Diversity in specificity, abundance, and composition of anti-Neu5Gc antibodies in normal humans: Potential implications for disease

Abstract

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.

Sexual selection by female immunity against paternal antigens can fix loss of function alleles

“…Additionally, microbial presentation of dietary Neu5Gc could have provided consistent immunization of most individuals (17). Circulating anti-Neu5Gc antibodies have been detected in all humans tested so far (16). Second, females must come into close contact with antigens expressed on sperm or embryonic tissues.…”

“…Antibody titers in mouse serum and mouse uterine fluids were determined by ELISA using Neu5Gc-and Neu5Ac-polyacrylamide beads as plated targets (16). Sia dependence of antibody binding was confirmed by mild periodate treatment to destroy the side chain of Sia and controlled for by mock treatment without the premixed periodate and sodium borohydride (16). Uterine fluid was collected by immediate postsacrifice flushing of the uteri with PBS.…”

“…Sera included human sera with high or low tiers of anti-Neu5Gc and chimpanzee sera (no anti-Neu5Gc antibodes). AntiNeu5Gc content of human sera had been previously determined by ELISA using Neu5Gc target probes (16). Samples were then washed and stained for cell death with propidium iodine (PI, 15 min at RT) or for complement deposition with anti-C3 monoclonal antibody [1 h at RT, C3 (6C9), Santa Cruz Biotechnology].…”

“…1). Human antibody profiles against Neu5Gc are varied in level and composition, but all humans have circulating antibodies against Neu5Gc, which appear early in life (15)(16)(17). The antibody repertoire of females also extends to their reproductive tract, through which the sperm must pass and in which embryos must implant (18).…”

Monitoring Glycosylation Profile and Protein Titer in Cell Culture Samples Using ZipChip CE-MS

“…Glycosylation is one of very important post-translational modifications because it can have significant impact on antibody-dependent cell-mediated cytotoxicity (ADCC) activity [3,4], complement-dependent cytotoxicity (CDCC) activity [5], clearance [6] and immunogenicity [7,8]. Protein glycosylation can be significantly impacted by the host cell line, clone, media composition, feeding strategy and downstream processing conditions [9][10][11].…”