Differential Recognition of Diet-Derived Neu5Gc-Neoantigens on Glycan Microarrays by Carbohydrate-Specific Pooled Human IgG and IgA Antibodies.
ABSTRACT: 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.
Project description: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 1 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 noncancer patients' sera, using a novel sialoglycan microarray presenting multiple Neu5Gc-glycans and control Neu5Ac-glycans. Antibodies against Neu5Gc?2-6GalNAc?1-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 xeno-autoantigens have potential for novel diagnostics, prognostics, and therapeutics in human carcinomas.
Project description:BACKGROUND:High consumption of red and processed meat is commonly associated with increased cancer risk, particularly colorectal cancer. Antibodies against the red meat-derived carbohydrate N-glycolylneuraminic acid (Neu5Gc) exacerbate cancer in "human-like" mice. Human anti-Neu5Gc IgG and red meat are both independently proposed to increase cancer risk, yet how diet affects these antibodies is largely unknown. METHODS:We used world global data to demonstrate that colorectal cancer incidence and mortality are associated with increased national meat consumption. In a well-defined large cohort, we used glycomics to measure daily Neu5Gc intake from red meat and dairy, and investigated serum as well as affinity-purified anti-Neu5Gc antibodies. Based on 24-h dietary records, daily Neu5Gc intake was calculated for 19,621 subjects aged ??18?years of the NutriNet-Santé study. Serum and affinity-purified anti-Neu5Gc antibodies were evaluated by ELISA and glycan microarrays in representative 120 individuals, each with at least eighteen 24-h dietary records (aged 45-60, Q1-Q4; aged >?60, Q1 and Q4; 10 men/women per quartile). RESULTS:We found that high-Neu5Gc diet, gender, and age affect the specificity, levels, and repertoires of anti-Neu5Gc IgG immune responses, but not their affinity. Men consumed more Neu5Gc than women, mostly from red meat (p?=?0.0015), and exhibited higher overall serum anti-Neu5Gc IgG levels by ELISA (3.94?ng/?l versus 2.22?ng/?l, respectively; p?=?0.039). Detailed glycan microarray analysis against 56 different glycans revealed high Neu5Gc-specificity with increased anti-Neu5Gc IgG and altered repertoires, associated with higher consumption of Neu5Gc from red meat and cow dairy. Affinity purification of serum anti-Neu5Gc antibodies revealed increased levels and biased array repertoire patterns, without an increase in antibody affinity, in individuals consuming higher Neu5Gc levels. Furthermore, in a high-meat diet, antibody diversity patterns on glycan microarrays shifted towards Neu5Gc?3-linked glycans, increasing the ?3/?6-glycans ratio score. CONCLUSIONS:We found a clear link between the levels and repertoire of serum anti-Neu5Gc IgG and Neu5Gc intake from red meat and dairy. These precise rational methodologies allowed to develop a Gcemic index to simplify the assessment of Neu5Gc in foods that could potentially be adapted for dietary recommendations to reduce cancer risk.
Project description: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.
Project description:The full potential of recombinant Immunoglobulin A as therapeutic antibody is not fully explored, owing to the fact that structure-function relationships of these extensively glycosylated proteins are not well understood. Here monomeric IgA1, IgA2m(1), and IgA2m(2) variants of the anti-HER2 antibody (IgG1) trastuzumab were expressed in glyco-engineered Nicotiana benthamiana plants and in human HEK293-6E cells. All three IgA isotypes were purified and subjected to biophysical and biochemical characterization. While no differences in assembly, antigen binding, and glycosylation occupancy were observed, both systems vary tremendously in terms of glycan structures and heterogeneity of glycosylation. Mass-spectrometric analysis of site-specific glycosylation revealed that plant-produced IgAs carry mainly complex-type biantennary N-glycans. HEK293-6E-produced IgAs, on the contrary, showed very heterogeneous N-glycans with high levels of sialylation, core-fucose, and the presence of branched structures. The site-specific analysis revealed major differences between the individual N-glycosylation sites of each IgA subtype. Moreover, the proline-rich hinge region from HEK293-6E cell-derived IgA1 was occupied with mucin-type O-glycans, whereas IgA1 from N. benthamiana displayed numerous plant-specific modifications. Interestingly, a shift in unfolding of the CH2 domain of plant-produced IgA toward lower temperatures can be observed with differential scanning calorimetry, suggesting that distinct glycoforms affect the thermal stability of IgAs.
Project description:Human heterophile antibodies that agglutinate animal erythrocytes are known to detect the nonhuman 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 vary 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 showed 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.
