Search

Purpose: Interactions with tumor-associated microglia and macrophages (TAM) are critical for glioblastoma progression. Polysialic acid (polySia) is a tumor-associated glycan, but its frequency of occurrence and its prognostic value in glioblastoma are disputed. Through interactions with the opposing immune receptors Siglec-11 and Siglec-16, polySia is implicated in the regulation of microglia and macrophage activity. However, due to a nonfunctional SIGLEC16P allele, SIGLEC16 penetrance is less than 40%. Here, we explored possible consequences of SIGLEC16 status and tumor cell–associated polySia on glioblastoma outcome. Experimental Design: Formalin-fixed paraffin-embedded specimens of two independent cohorts with 70 and 100 patients with newly diagnosed glioblastoma were retrospectively analyzed for SIGLEC16 and polySia status in relation to overall survival. Inflammatory TAM activation was assessed in tumors, in heterotypic tumor spheroids consisting of polySia-positive glioblastoma cells and Siglec-16–positive or Siglec-16–negative macrophages, and by exposing Siglec-16–positive or Siglec-16–negative macrophages to glioblastoma cell–derived membrane fractions. Results: Overall survival of SIGLEC16 carriers with polySia-positive tumors was increased. Consistent with proinflammatory Siglec-16 signaling, levels of TAM positive for the M2 marker CD163 were reduced, whereas the M1 marker CD74 and TNF expression were increased, and CD8+ T cells enhanced in SIGLEC16/polySia double-positive tumors. Correspondingly, TNF production was elevated in heterotypic spheroid cultures with Siglec-16–expressing macrophages. Furthermore, a higher, mainly M1-like cytokine release and activating immune signaling was observed in SIGLEC16-positive as compared with SIGLEC16-negative macrophages confronted with glioblastoma cell–derived membranes. Conclusions: Collectively, these results strongly suggest that proinflammatory TAM activation causes the better outcome in patients with glioblastoma with a functional polySia-Siglec-16 axis.

Supplementary Table S1.Demographic and clinical parameters of GB patient cohorts.Supplementary Table S2.Characteristics of the two cohorts of n = 70 (MHH) and n = 100 (UMG) GB patients stratified for the presence of a functional SIGLEC16 allele (A) or for the occurrence of polySia on tumor cells (B).Supplementary Table S3.Cox proportional hazards regression analysis of covariates for SIGLEC16 and polySia status in relation to OS (MHH and UMG cohorts combined).Supplementary Table S4.Median age of GB cases stratified for SIGLEC16 genotype and polySia on tumor cells.Supplementary Table S5.OS analysis of GB cases receiving (A) or not receiving gross total resection (B) stratified for SIGLEC16 genotype and polySia on tumor cells (MHH and UMG cohorts combined).Supplementary Table S6.OS analysis of GB cases receiving (A) or not receiving chemotherapy (B) stratified for SIGLEC16 genotype and polySia on tumor cells (MHH cohort only).Supplementary Table S7.OS analysis of GB cases receiving gross total resection and chemotherapy stratified for SIGLEC16 genotype and polySia on tumor cells (MHH and UMG cohorts combined, n=101).Supplementary Table S8.OS analysis of IDH-wildtype GB cases (UMG cohort) stratified for SIGLEC16 genotype and polySia on tumor cells.Supplementary Table S9.OS analysis of UMG cohort stratified for MGMT gene promoter methylation and SIGLEC16 genotype, polySia on tumor cells, CD163 and CD74 status.Supplementary Table S10.Characteristics of the two cohorts of n = 70 (MHH) and n = 100 (UMG) GB patients stratified for CD74low and CD74high.Supplementary Table S11. Median age at resection of GB cases stratified for CD163 to CD68 (A) or CD74 to IBA1 ratios (B) alone or in combination with the SIGLEC16 genotype.Supplementary Table S12. OS analysis of GB cases receiving (A) or not receiving gross total resection (B) stratified for CD163 to CD68 or CD74 to IBA1 ratios alone or in combination with the SIGLEC16 genotype (MHH and UMG cohorts combined).Supplementary Table S13. OS analysis of GB cases receiving (A) or not receiving chemotherapy (B) stratified for CD163 to CD68 or CD74 to IBA1 ratios alone or in combination with the SIGLEC16 genotype (MHH cohort only).Supplementary Table S14.OS analysis of GB cases receiving both gross total resection and chemotherapy stratified for CD163 to CD68 or CD74 to IBA1 ratios alone or in combination with the SIGLEC16 genotype (MHH and UMG cohorts combined, n=101).

