Differential recognition of diet-derived Neu5Gc-Neoantigens on glycan microarrays by carbohydrate-specific pooled human IgG and IgA antibodies

Article abstract of DOI:10.1021/acs.bioconjchem.9b00273

Sialic acids (Sias) cover vertebrate cell surface glycans. N-Acetylneuraminic acid (Neu5Ac) and its hydroxylated form N-glycolylneuraminic acid (Neu5Gc) are common Sia in mammals. Humans cannot synthesize Neu5Gc but accumulate it on cells through red-meat rich diets, generating numerous immunogenic Neu5Gc-neoantigens. Consequently, humans have diverse anti-Neu5Gc antibodies affecting xenotransplantation, cancer, atherosclerosis, and infertility. Anti-Neu5Gc antibodies circulate as IgG, IgM, and IgA isotypes; however, repertoires of the different isotypes in a large population have not been studied yet. Here, we used glycan microarrays to investigate anti-Neu5Gc IgGs and IgAs in intravenous immunoglobulin (IVIG) or pooled human IgA, respectively. Binding patterns on microarrays fabricated with Neu5Gc- and Neu5Ac-glycans, together with inhibition assays, revealed that different IVIG preparations have highly specific anti-Neu5Gc IgG reactivity with closely related repertoires, while IgAs show cross-reactivity against several Neu5Ac-glycans. Such different anti-Neu5Gc IgG/IgA repertoires in individuals could possibly mediate distinctive effects on human diseases.

Full text of DOI:10.1021/acs.bioconjchem.9b00273

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Article
Differential recognition of diet-derived Neu5Gc-neoantigens on glycan
microarrays by carbohydrate-specific pooled human IgG and IgA antibodies
Shani Leviatan Ben-Arye, Christoph Schneider, Hai Yu, Salam
Bashir, Xi Chen, stephan von gunten, and Vered Padler-Karavani
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00273 • Publication Date (Web): 17 Apr 2019
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Differential recognition of diet-derived Neu5Gc-neoantigens on glycan microarrays by
carbohydrate-specific pooled human IgG and IgA antibodies
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Shani Leviatan Ben-Arye†, Christoph Schneider§,#, Hai Yu‡, Salam Bashir†, Xi Chen‡, Stephan
von Gunten§ and Vered Padler-Karavani†*
†The George S. Wise Faculty of Life Sciences, Department of Cell Research and Immunology,
Tel Aviv University, Tel Aviv, 69978, Israel; §Institute of Pharmacology, University of Bern,
Bern, 3010, Switzerland; ‡ Department of Chemistry, University of California-Davis, Davis, CA
95616, USA
# Currently at CSL Behring AG, Switzerland
Keywords: anti-carbohydrate antibodies, IgG, IgA, sialic acids, N-glycolylneuraminic acid
(Neu5Gc), glycan microarrays.
Corresponding author address: Department of Cell Research & Immunology, The George S.
Wise Faculty of Life Sciences, Tel-Aviv University, Tel Aviv 69978 Israel. Tel: +972-3-640-6737.
Fax: +972-3-642-2046. E-mail address: vkaravani@tauex.tau.ac.il
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Abstract
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Sialic acids (Sias) cover vertebrate cell surface glycans. N-acetylneuraminic acid (Neu5Ac)
and its hydroxylated form N-glycolylneuraminic acid (Neu5Gc) are common Sia in mammals.
Humans cannot synthesize Neu5Gc, but accumulate it on cells through red-meat rich diets,
generating numerous immunogenic Neu5Gc‐neoantigens. Consequently, humans have diverse
anti-Neu5Gc antibodies affecting xenotransplantation, cancer, atherosclerosis and infertility. Anti-
Neu5Gc antibodies circulate as IgG, IgM and IgA isotypes, however repertoires of the different
isotypes in a large population had not been studied yet. Here, we used glycan microarrays to
investigate anti-Neu5Gc IgGs and IgAs in intravenous immunoglobulin (IVIG) or pooled human
IgA, respectively. Binding patterns on microarrays fabricated with Neu5Gc- and Neu5Ac-glycans,
together with inhibition assays, revealed that different IVIG preparations have highly specific anti-
Neu5Gc IgG reactivity with closely related repertoires, while IgAs show cross-reactivity against
several Neu5Ac-glycans. Such different anti-Neu5Gc IgG/IgA repertoires in individuals could
possibly mediate distinctive effects on human diseases.
