growth Factor
2 Binding Proteins (IGFBPs) 7.50E-05

3 Post-translational protein phosphorylation 5.54E-04

4 Interleukin-4 and 13 signaling 6.36E-04

5 Toll-Like Receptors Cascades 2.27E-03

6 Extracellular matrix organization 2.13E-02

1 2 3 4 5 6
Plasminogen P00747 PLG
Antithrombin-III P01008 SERPINC1
Fibrinogen alpha chain P02671 FGA
Lactotransferrin P02788 LTF
Thioredoxin P10599 TXN
Integrin alpha-M P11215 ITGAM
Endoplasmin P14625 HSP90B1
Matrix metalloproteinase-9 P14780 MMP9
Bactericidal permeability-increasing protein P17213 BPI
Azurocidin P20160 AZU1
Peroxiredoxin-4 Q13162 PRDX4
Protein disulfide-isomerase A6 Q15084 PDIA6
Calmodulin-like protein 5 Q9NZT1 CALML5

F I G U R E 1 Pathway analysis of significantly altered proteins in AgP. The list of affected pathways (p < 0.05) was obtained by an
overrepresentation test of 38 proteins with the Reactome database (up). The proteins involved in the affected pathways are listed (down).
Numbers (1–6) indicate the pathways mentioned in the table above, and dark boxes show whether the protein is involved in the indicated
pathway.

After reviewing the English-language literature, it was
seen that case reports of aggressive periodontitis can
be traced back to the 1920s to the 1940s. The earliest
report, by Gottlieb, described a 22-year-old man with fatal
influenza who exhibited “diffuse atrophy of alveolar bone”,
which the author speculated was a manifestation of a
systemic condition.20 Over the time, this condition I has
been known by various names including, deep cemen-
topathy, progressive marginal periodontitis, periodontosis,
periodontal syndrome, juvenile periodontitis, and then,
aggressive periodontitis.20 The World Workshop on the
Classification of Periodontal and Peri-implant Diseases
and Conditions in 2017 did abandon the terms “aggres-
sive” and “chronic” periodontitis and instead grouped all
cases with progressive clinical periodontal attachment loss
and radiological loss of bone support under the title of
“periodontitis”.16 The workshop also defined stages and
grades for periodontitis cases to aid in diagnosis and treat-
ment planning. This updated classification system reflects
the latest scientific evidence and clinical applicability15
and the findings of this study together with our previ-
ous studies2,3 are indicative of supporting the alteration in
classification.
The present study, which is part of a research project,
was planned and started before the latest classification
system was accepted. The aim of our study was to reveal
the pathophysiological pathways of chronic and aggres-
sive periodontitis on a molecular basis. For this purpose,
transcriptomics and proteomics analyses were performed
on gingival tissues obtained from patients with chronic
and aggressive periodontitis, and the results of these
studies were combined and confirmed using immunohis-