Purpose:Interactions with tumor-associated microglia and macrophages (TAM) are critical for glioblastoma progression. Polysialic acid (polySia) is a tumor-associated glycan, but its frequency of occurrence and its prognostic value in glioblastoma are disputed. Through interactions with the opposing immune receptors Siglec-11 and Siglec-16, polySia is implicated in the regulation of microglia and macrophage activity. However, due to a nonfunctional SIGLEC16P allele, SIGLEC16 penetrance is less than 40%. Here, we explored possible consequences of SIGLEC16 status and tumor cell–associated polySia on glioblastoma outcome.Experimental Design:Formalin-fixed paraffin-embedded specimens of two independent cohorts with 70 and 100 patients with newly diagnosed glioblastoma were retrospectively analyzed for SIGLEC16 and polySia status in relation to overall survival. Inflammatory TAM activation was assessed in tumors, in heterotypic tumor spheroids consisting of polySia-positive glioblastoma cells and Siglec-16–positive or Siglec-16–negative macrophages, and by exposing Siglec-16–positive or Siglec-16–negative macrophages to glioblastoma cell–derived membrane fractions.Results:Overall survival of SIGLEC16 carriers with polySia-positive tumors was increased. Consistent with proinflammatory Siglec-16 signaling, levels of TAM positive for the M2 marker CD163 were reduced, whereas the M1 marker CD74 and TNF expression were increased, and CD8+ T cells enhanced in SIGLEC16/polySia double-positive tumors. Correspondingly, TNF production was elevated in heterotypic spheroid cultures with Siglec-16–expressing macrophages. Furthermore, a higher, mainly M1-like cytokine release and activating immune signaling was observed in SIGLEC16-positive as compared with SIGLEC16-negative macrophages confronted with glioblastoma cell–derived membranes.Conclusions:Collectively, these results strongly suggest that proinflammatory TAM activation causes the better outcome in patients with glioblastoma with a functional polySia-Siglec-16 axis.

PolySia on GB tumor cells is associated with NCAM

Supplementary Methods

PolySia on CD11c-positive and on CD45-positive, IBA1-negative cells

Antibody array detection of cytokines and chemokines

PolySia is not associated with CD3- or CD34-positive cells

OS analysis of the two GB cohorts individually stratified for CD163 to CD68 or CD74 to IBA1 ratios

PolySia is associated with GFAP-positive cells

SummaryLate-onset Alzheimer’s Disease (LOAD) pathology is rare in our closest living evolutionary relatives (chimpanzees), which also express much lower microglial levels of CD33(Siglec-3)–a myelomonocytic receptor inhibiting innate immune reactivity by extracellular V-set domain recognition of sialic acid(Sia)-containing “self-associated molecular patterns” (SAMPs). We earlier showed that V-set domain-deficient CD33-variant allele, protective against LOAD, is derived and specific to hominin-lineage. We now report that CD33 also harbors multiple hominin-specific V-set domain mutations and explore selection forces that may have favored such genomic changes. N-glycolylneuraminic acid (Neu5Gc), the preferred Sia-ligand of ancestral CD33 is absent in humans, due to hominin-specific, fixed loss-of-function mutation in CMAH, which generates CMP-Neu5Gc from its precursor, CMP-N-acetylneuraminic acid (Neu5Ac). Extensive mutational analysis and MD-simulations indicate that fixed change in amino acid 21 of hominin V-set domain and conformational changes related to His45 corrected for Neu5Gc-loss by switching to Neu5Ac-recognition. Considering immune-evasive “molecular mimicry” of SAMPs by pathogens, we found that human-specific pathogens Neisseria gonorrhoeae and Group B Streptococcus (affecting fertility and fetuses/neonates respectively) selectively bind huCD33 and this binding is significantly impacted by amino acid 21 modification. Alongside LOAD-protective CD33 alleles, humans harbor additional, derived, population-universal, cognition-protective variants absent in “great ape” genomes. Interestingly, 11 of 13 SNPs in these human genes (including CD33), that protect the cognitive health of elderly populations, are not shared by genomes of archaic hominins: Neanderthals and Denisovans. Finally, we present a plausible evolutionary scenario to compile, correlate and comprehend existing knowledge about huCD33 evolution and suggest that grandmothering emerged in humans.