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Introduction
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Glycans are fundamental building blocks of cells1 and their “signature” on proteins, lipids,
and surfaces of cells or pathogens have profound biological roles.2 In vertebrates, cell surface
glycans are commonly terminated with sialic acids (Sias) that are acidic carbohydrates with a 9-
carbon backbone.3 The two major Sias in mammals are N-acetylneuraminic acid (Neu5Ac) and its
hydroxylated form N-glycolylneuraminic acid (Neu5Gc), that differ only by a single oxygen
atom.4 Humans lack the CMAH gene encoding cytidine 5’-monophosphate-N-acetylneuraminic
acid (CMP-Neu5Ac) hydroxylase, and have no alternative Neu5Gc-synthesis pathway,5 humans
cannot synthesize Neu5Gc. However, Neu5Gc appears at low levels on the surfaces of human
epithelia and endothelia cells, and it is especially enriched on carcinoma.6–8 Neu5Gc can be
consumed from dietary mammalian foods,7, 9, 10 and although the amounts incorporated are very
small, the human immune system recognize Neu5Gc-glycans as foreign, generating a polyclonal
and diverse anti-Neu5Gc antibody response,7, 11–13 even in infants upon exposure to mammalian-
derived foods.14 Sia-containing glycans and glycoconjugates are highly diverse, owing to various
modifications of Sia and differences of linkages or the identity of underlying glycans and carrier
molecules (proteins, lipids etc.), as well as their geometric organization, that altogether generate a
huge range of diet-derived Neu5Gc‐neoantigens.4, 15 As a result, diverse anti-Neu5Gc antibodies
are present and circulate in all human sera examined thus far.7, 11–13, 16, 17
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Anti-Neu5Gc antibodies have diverse effects in humans. They are involved in
xenotransplantation immunological responses.4, 18–20 In cancer, they have dose-dependent dual
effects, facilitating tumor progression via chronic inflammation at a low concentration,21 but
inhibit tumor growth at higher concentrations,22 and these effects can switch even at two-fold
differences of antibody dose.23 Anti-Neu5Gc antibodies were recently suggested to be the link
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between increased cancer risk and red meat consumption.16, 24 Likewise, anti-Neu5Gc antibodies
have been suggested to exacerbate vascular inflammatory diseases, such as atherosclerosis.25
Human infertility could also be mediated by anti-Neu5Gc antibodies in semen and uterine fluids
that bind Neu5Gc on sperm and/or endometrium.26 In addition, Neu5Gc can be found on some
glycosylated biotherapeutics, since many of those are produced in non-human mammalian cell-
lines and/or supplemented with animal-derived additives during their production.27 Treatment with
such drugs could alter the repertoire of anti-Neu5Gc antibodies in patients.28 Furthermore,
circulating anti-Neu5Gc antibodies could capture Neu5Gc-glycosylated drugs to generate immune
complexes and expedite their clearance.29 Thus, anti-Neu5Gc antibodies could have detrimental
effects on human health. It had previously been shown that different healthy human sera contain
anti-Neu5Gc antibodies of IgG, IgM or IgA isotypes against seven different Neu5Gc-glycans.12
However, the relationship between the repertoires of anti-Neu5Gc antibodies of different antibody
isotypes in a larger cohort had not been studied thus far.
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Intravenous immunoglobulin (IVIG) preparations are purified from plasma of thousands
of healthy human donors,30 and hence are optimal for investigating antibody repertoires. IVIG
preparations are collected and manufactured in several countries around the world, including
diverse populations that likely differ in their dietary habits. In addition, manufacturers use slightly
different production methods,30 that overall could influence the pooled antibodies arsenal, diversity
and specificity.
Detection of anti-Neu5Gc antibodies is challenging, not only due to the high diversity of
Neu5Gc-glycans, but also because of the on-assay glycans presentation, which have led to
establishment of different methods for their assessment.16, 31 ELISA assays allow the investigation
of individual Neu5Gc-glycan epitopes at a time,16 while EIA assays with a mixture of Neu5Gc-
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glycoproteins are commonly used for the detection of overall anti-Neu5Gc reactivity.16, 31–35 In
comparison, dedicated sialoglycan microarrays allow high resolution detection of antibody
specificity against diverse glycans simultaneously.36–40 Here, we examined the repertoires of
human IgG and IgA in IVIG and Serum-derived pooled IgA against glycan microarrays fabricated
with a large collection of sialic acid-containing glycans. We compare different preparations and
lots of IVIG and pooled IgAs, and investigate their binding patterns and specificity against diverse
Neu5Gc-glycans and their matched Neu5Ac-glycans. We show that different preparations of IVIG
have closely related anti-Neu5Gc IgG repertoires, whereas pooled IgA have cross-reactivity with
selected Neu5Ac-glycans.
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Results and Discussion
IVIG and IgA pool recognize diverse Neu5Gc-glycans
To assess binding repertoires of pooled human serum IgGs or IgAs, the different polyclonal
antibody-preparations were screened on glycan microarrays that contain diverse Neu5Gc-glycans
and matching Neu5Ac-glycans (Table 1). Matrix analysis revealed high correlation between IVIG-
1, IVIG -2 and IVIG -5, slightly less to IVIG-3 and IVIG-4, with least correlation between all
IVIG and pooled IgA (Figure 1A). While there is no current indication that these preparations
differ in their clinical efficacy, differences in IVIG repertoires could be affected by the
manufacturing processes (as described in drug data sheets), and level of purity. For example, IVIG
preparation can differ in purification process, stabilizers and IgA content (IVIG1-5 IgA reported
to be 37 µg/ml, 4 µg/ml, <25 µg/ml, <50 µg/ml, 1500 µg/ml, respectively). Polyclonal IgA pools
were provided by CSL Behring and do not reflect a marketed product. While interesting, we did
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not have access for pooled human IgM for similar analysis as such preparation are not used
clinically.
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Figure 1. Diverse polyclonal human IgA and IgG preparations show pronounced recognition
of Neu5Gc. Dendrogrammed correlation matrix for polyclonal IgA and IgG preparations in color
code representation showing Isotype-dependent clustering of glycan-recognition.
