Advances in Cancer Biology - Metastasis 3 (2021) 100016

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Advances in Cancer Biology - Metastasis

journal homepage: www.journals.elsevier.com/advances-in-cancer-biology-metastasis

Original article

Regulation of cell adhesion to galectins by glycosylation: A new concept in

lymphoma cell adhesion
Osamu Suzuki
Department of Diagnostic Pathology, Fukushima Medical University, School of Medicine, 1 Hikariga-oka, Fukushima, 960-1295, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: To investigate the biological roles of glycosylation in human lymphoma cell adhesion to galectins, I performed the
Galectins cell adhesion assay using neuraminidase or glycosylation inhibitors on B cell lymphoma cell line. I show that
B cell receptor desialylation by neuraminidase treatment results in enhancement of cell adhesion to Phaseolus vulgaris leukoag-
CD45
glutinating lectin (L-PHA), suggesting that neuraminidase treatment removes sialic acids from N-glycans, which
B cell lymphoma
we have shown react to L-PHA in a lymphoma cell line. Neuraminidase treatment resulted in enhancement of cell
adhesion to galectin-3, an effect that was especially pronounced following pre-stimulation of B cell receptors
(BCR) by anti-IgM antibody treatment. This suggests that sialic acid may be a candidate regulator of interactions
between glycans on BCR and galectins. From treatment with tunicamycin (TM), a potent inhibitor of N-glyco-
sylation, cell adhesion capacity decreased. Furthermore, it is suggested that IgM and CD45 have N-glycans for
binding to galectins in western blot analysis. In a glycoengineering approach, I found that treatment with the
sialic acid mimetic analogue 3Fax-peracetyl Neu5Ac, which is a potent inhibitor of sialyltransferases, markedly
enhanced lymphoma cell adhesion to galectin-3. In conclusion, sialic acids and N-glycosylation appeared to in-
fluence lymphoma cell adhesion to galectins.

1. Introduction
Galectin is known to be a member of a family of beta-galactoside
binding lectin and it plays an important regulator in cancer biology,
including apoptosis, metastasis, immune surveillance [1]. Galectin reacts
to N-glycans which possess beta1,6-branched N-glycans recognized by
L-PHA lectin. The human proteins with beta1,6-branched N-glycans have
already been identified as integrin, N-cadherin, lysosomal associated
membrane proteins, L1, Mac-2 binding protein, CD166, melano-
transferrin [2,3] and such a protein could be an attractive target for
therapeutic treatment. Beta1,6-branched N-glycans are already identified
in melanoma [4]. In recent studies, I found that alpha 2,6 sialylation of
beta1,6-branched N-glycans in Diffuse large B cell lymphoma (DLBCL),
sialic acids and beta1,6-branched N-glycans modulate lymphoma cell
adhesion to the extracellular matrix (ECM) [5–8]. Glycosylation of the
variable region of IgM may affect antigen affinity to immunoglobulin [9].
In synapse formation, galectin-1 is known to be a stromal cell ligand for
the pre-B cell receptor [10]. Galectin-glycan lattice regulate cell-surface
glycoprotein organization and signaling [11]. Galectin-glycan lattice is
known to regulate B cell receptor signaling in lymphocytes [12]. B cell
receptor acts as galectin ligand, and in addition to B cell receptor CD45 is
also galectin ligand in relation to B cell receptor signaling. B cell receptor
interacts with CD45 through galectin bridging between B cell receptor
and CD45.
Sialic acids and glycosylation are known to play major metabolic,
structural and physical roles in biological system [13]. Sialic acids are
closely associated with malignant tumors [14–16]. In our previous
analysis we found that alpha-2,6 sialylation of N-glycans, and in partic-
ular sialylation of beta1,6-branched N-glycans, are closely associated
with a poor prognosis for patients with diffuse large B cell lymphoma
(DLBCL) [14]. Human Burkitt lymphoma cell clones showed a different
metastatic capacity in severe combined immunodeficiency mouse models
[17] and it suggested that sialic acids may play an important role in
lymphoma cell metastasis. Galectin is known to regulate cell adhesion in
lymphoma cells and sialic acids inhibit cell adhesion to galectin in my
previous analysis [6].
In the present study I clarify that galectin-glycan lattice may be a new
concept in cell adhesion in human malignant lymphoma.

E-mail address: osuzuki@fmu.ac.jp.