Compared with our closest living evolutionary cousins, humans appear unusually prone to develop carcinomas (cancers arising from epithelia). The SIGLEC12 gene, which encodes the Siglec‐XII protein expressed on epithelial cells, has several uniquely human features: a fixed homozygous missense mutation inactivating its natural ligand recognition property; a polymorphic frameshift mutation eliminating full‐length protein expression in ~60%–70% of worldwide human populations; and, genomic features suggesting a negative selective sweep favoring the pseudogene state. Despite the loss of canonical sialic acid binding, Siglec‐XII still recruits Shp2 and accelerates tumor growth in a mouse model. We hypothesized that dysfunctional Siglec‐XII facilitates human carcinoma progression, correlating with known tumorigenic signatures of Shp2‐dependent cancers. Immunohistochemistry was used to detect Siglec‐XII expression on tissue microarrays. PC‐3 prostate cancer cells were transfected with Siglec‐XII and transcription of genes enriched with Siglec‐XII was determined. Genomic SIGLEC12 status was determined for four different cancer cohorts. Finally, a dot blot analysis of human urinary epithelial cells was established to determine the Siglec‐XII expressors versus non‐expressors. Forced expression in a SIGLEC12 null carcinoma cell line enriched transcription of genes associated with cancer progression. While Siglec‐XII was detected as expected in ~30%–40% of normal epithelia, ~80% of advanced carcinomas showed strong expression. Notably, >80% of late‐stage colorectal cancers had a functional SIGLEC12 allele, correlating with overall increased mortality. Thus, advanced carcinomas are much more likely to occur in individuals whose genomes have an intact SIGLEC12 gene, likely because the encoded Siglec‐XII protein recruits Shp2‐related oncogenic pathways. The finding has prognostic, diagnostic, and therapeutic implications.

Many pathogenic bacteria secrete AB5 toxins that can be virulence factors. Cytotoxic A subunits are delivered to the cytosol following B subunit binding to specific host cell surface glycans. Some B subunits are not associated with A subunits, for example, YpeB of Yersinia pestis, the etiologic agent of plague. Plague cannot be eradicated because of Y.pestis' adaptability to numerous hosts. We previously showed selective binding of other B5 pentamers to a sialoglycan microarray, with sialic acid (Sia) preferences corresponding to those prominently expressed by various hosts, for example, N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Here, we report that A subunit phylogeny evolved independently of B subunits and suggest a future B subunit nomenclature based on bacterial species names. We also found via phylogenetic analysis of B subunits, which bind Sias, that homologous molecules show poor correlation with species phylogeny. These data indicate ongoing lateral gene transfers between species, including mixing of A and B subunits. Consistent with much broader host range of Y.pestis, we show that YpeB recognizes all mammalian Sia types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, which is abolished by a mutation (Y77F) eliminating Sia recognition, suggesting that cell proliferation and death are promoted via lectin-like crosslinking of cell surface sialoglycoconjugates. These findings help explain the host range of Y.pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties.

Vertebrate sialic acids (Sias) display much diversity in modifications, linkages, and underlying glycans. Slide microarrays allow high-throughput explorations of sialoglycan–protein interactions. A microarray presenting ~150 structurally defined sialyltrisaccharides with various Sias linkages and modifications still poses challenges in planning, data sorting, visualization, and analysis. To address these issues, we devised a simple 9-digit code for sialyltrisaccharides with terminal Sias and underlying two monosaccharides assigned from the nonreducing end, with 3 digits assigning a monosaccharide, its modifications, and linkage. Calculations based on the encoding system reveal >113,000 likely linear sialyltrisaccharides in nature. Notably, a biantennary N-glycan with 2 terminal sialyltrisaccharides could thus have >1010 potential combinations and a triantennary N-glycan with 3 terminal sequences, >1015 potential combinations. While all possibilities likely do not exist in nature, sialoglycans encode enormous diversity. While glycomic approaches are used to probe such diverse sialomes, naturally occurring bacterial AB5 toxin B subunits are simpler tools to track the dynamic sialome in biological systems. Sialoglycan microarray was utilized to compare sialoglycan-recognizing bacterial toxin B subunits. Unlike the poor correlation between B subunits and species phylogeny, there is stronger correlation with Sia-epitope preferences. Further supporting this pattern, we report a B subunit (YenB) from Yersinia enterocolitica (broad host range) recognizing almost all sialoglycans in the microarray, including 4-O-acetylated-Sias not recognized by a Yersinia pestis orthologue (YpeB). Differential Sia-binding patterns were also observed with phylogenetically related B subunits from Escherichia coli (SubB), Salmonella Typhi (PltB), Salmonella Typhimurium (ArtB), extra-intestinal E.coli (EcPltB), Vibrio cholera (CtxB), and cholera family homologue of E. coli (EcxB).