Further detailed analysis of binding patterns at serial dilutions revealed that all the tested
antibodies preparations recognized Sia epitopes with strong preference to Neu5Gc-glycans (Figure
2A-B). In IVIG-1,5 there were higher intensities against some glycans compared to IVIG-2,3,4,
although the overall patterns seemed to be similar. While, all IVIG preparations strongly
recognized diverse Neu5Gc-glycans with minimal reactivity against Neu5Ac-glycans, pooled IgA
also recognized some Neu5Ac-glycans with underlying skeleton structures of Type-1 or GalNAc
(ID #5, 7, 13, 23), or lactose (ID #21, 39, 66, 68, 76; Figure 2A). IgA recognition of Neu5Ac-
glycans could represent cross-reactivity with Neu5Gc-glycans, especially in light of the fact that
they differ only by the additional hydroxyl group. Neu5Ac is a common ‘self’ carbohydrate,41
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hence IgA cross-reactivity with such glycans could result in auto-reactivity on cells. Further
examination of seven different lots of IVIG-1 and two lots of pooled IgA showed minimal lot-to-
lot variability in both IVIG and IgA, with overall similar binding reactivity and intensity (Figure
2C). This analysis revealed that different preparations of IVIG or IgA from a large collection of
human donors have similar Neu5Gc-glycans recognition patterns.
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Figure 2. Human IgG and IgA have pronounced recognition of Neu5Gc-glycans with
minimal lot-to-lot variability. (A) Each IVIG brand and pooled IgA were examined at serial
dilutions of 1, 0.5, 0.25, 0.125 mg/ml total protein, at 100 µl/array, and glycan binding reactivity
detected with Cy3-anti human IgG (1.2 µg/ml) or Cy3-anti human IgA (1.6 µg/ml), respectively,
then scanned and relative fluorescent units (RFU) calculated. Binding patterns were examined in
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accordance with glycans type, skeleton and linkage, as described in Table 1. (B) Mean RFU of the
4 dilutions of each IVIG brand and pooled IgA against all Neu5Gc-glycans and all Neu5Ac-
glycans that are described in A (mean  sem). (C) Glycan microarray analysis of seven IVIG-1
and two pooled IgA lots, were tested at 0.5 mg/ml total protein, 100 µl/array (In IVIG-1 Pearson
correlation coefficient to lot1 was 0.93, 0.92, 0.85, 0.88, 0.9, 0.82 and 0.82 between the two lots
of IgA).
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Specificity of IVIG and IgA against Neu5Gc-glycans
To evaluate the specificity against Neu5Gc-glycans, we used competitive glycan
microarray assays with free Neu5Ac or Neu5Gc. To better control the efficacy of binding
inhibition, we fixed the sialic acids ring structure in the natural Siaα2-linkage in the form of 2-O-
methyl-α-Neu5Ac (Ac2Me) or 2-O-methyl-α-Neu5Gc (Gc2Me), thereby preventing mutarotation
to the favorable unnatural β-anomers.12 Binding patterns of three IVIG preparations and pooled
IgA were tested on glycan microarrays, in the presence or absence of Ac2Me or Gc2Me. Binding
of all IVIG preparations against Neu5Gc-glycans was strongly inhibited with Gc2Me, but not with
Ac2Me (Figure 3A). Since Neu5Gc differ from Neu5Ac by a single oxygen atom in its additional
hydroxyl at C5, this differential inhibition suggest strong specificity against Neu5Gc-glycans. In
contrast, pooled IgA binding to both Neu5Gc-/Neu5Ac-glycans were inhibited with either Gc2Me
or Ac2Me (Figure 3B). These observations suggest that human IgA pool contain antibodies that
cross-reacts with the two glycan isoforms. Hence, in normal human serum anti-Neu5Gc IgGs seem
to be highly specific, whereas anti-Neu5Gc IgAs show cross-reactivity with related Neu5Ac-
glycans. Serum/plasma IgA is the second most abundant isotype after IgG, and although largely
considered anti-infectious and anti-inflammatory,42 some reports also suggest it can be pro-
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inflammatory.43 While serum IgA is mostly monomeric, in the mucosa it is more commonly found
as polymeric IgA where it is the most privileged isotype.30, 44 Emerging evidence suggest that
serum/plasma IgA play diverse roles in immune functions in health and disease conditions.43
Hence the cross-reactivity of anti-Neu5Gc IgAs with Neu5Ac-glycans commonly expressed on all
human cells could have detrimental immunological effects such as in autoimmune diseases.
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Figure 3. Competitive glycan microarray inhibition assays of human anti-Neu5Gc IgG and
IgA. IVIG (A) and pooled IgA (B) were analyzed. IVIG-1, -3, -5 and pooled IgA were examined
at 0.5 mg/ml total protein in PBS/OVA pH 7 or mixed with 2 mM of 2-O-methyl-α-Neu5Ac
(Ac2Me), 2-O-methyl-α-Neu5Gc (Gc2Me) in PBS/OVA pH 7, at 100 µl/tube, and incubated on
ice for 2 hours. Then, samples were each examined on glycan microarrays, and glycan binding
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reactivity detected with Cy3-anti human IgG (1.2 µg/ml) or Cy3-anti human IgA (1.6 µg/ml),
respectively, then scanned and relative fluorescent units (RFU) calculated. (A) RFU IgG
reactivities against all Neu5Gc-glycans in the presence of Ac2Me, Gc2Me or without inhibitor
were calculated (Box and Whiskers showing Min to Max; One-way ANOVA, **** p<0.0001).
