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Background The two common sialic acid (Sia) in mammals are N-Acetylneuraminic acid (Neu5Ac) and its hydroxylated form N-Glycolylneuraminic acid (Neu5Gc). Unlike most mammals, humans cannot synthesize Neu5Gc that is considered foreign and recognized by circulating antibodies. Thus, Neu5Gc is a potential xenogenic carbohydrate antigen in bioprosthetic heart valves (BHV) that tend to deteriorate in time within human patients. Methods We investigated Neu5Gc expression in non-engineered animal-derived cardiac tissues and in clinically used commercial BHV, and evaluated Neu5Gc immunogenicity on BHV through recognition by human anti-Neu5Gc IgG. Results Neu5Gc was detected by immunohistochemistry in porcine aortic valves and in porcine and bovine pericardium. Qualitative analysis of Sia-linkages revealed Siaα2–3>Siaα2–6 on porcine/bovine pericardium while the opposite in porcine aortic/pulmonary valve cusps. Similarly, six commercial BHV containing either porcine aortic valve or porcine/bovine/equine pericardium revealed Siaα2–3>Siaα2–6 expression. Quantitative analysis of Sia by HPLC showed porcine/bovine pericardium express four-fold higher Neu5Gc levels compared to the porcine aortic/pulmonary valves, with Neu5Ac at six-fold over Neu5Gc. Likewise, Neu5Gc was expressed on commercial BHV (186.3±16.9 pmol Sia/μg protein), with Neu5Ac at eight-fold over Neu5Gc. Affinity-purified human anti-Neu5Gc IgG showing high specificity towards Neu5Gc-glycans (with no binding to Neu5Ac-glycans) on a glycan microarray, strongly bound to all tested commercial BHV, demonstrating Neu5Gc immune recognition in cardiac xenografts. Conclusions We conclusively demonstrated Neu5Gc expression in native cardiac tissues, as well as in six commercial BHV. These Neu5Gc xeno-antigens were recognized by human anti-Neu5Gc IgG, supporting their immunogenicity. Altogether, these findings suggest BHV-Neu5Gc/anti-Neu5Gc may play a role in valve deterioration warranting further investigation.

Humans have circulating antibodies against diverse glycans containing N-glycolylneuraminic acid (Neu5Gc) due to function-loss mutation of the CMAH gene. This xenogenic non-human carbohydrate is abundant in red meat, xenografts and biotherapeutics. Low levels of diet-derived Neu5Gc is also present on normal human endothelial cells, and together with anti-Neu5Gc antibodies could potentially mediate “xenosialitis” chronic-inflammation. Rabbit anti-human thymocyte globulin (ATG) is a drug containing polyclonal IgG glycoproteins commonly used as an immunosuppressant in human transplantation and autoimmune diseases. In type-1 diabetes patients, infusion of Neu5Gc-glycosylated ATG caused increased global anti-Neu5Gc response. Here, for the first time we explore changes in anti-Neu5Gc IgG repertoire following the immunization elicited by ATG, compared with the basal antibodies repertoire that reflect exposure to dietary-Neu5Gc. We used glycan microarrays with multiple Neu5Gc-glycans and controls to elucidate eventual differences in ATG-elicited repertoire, before/after ATG administration and track their kinetics (0, 1, 18 and 24 months). Response of all basal-pre-existing Neu5Gc-specific antibodies rapidly increased. This response peaked at one month post-ATG, with enhanced affinity, then resolved at 18–24 months. Induced-antibodies showed expanded diversity and de-novo recognition of different Neu5Gc-glycans, including endogenous glycolipids, that was further validated by affinity-purified anti-Neu5Gc antibodies from patients’ sera. These findings strongly suggest that ATG-induced anti-Neu5Gc IgGs represent a secondary exposure to this dietary carbohydrate-antigen in humans, with immune memory. Given their modified recognition patterns, ATG-evoked anti-Neu5Gc antibodies could potentially mediate biological effects different from pre-existing antibodies.

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5'monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format.

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