Many pathogenic bacteria secrete AB

Vertebrate sialic acids (Sias) display much diversity in modifications, linkages and underlying glycans. Slide microarrays allow high-throughput explorations of sialoglycan-protein interactions. A microarray presenting ∼150 structurally-defined sialyltrisaccharides with various Sias linkages and modifications still poses challenges in planning, data sorting, visualization and analysis. To address these issues, we devised a simple 9-digit code for sialyltrisaccharides with terminal Sias and underlying two monosaccharides assigned from the non-reducing end, with three digits assigning a monosaccharide, its modifications, and linkage. Calculations based on the encoding system reveals >113,000 likely linear sialyltrisaccharides in nature. Notably a biantennary N-glycan with two terminal sialyltrisaccharides could thus have >1010 potential combinations and a triantennary N-glycan with three terminal sequences, >1015 potential combinations. While all possibilities likely do not exist in nature, sialoglycans encode enormous diversity. While glycomic approaches are used to probe such diverse sialomes, naturally-occurring bacterial AB5 toxin B subunits are simpler tools to track the dynamic sialome in biological systems. Sialoglycan microarray was utilized to compare sialoglycan-recognizing bacterial toxin B subunits. Unlike the poor correlation between B subunits and species phylogeny, there is stronger correlation with Sia-epitope preferences. Further supporting this pattern, we report a B subunit (YenB) from Yersinia enterocolitica (broad host range) recognizing almost all sialoglycans in the microarray, including 4-O-acetylated-Sias not recognized by a Y. pestis orthologue (YpeB). Differential Sia-binding patterns were also observed with phylogenetically-related B subunits from Escherichia coli (SubB), Salmonella Typhi (PltB), S. Typhimurium (ArtB), extra-intestinal E.coli (EcPltB), Vibrio cholera (CtxB), and cholera family homologue of E. coli (EcxB).

Cytotoxic A subunits of bacterial AB5 toxins enter the cytosol following B subunit binding to host cell glycans. We report that A subunit phylogeny evolves independently of B subunits and suggest a future B subunit nomenclature based on species name. Phylogenetic analysis of B subunits that bind sialic acids (Sias) with homologous molecules in databases also show poor correlation with phylogeny. These data indicate ongoing lateral gene transfers between species, with mixing of A and B subunits. Some B subunits are not even associated with A subunits e.g., YpeB of Yersinia pestis, the etiologic agent of plague epidemics. Plague cannot be eradicated because of Y. pestis’ adaptability to numerous hosts. YpeB shares 58% identity/79% similarity with the homo-pentameric B subunit of E. coli Subtilase cytotoxin, and 48% identity/68% similarity with the B subunit of S. Typhi typhoid toxin. We previously showed selective binding of B5 pentamers to a sialoglycan microarray, with Sia preferences corresponding to hosts e.g., N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Consistent with much broader host range of Y. pestis, YpeB binds all mammalian sialic acid types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, abolished by a mutation (Y77F) eliminating Sia recognition, suggesting cell proliferation and death via lectin-like cross-linking of cell surface sialoglycoconjugates. These findings help explain the host range of Y. pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a new virus that has higher contagious capacity than any other previous human coronaviruses (HCoVs) and causes the current coronavirus disease 2019 pandemic. Sialic acids are a group of nine-carbon acidic α-keto sugars, usually located at the end of glycans of cell surface glycoconjugates and serve as attachment sites for previous HCoVs. It is therefore speculated that sialic acids on the host cell surface could serve as co-receptors or attachment factors for SARS-CoV-2 cell entry as well. Recent in silico modeling, molecular modeling predictions and microscopy studies indicate potential sialic acid binding by SARS-CoV-2 upon cell entry. In particular, a flat sialic acid-binding domain was proposed at the N-terminal domain of the spike protein, which may lead to the initial contact and interaction of the virus on the epithelium followed by higher affinity binding to angiotensin-converting enzyme 2 (ACE2) receptor, likely a two-step attachment fashion. However, recent in vitro and ex vivo studies of sialic acids on ACE2 receptor confirmed an opposite role for SARS-CoV-2 binding. In particular, neuraminidase treatment of epithelial cells and ACE2-expressing 293T cells increased SARS-CoV-2 binding. Furthermore, the ACE2 glycosylation inhibition studies indicate that sialic acids on ACE2 receptor prevent ACE2–spike protein interaction. On the other hand, a most recent study indicates that gangliosides could serve as ligands for receptor-binding domain of SARS-CoV-2 spike protein. This mini-review discusses what has been predicted and known so far about the role of sialic acid for SARS-CoV-2 infection and future research perspective., Graphical Abstract Graphical Abstract