https://doi.org/10.1016/j.adcanc.2021.100016
Received 23 August 2021; Received in revised form 29 October 2021; Accepted 29 October 2021
Available online 3 November 2021
2667-3940/© 2021 The Author. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
O. Suzuki
2. Materials and methods
2.1. Cell lines
Use of a cell line derived from patients was approved by the Bioethics
Committee of Fukushima Medical University. Informed consent was
obtained by disclosure of the research concepts on the homepage of the
Fukushima Medical University website. The HBL-2 cell line was estab-
lished in our laboratory from a patient who had diffuse large B cell
lymphoma. HBL-2 cells were grown in a culture medium of RPMI1640
containing 15% fetal calf serum in 5% CO2 at 37
C. HBL-8 is a human
Burkitt lymphoma cell line. Two clones of HBL-8, 3G3 and 3D2: Burkitt
lymphoma cell lines.
2.2. Cell surface lectin binding assays
HBL-2 cells were treated with or without neuraminidase from
Arthrobacter ureafaciens (No. 10269611001, Roche, Germany) at 0.2 U/
ml, at 37
C for 30 min. A 96-well plate was coated by Phaseolus vulgaris
(L-PHA) lectin, specific for beta1,6-branched N-glycans (EY Laboratory,
USA, L-1801-2, 10 μl/well from 1 mg/ml solution), Helix pomatia (HPA)
lectin, specific for GalNAc (EY Laboratory, USA, L-3601-1, 10 μl/well
from 1 mg/ml solution) or galectin-3 (PROSPEC, recombinant human
galectin-3, Cat# CYT-606, Po. BOX 4157, Ness Ziona 7414003, Israel)
(10 μl/well from 50 μg/250 μl solution) or LGALS 1 (galectin-1) (human,
recombinant, ABN, P4390, Funakoshi, Japan) (10 μl/well from 50 μg/
500 μl solution) and air dried. The neuraminidase treated or non-treated
lymphoma cells (1  106/2 ml) were applied to each well (100 μl/well)
and incubated at 37
C for 60 min. After aspiration of the medium, PBS
was added to each well and then aspirated to remove non-adhered cells.
Next, 100 μl of 3.7% formaldehyde was added to each well and incubated
at RT for 5 min to fix the adhesive cells. After aspiration of formaldehyde,
100 μl of 0.1% crystal violet was added to each well and the plates were
incubated at RT for 2 min. After washing with PBS, 100 μl of 10% acetic
acid was added to each well and the absorbance at 570 nm was deter-
mined using an ELISA plate reader (iMark™, Microplate Reader, Bio-Rad,
Hercules, CA) [7]. To analyze the contribution of phosphatidylinositol 3
kinase (PI3K) in HBL-2 cell adhesion to galectin-3 or L-PHA, cells were
pre-incubated with 0.2 U/ml neuraminidase from Arthrobacter ure-
afaciens (No. 10269611001, Roche, Germany) at 37
C for 30 min and
then with 1.7 μM of the PI3K inhibitor wortmannin (CALBIOCHEM,
#681675, Merck, Japan) or 25 μM of the MEK inhibitor PD98059
(CALBIOCHEM, #513000, Merck, Japan) at 37
C for 30 min PI3K and
MEK were detected in the cytoplasm of HBL-2 cells by immunohisto-
chemical staining using anti-PI3K or MEK Ab. Akt phosphorylation upon
stimulation by anti-IgM Ab was detected by western blot analysis (not
shown). To evaluate the effects of the dual Rac1 and Cdc42 inhibitor
AZA1 (#530152, Calbiochem, USA), lymphoma cells were pre-incubated
with 200 μM AZA1 for 2 h. AZA1 is a potent inhibitor of Cdc42 and Rac1.
To inhibit N-glycosylation, HBL-2 cells were pre-treated with 0.5 μg/ml
swainsonine (SW) (S9263, Sigma, Japan) at 37
C for 3 days, or 5 μg/ml
tunicamycin (TM) (T7765, Sigma, Japan) at 37
C for 3 days. To analyze
the effects of anti-IgM antibody, HBL-2 cells which is known to express
IgM on cell surface were pre-incubated with anti-IgM antobody (5 μg/ml,
NBP2-34649, Novus, USA) at 37
C for 45min before cell adhesion
analysis.
2.3. Western blotting
HBL-2 cells were treated with 5 μg/ml tunicamycin (TM) for 3 days at
37
C, then cell lysates were prepared. Briefly, cell pellets were sus-
pended in lysis buffer (0.5% Nonidet P-40 (Sigma) in 0.0 1 M PBS) and
lysed by gently pipetting with micropipette. The lysates were then
centrifuged at 11,000 rpm for 25 min at 4
C. The same protein con-
centration from each sample, as determined using a TaKaRa BCA protein
assay kit (T9300A, TaKaRa, Japan), was heated at 100
C for 5 min, then
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Advances in Cancer Biology - Metastasis 3 (2021) 100016
30 μg was loaded in each lane of a 7.5% SDS-PAGE gel and electro-
phoresed under reducing conditions at a constant current of 20 mA.
Proteins were transferred from the gel to a PVDF membrane (Millipore)
in buffer at a constant current of 120 mA for 60 min and then 180 mA for
30 min. To block non-specific binding, the membrane was incubated
overnight at 4
C in 0.05 M Tris buffer, pH 7.6, containing 5% bovine
serum albumin. The membrane was then incubated with primary anti-
bodies, including anti-IgM (1:500, DM074-05, Acris Antibodies, USA),
anti-CD45 (1:500, H0408, Nichirei, Japan) and β-actin for 30 min at RT.
After washing, the blots were incubated with biotinylated anti-mouse Ig
antibody (1:1000 dilution, BA-9200, VECTOR). After an additional wash
step, the membrane was incubated with a streptavidin-biotin peroxidase
complex (VECSTATIN, Elite ABC standard kit, VECTOR) for 20 min and
then with a DAB-H2O2 solution for 5 min at RT. After each incubation
step, the membrane was washed three times for 5 min in Tris buffer, pH
7.6, containing 0.01% Tween 20. Western blotting of β-actin provided an
internal control and was detected using an anti-β-actin antibody
(1:10000 dilution, clone AC74, Sigma-Aldrich, Japan).
To evaluate the effect of neuraminidase treatment, HBL-2 cells were
incubated with neuraminidase from Arthrobacter ureafaciens at a final
concentration of 0.2 U/ml at 37
C for 30 min (Roche, Germany). For
inhibition of N-glycosylation, HBL-2 cells were pre-treated with 5 μg/ml
tunicamycin (TM) (T7765, Sigma, Japan) at 37
C for 3 days. The amount
of cells with TM treatment was normalized in all assay because TM
mediated ER stress induces growth inhibition.
2.4. Protein tyrosine phosphatase (PTP) activity
The PTP activity of HBL-2 cells was measured using a p-Nitrophenyl
phosphate (pNPP) protein phosphatase assay kit according to the man-
ufacturer's instructions (ANA SPEC, No. AS-71105, USA). Cell lysate for
PTP activity assay was obtained from the lysate used for western blotting
as described above. For treatment with tunicamycin, HBL-2 cells were
treated as described above.
2.5. Fluorinated sialic acid analogue (3 Fax-peracetyl Neu5Ac) treatment
Fluorinated sialic acid analogue (3 Fax-peracetyl Neu5Ac: 566224,
Millipore USA) is a potent inhibitor of sialyltransferases. HBL-8 3G3
clone lymphoma cells were treated with 200 μM 3 Fax-peracetyl Neu5Ac
for 3 days. After treatment, a cell surface lectin binding assay was per-
formed using Helix pomatia (HPA) lectin (L-3601-1, EY Laboratory, USA)
and galectin-3 (PROSPEC, recombinant human galectin-3, Cat# CYT-
606, Po. BOX 4157, Ness Ziona 7414003, Israel), as described above. A
96-well plate was coated by Helix pomatia (HPA) lectin (EY Laboratory,
USA, L-3601-1, 10 μl/well from 1 mg/ml solution) or galectin-3
(PROSPEC, recombinant human galectin-3, Cat# CYT-606, Po. BOX
4157, Ness Ziona 7414003, Israel) and air dried. The lymphoma cells
(1  106/2 ml) were applied to each well (100 μl/well) and incubated at
37
C for 60 min. After aspiration of the medium, PBS was added to each
well and then aspirated to remove non-adhered cells. Next, 100 μl of
3.7% formaldehyde was added to each well and incubated at RT for 5 min
to fix the adhesive cells. After aspiration of formaldehyde, 100 μl of 0.1%
crystal violet was added to each well and the plates were incubated at RT
for 2 min. After washing with PBS, 100 μl of 10% acetic acid was added to
each well and the absorbance at 570 nm was determined using an ELISA
plate reader (iMark™, Microplate Reader, Bio-Rad, Hercules, CA) [7]. I
performed the sialic acid analogue assay using HPA lectin and galectin-3.
HPA lectin binding capacity is known to be strongly inhibited by sialic
acids. Therefore, regardless of N- or O-glycans, I want to confirm the
desialylated effects of sialic acid analogue in relation to adhesion to
galectin-3. In future investigation, I will try to perform the L-PHA, DSA
lectin (which are specific for N-glycans) binding assay with sialic acid
analogue treatment.
O. Suzuki
2.6. Immunohistochemical detection of PI3K and MEK
