Human heterophile antibodies that agglutinate animal erythrocytes are known to detect the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc). This monosaccharide cannot by itself fill the binding site (paratope) of an antibody and can also be modified and presented in various linkages, on diverse underlying glycans. Thus, we hypothesized that the human anti-Neu5Gc antibody response is diverse and polyclonal. Here we use a novel set of natural and chemoenzymatically-synthesized glycans to show that normal humans have an abundant and diverse spectrum of such anti-Neu5Gc antibodies, directed against a variety of Neu5Gc-containing epitopes. High sensitivity and specificity assays were achieved by using N-acetylneuraminic acid (Neu5Ac)-containing probes (differing from Neu5Gc by one less oxygen atom) as optimal background controls. The commonest anti-Neu5Gc antibodies are of the IgG class. Moreover, the range of reactivity and Ig classes of antibodies varies greatly amongst normal humans, with some individuals having remarkably large amounts, even surpassing levels of some well-known natural blood group and xenoreactive antibodies. We purified these anti-Neu5Gc antibodies from individual human sera using a newly developed affinity method and show that they bind to wild-type but not Neu5Gc-deficient mouse tissues. Moreover, they bind back to human carcinomas that have accumulated Neu5Gc in vivo. As dietary Neu5Gc is primarily found in red meat and milk products, we suggest that this ongoing antigen-antibody reaction may generate chronic inflammation, possibly contributing to the high frequency of diet-related carcinomas and other diseases in humans.

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Human carcinomas can metabolically incorporate and present the dietary non-human sialic acid Neu5Gc, which differs from the human sialic acid N-acetylneuraminic acid (Neu5Ac) by one oxygen atom. Tumor-associated Neu5Gc can interact with low levels of circulating anti-Neu5Gc antibodies, thereby facilitating tumor progression via chronic inflammation in a human-like Neu5Gc-deficient mouse model. Here we show that human anti-Neu5Gc antibodies can be affinity-purified in substantial amounts from clinically-approved intravenous IgG (IVIG) and used at higher concentrations to suppress growth of the same Neu5Gc-expressing tumors. Hypothesizing that this polyclonal spectrum of human anti-Neu5Gc antibodies also includes potential cancer biomarkers, we then characterize them in cancer and non-cancer patients’ sera, using a novel sialoglycan-microarray presenting multiple Neu5Gc-glycans and control Neu5Ac-glycans. Antibodies against Neu5Gcα2–6GalNAcα1-O-Ser/Thr (GcSTn) were found to be more prominent in patients with carcinomas than with other diseases. This unusual epitope arises from dietary Neu5Gc incorporation into the carcinoma marker Sialyl-Tn, and is the first example of such a novel mechanism for biomarker generation. Finally, human serum or purified antibodies rich in anti-GcSTn-reactivity kill GcSTn-expressing human tumors via complement-dependent-cytotoxicity or antibody-dependent-cellular-cytotoxicity. Such xeno-autoantibodies and xenoautoantigens have potential for novel diagnostics, prognostics and therapeutics in human carcinomas.

Control of inflammation is critical for therapy of infectious diseases. Pathogen-associated and/or danger-associated molecular patterns (PAMPs and DAMPs, respectively) are the two major inducers of inflammation. Because the CD24-Siglec G/10 interactions selectively repress inflammatory response to DAMPs, microbial disruption of the negative regulation would provide a general mechanism to exacerbate inflammation. Here we show that the sialic acid-based pattern recognitions of CD24 by Siglec G/10 are targeted by sialidases in polybacterial sepsis. Sialidase inhibitors protect mice against sepsis by a CD24-Siglecg-dependent mechanism, whereas a targeted mutation of either CD24 or Siglecg exacerbates sepsis. Bacterial sialidase and host CD24 and Siglecg genes interact to determine pathogen virulence. Our data demonstrate a critical role for disrupting sialic acid-based pattern recognitions in microbial virulence and suggest a therapeutic approach to dampen harmful inflammatory response during infection.

Sialic acid aldolases or N-acetylneuraminate lyases (NanAs) catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-D-mannosamine (ManNAc). A capillary electrophoresis (CE) assay was developed to directly characterize the activities of NanAs in both Neu5Ac cleavage and Neu5Ac synthesis directions. The assay was used to obtain the pH profile and the kinetic data of a NanA cloned from Pasteurella multocida P-1059 (PmNanA) and a previously reported recombinant Escherichia coli K12 NanA (EcNanA). Both enzymes are active in a broad pH range of 6.0-9.0 in both reaction directions and have similar kinetic parameters. Substrates specificity studies showed that 5-O-methyl-ManNAc, a ManNAc derivative, can be used efficiently as a substrate by PmNanA, but not efficiently by EcNanA, for the synthesis of 8-O-methyl Neu5Ac. In addition, PmNanA (250 mg per liter culture) has a higher expression level (2.5 fold) than EcNanA (94 mg per liter culture). The higher expression level and a broader substrate tolerance make PmNanA a better catalyst than EcNanA for the chemoenzymatic synthesis of sialic acids and their derivatives.

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. Binary X-ray crystal structures of a multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1) with a donor analogue CMP-3F(a)Neu5Ac or CMP-3F(e)Neu5Ac were determined at 2.0 and 1.9 A resolutions, respectively. Ternary X-ray structures of the protein in complex with CMP or a donor analogue CMP-3F(a)Neu5Ac and an acceptor lactose have been determined at 2.0 and 2.27 A resolutions, respectively. This represents the first sialyltransferase structure and the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptor simultaneously. The four structures presented here reveal that binding of the nucleotide-activated donor sugar causes a buried tryptophan to flip out of the protein core to interact with the donor sugar and helps define the acceptor sugar binding site. Additionally, key amino acid residues involved in the catalysis have been identified. Structural and kinetic data support a direct displacement mechanism involving an oxocarbenium ion-like transition state assisted with Asp141 serving as a general base to activate the acceptor hydroxyl group.

Humans are genetically unable to synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc). However, Neu5Gc can be metabolically incorporated and covalently expressed on cultured human cell surfaces. Meanwhile, humans express varying and sometimes high titers of polyclonal anti-Neu5Gc antibodies. Here, a survey of human tissues by immunohistochemistry with both a monospecific chicken anti-Neu5Gc antibody and with affinity-purified human antiNeu5Gc antibodies demonstrates endothelial expression of Neu5Gc, likely originating from Neu5Gc-rich foods like red meats. We hypothesized that the combination of Neu5Gc incorporation and anti-Neu5Gc antibodies can induce endothelial activation. Indeed, the incubation of high-titer human sera with Neu5Gc-fed endothelial cells led to Neu5Gc-dependent antibody binding, complement deposition, endothelial activation, selectin expression, increased cytokine secretion, and monocyte binding. The proinflammatory cytokine tumor necrosis factor-␣ also selectively enhanced human anti-Neu5Gc antibody reactivity. AntiNeu5Gc antibodies affinity-purified from human serum also directed Neu5Gc-dependent complement deposition onto cultured endothelial cells. These data indicate a novel human-specific mechanism in which Neu5Gc-rich foods deliver immunogenic Neu5Gc to the endothelium, giving anti-Neu5Gc antibody-and complement-dependent activation, and potentially contributing to human vascular pathologies. In the case of atherosclerosis, Neu5Gc is present both in endothelium overlying plaques and in subendothelial regions, providing multiple pathways for accelerating inflammation in this disease.

Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in ␤-linked galactose at the nonreducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate ␣2-3-and ␣2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting ␣2-6-and ␣2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either ␣2-6-linked Neu5Ac9Lt or ␣2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.