All vertebrate cell surfaces display a dense glycan layer often terminated with sialic acids, which have multiple functions due to their location and diverse modifications. The major sialic acids in most mammalian tissues are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), the latter being derived from Neu5Ac via addition of one oxygen atom at the sugar nucleotide level by CMP-Neu5Ac hydroxylase (Cmah). Contrasting with other organs that express various ratios of Neu5Ac and Neu5Gc depending on the variable expression of Cmah, Neu5Gc expression in the brain is extremely low in all vertebrates studied to date, suggesting that neural expression is detrimental to animals. However, physiological exploration of the reasons for this long term evolutionary selection has been lacking. To explore the consequences of forced expression of Neu5Gc in the brain, we have established brain-specific Cmah transgenic mice. Such Neu5Gc overexpression in the brain resulted in abnormal locomotor activity, impaired object recognition memory, and abnormal axon myelination. Brain-specific Cmah transgenic mice were also lethally sensitive to a Neu5Gc-preferring bacterial toxin, even though Neu5Gc was overexpressed only in the brain and other organs maintained endogenous Neu5Gc expression, as in wild-type mice. Therefore, the unusually strict evolutionary suppression of Neu5Gc expression in the vertebrate brain may be explained by evasion of negative effects on neural functions and by selection against pathogens.

In 2011, genome wide association studies implicated a polymorphism near CD33 as a genetic risk factor for Alzheimer’s disease. This finding sparked interest in this member of the sialic acid-binding immunoglobulin-type lectin family which is linked to innate immunity. Subsequent studies found that CD33 is expressed in microglia in the brain and then investigated the molecular mechanism underlying the CD33 genetic association with Alzheimer’s disease. The allele that protects from Alzheimer’s disease acts predominately to increase a CD33 isoform lacking exon 2 at the expense of the prototypic, full-length CD33 that contains exon 2. Since this exon encodes the sialic acid ligand binding domain, the finding that loss of exon 2 was associated with decreased Alzheimer’s disease risk was interpreted as meaning that a decrease in functional CD33 and its associated immune suppression was protective from Alzheimer’s disease. However, this interpretation may need to be reconsidered given current findings that a genetic deletion which abrogates CD33 is not associated with Alzheimer’s disease risk. Therefore, integrating currently available findings leads us to propose a model wherein the CD33 isoform lacking the ligand binding domain represents a gain of function variant that reduces Alzheimer’s disease risk.

The immunomodulatory receptor Siglec-3/CD33 influences risk for late-onset Alzheimer's disease (LOAD), an apparently human-specific post-reproductive disease. CD33 generates two splice variants: a full-length CD33M transcript produced primarily by the “LOAD-risk” allele and a shorter CD33m isoform lacking the sialic acid-binding domain produced primarily from the “LOAD-protective” allele. An SNP that modulates CD33 splicing to favor CD33m is associated with enhanced microglial activity. Individuals expressing more protective isoform accumulate less brain β-amyloid and have a lower LOAD risk. How the CD33m isoform increases β-amyloid clearance remains unknown. We report that the protection by the CD33m isoform may not be conferred by what it does but, rather, from what it cannot do. Analysis of blood neutrophils and monocytes and a microglial cell line revealed that unlike CD33M, the CD33m isoform does not localize to cell surfaces; instead, it accumulates in peroxisomes. Cell stimulation and activation did not mobilize CD33m to the surface. Thus, the CD33m isoform may neither interact directly with amyloid plaques nor engage in cell-surface signaling. Rather, production and localization of CD33m in peroxisomes is a way of diminishing the amount of CD33M and enhancing β-amyloid clearance. We confirmed intracellular localization by generating a CD33m-specific monoclonal antibody. Of note, CD33 is the only Siglec with a peroxisome-targeting sequence, and this motif emerged by convergent evolution in toothed whales, the only other mammals with a prolonged post-reproductive lifespan. The CD33 allele that protects post-reproductive individuals from LOAD may have evolved by adaptive loss-of-function, an example of the less-is-more hypothesis.

Certain pathogenic bacteria are known to modulate the innate immune response by decorating themselves with sialic acids, which can engage the myelomonocytic lineage inhibitory receptor Siglec-9, thereby evading immunosurveillance. We hypothesized that the well-known up-regulation of sialoglycoconjugates by tumors might similarly modulate interactions with innate immune cells. Supporting this hypothesis, Siglec-9-expressing myelomonocytic cells found in human tumor samples were accompanied by a strong up-regulation of Siglec-9 ligands. Blockade of Siglec-9 enhanced neutrophil activity against tumor cells in vitro. To investigate the function of inhibitory myelomonocytic Siglecs in vivo we studied mouse Siglec-E, the murine functional equivalent of Siglec-9. Siglec-E-deficient mice showed increased in vivo killing of tumor cells, and this effect was reversed by transgenic Siglec-9 expression in myelomonocytic cells. Siglec-E-deficient mice also showed enhanced immunosurveillance of autologous tumors. However, once tumors were established, they grew faster in Siglec-E-deficient mice. In keeping with this, Siglec-E-deficient macrophages showed a propensity toward a tumor-promoting M2 polarization, indicating a secondary role of CD33-related Siglecs in limiting cancer-promoting inflammation and tumor growth. Thus, we define a previously unidentified impact of inhibitory myelomonocytic Siglecs in cancer biology, with distinct roles that reflect the dual function of myelomonocytic cells in cancer progression. In keeping with this, a human polymorphism that reduced Siglec-9 binding to carcinomas was associated with improved early survival in non-small-cell lung cancer patients, which suggests that Siglec-9 might be therapeutically targeted within the right time frame and stage of disease.