To detect PI3K and mitogen-activated protein kinase kinase (MEK) in
HBL-2 cells, HBL-2 cells were cytospun to the slide glass, the specimen
was fixed with 100% acetone at 4
C for 30 min, and then air dried for
30 min. Immunohistochemical staining was done using an anti-PI3K
antibody (dilution 1:100, Santa Cruz, clone B-9, sc-1637) or anti-MEK
antibody (dilution 1:100, MEK1/2 (L38C12), 4694S, Cell Signaling
technology, Japan). Next, the cell preparations were incubated with
biotinylated anti-mouse immunoglobulin. After washing three times in
PBS, the preparations were incubated at room temperature for 20 min
with an Avidin-Biotin peroxidase complex (DAKO, Tokyo, Japan). Then,
they were incubated for 5 min at room temperature with dia-
minobenzidine (DAB)-H2O2 solution (60 μg DAB (Wako, DOJINDO, 347-
00904, Japan) in 150 ml PBS). The preparations were counterstained
with hematoxylin and mounted.
2.7. Statistical analysis
The P values for experimental results were calculated using the Stu-
dent's t-test. P < 0.05 was considered statistically significant. Data
analysis was performed using Microsoft Office Excel 2007.
3. Results
I first assayed cell adhesion of a lymphoma cell line derived in our lab,
HBL-2. I assayed adhesion of these cells to L-PHA and galectin-3. On the
cell surface lectin binding assay using L-PHA lectin and galectin-3, de-
sialylation by neuraminidase treatment resulted in enhancement of cell
adhesion to L-PHA lectin or galectin-3. Furthermore, as anti-IgM anti-
body (Ab) stimulates IgM, the enhanced effect of anti-IgM Ab treatment
on cell adhesion to L-PHA or galectin-3 is due to IgM-induced signaling,
suggesting that sialic acid may inhibit interaction between glycans on
IgM and galectin-3 (Fig. 1a and b). Moreover, there is a possibility that
anti-IgM antibody may stimulate interaction between glycan on IgM and
galectins. Following pre-treatment with wortmannin to inhibit PI3K,
anti-IgM Ab-mediated enhancement of cell adhesion to galectin-3 dis-
appeared (Fig. 1c). This result suggests that anti-IgM Ab-mediated
enhancement of cell adhesion to galectin-3 may be regulated by PI3K in
the HBL-2 cell line. As shown in Fig. 1d, PI3K protein is detectable in the
cytoplasm of HBL-2 cells. In our preliminary data, pre-treatment with
wortmannin abrogated anti-IgM Ab-mediated enhancement of cell
adhesion to L-PHA (data not shown). As observed by immunohisto-
chemistry, MEK is expressed in the cytoplasm in HBL-2 cells; however,
treatment with the MEK inhibitor PD98059 did not alter IgM-mediated
cell adhesion in HBL-2 cells (not shown).
In the absence of neuraminidase treatment, adhesion of HBL-2 cells to
galectin-3 could be inhibited by treatment with the dual Rac1 and Cdc42
inhibitor AZA1 (25 μM for 30 min). In contrast, following cleavage of cell
surface sialic acids by neuraminidase treatment, enhanced cell adhesion
to galectin-3 was markedly inhibited by AZA1 (Fig. 2a). Cell adhesion to
galectin-1 was enhanced following anti-IgM Ab treatment and this effect
was abrogated by pre-treatment with AZA1 (Fig. 2b). Galectin-1 medi-
ated cell adhesion to anti-IgM Ab is linked to Cdc42 signaling in HBL-2.
To evaluate participation of N-glycans in cell adhesion, HBL-2 cells
were pre-treated with tunicamycin (TM) then cell adhesion was assayed.
After treatment with TM, the enhanced effects of cell adhesion to
galectin-3 normally mediated by anti-IgM Ab disappeared (Fig. 3a). I also
tested the effects of swainsonine (SW), a potent inhibitor for elongation
of N-glycans. I found that treatment with SW enhanced cell adhesion to
galectin-3 (data not shown). The enhancement effect by SW is still
controversial result, because SW inhibited complex type N-glycans. The
phenomena still remain unclear. This data may be confirmed in future
investigations.
As observed by western blot, CD45 in the HBL-2 cell line was
degraded (control: ~200 kDa, TM treatment: not detected) and the
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Advances in Cancer Biology - Metastasis 3 (2021) 100016
molecular weight of IgM decreased after TM treatment (control: ~
70 kDa, TM treatment: ~40 kDa) (Fig. 3b). CD45 may be deglycosylated
after TM treatment because glycoprotein tends to be degraded after
deglycosylation with TM treatment. In addition, IgM may be deglyco-
sylated after TM treatment because the molecular weight of IgM is
decreased due to removal of N-glycans after TM treatment. These data
suggest that N-glycosylation of CD45 and IgM are inhibited following TM
treatment. We next measured PTP activity using a PTP activity kit
(Fig. 3c). PTP activity was inhibited following tunicamycin treatment. In
our preliminary experiments, we found that PTP activity was not
changed following treatment of galectin-3 (20 μM, 1 h) in HBL-2 cells
(not shown).
Following treatment of HBL-8 cells with a fluorinated sialic acid
analogue (3 Fax-peracetyl Neu5Ac), cell adhesion to HPA lectin was
enhanced. Cell adhesion to galectin-3 also appeared to be enhanced
(Fig. 3d). We confirmed that the number of cells was not altered after
treatment with the sialic acid analogue for 3 days using a WST-1 cell
viability assay (not shown). We did not observe any effect of the sialic
acid analogue on cell adhesion of HBL-2 cells (not shown).
4. Discussion
In the present study, I found that anti-IgM Ab enhanced cell adhesion
to galectin-3. I reason that this enhancement effect may be due to stim-
ulation of IgM or stimulation of interaction between glycans on IgM and
galectin-3, as detectable in a cell adhesion assay. This enhancement effect
of anti-IgM Ab on cell adhesion suggests that HBL-2 cells may adhere to
galectin-3 through interaction between glycans on BCR (IgM) and
galectin-3.
By western blot analysis, the observed molecular weight of IgM
decreased following neuraminidase treatment, suggesting that glycans of
BCR are conjugated with sialic acids (in our preliminary data). Galectins
produced by macropahges, are secreated to ECM [18]. Therefore, there is
a possibility that DLBCL cells may interact with macrophages and ECM
via interaction between glycans of BCR and galectin-3, and sialic acids on
glycans of BCR may control cell adhesion capacity to galectin-3 of lym-
phoma cells. Sialic acids may inhibit cell adhesion to galectins and
facilitate cell invasive capacity, because lymphoma cells cannot move
smoothly due to the marked enhancement in cell adhesion induced by
the desialylation that results from neuraminidase treatment. In a previ-
ous study using the Anaplastic large cell lymphoma cell line H-ALCL, I
found that H-ALCL show low invasive capacity upon neuraminidase
treatment due to marked enhancement of cell adhesion to galectin-1 [6].
I next asked if disruption of adhesion by anti-IgM Ab treatment affects
cell adhesion to galectins. In a cell adhesion assay, I found that treatment
with an anti-IgM Ab led to enhanced cell adhesion to galectins. Although
the detailed mechanisms remain unclear, this enhanced effect may be
due to stimulation or changes in the molecular conformation of IgM
induced by anti-IgM Ab treatment. Thus, interaction between galectins
and BCR might be a foundation of BCR-mediated cell adhesion mecha-
nism. In previous reports, CD45 was proposed as a ligand for galectin-1
involved in regulation of cell death [19,20]. Moreover, CD45 has been
shown to be a ligand for both galectin-3 and galectin-1 [21,22]. They
showed that sialic acids may inhibit interaction between CD45 and
galectin-3, and this suggested that sialic acids may be a key regulator for
interaction between CD45 and galectin. In this work, I found evidence
that CD45 and BCR are modified by N-glycans. Namely, I found that
CD45 was degraded with TM treatment, and molecular weight of BCR
decreased with TM treatment on western blot analysis, suggesting that
N-glycans may be removed from CD45 and N-glycans may be also
removed from BCR, and N-glycans may also be a regulator for interaction
between CD45 or BCR and galectins.
Interaction between CD45 and BCR through the glycan-galectin lat-
tice might be associated with firm cell adhesion to galectin due to glycan-
galectin lattice effects, and loss of glycans on these molecules might lead
to weak cell adhesion to galectins even in the absence of an interaction
O. Suzuki Advances in Cancer Biology - Metastasis 3 (2021) 100016