(B) RFU IgA reactivities against all Neu5Gc-/Neu5Ac-glycans in the presence of Ac2Me, Gc2Me
or without inhibitor were calculated (Box and Whiskers showing Min to Max; One-way ANOVA,
**** p<0.0001, *** p<0.001, ** p<0.01).
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To further investigate the differences between reactivities of anti-Neu5Gc IgG/IgA, we
tested IVIG and pooled IgA on glycan microarrays with increasing concentrations of Ac2Me,
Gc2Me, or Neu5Gc-glycopeptides. Neu5Gc-glycopeptides were digested from serum sialo-
glycoproteins from wild-type C57BL/6 mouse31 and contain a diverse collection of naturally-
expressed Neu5Gc-glycan structures, therefore could authentically represent the competing
entities for circulating anti-Neu5Gc antibodies. Using Ac2Me as a competitive inhibitor, anti-
Neu5Gc IgG reactivities were not inhibited even at 8 mM, while anti-Neu5Gc IgA reactivities
were inhibited at 4 mM and 8mM (Figure 4A, D). Using Gc2Me as a competitive inhibitor, both
anti-Neu5Gc IgG/IgA reactivities reached >50% inhibition already at 0.06 mM, however while
anti-Neu5Gc IgG reactivities were completely inhibited at 0.25 mM Gc2Me, anti-Neu5Gc IgA
reactivities were completely inhibited only at 4 mM (Figure 4B, D). Similar differential IgG/IgA
inhibition was observed when Neu5Gc-glycopeptides were used as competitive inhibitors,
maximally inhibiting anti-Neu5Gc IgG reactivities at 0.05 mM, while at 0.1 mM for IgA (Figure
4C, D). These results emphasize the differences between serum IgG and IgA recognition of
Neu5Gc-glycans, and the cross-reactive nature of serum anti-Neu5Gc IgA.
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Figure 4. Differential inhibition of anti-Neu5Gc IgG and IgA reactivities. Glycan microarray
binding of IVIG or serum IgA against all Neu5Gc-glycans were investigated with serial dilutions
of 8 mM to 0.06 mM Ac2Me (A) or Gc2Me (B), or 0.9 mM to 0.025 mM Neu5Gc-glycopeptides,
in PBS/OVA pH 7 (C). Inhibitors were serially diluted in PBS buffer (pH 7.0), and IVIG-1 or
pooled IgA added (0.5 mg/ml total protein, PBS/OVA; 100 µl/tube), then incubated on ice for 2
hours. Samples were then each examined on glycan microarrays, and binding detected with Cy3-
anti human IgG (1.2 µg/ml) or Cy3-anti human IgA (1.6 µg/ml), then scanned and relative
fluorescent units (RFU) calculated. Statistical analysis was performed with Prism version 8 and
outlier values were excluded (A) Compared to binding with no inhibition, Ac2Me at 4 mM and 8
mM inhibits reactivity of anti-Neu5Gc IgA for each glycan, while IgG reactivity was not inhibited
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at all (**, p = 0.0011; ***, p = 0.0002; ns, respectively; Two-way ANOVA, Dunnett posttest). (B)
Compared to binding with no inhibition, Gc2Me inhibits both anti-Neu5Gc IgA/IgG reactivity
(****, p < 0.0001; Two-way ANOVA, Dunnett posttest). (C) Compared to binding with no
inhibition, Neu5Gc-glycopeptides inhibits both anti-Neu5Gc IgA/IgG reactivity (****, p <
0.0001; Two-way ANOVA, Dunnett posttest). (D) Normalized Mean RFU comparing anti-
Neu5Gc IVIG-1 and IgA shows that only in IgA Ac2Me inhibit reactivity against Neu5Gc-
glycans, while IgG shows no inhibition (Two-way ANOVA, Sidak posttest; 4 mM p = 0.0015, 8
mM p < 0.0001).