SUNNETCI-AKKOYUNLU et al.
F I G U R E 2 The network of the proteins which have roles in
the indicating pathways.
tochemical analyses to match the clinical features of the
cases. This study presented here is a part of this series and
aims to confirm the previously obtained transcriptomics
data using proteomics and IHC analyses in aggressive
periodontitis samples. The association between MZB1 and
chronic periodontitis has been previously demonstrated
through genomic, proteomic, and IHC analyses.3 When
considering this finding alongside the results of the
present study, it can be concluded that these findings sup-
port the inclusion of aggressive and chronic periodontitis
under the term “periodontitis” in the latest classification
of periodontal and peri-implant diseases and conditions.16
In the first phase of the study, differentially expressed
genes were identified in aggressive periodontitis than in
periodontally healthy individuals by high throughput anal-
ysis of whole transcriptomes by microarray.3 As a result of
this microarray gene expression profile, 125 downregulated
and 389 upregulated genes were identified. There were
notable variations in the expression of the tumor necro-
sis factor receptor superfamily member 17 (TNFRSF17), Fc
receptor-like 5 (FCRL5), MZB1, and desmocollin-1 (DSC1)
genes. Specifically, TNFRSF17, FCRL5, and MZB1 genes
demonstrated increased expression, while the DSC1 gene
exhibited decreased expression.
In the second part of the study, and inclusion of this
article, the nanoLC-ESI- qTOF system was employed
19433670, 2023, 11, Downloaded from https://aap.onlinelibrary.wiley.com/doi/10.1002/JPER.23-0224, Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1291
to investigate changes in protein expressions and iden-
tify proteins. Of these differentially expressed proteins,
715 were identified through at least three distinct pep-
tide sequences. Subsequent analysis using Reactome and
STRING online tools revealed that out of the 147 pro-
teins, 85 were upregulated and 62 were downregulated
in patients when compared to controls (see Table S1
in the online Journal of Periodontology). Differentially
expressed proteins were related to the innate immune
system, post-translational protein phosphorylation, inter-
leukin (IL)-4 and IL-13 signaling, and toll-like receptors
cascades (Figure 1).
Last, after integrating and validating the transcriptomics
and proteomics data, it was identified that one gene and
protein, MZB1, were common shared. This result is con-
sistent with the previous studies that have demonstrated
the upregulation of MZB1 in patients with aggressive
periodontitis and chronic periodontitis.2,3,8,21–23
The MZB1 gene (marginal zone B- and B1-cell-specific
protein) encodes for the protein MZB1 and is defined
in the human gene database24 as a gene that interacts
with both heavy and light chains of IgM (immunoglobulin
M) to facilitate its assembly and secretion. The pathways
related to the MZB1 gene encompass positive regulation
of the biosynthesis process of immunoglobulin, apoptosis,
cell proliferation, B-cell proliferation regulation, and inte-
grin activation. MZB1 is a component of the endoplasmic
reticulum (ER) chaperone complex, which is responsible
for controlling the surface presentation and secretion of
IgM that plays a role in antibody secretion and B cell
differentiation25–27 and increased expression of MZB1 is
associated with a variety of human diseases such as cancer,
rheumatoid arthritis, lupus, HIV. Isoform 2 of this protein
may be involved in regulating apoptosis and contributes to
the diversification of peripheral B-cell functions by con-
trolling Ca(2+) stores, antibody secretion, and integrin
activation. Additionally, MZB1 functions as a hormone-
regulated adipokine/proinflammatory cytokine that plays
a role in causing chronic inflammation and influenc-
ing cellular expansion. It is primarily expressed in the
marginal zone and B1 B cells, which are specialized sub-
sets of B cells that respond rapidly to infections. MZB1 is
not only affected immune protein synthesis but also affects
the maturation of immune cells.26 As a target of MZB1,
microRNA-185 causes the developmental arrest of T cells.28
The findings from the gene expression profile, micro-
CT analysis, and hematoxylin-eosin staining suggest that
MZB1 may play a role in the development and progres-
sion of periodontitis.22 The observed increase in MZB1
gene expression in periodontitis supports the notion that
MZB1 upregulation is a common feature of inflamma-
tory diseases, including periodontitis, lupus, cancer, and
immune diseases. Moreover, the knockdown of MZB1 gene
 19433670, 2023, 11, Downloaded from https://aap.onlinelibrary.wiley.com/doi/10.1002/JPER.23-0224, Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1292 SUNNETCI-AKKOYUNLU et al.

FIGURE 3 The co-expression analysis of the proteins which have roles in the indicating pathways.

expression appears to have a beneficial effect on alveo-

F I G U R E 4 Immunohistopathological section from (A) a
patient with aggressive periodontitis for MZB1 primary antibody
(×400) and (B) a control individual for MZB1 primary antibody
(×400).
lar bone loss, indicating that MZB1 may contribute to the
pathogenesis of periodontitis.22
MZB1 appears to have a role in regulating mitochondrial
function and apoptosis,29 as well as calcium homeostasis,
antibody secretion, and integrin activation.26 Overexpres-
sion of MZB1 was found to improve mitochondrial func-
tion, increase ATP levels, reduce inflammation, and inhibit
apoptosis, leading to cardioprotective effects in mice. On
the other hand, the knockdown of MZB1 decreased mito-
chondrial membrane potential and impaired mitochon-
drial function, which may have detrimental effects. It was
also found to affect calcium homeostasis, antibody secre-
tion, and integrin-mediated cell adhesion, which suggests
a broader role for MZB1 beyond mitochondrial function
and apoptosis regulation.
The upregulation of MZB1 expression is a complex phe-
nomenon that likely reflects the involvement of multiple
signaling pathways and cellular processes. MZB1 may con-
tribute to expanding the range of functions of various
peripheral B cell subsets.26 MZ B cells and B1 cells have
a rapid ability to secrete antibodies in response to stimu-
lation by lipopolysaccharide, and B1 cells have even been
observed to secrete antibodies spontaneously.30 MZ B and
B1 cells are a type of B cells that have been referred to as
“innate-like B cells” because they can rapidly differentiate
into antibody-secreting cells when stimulated by ligands
of toll-like receptors (TLRs). These cells produce polyreac-
tive antibodies, which are considered “natural” antibodies,
and this function allows them to play an important role
in the early defense against infections. This term was
coined due to the ability of these cells to function similarly
to innate immune cells, such as macrophages and neu-
trophils, in their ability to rapidly respond to pathogens.