Fig. 1. Cell adhesion assay to galectins. Cell adhesion assay was performed using L-PHA and galectin-3. Adherent cells were measured on 96-well lectin-coated plates
and stained with crystal violet. The number of adhered cells was evaluated by crystal violet staining (i.e. 570 nm absorbance) using an ELISA plate reader. Following
neuraminidase neuraminidase treatment, cell adhesion to L-PHA lectin ((a), *: p < 0.05, **: p < 0.05) or galectin-3 ((b), *: p < 0.005, **: p < 0.005). N(): Non-
treatment with neuraminidase, N(þ): Treatment with neuraminidase. Anti-IgM Ab treatment enhanced in cell adhesion to L-PHA and galectin-3. There is an addi-
tional effect by anti-IgM antibody treatment. N: Neuraminidase treatment, iso: Isotype control antibody, anti-IgM: Anti-IgM antibody. P values are calculated based on
Student's t-test. Bars represent mean  SD. Data are representative of two independent experiments performed in triplicate.
Contribution of PI3K to adhesion of cells to galectin-3. Shown, results of PI3K-mediated cell adhesion to galectin-3 with anti-IgM Ab treatment. Briefly, HBL-2 cells
were treated with neuraminidase to remove cell surface sialic acids. Next, cells were treated with or without the PI3K inhibitor wortmannin. After washing, cells were
incubated in 96-well plates coated with galectin-3 and cell adhesion was assayed. Adhesion to galectin-3 with treatment of anti-IgM Ab (*: p < 0.005, (c)). Data shown
are representative of two independent experiments performed in triplicate. For samples pre-treated with wortmannin, anti-IgM Ab-mediated enhancement of cell
adhesion to galectin-3 compared to isotype control Ab was abrogated (NS: not significant). Immunohistochemical detection reveals detection of PI3K in the cytoplasm
of HBL-2 cells (original magnification x400; a representative PI3K positive cell was selected for panel (d). Treatment with the MEK inhibitor PD98059, by contrast, did
not alter IgM-mediated cell adhesion to galectin in HBL-2 (not shown). P values were calculated using the Student's t-test. Bars represent mean  SD. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