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IVIG is widely used for a growing number of clinical conditions,30 while plasma-derived
IgA (pd-IgA; IgAbulin) has been largely examined for prophylaxis and therapy of infectious
diseases as intranasal or oral treatments.30, 45 Thus, plasma-derived IgA currently has a rather
limited clinical use, mainly due to difficulties in its large scale production and lack of a clear
clinical advantage.30 In fact, as found in plasma/serum, both IVIG and pooled IgA contain an
enormous collection of antibodies against diverse protein- and carbohydrate-antigens.17, 46–48 Here,
the specific differences between anti-Neu5Gc IgG and IgA were examined in a system that allows
to investigate recognition of antibodies against unique antigens, even within a large pool of
antibodies of diverse specificities. These unique glycan microarrays were printed with a large
collection of carbohydrate antigens with terminal Neu5Gc or Neu5Ac. To further demonstrate and
emphasize the presence of other anti-carbohydrate antibodies within the pools of IVIG and IgA,
we used enzymatic cleavage of sialic acids on the arrays, followed by their binding assays (Figure
5). While there was no (IgG) or low (IgA) recognition of Neu5Ac-glycans against the native
sialoglycan microarrays, after the sialidase enzymatic treatment (that peel-off terminal sialic acid
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moieties from the array-printed glycans), there was a dramatic increase in binding of both IVIG
and pooled IgA, representing robust antibodies recognition of the resulting non-sialylated glycans
(Figure 5). This increased IgG/IgA binding clearly demonstrate the full capacity of IVIG and
pooled IgA to bind multiple carbohydrate antigens. More importantly, it also suggests that within
the plasma/serum, antibodies against Neu5Gc/Neu5Ac-glycans compete with all the other anti-
carbohydrate antibodies. Altogether, these data support the notion that sialic acids serve as ‘self-
associated molecular patterns’ or SAMPs,41 that actually provide a ‘shield’ against a potential
attack on self-carbohydrates by circulating antibodies. Hence in that respect, the cross-reactive
recognition of ‘self’ Neu5Ac-glycans by IgA could potentially undermine the sialic acids
protective defense and represent potential autoreactive detrimental effects, not only by therapeutic
pooled IgA, but also by circulating IgAs in the blood of individual humans.
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Figure 5. IVIG and pooled IgA contain diverse anti-carbohydrate antibodies. Glycan
microarray slides were treated with 50 mU/well AUS sialidase, washed then incubated with IVIG-
1, IVIG-5 or pooled IgA (0.25 mg/ml at 100 µl/array). Glycan binding reactivity was detected with
Cy3-anti human IgG (1.2 µg/ml) or Cy3-anti human IgA (1.6 µg/ml), respectively, then scanned
and relative fluorescent units (RFU) calculated and analyzed (scatter plots show mean  sem;
paired t-test, **** p<0.00001).
Conclusions
Anti-Neu5Gc antibodies play important biological roles in human health and disease.4, 27,
49, 50 Sialic acid-focused glycan microarrays provide excellent tools to investigate the full repertoire
of such antibodies,36–40 and their isotype relationship. Using sialoglycan-dedicated microarrays,
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we showed that pooled human IgG or IgA preparations contain diverse anti-Neu5Gc reactivities.
Regardless of origin and/or methodology of purification, different IVIG preparations contain
highly specific anti-Neu5Gc IgG antibodies with low lot-to-lot variability, and high specificity
inhibited with Gc2Me or Neu5Gc-glycopeptides, but not with Ac2Me. In contrast, pooled IgA
samples contain anti-Neu5Gc IgA antibodies that have cross-reactivity with Neu5Ac-glycans, and
hence may be involved with autoreactivity in some individuals. While IgA is more commonly
regarded as anti-inflammatory, it had been suggested that circulating IgA immune complexes
could have deleterious roles to the host by sustained activation of IgA receptors.51 Furthermore,
multiplex assays with glycan microarrays suggested that anti-glycan IgA repertoires are highly
correlated with IgG repertoires within an individual,52 and that measurements of serum anti-glycan
antibodies could be significantly influenced by the levels of the different antibody isotypes.53
Altogether these findings warrant further investigation into the roles of anti-Neu5Gc IgA and their
differential effects from anti-Neu5Gc IgG in humans.
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Materials and Methods
Materials. PBS ×10 was purchased from Hy-labs, Ethanol Amine from Fisher, Calcium Chloride,
Ovalbumin (Grade V) Sodium phosphate monobasic monohydrate, Sodium phosphate dibasic
heptahydrate, Tween-20 and Tris/HCl were purchased from Sigma-Aldrich. BCA kit was
purchased from Thermo. Antibodies were purchased from Jackson ImmunoResearch: Cy3-anti
Human IgA, Cy3-goat-anti-human IgG H+L, Chromopure Whole Human IgG. Five IVIG
preparations were examined: GammaGard (Baxter, USA; IVIG-1) was a kind gift from Prof.
Adriana Tremolot, Rady Children’s Hospital, San Diego. GammaPlex, (Bio Products Laboratory
UK; IVIG-2), Privigen, Hizentra (both from CSL Behring USA; IVIG-3, IVIG-4, respectively)
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and Immunovenin-Intact (BB-NCIPD Bulgaria; IVIG-5) were used. IVIG preparations can differ
in purification process, stabilizers and IgA content (e.g. IgA content is reported to be 37 µg/ml in
IVIG-1, 4 µg/ml in IVIG-2, <25 µg/ml in IVIG-3, <50 µg/ml in IVIG-4, and 1500 µg/ml in IVIG-
5). Serum-derived pooled IgA preparations were kindly provided by CSL Behring, Bern
(Switzerland).