Dense and complex glycans on vertebrate cell surfaces and secreted molecules often terminate with sialic acids (Sias), a diverse family of sugars involved in numerous biological processes, including cell-cell interactions, immune regulation, cellular homeostasis, host-pathogen interactions and microbial pathogeneses. Due to immense structural variations, a major fraction of sialoglycans remain poorly characterized by current inadequate conventional techniques. While traditional glycomic methods are useful, they can destroy or overlook crucial aspects of Sia diversity, or fail to elucidate native structures as they exist in biological systems. To date, in situdetection and analysis of sialoglycans have been largely based on plant lectins, sialidases or antibodies whose preferences are limited and/or uncertain. We defined 9 classes of Sialoglycan Recognizing Probes (SGRPs: SGRP1- SGRP9) aiming to provide a simple and systematic approach to track mammalian sialome changes, using techniques common in most laboratories. Our approach utilizes molecules naturally evolved with maximized specificity towards sialoglycans such as bacterial adhesins, toxin subunits and viral hemagglutinin-esterases. We selected the experimentally proven best candidates for SGRPs and engineered each with a corresponding non-binding mutant as a negative control. Following sialoglycan microarray studies to confirm the binding specificity of our SGRPs, the optimized panel of SGRPs was subsequently applied in common detection methods such as ELISA, Western Blot and FACS analysis. While further work is needed to define a comprehensive suite of SGRPs, our efforts provide a reliable toolkit with specificity to track the mammalian sialoglycome.

CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer.

All vertebrate cell surfaces display a dense glycan layer often terminated with sialic acids, which have multiple functions due to their location and diverse modifications. The major sialic acids in most mammalian tissues are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), the latter being derived from Neu5Ac via addition of one oxygen atom at the sugar nucleotide level by CMP-Neu5Ac hydroxylase (Cmah). Contrasting with other organs that express various ratios of Neu5Ac and Neu5Gc depending on the variable expression of Cmah, Neu5Gc expression in the brain is extremely low in all vertebrates studied to date, suggesting that neural expression is detrimental to animals. However, physiological exploration of the reasons for this long term evolutionary selection has been lacking. To explore the consequences of forced expression of Neu5Gc in the brain, we have established brain-specific Cmah transgenic mice. Such Neu5Gc overexpression in the brain resulted in abnormal locomotor activity, impaired object recognition memory, and abnormal axon myelination. Brain-specific Cmah transgenic mice were also lethally sensitive to a Neu5Gc-preferring bacterial toxin, even though Neu5Gc was overexpressed only in the brain and other organs maintained endogenous Neu5Gc expression, as in wild-type mice. Therefore, the unusually strict evolutionary suppression of Neu5Gc expression in the vertebrate brain may be explained by evasion of negative effects on neural functions and by selection against pathogens.

Lectin galactoside-binding soluble 3 binding protein (LGALS3BP, also called Mac-2 binding protein) is a heavily glycosylated secreted molecule that has been shown previously to be up-regulated in many cancers and has been implicated in tumor metastatic processes, as well as in other cell adhesion and immune functions. The CD33-related subset of sialic acid-binding immunoglobulin-like lectins (Siglecs) consists of immunomodulatory molecules that have recently been associated with the modulation of immune responses to cancer. Because up-regulation of Siglec ligands in cancer tissue has been observed, the characterization of these cancer-associated ligands that bind to inhibitory CD33-related Siglecs could provide novel targets for cancer immunomodulatory therapy. Here we used affinity chromatography of tumor cell extracts to identify LGALS3BP as a novel sialic acid-dependent ligand for human Siglec-9 and for other immunomodulatory Siglecs, such as Siglec-5 and Siglec-10. In contrast, the mouse homolog Siglec-E binds to murine LGALS3BP with lower affinity. LGALS3BP has been observed to be up-regulated in human colorectal and prostate cancer specimens, particularly in the extracellular matrix. Finally, LGALS3BP was able to inhibit neutrophil activation in a sialic acid- and Siglec-dependent manner. These findings suggest a novel immunoinhibitory function for LGALS3BP that might be important for immune evasion of tumor cells during cancer progression.