4
O. Suzuki
Fig. 2. Adhesion of HBL-2 cells to galectin-3 following AZA1 treatment (25 μM
for 30 min). Briefly, HBL-2 cells were treated with or without neuraminidase to
remove cell surface sialic acids. Then, cells were treated with or without AZA1.
After washing, cells were incubated in 96-well plates coated with galectin-3 and
cell adhesion was assayed. Adhesion to galectin-3 with neuraminidase by HBL-
2 cells (*: p < 0.005: no neuraminidase [sialylation]; **: p < 0.01: neuramini-
dase treatment [desialylation]) (a). Briefly, HBL-2 cells were treated with
neuraminidase to remove cell surface sialic acids. Then, cells were treated with
or without the Cdc42 inhibitor AZA1. After washing, cells were incubated in 96-
well plates coated with galectin-1 and cell adhesion was assayed. Cell adhesion
to galectin-1 was enhanced by anti-IgM Ab treatment and this effect was
abrogated by AZA1 pre-treatment (*: P < 0.005, NS: not significant, (b)).
Galectin-mediated IgM signaling is linked to Cdc42 signaling in HBL-2. Data
shown are representative of two independent experiments performed in tripli-
cate. Following treatment with AZA1, we confirmed that AZA1-treated cells did
not show signs of cell death, as determined using a Trypan-blue exclusion assay.
P values were calculated using the Student's t-test. Bars represent mean  SD.
between glycans of CD45 and BCR. We based this idea on the assumption
that when adhesion of tumor cells to ECM is weakened, cells are more
able to move through galectins.
The galactose residue of N-glycans may be ligand for galectins that
can regulate interactions between galectins and glycans, and CD45-
mediated intracellular signaling, such as regulation of CD45 protein
tyrosine phosphatase activity. In the recent research interaction between
5
Advances in Cancer Biology - Metastasis 3 (2021) 100016
CD45 and T cell receptor through glycan-galectin interaction was pro-
posed in T cell receptor signaling [23]. Furthermore, CD45 positively
controls BCR signaling [24] and another phosphatase, Src homology
region-2 domain-containing phosphatase-1 (SHP-1), is known to control
B cell receptor signaling [25]. I propose that galectin might interact with
glycans on CD45 and BCR, bringing about firm cell adhesion to galectins
(Fig. 4a). In a previous report, N-glycosylation was shown to be related to
CD45 protein stability and phosphatase activity [26]. PTP activity rep-
resents whole PTP including CD45PTP or SHP-1. Specific PTP analysis
can not be available in our laboratory. Therefore, it is needed to consider
whole PTP activity influencing cell signaling. PTP activity was not
observed to change following treatment with galectin-3 of HBL-2 cells
(unpublished data), suggesting that galectin-3 cannot inhibit CD45 PTP
activity. But I speculate that decreasing of PTP activity by loss of N-gly-
cans on CD45 may influence BCR signaling (Fig. 4a).
Sialylation of the cell surface of lymphoma cells may be associated
with a poor prognosis [14] and influence cell adhesion to or invasion of
ECM, leading to increased aggressiveness [27]. In our speculation, sialic
acids may be key molecules in lymphoma cell adhesion or invasion
through glycan-galectin interaction. In a recent report, the authors re-
ported that galectin-1 interacts with BCR [10]. Another report showed
that altered N-glycans on immunoglobulin interact with lectin derived
from dendritic cells and further, that this interaction induces immuno-
globulin signaling in follicular lymphoma [28,29]. Moreover, previous
reports showed that the protein tyrosine phosphatase is involved in B cell
receptor signaling [30]. PI3K is known to be involved in galectin
signaling [31]. Upon treatment with the PI3K inhibitor wortmannin, the
enhanced effects in cell adhesion to galectin-3 normally brought about by
anti-IgM Ab were abrogated, suggesting that cell adhesion to galectin-3
on treatment with anti-IgM Ab is dependent on PI3K.
BCR signaling is known to be involved in cell adhesion or migration
[25]. Another report showed that BCR signaling controls integrin activity
and cell adhesion to fibronectin [32]. In the present study, I found that
treatment with anti-IgM Ab markedly enhanced cell adhesion to
galectin-3 with or without neuraminidase treatment. Upon treatment
with neuraminidase to remove sialic acids from the beta-galactose res-
idue, which is a ligand for galectins, beta-galactose was exposed and cell
adhesion to galectins was enhanced. Therefore, I conclude that sialic acid
masks beta-galactose, resulting in inhibition of cell adhesion. But in
future, it still remains unclear whether integrins may be involved in this
adhesion.
PI3K is a candidate for intracellular signaling that mediates the
interaction between N-glycans of BCR and galectin. In addition, members
of the Rho GTPase family are known to modulate lymphoma cell motility
[6]. Rho has been associated with a prognosis for patients with follicular
lymphoma [33]. In addition, Rho GTPase is thought to be involved in the
malignant phenotype by affecting the invasive capacity of lymphoma
cells. In the previous report Cdc42 and Rac1 activation is known to
associated with integrin mediated cell adhesion [34]. Therefore, in the
present study, it is performed whether cell adhesion to galectin-3 may be
inhibited with pre-treatment with Rac1 or Cdc42 inhibitor, AZA1 using
HBL-2 cells, or not. AZA1 treatment inhibited cell adhesion to galectin-3.
These data suggest that Rac1 and Cdc42 are involved in regulating the
capacity of HBL-2 cells to adhere to galectin-3. In the galectin-1 adhesion
assay, cell adhesion to galectin-1 was enhanced following treatment with
anti-IgM Ab. Moreover, this enhanced effect was completely inhibited by
pretreatment with AZA1, suggesting that the galectin-IgM interaction is
mediated through one or more Cdc42, Rho family protein as well as
through PI3K. Based on these results, activation of PI3K and Cdc42
signaling via adhesion of lymphoma cells to galectins likely remodels the
cytoskeleton, which in turn can affect adhesion.
Our recent data suggest that Rho regulates cell invasion through
galectins [6]. Collectively, these data indicate that Rho family activation
is required for lymphoma cell invasion via galectin. Enhanced adhesion
of HBL-2 cells to galectin-3 following neuraminidase treatment was
abolished by AZA1 treatment. Alteration of sialic acid on the cell surface
O. Suzuki Advances in Cancer Biology - Metastasis 3 (2021) 100016