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Preparation of 2-O-Methyl--Neu5Gc (Gc2Me) and 2-O-methyl--Neu5Ac (Ac2Me). 2-O-
Methyl--Neu5Ac (Ac2Me) was prepared from commercially available Neu5Ac using reported
procedures (Figure 6).12, 54, 55 Briefly, Neu5Ac was converted into the corresponding per-O-
acetylated methyl ester by treatment with trifluoroacetic acid (TFA) in dry methanol followed by
reacting with acetic anhydride in dry pyridine. The obtained compound was treated with acetyl
chloride in acetic acid (AcCl/HOAc) to produce acetochloroneuraminic methyl ester. The crude
product was dissolved in anhydrous methanol and the mixture was kept at room temperature for 1
h to form the crude acetylated Neu5Ac methyl glycoside. The product was then treated with
NaOMe/MeOH and NaOH (2 N), neutralized with H+ resin, and purified by silica gel
chromatography to produce Ac2Me. 2-O-Methyl--Neu5Gc (Gc2Me) was prepared from
acetylated Neu5Ac methyl glycoside, which was treated with NaOH (2 N) and reflux for 8 h to
hydrolyze the ester bonds and remove the N-acetyl group. Selective acylation of the obtained free
amino group using acetoxyacetyl chloride in the presence of NaHCO3 in CH3CN/H2O (1:1)
followed by de-O-acetylation using NaOMe/MeOH produced the desired Gc2Me. The NMR data
of both Ac2Me and Gc2Me were consistent with those reported previously.12
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Figure 6. Preparation of 2-O-Methyl--Neu5Gc (Gc2Me) and 2-O-methyl--Neu5Ac
(Ac2Me).
Sialoglycan microarray fabrication. Arrays were fabricated with NanoPrint LM-60 Microarray
Printer (Arrayit) on epoxide-derivatized slides (Corning) with 16 sub-array blocks on each slide.
Glycoconjugates were distributed into one 384-well source plates using 4 replicate wells per
sample and 8 µl per well (Version 2.0). Each glycoconjugate was prepared at 100 µM in an
optimized print buffer (300 mM phosphate buffer, pH 8.4). To monitor printing quality, replicate-
wells of human IgG (80, 40, 20, 10, 5, 0.25 ng/µl in PBS with 10% glycerol) and AlexaFlour-555-
Hydraside (Invitrogen, at 1 ng/µl in 178 mM phosphate buffer, pH 5.5) were used for each printing
run. The arrays were printed with four SMP3 pins (5 µm tip, 0.25 µl sample channel, ~100 µm
spot diameter; Arrayit). Each block (sub-array) has 18 spots/row, 20 columns with spot to spot
spacing of 225 µm. The humidity level in the arraying chamber was maintained at about 70%
during printing. Printed slides were left on arrayer deck over-night, allowing humidity to drop to
ambient levels (40-45%). Next, slides were packed, vacuum-sealed and stored at room temperature
(RT) until used.
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Sialoglycan microarray binding assay. Slides were developed and analyzed as previously
described,36 with some modifications. Slides were rehydrated with dH2O and incubated for 30 min
in a staining dish with 50C pre-warmed ethanolamine (0.05 M) in Tris-HCl (0.1 M, pH 9.0) to
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block the remaining reactive epoxy groups on the slide surface, then washed with 50 C pre-
warmed dH2O. Slides were centrifuged at 200 × g for three min then fitted with ProPlate™ Multi-
Array 16-well slide module (Invitrogen) to divide into the sub-arrays (blocks). Slides were washed
with PBST (0.1% Tween 20), aspirated and blocked with 200 µl/sub-array of blocking buffer
(PBS/OVA, 1% w/v ovalbumin, in PBS, pH 7.3) for 1 hour at RT with gentle shaking. Next, the
blocking solution was aspirated and 100 µl/block of IVIG or IgA pools (diluted to 1, 0.5, 0.25 or
0.125 mg/ml total protein in PBS/OVA, or as listed in the Figure legends) were incubated with
gentle shaking for 2 hours at RT. Slides were washed three times with PBST, then with PBS for 2
min. Bound antibodies were detected by incubating with secondary antibody diluted in PBS, 200
µl/block at RT for 1 hour: Cy3-anti Human IgG (1.2 µg/ml) or Cy3-anti Human IgA (1.6 µg/ml).
Slides were washed three times with PBST then with PBS for 10 min followed by removal from
ProPlate™ Multi-Array slide module and immediately dipping in a staining dish with dH2O for 10
min with shaking, then centrifuged at 200 × g for 3 min. Dry slides immediately scanned
Array slide processing. Processed slides were scanned and analyzed as described at 10 μm
resolution with a Genepix 4000B microarray scanner (Molecular Devices) using 350 gain. Image
analysis was carried out with Genepix Pro 6.0 analysis software (Molecular Devices). Spots were
defined as circular features with a variable radius as determined by the Genepix scanning software.
Local background subtraction was performed.
Preparation of Neu5Gc-glycopeptides. Neu5Gc-Glycopeptides were prepared by Pronase
digestion of wild-type C57BL/6 mouse sera, as described.31 Briefly, mouse sera (80 mg) was
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diluted in sterilized digestion buffer (0.1 M Tris-HCl pH 8.0 and 10 mM CaCl2). Simultaneously,
filter sterilized Pronase solution (Calbiochem) was prepared at a final concentration of 10 mg/ml
in distilled water and incubated at 60 °C for 30 min to remove any contaminating sialidase
activities. Serum was digested with 500 μl sterile Pronase solution at 37 °C, up to 5 days, with
daily additions of 250 μl sterile Pronase. Subsequently, Pronase digest was filtered (Amicon 3 kDa
filters; Sigma-Aldrich), the top fraction collected and sialic acids content analyzed, then stored at
20 °C until used.