SUNNETCI-AKKOYUNLU et al.
This characteristic distinguishes them from conventional
B cells, which require T cell help to produce specific
antibodies.30,31
The main strength of this study and our previous study2
is the utilization of advanced techniques for genome and
proteome analysis. The LC-MS/MS technique employed
for protein expression analysis is known for its superior
sensitivity, specificity, and accuracy compared to nonquan-
titative immunohistochemistry.
The consistency of the sampling location is another
noteworthy strength of this study. By obtaining specimens
from the clearly inflamed area before any periodontal
procedure, we ensured consistency in the sampling loca-
tion and depth throughout the inflamed tissue. This, in
turn, enhances the reliability and validity of the study’s
findings. Furthermore, the study involved a clear and
well-defined diagnosis of aggressive periodontitis, with the
patients fully representing the clinical phenotype of the
disease. This was ensured through examination and diag-
nosis by an experienced periodontist (E.G.-A.), a detailed
review of participants’ medical history, and collection
of specimens by the same periodontist. Additionally, all
participants were confirmed to be free of any systemic dis-
eases, which further strengthens the validity and reliability
of the study’s findings.
One potential limitation is the manual integra-
tion of transcriptome and proteomic data. However,
a new version of the Human Protein Atlas has been
released that integrates data from transcriptomic and
antibody-based proteomic analyses at the individual gene
level.32
The study utilized a proteomic analysis approach
employing Data-Independent Acquisition (DIA) instead
of Data-Dependent Acquisition (DDA). This enabled the
examination of all peptide ions present in the sample with-
out any precursor ion preselection. While adjustments in
chromatographic conditions, such as prolonged run time
or added fractionation steps, may accelerate the identifica-
tion of proteins, the methodology employed in the study
was deemed optimal for this type of investigation.
The overexpression of MZB1, which was found to be
present in the gingival tissue of periodontitis patients,
could indicate a potential role of B cells in the pathogen-
esis of periodontitis. Further research on the role of B cells
in periodontitis may be warranted based on the present
findings.
5 CONCLUSION
The similarity of the results between this study and previ-
ous studies with a similar focus on periodontitis supports
the amalgamation of “chronic periodontitis” and “gener-
19433670, 2023, 11, Downloaded from https://aap.onlinelibrary.wiley.com/doi/10.1002/JPER.23-0224, Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1293
alized aggressive periodontitis” in the 2017 Classification,
despite differences in their clinical presentations.
The findings suggest that targeting MZB1 may be a
promising strategy for the treatment of periodontitis.
When approached from this perspective, our next step will
be microRNAs that play a role in gene silencing through
RNA-mediated mechanisms that involve the inhibition of
transcription and translation. However, further research
is needed to fully understand the underlying mechanisms
by which MZB1 contributes to periodontal tissue destruc-
tion and to develop targeted therapies that can modulate
its expression and activity.
AU T H O R CO N T R I B U T I O N S
Esra Guzeldemir-Akcakanat, Deniz Sunnetci-Akkoyunlu,
Busra Gurel, and Naci Cine have made contributions to
the conception and design of the study. Esra Guzeldemir-
Akcakanat, V. Merve Balta-Uysal, and Begum Alkan
have been involved in sample collection. Deniz Sunnetci-
Akkoyunlu, Busra Gurel, Emel Akgun, and Vakur Olgac
have been involved in the analysis of the samples, Esra
Guzeldemir-Akcakanat, Deniz Sunnetci-Akkoyunlu,
Busra Gurel, and Ahmet Tarik Baykal have been
involved in the analysis and interpretation of data,
Esra Guzeldemir-Akcakanat has been involved in drafting
the manuscript and Deniz Sunnetci-Akkoyunlu and Busra
Gurel have been involved in revising it critically and all
authors have given final approval of the version to be
published.
AC K N OW L E D G M E N T S
This study was supported kindly by a grant from the
Technological Research Council of Turkey (TUBITAK)
(214S008), Ankara, Turkey, and Kocaeli University the
Scientific Research Projects Unit Grant (KOU 2013/5),
Kocaeli, Turkey. The authors thank TUBITAK and Kocaeli
University for their generous support.
C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors report no conflicts of interest related to this
study.
ORCID
Esra Guzeldemir-Akcakanat https://orcid.org/0000-
0002-0204-9487
V. Merve Balta-Uysal https://orcid.org/0000-0001-8329-
6895
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S U P P O RT I N G I N F O R M AT I O N
Additional supporting information can be found online
in the Supporting Information section at the end of this
article.
How to cite this article: Sunnetci-Akkoyunlu D,
Guzeldemir-Akcakanat E, Alkan B, et al. Altered
expression of MZB1 in periodontitis: A possible link
to disease pathogenesis. J Periodontol.
2023;94:1285–1294.
https://doi.org/10.1002/JPER.23-0224