Compelling evidence for naturally occurring immunosurveillance against malignancies informs and justifies some current approaches toward cancer immunotherapy. However, some types of immune reactions have also been shown to facilitate tumor progression. For example, our previous studies showed that although experimental tumor growth is enhanced by low levels of circulating antibodies directed against the nonhuman sialic acid N-glycolyl-neuraminic acid (Neu5Gc), which accumulates in human tumors, growth could be inhibited by anti-Neu5Gc antibodies from a different source, in a different model. However, it remains generally unclear whether the immune responses that mediate cancer immunosurveillance vs. those responsible for inflammatory facilitation are qualitatively and/or quantitatively distinct. Here, we address this question using multiple murine tumor growth models in which polyclonal antibodies against tumor antigens, such as Neu5Gc, can alter tumor progression. We found that although growth was stimulated at low antibody doses, it was inhibited by high doses, over a linear and remarkably narrow range, defining an immune response curve (IRC; i.e., inverse hormesis). Moreover, modulation of immune responses against the tumor by altering antibody avidity or by enhancing innate immunity shifted the IRC in the appropriate direction. Thus, the dualistic role of immunosurveillance vs. inflammation in modulating tumor progression can be quantitatively distinguished in multiple model systems, and can occur over a remarkably narrow range. Similar findings were made in a human tumor xenograft model using a narrow range of doses of a monoclonal antibody currently in clinical use. These findings may have implications for the etiology, prevention, and treatment of cancer.

Siglecs are sialic acid-binding Ig-like lectins that recognize sialoglycans via amino-terminal V-set domains. CD33-related Siglecs (CD33rSiglecs) on innate immune cells recognize endogenous sialoglycans as “self-associated molecular patterns” (SAMPs), dampening immune responses via cytosolic immunoreceptor tyrosine-based inhibition motifs that recruit tyrosine phosphatases. However, sialic acid-expressing pathogens subvert this mechanism through molecular mimicry. Meanwhile, endogenous host SAMPs must continually evolve to evade other pathogens that exploit sialic acids as invasion targets. We hypothesized that these opposing selection forces have accelerated CD33rSiglec evolution. We address this by comparative analysis of major CD33rSiglec (Siglec-3, Siglec-5, and Siglec-9) orthologs in humans, chimpanzees, and baboons. Recombinant soluble molecules displaying ligand-binding domains show marked quantitative and qualitative interspecies differences in interactions with strains of the sialylated pathogen, group B Streptococcus, and with sialoglycans presented as gangliosides or in the form of sialoglycan microarrays, including variations such as N-glycolyl and O-acetyl groups. Primate Siglecs also show quantitative and qualitative intra- and interspecies variations in expression patterns on leukocytes, both in circulation and in tissues. Taken together our data explain why the CD33rSiglec-encoding gene cluster is undergoing rapid evolution via multiple mechanisms, driven by the need to maintain self-recognition by innate immune cells, while escaping 2 distinct mechanisms of pathogen subversion.—Padler-Karavani, V., Hurtado-Ziola, N., Chang, Y.-C., Sonnenburg, J. L., Ronaghy, A., Yu, H., Verhagen, A., Nizet, V., Chen, X., Varki, N., Varki, A., Angata, T. Rapid evolution of binding specificities and expression patterns of inhibitory CD33-related Siglecs in primates.

Sialic acids are dominant glycans on vertebrate cell surfaces, mediating roles as diverse as influenza infection, leukocyte trafficking and neural plasticity. It has long been known that malignant cells upregulate sialic acids, but few reasons for this have so far been elucidated. In apparently unrelated work, we have recently found that certain pathogenic bacteria coat themselves with sialic acids, thereby dampening innate immune responses, via engagement of inhibitory Siglecs (sialic acid binding Ig-like lectins). We therefore hypothesized that hypersialylated carcinoma cells might similarly engage Siglecs and modulate immune cell function. Confirming this hypothesis, we now have the first demonstration of the involvement of Siglecs in cancer progression in vivo. Siglec-9 is the most abundant inhibitory Siglec on human neutrophils and monocytes/macrophages. We first demonstrate that ligands for Siglec-9 are strongly upregulated in human carcinomas. We further provide evidence that ligands on carcinoma cells can inhibit neutrophil activation and killing of tumor cells by neutrophils. We then show that mice lacking Siglec-E (the murine functional equivalent of Siglec-9) have an increased immunosurveillance in autochthonous and transplantation models, which could be reversed by transgenic expression of Siglec-9 in myelomonocytic cells. Studies of later phases of tumor progression however showed that Siglec-E/-9 inhibits polarization of M2 macrophages and therefore impairs angiogenesis and tumor growth. In keeping with this dualistic role of myelomonocytic Siglecs during different phases of cancer progression, survival of non-small cell lung cancer patients with a polymorphism that reduced ligand binding to Siglec-9 had initially an improved survival, suggesting an increased immunosurveillance, but this effect was lost during longer followup. Our results identify inhibitory CD33-related myelomonocytic Siglecs as important players in cancer biology and as potential targets for immunomodulatory therapy. We show that hypersialylated carcinoma cells modulate the innate immune response by engaging Siglec-9 and inhibit either immunosurveillance or cancer-related inflammation. Our data also exemplify the dualistic role of innate immune cells and their receptors in cancer progression depending on the context and the microenvironment, i.e., growth control by immunosurveillance or growth support by cancer-related inflammation. Citation Format: Heinz Läubli, Oliver M. T. Pearce, Flavio Schwarz, Lingquan Deng, Michal Stanczak, Liwen Deng, Andrea Verhagen, Patrick Secrest, Chrissy Lusk, Ann G. Schwartz, Nissi Varki, Jack Bui, Ajit Varki. Engagement of myelomonocytic siglecs by tumor-associated ligands modulates innate immune responses to cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3659. doi:10.1158/1538-7445.AM2014-3659

