Search

Many pathogenic bacteria secrete AB 5 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 B 5 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Sialic acids (Sias) mediate many biological functions, including molecular recognition during development, immune response, and fertilization. A Sia-rich glycocalyx coats the surface of sperm, allowing them to survive as allogeneic cells in the female reproductive tract despite female immunity. During capacitation, sperm lose a fraction of their Sias. We quantified shed Sia monosaccharides released from capacitated sperm and measured sperm sialidase activity. We report the presence of two sialidases (neuraminidases Neu1 and Neu3) on mammalian sperm. These are themselves shed from sperm during capacitation. Inhibiting sialidase activity interferes with sperm binding to the zona pellucida of the ovum. A survey of human sperm samples for the presence of sialidases NEU1 and NEU3 identified a lack of one or both sialidases in sperm of some male idiopathic infertility cases. The results contribute new insights into the dynamic remodeling of the sperm glycocalyx prior to fertilization.

The primate SIGLEC12 gene encodes one of the CD33-related Siglec family of signaling molecules in immune cells. We had previously reported that this gene harbors a human-specific missense mutation of the codon for an Arg residue required for sialic acid recognition. Here we show that this R122C mutation of the Siglec-XII protein is fixed in the human population, i.e. it occurred prior to the origin of modern humans. Additional mutations have since completely inactivated the SIGLEC12 gene in some but not all humans. The most common inactivating mutation with a global allele frequency of 58% is a single nucleotide frameshift that markedly shortens the open reading frame. Unlike other CD33-related Siglecs that are primarily found on immune cells, we found that Siglec-XII protein is expressed not only on some macrophages but also on various epithelial cell surfaces in humans and chimpanzees. We also found expression on certain human prostate epithelial carcinomas and carcinoma cell lines. This expression correlates with the presence of the nonframeshifted, intact SIGLEC12 allele. Although SIGLEC12 allele status did not predict prostate carcinoma incidence, restoration of expression in a prostate carcinoma cell line homozygous for the frameshift mutation induced altered regulation of several genes associated with carcinoma progression. These stably transfected Siglec-XII-expressing prostate cancer cells also showed enhanced growth in nude mice. Finally, monoclonal antibodies against the protein were internalized by Siglec-XII-expressing prostate carcinoma cells, allowing targeting of a toxin to such cells. Polymorphic expression of Siglec-XII in humans thus has implications for prostate cancer biology and therapeutics.

Numerous vertebrate genes are involved in the biology of the oligosaccharide chains attached to glycoconjugates. These genes fall into diverse groups within the conventional Gene Ontology classification. However, they should be evaluated together from functional and evolutionary perspectives in a "biochemical systems" approach, considering each monosaccharide unit's biosynthesis, activation, transport, modification, transfer, recycling, degradation, and recognition. Sialic acid (Sia) residues are monosaccharides at the outer end of glycans on the cell-surface and secreted molecules of vertebrates, mediating recognition by intrinsic or extrinsic (pathogen) receptors. The availability of multiple genome sequences allows a system-wide comparison among primates and rodents of all genes directly involved in Sia biology. Taking this approach, we present further evidence for accelerated evolution in Sia-binding domains of CD33-related Sia-recognizing Ig-like lectins. Other gene classes are more conserved, including those encoding the sialyltransferases that attach Sia residues to glycans. Despite this conservation, tissue sialylation patterns are shown to differ widely among these species, presumably because of rapid evolution of sialyltransferase expression patterns. Analyses of N- and O-glycans of erythrocyte and plasma glycopeptides from these and other mammalian taxa confirmed this phenomenon. Sia modifications on these glycopeptides also appear to be undergoing rapid evolution. This rapid evolution of the sialome presumably results from the ongoing need of organisms to evade microbial pathogens that use Sia residues as receptors. The rapid evolution of Sia-binding domains of the inhibitory CD33-related Sia-recognizing Ig-like lectins is likely to be a secondary consequence, as these inhibitory receptors presumably need to keep up with recognition of the rapidly evolving "self"-sialome.

Siglecs are immunoglobulin superfamily member lectins that selectively recognize different types and linkages of sialic acids, which are major components of cell surface and secreted glycoconjugates. We report here a human Siglec-like molecule (Siglec-L1) that lacks a conserved arginine residue known to be essential for optimal sialic acid recognition by previously known Siglecs. Loss of the arginine from an ancestral molecule was caused by a single nucleotide substitution that occurred after the common ancestor of humans with the great apes but before the origin of modern humans. The chimpanzee Siglec-L1 ortholog remains fully functional and preferentially recognizes N-glycolylneuraminic acid, which is a common sialic acid in great apes and other mammals. Reintroducing the ancestral arginine into the human molecule regenerates the same properties. Thus, the single base pair mutation that replaced the arginine on human Siglec-L1 is likely to be evolutionarily related to the previously reported loss of N-glycolylneuraminic acid expression in the human lineage. Siglec-L1 and its chimpanzee Siglec ortholog also have a different expression pattern from previously reported Siglecs because they are found on the lumenal edge of epithelial cell surfaces. Notably, the human genome contains several Siglec-like pseudogenes that have independent mutations that would have replaced the arginine residue required for optimal sialic acid recognition. Thus, additional changes in the biology of sialic acids may have taken place during human evolution.