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Competitive glycan microarray inhibition assays. Slides were developed as described above
with the following modification: IVIG or pooled IgA were pre-incubated with either PBS/OVA,
2-O-methyl--Neu5Ac (Ac2Me), 2-O-methyl--Neu5Gc (Gc2Me), or Neu5Gc-glycopeptides,
diluted in PBS/OVA buffer (pH 7.0) for 2 hours on ice (inhibitors concentrations are described in
context). The inhibitor-antibodies complexes were then applied onto the microarray for 2 hours,
washed and detected with Cy3-anti-human IgG or IgA secondary antibodies, respectively. Slides
were scanned and analyzed using GenePix Pro software as described.
Sialidase glycan microarray assay. Microarray slides were blocked with ethanolamine and
washed, as described above, then treated with Arthrobacter Ureafaciens Sialidase (AUS) solution
(0.25 mU/µl in reaction buffer containing 50 mM sodium acetate pH 5.5, at 100 µl/well) or control
treated without sialidase (PBS buffer). The slides were then washed, incubated in blocking buffer,
then IVIG or pooled plasma IgA were applied and analyzed, as described above.
Statistical analysis. Statistical analyses were performed using GraphPad Prism 8.2 and “R”, and
described in context in the Figure legends.
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Supporting Information. Glycan microarray data file is included in the supplementary file.
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Glycan microarray data.xlsx (Excel)
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Author Contributions
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V.P-K. designed the experiments, S.L.B-A conducted the research with assistance of C.S. and
S.V.G.. H.Y., X.C. and S.V.G. provided critical reagents. V.P-K. and S.L.B-A wrote the
manuscript, and all authors read and approved the final version of the manuscript
Notes
The authors have no conflict of interest.
Acknowledgment
This work was supported by the European Union H2020 Program grants (ERC-2016-STG-
716220) (to V.P-K.). Research in the laboratory of S.V.G was supported by Swiss National Science
Foundation (SNSF) grants 310030_184757 and 310030_162552, and by the Swiss Cancer
League/Swiss Cancer Research grant KFS-3941-08-2016.
Abbreviations
Neu5Gc, N-glycolylneuraminic acid; Neu5Ac, N-acetylneuraminic acid; Gc2Me, 2-O-methyl--
Neu5Gc; Ac2Me, 2-O-methyl--Neu5Ac; CMP-Neu5Ac, cytidine 5’-monophosphate-N-
acetylneuraminic acid; Sia, sialic acid.
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Table 1. List of glycans on array.
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9
ID TYPE
Skeleton Linkage Modification Structure
1
2
3
4
5
6
7
9
Ac
Gc
Ac
Gc
Ac
Gc
Ac
Ac
LacNAc
LacNAc
LacNAc
LacNAc
GalNAc
GalNAc
Type1
Core1
Core1
LacNAc
LacNAc
Type1
α3
α3
α6
α6
α6
α6
α3
α3
α3
α3
α3
α3
α3
α3
α3
α6
α6
α6
α6
α3
α3
α6
α6
9OAc
9OAc
9OAc
9OAc
0
0
9OAc
9OAc
9OAc
0
0
0
0
0
0
0
0
0
0
0
0
Neu5-9Ac2α2-3Galβ1-4GlcNAcβProNH2
Neu5Gc9Acα2-3Galβ1-4GlcNAcβProNH2
Neu5,9Ac2α2-6Galβ1-4GlcNAcβProNH2
Neu5Gc9Acα2-6Galβ1-4GlcNAcβProNH2
Neu5Acα2-6GalNAcαProNH2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Neu5Gcα2-6GalNAcαProNH2
Neu5,9Ac2α2-3Galβ1-3GlcNAcβProNH2
Neu5,9Ac2α2-3Galβ1-3GalNAcαProNH2
Neu5Gc9Acα2-3Galβ1-3GalNAcαProNH2
Neu5Acα2-3Galβ1-4GlcNAcβProNH2