Compelling evidence for naturally occurring immunosurveillance against malignancies informs and justifies some current approaches towards cancer immunotherapy. However, some types of immune reactions have also been shown to facilitate tumor progression. For example, our previous studies showed that experimental tumor growth in mice can be enhanced by low levels of circulating antibodies directed against the non-human sialic acid N-glycolyl-neuraminic acid (Neu5Gc, which accumulates in human tumors from dietary sources). However, tumor growth could instead be inhibited by anti-Neu5Gc antibodies from a different source, in a different model. Thus, anti-Neu5Gc antibodies appeared to be showing differential effects on tumor growth. However it remains generally unclear whether the immune responses that mediate cancer immunosurveillance versus those responsible for inflammatory facilitation are qualitatively and/or quantitatively distinct. Here, we address this question using multiple murine tumor growth models in which polyclonal antibodies against tumor antigens such as Neu5Gc can alter tumor progression. We found that while growth was stimulated at low antibody doses it was inhibited by high doses, over a linear and remarkably narrow range, defining an immune response curve (IRC), i.e., inverse hormesis. Moreover, modulation of immune responses against the tumor by altering antibody avidity or by enhancing innate immunity shifted the IRC in the appropriate direction. Thus, the dualistic role of immunosurveillance versus inflammation in modulating tumor progression can be quantitatively distinguished in multiple model systems, and can occur over a remarkably narrow range. We also reproduced the biphasic IRC in a model using the monoclonal anti-CD20 antibody rituximab. Furthermore, depletion of macrophages abrogated the growth promotion at low antibody concentrations and depletion of NK cells reduced growth inhibition at high antibody concentrations. These findings may have implications for the etiology and prevention of cancer, and for immunotherapeutic approaches utilizing antibodies. Citation Format: Oliver M.T. Pearce, Heinz Läubli, Andrea Verhagen, Patrick Secrest, Jiquan Zhang, Paul R. Crocker, Nissi Varki, Jack Bui, Ajit Varki. Inverse hormesis of cancer growth mediated by narrow ranges of tumor-directed antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1063. doi:10.1158/1538-7445.AM2014-1063

Siglecs (Sialic acid-binding Immunoglobulin-like Lectins) are vertebrate sialic acid (Sia) binding proteins mostly expressed in hematopoietic and immune cells. They are type-I proteins with an extracellular N-terminal Sia-binding Ig-like V-set domain, one or more C2-set Ig-like domains, a transmembrane domain and a C-terminal cytoplasmic tail. CD33-related Siglecs (CD33rSiglecs) are a genetically clustered subgroup. Prior comparative genomic analysis indicated accelerated evolution of CD33rSiglecs selectively occurring in the Sia-binding V-set domain, suggesting that this domain is under the greatest selection pressure. To gain further insight into this rapid evolution of CD33rSiglecs, we prepared recombinant form of the N-terminal domains of CD33rSiglecs from human, chimpanzee, and baboon, and compared their sialoglycan binding-specificities. Using ELISA on a panel of gangliosides or analysis on a sialoglycan microarray we show that binding patterns are widely divergent between the three species. Some show preference to Neu5Gc over Neu5Ac, especially in chimpanzees and baboons, indicating an evolutionary adjustment to the loss of Neu5Gc expression in humans. Expression patterns of CD33rSiglecs on circulating blood cells of humans and related great apes also show evidence for rapid evolution, and overall expression is greater in apes compared to human. The selection mechanisms driving this rapid evolution will be discussed.