Fig. 3. Inhibitor of glycosylation. HBL-2 cells were pre-treated with 5 μg/ml TM for 3 days at 37
C. Then, a cell adhesion was assayed. After treatment with TM, the
enhanced cell adhesion to galectin-3 normally induced by anti-IgM Ab was not observed after treatment with TM (*: P < 0.005, NS: not significant). These data
indicate that N-glycans are needed to induce IgM-mediated intracellular signaling in HBL-2 cells. P values were calculated using the Student's t-test. Bars represent
mean  SD. To evaluate N-glycans in cell adhesion to galectin-3 as induced by SW treatment, HBL-2 cells were pre-treated with 0.5 μg/ml SW for 3 days at 37
C. Then,
cell adhesion was assayed. After treatment with SW, cell adhesion to galectin-3 was enhanced (not shown). Data shown are representative of two independent ex-
periments performed in triplicate (a).
Western blot analysis of CD45 and IgM. HBL-2 cells were treated with 5 μg/ml TM for 72 h, then cell lysate was prepared and separated by SDS-PAGE, and transferred
to PVDF membrane. The membrane was then blotted with anti-CD45 or anti-IgM Ab. CD45 was degraded (control: ~200 kDa, TM treatment: not detected) and the
observed molecular weight of IgM decreased (control: ~70 kDa, TM treatment: ~40 kDa) after TM treatment (b). These data suggest that N-glycosylation of CD45 and
IgM is inhibited following TM treatment of HBL-2 cells. PTP activity was measured using a PTP activity kit. PTP activity was inhibited by treatment with TM (*:
p < 0.005). Vehicle control for TM treatment, ethanol. The protein concentration in each assay lysate was 1.5 μg/μl per well of a 96-well plate and protein con-
centration was measured using a TaKaRa BCA protein assay kit (T9300A, TaKaRa, Japan) (c). The data shown are representative of two independent experiments.
Effects on cell adhesion of treatment with a sialic acid analogue. Shown, results of treatment of HBL-8 cells with fluorinated sialic acid analogue (3 Fax-peracetyl
Neu5Ac ¼ sialyltransferase inhibitor (STI)). Cell adhesion to HPA lectin following 3 Fax-peracetyl Neu5Ac pre-treatment (*: P < 0.01). Cell adhesion to galectin-3
(**: P < 0.05). The number of cells did not change following treatment with the sialic acid analogue for 3 days at 37
C, as assayed using a WST-1 cell viability
assay (not shown). P values were calculated using the Student's t-test. Bars represent mean  SD. Data are representative of two independent experiments performed in
triplicate. No effect of the sialic acid analogue on cell adhesion was observed for the HBL-2 cell line (not shown) (d).