Neu5Gcα2-3Galβ1-4GlcNAcβProNH2
Neu5Acα2-3Galβ1-3GlcNAcβProNH2
Neu5Gcα2-3Galβ1-3GlcNAcβProNH2
Neu5Acα2-3Galβ1-3GalNAcαProNH2
Neu5Gcα2-3Galβ1-3GalNAcαProNH2
Neu5Acα2-6Galβ1-4GlcNAcβProNH2
Neu5Gcα2-6Galβ1-4GlcNAcβProNH2
Neu5Acα2-6LacβProNH2
10 Gc
11 Ac
12 Gc
13 Ac
14 Gc
15 Ac
16 Gc
17 Ac
18 Gc
19 Ac
20 Gc
21 Ac
22 Gc
23 Ac
24 Gc
25 Ac
26 Gc
27 Ac
28 Gc
29 Ac
30 Gc
31 Ac
32 Gc
33 Ac
34 Gc
35 Ac
36 Gc
37 Ac
38 Gc
39 Ac
40 Gc
41 Ac-Ac
Type1
Core1
Core1
LacNAc
LacNAc
Lactose
Lactose
Lactose
Lactose
GalNAc
GalNAc
Galactose α3
Galactose α3
Galactose α6
Galactose α6
Galactose α3
Galactose α3
Galactose α6
Galactose α6
Core1
Core1
Core1
Core1
Lactose
Lactose
Lactose
Lactose
Lactose
Neu5Gcα2-6LacβProNH2
Neu5Acα2-3Galβ1-4GlcβProNH2
Neu5Gcα2-3Galβ1-4GlcβProNH2
Neu5,9Ac2α2-6GalNAcαProNH2
Neu9Ac5Gcα2-6GalNAcαProNH2
Neu5Acα2-3GalβProNH2
Neu5Gcα2-3GalβProNH2
Neu5Acα2-6GalβProNH2
Neu5Gcα2-6GalβProNH2
Neu5,9Ac2α2-3GalβProNH2
9OAc
9OAc
0
0
0
0
9OAc
9OAc
9OAc
9OAc
0
Neu9Ac5Gcα2-3GalβProNH2
Neu5,9Ac2α2-6GalβProNH2
Neu9Ac5Gcα2-6GalβProNH2
α3
α3
α3
α3
α6
α6
α3
α3
Neu5Acα2-3Galβ1-3GalNAcβProNH2
Neu5Gcα2-3Galβ1-3GalNAcβProNH2
Neu5,9Ac2α2-3Galβ1-3GalNAcβProNH2
Neu9Ac5Gcα2-3Galβ1-3GalNAcβProNH2
Neu5,9Ac2α2-6Galβ1-4GlcβProNH2
Neu9Ac5Gcα2-6Galβ1-4GlcβProNH2
Neu5,9Ac2α2-3Galβ1-4GlcβProNH2
Neu9Ac5Gcα2-3Galβ1-4GlcβProNH2
0
9OAc
9OAc
9OAc
9OAc
9OAc
9OAc
0
α8-α3
Neu5Acα2-8Neu5Acα2-3Galβ1-4GlcβProNH2
Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3Galβ1-
4GlcβProNH2
Ac-Ac-
Ac
42
Lactose
α8-α3
0
55 Ac
56 Gc
Lex
Lex
α3
α3
0
0
Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβProNH2
Neu5Gcα2-3Galβ1-4(Fucα1-3)GlcNAcβProNH2
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Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc6S-
βProNH2
Neu5Gcα2-3Galβ1-4(Fucα1-3)GlcNAc6S-
βProNH2
Neu5Acα2-3Galβ1-3GlcNAcβ1-3LacβProNH2
-
-
57 Ac
58 Gc
6S-Lex
α3
α3
6OSO3
6OSO3
6S-Lex
LNT
LNT
6S-
LacNAc
6S-
LacNAc
60 Ac
61 Gc
α3
α3
0
0
Neu5Gcα2-3Galβ1-3GlcNAcβ1-3LacβProNH2
-
-
62 Ac
α3
6OSO3
6OSO3
0
Neu5Acα2-3Galβ1-4GlcNAc6SβProNH2
10
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13
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25
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27
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63 Gc
α3
Neu5Gcα2-3Galβ1-4GlcNAc6SβProNH2
Neu5Acα2-8Neu5Acα2-3Galβ1-4GlcβPro-HEG-
NH2
64 Ac-Ac
Lactose
α8-α3
Ac-Ac-
Ac
Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3Galβ1-
4GlcβPro-HEG-NH2
65
Lactose
α8-α3
0
66 Ac(Ac)
67 Gc(Ac)
68 KDN(Ac) Lactose
69 Gc-Ac Lactose
70 KDN-Ac Lactose
71 Ac-KDN Lactose
72 Ac-Gc
73 Ac-Gc
Lactose
Lactose
α3/α6
α3/α6
α3/α6
α8-α3
α8-α3
α8-α6
α8-α3
α8-α6
α8-α3
α8-α3
α8-α6
0
0
0
0
0
0
0
0
0
0
0
Neu5Acα2-3(Neu5Acα2-6)Galβ1-4GlcβProNH2
Neu5Gcα2-3(Neu5Acα-2-6)Galβ1-4GlcβProNH2
KDNα2-3(Neu5Acα2-6)Galβ1-4GlcβProNH2
Neu5Gcα2-8Neu5Acα2-3Galβ1-4GlcβProNH2
KDNα-2-8Neu5Acα-2-3Galβ1-4GlcβProNH2
Neu5Acα-2-8KDNα2-6Galβ1-4GlcβProNH2
Neu5Acα2-8Neu5Gcα2-3Galβ1-4GlcβProNH2
Neu5Acα2-8Neu5Gcα2-6Galβ1-4GlcβProNH2
KDNα2-8Neu5Gcα2-3Galβ1-4GlcβProNH2
Neu5Gcα2-8Neu5Gcα2-3Galβ1-4GlcβProNH2
Lactose
Lactose
74 KDN-Gc Lactose
75 Gc-Gc
76 Ac-Ac
Lactose
Lactose
Neu5Acα2-8Neu5Acα2-6Galβ1-4GlcβProNH2
Neu5GcMeα2-8Neu5Acα2-3Galβ1-
4GlcβProNH2
77 GcMe-Ac Lactose
α8-α3
Me
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