Project description:Rotavirus is a major cause of gastroenteritis in children, with infection typically inducing high levels of protective antibodies. Antibodies targeting the middle capsid protein VP6 are particularly abundant, and as VP6 is only exposed inside cells, neutralisation must be post-entry. However, while a system of poly immune globulin receptor (pIgR) transcytosis has been proposed for anti-VP6 IgAs, the mechanism by which VP6-specific IgG mediates protection remains less clear. We have developed an intracellular neutralisation assay to examine how antibodies neutralise rotavirus inside cells, enabling comparison between IgG and IgA isotypes. Unexpectedly we found that neutralisation by VP6-specific IgG was much more efficient than by VP6-specific IgA. This observation was highly dependent on the activity of the cytosolic antibody receptor TRIM21 and was confirmed using an in vivo model of murine rotavirus infection. Furthermore, mice deficient in only IgG and not other antibody isotypes had a serious deficit in intracellular antibody-mediated protection. The finding that VP6-specific IgG protect mice against rotavirus infection has important implications for rotavirus vaccination. Current assays determine protection in humans predominantly by measuring rotavirus-specific IgA titres. Measurements of VP6-specific IgG may add to existing mechanistic correlates of protection.
Project description:Hypogammaglobulinemia is the most frequently observed immune defect in chronic lymphocytic leukemia (CLL). Although CLL patients usually have low serum levels of all isotypes (IgG, IgM and IgA), standard immunoglobulin (Ig) preparations for replacement therapy administrated to these patients contain more than 95% of IgG. Pentaglobin is an Ig preparation of intravenous application (IVIg) enriched with IgM and IgA (IVIgGMA), with the potential benefit to restore the Ig levels of all isotypes. Because IVIg preparations at high doses have well-documented anti-inflammatory and immunomodulatory effects, we aimed to evaluate the capacity of Pentaglobin and a standard IVIg preparation to affect leukemic and T cells from CLL patients. In contrast to standard IVIg, we found that IVIgGMA did not modify T cell activation and had a lower inhibitory effect on T cell proliferation. Regarding the activation of leukemic B cells through BCR, it was similarly reduced by both IVIgGMA and IVIgG. None of these IVIg preparations modified spontaneous apoptosis of T or leukemic B cells. However, the addition of IVIgGMA on in vitro cultures decreased the apoptosis of T cells induced by the BCL-2 inhibitor, venetoclax. Importantly, IVIgGMA did not impair venetoclax-induced apoptosis of leukemic B cells. Overall, our results add new data on the effects of different preparations of IVIg in CLL, and show that the IgM/IgA enriched preparation not only affects relevant mechanisms involved in CLL pathogenesis but also has a particular profile of immunomodulatory effects on T cells that deserves further investigation.
Project description:Salmonella Typhi is an exclusive human pathogen that causes typhoid fever. Typhoid toxin is a S. Typhi virulence factor that can reproduce most of the typhoid fever symptoms in experimental animals. Toxicity depends on toxin binding to terminally sialylated glycans on surface glycoproteins. Human glycans are unusual because of the lack of CMAH, which in other mammals converts N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc). Here, we report that typhoid toxin binds to and is toxic toward cells expressing glycans terminated in Neu5Ac (expressed by humans) over glycans terminated in Neu5Gc (expressed by other mammals). Mice constitutively expressing CMAH thus displaying Neu5Gc in all tissues are resistant to typhoid toxin. The atomic structure of typhoid toxin bound to Neu5Ac reveals the structural bases for its binding specificity. These findings provide insight into the molecular bases for Salmonella Typhi's host specificity and may help the development of therapies for typhoid fever.
Project description: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.
Project description:Sialic acids are common monosaccharides that are widely expressed as outer terminal units on all vertebrate cell surfaces, and play fundamental roles in cell-cell and cell-microenvironment interactions. The predominant sialic acids on most mammalian cells are N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Neu5Gc is notable for its deficiency in humans due to a species-specific and species-universal inactivating deletion in the CMAH gene encoding the hydroxylase that converts CMP-Neu5Ac to CMP-Neu5Gc. However, Neu5Gc is metabolically incorporated into human tissues from dietary sources (particularly red meat), and detected at even higher levels in some human cancers. Early life exposure to Neu5Gc-containing foods in the presence of certain commensal bacteria that incorporate dietary Neu5Gc into lipooligosaccharides can lead to generation of antibodies that are also cross-reactive against Neu5Gc-containing glycans in human tissues ("xeno-autoantigens"). Such anti-Neu5Gc "xeno-autoantibodies" are found in all humans, although ranging widely in levels among individuals, and displaying diverse and variable specificities for the underlying glycan. Experimental evidence in a human-like Neu5Gc-deficient Cmah(-) (/) (-) mouse model shows that inflammation due to "xenosialitis" caused by this antigen-antibody interaction can promote tumor progression, suggesting a likely mechanism for the well-known epidemiological link between red meat consumption and carcinoma risk. In this review, we discuss the history of this field, mechanisms of Neu5Gc incorporation into tissues, the origin and specificities of human anti-Neu5Gc antibodies, their use as possible cancer biomarkers, implications of xenosialitis in cancer initiation and progression, and current and future approaches toward immunotherapy that could take advantage of this unusual human-specific phenomenon.