appeared to be linked to activation of Rac1 and Cdc42. Members of the
Rho GTPase family are known to regulate cell motility and chemotaxis
[35,36]. Sialic acids may regulate the Rho GTPase family in relation to
cell motility in lymphoma cells.
PI3K is known to regulate integrin signaling and PI3K is involved in
Akt signaling. In our preliminary data, I found that treatment with an
anti-IgM Ab induces phosphorylation of Akt as detected by western blot
analysis. In a cell adhesion assay, Akt inhibition enhanced anti-IgM
mediated cell adhesion to galectin-3 suggesting that Akt may nega-
tively regulate cell adhesion to galectin-3 through IgM signaling (not
shown). I observed differences between PI3K and Akt with regards to
regulation of IgM-mediated cell adhesion to galectin-3. In relation to cell
adhesion to galectin-3, anti-IgM mediated cell adhesion to galectin-3 may
be positively regulated by PI3K and negatively regulated by Akt.
Therefore, a balance between PI3K and Akt signaling may be critical for
adhesion of HBL-2 cells to galectin-3. In a recent report, PI3K signaling
was linked to anti-IgM mediated cell adhesion to fibronectin in chronic
lymphocytic leukemia [37]. Sialylation of the glycans in integrins or BCR
may regulate galectin-mediated intracellular signal transduction, in turn
modulating the adhesive or invasive capacity of lymphoma cells as
shown in Fig. 4b. It is therefore possible that treatment with neuramin-
idase or with siRNA targeting the sialyltransferase enzyme ST6GalI
which induces sialylation of cell surface N-glycans, could be used in
lymphoma therapy. Specifically, treatment with these molecules might
modulate cell surface sialylation, thereby inhibiting lymphoma cell in-
vasion and slowing disease progression.
From our previous data we have clarified that O-glycans are associ-
ated with galectin-1 mediated cell death, and O-glycan inhibitor, Benzyl-

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O. Suzuki Advances in Cancer Biology - Metastasis 3 (2021) 100016

Fig. 4. Model of the effects of glycan-galectin interaction on IgM- and CD45-mediated cell adhesion in DLBCL. The glycan-galectin lattice between IgM and CD45 leads
to firm cell adhesion to galectins, and loss of N-glycans, for example as experimentally induced by tunicamycin treatment, and the subsequent loss of a glycan-galectin
lattice may be associated with weak cell adhesion to galectins. CD45 PTP might regulate B cell receptor (IgM) signaling and deglycosylation of CD45 might decrease
CD45 PTP activity, influencing B cell receptor signaling. Furthermore, the galectin receptors IgM and CD45 may interact via the glycan-galectin lattice. These
mechanistic functions of glycan-galectin interaction may be relevant to clinical outcomes of DLBCL (a).
Model of biological roles of sialylation in glycan-galectin interaction. Changes to sialylation of glycans might regulate IgM-mediated intracellular signaling and cell
adhesion properties associated with invasion and metastasis of lymphoma cells. Moreover, sialylation of glycans on IgM may be regulated by sialyltransferase (b).

GalNAc (BZ) treatment prevents cell death induced by galectin-1 [19].
On western blot BZ treatment led to decreasing of CD45 molecular
weight and it is suggested that CD45 has some O-glycans. There is a
possibility that galectins may interact to CD45 O-glycans as well as
N-glycans. In future investigations it is needed to clarify the biological
roles of CD45 O-glycans in galectin mediated cell adhesion and signaling
in lymphoma cells.
In glycoengineering experiment, sialic acid analogue was found to
alter cell surface sialylation status [38]. A fluorinated sialic acid
analogue, 3Fax-peracetyl Neu5Ac, inhibits sialyltransferase, tumor cell
invasion, and growth [39]. Expression of the mRNA for ST6GalI and
ST3Gal IV was detected in the HBL-8 3G3 clone [27]. 3Fax-peracetyl
Neu5Ac treatment induced enhancement of cell adhesion to HPA lec-
tin, suggesting that N-acetylgalactosamine increased after treatment.
3Fax-peracetyl Neu5Ac is a potent inhibitor of sialyltransferases such as
ST3Gal IV. Thus, 3Fax-peracetyl Neu5Ac may inhibit ST3Gal IV, thereby
leading to reduced sialylation of cell surface glycans. Moreover, this
might subsequently expose beta-galactose and inhibit
beta-galactosylation (in our speculation, probably by endogenous
beta-galactosidase digestion), resulting in exposure of N-acetylgalactos-
amine. In support of this idea, I note that endogenous beta-galactosidase
was shown to be activated after neuraminidase activation in living cells
[40].
Nevertheless, I recognize that there is a possibility that 3Fax-peracetyl
Neu5Ac may block attaching of sialic acids to N-acetylgalactosamine. I
note that HPA lectin can react to N-acetylgalactosamine after neur-
aminidase treatment and strongly reacts to desialylated glycans [41]. As
galectin-3 also recognizes N-acetylgalactosamine and beta-galactose
[42], removal of sialic acids from glycans may enhance cell adhesion
to galectin-3. Based on the present results, I predict that 3Fax-peracetyl
Neu5Ac may be a useful tool for alteration of cell surface sialylation,
leading to regulation of cell adhesion to galectin-3. Therefore, I speculate
that inhibition of cell surface sialylation by 3Fax-peracetyl Neu5Ac may
lead to a marked enhancement of cell adhesion to galectins and inhibi-
tion of cell invasive capacity and metastasis, as invasion would be sup-
pressed by enhancement of cell adhesion. As mentioned, in an SCID
mouse animal model using Burkitt lymphoma cell clones, the highly
sialylated 3G3 clone showed a higher metastatic capacity than the 3D2
clone with low levels of sialylation [17]. Based on this model, I further
speculate that sialylation is closely associated with high metastatic rate
due to low adhesiveness to extracellular matrix and high invasive ca-
pacity [43]. The low adhesion capacity to extracellular matrix due to
negative charged sialic acids on the cell surface may result in high
invasive capacity of lymphoma cells.
A recent report showed that treatment with a sialic acid glycomimetic
inhibited metastasis of melanoma cells in an animal model [44].
Furthermore, intratumoral injections with sialic acid glycomimetic in-
hibits tumor cell growth and permits cytotoxic T cell-mediated tumor
immunity to tumor cells [45]. This inhibition of tumor cell growth may
be associated with desialylation-induced killing of tumor cells by cyto-
toxic T cells. Thus, these processes, including inhibition of tumor cell
metastasis or growth, may be candidates for lymphoma therapy based on
glycoengineering.
In conclusion, N-glycosylation or sialic acids influence clinical out-
comes in lymphoma, and N-glycosylation alteration or glycoengineering
using a sialic acid analogue may be an available technique for inhibition
of cell invasion or metastasis, contributing to a strategy for lymphoma
glycotherapy.
Author contributions
OS performed all the experiments and wrote the manuscript. OS
approved the manuscript.
Declaration of competing interest
The author declares no conflict of interests.
Acknowledgement and Grant Support
The author would like to express deep appreciation for scientific
insight provided by Prof. Dr. Yuko Hashimoto in Department of Diag-
nostic Pathology, Fukushima Medical University.
This work was supported by JSPS KAKENHI grant number JP (No.
18K07240) from the Ministry of Education, Culture, Sports, Science and
Technology, Japan.

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O. Suzuki Advances in Cancer Biology - Metastasis 3 (2021) 100016

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