Kalyan Banda

A red meat-derived glycan promotes inflammation and cancer progression

Significance We present an unusual mechanism for the well-known association between red meat consumption and carcinoma risk involving the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc). We first evaluate the Neu5Gc content of various foods to show that red meats are particularly rich in orally bioavailable Neu5Gc and then investigate human-like Neu5Gc-deficient mice fed this form of Neu5Gc. When such mice were challenged with anti-Neu5Gc antibodies, they developed evidence of systemic inflammation. Long-term exposure to this combination resulted in a significantly higher incidence of carcinomas (five-fold increase) and an association with Neu5Gc accumulation in the tumors. Similar mechanisms may contribute to the association of red meat consumption with other diseases, such as atherosclerosis and type 2 diabetes, which are also exacerbated by inflammation. A well known, epidemiologically reproducible risk factor for human carcinomas is the long-term consumption of “red meat” of mammalian origin. Although multiple theories have attempted to explain this human-specific association, none have been conclusively proven. We used an improved method to survey common foods for free and glycosidically bound forms of the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc), showing that it is highly and selectively enriched in red meat. The bound form of Neu5Gc is bioavailable, undergoing metabolic incorporation into human tissues, despite being a foreign antigen. Interactions of this antigen with circulating anti-Neu5Gc antibodies could potentially incite inflammation. Indeed, when human-like Neu5Gc-deficient mice were fed bioavailable Neu5Gc and challenged with anti-Neu5Gc antibodies, they developed evidence of systemic inflammation. Such mice are already prone to develop occasional tumors of the liver, an organ that can incorporate dietary Neu5Gc. Neu5Gc-deficient mice immunized against Neu5Gc and fed bioavailable Neu5Gc developed a much higher incidence of hepatocellular carcinomas, with evidence of Neu5Gc accumulation. Taken together, our data provide an unusual mechanistic explanation for the epidemiological association between red meat consumption and carcinoma risk. This mechanism might also contribute to other chronic inflammatory processes epidemiologically associated with red meat consumption.

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups MECHANISMS UNDERLYING GASTROINTESTINAL INCORPORATION OF THE NON-HUMAN SIALIC ACID XENO-AUTOANTIGEN N-GLYCOLYLNEURAMINIC ACID

Background: Prior work implicated the sialic acid Neu5Gc as a xeno-autoantigen in humans. Results: Dietary Neu5Gc can be utilized by the murine gastrointestinal system and distributed for glycosylation in peripheral tissues. Conclusion: Diets containing Neu5Gc can lead to human-like tissue incorporation. Significance: Neu5Gc may be a dietary risk factor for progression of carcinomas, atherosclerosis, and immunologic diseases in humans. Although N-acetyl groups are common in nature, N-glycolyl groups are rare. Mammals express two major sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid (Neu5Gc). Although humans cannot produce Neu5Gc, it is detected in the epithelial lining of hollow organs, endothelial lining of the vasculature, fetal tissues, and carcinomas. This unexpected expression is hypothesized to result via metabolic incorporation of Neu5Gc from mammalian foods. This accumulation has relevance for diseases associated with such nutrients, via interaction with Neu5Gc-specific antibodies. Little is known about how ingested sialic acids in general and Neu5Gc in particular are metabolized in the gastrointestinal tract. We studied the gastrointestinal and systemic fate of Neu5Gc-containing glycoproteins (Neu5Gc-glycoproteins) or free Neu5Gc in the Neu5Gc-free Cmah−/− mouse model. Ingested free Neu5Gc showed rapid absorption into the circulation and urinary excretion. In contrast, ingestion of Neu5Gc-glycoproteins led to Neu5Gc incorporation into the small intestinal wall, appearance in circulation at a steady-state level for several hours, and metabolic incorporation into multiple peripheral tissue glycoproteins and glycolipids, thus conclusively proving that Neu5Gc can be metabolically incorporated from food. Feeding Neu5Gc-glycoproteins but not free Neu5Gc mimics the human condition, causing tissue incorporation into human-like sites in Cmah−/− fetal and adult tissues, as well as developing tumors. Thus, glycoproteins containing glycosidically linked Neu5Gc are the likely dietary source for human tissue accumulation, and not the free monosaccharide. This human-like model can be used to elucidate specific mechanisms of Neu5Gc delivery from the gut to tissues, as well as general mechanisms of metabolism of ingested sialic acids.

Specific inactivation of two immunomodulatory SIGLEC genes during human evolution

Sialic acid-recognizing Ig-like lectins (Siglecs) are signaling receptors that modulate immune responses, and are targeted for interactions by certain pathogens. We describe two primate Siglecs that were rendered nonfunctional by single genetic events during hominin evolution after our common ancestor with the chimpanzee. SIGLEC13 was deleted by an Alu-mediated recombination event, and a single base pair deletion disrupted the ORF of SIGLEC17. Siglec-13 is expressed on chimpanzee monocytes, innate immune cells that react to bacteria. The human SIGLEC17P pseudogene mRNA is still expressed at high levels in human natural killer cells, which bridge innate and adaptive immune responses. As both resulting pseudogenes are homozygous in all human populations, we resurrected the originally encoded proteins and examined their functions. Chimpanzee Siglec-13 and the resurrected human Siglec-17 recruit a signaling adapter and bind sialic acids. Expression of either Siglec in innate immune cells alters inflammatory cytokine secretion in response to Toll-like receptor-4 stimulation. Both Siglecs can also be engaged by two potentially lethal sialylated bacterial pathogens of newborns and infants, agents with a potential impact on reproductive fitness. Neanderthal and Denisovan genomes show human-like sequences at both loci, corroborating estimates that the initial pseudogenization events occurred in the common ancestral population of these hominins. Both loci also show limited polymorphic diversity, suggesting selection forces predating the origin of modern humans. Taken together, these data suggest that genetic elimination of Siglec-13 and/or Siglec-17 represents signatures of infectious and/or other inflammatory selective processes contributing to population restrictions during hominin origins.

SIGLEC12, a Human-specific Segregating (Pseudo)gene, Encodes a Signaling Molecule Expressed in Prostate Carcinomas

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.

Serum IgG Response to Cryptosporidium Immunodominant Antigen gp15 and Polymorphic Antigen gp40 in Children with Cryptosporidiosis in South India

ABSTRACT The surface-associated glycopeptides gp40, one of the most polymorphic Cryptosporidium antigens, and gp15, one of the most immunodominant Cryptosporidium antigens, are putative vaccine candidates because they mediate infection in vitro and induce immune responses in vivo. We evaluated antibody responses to these antigens before and after the first episode of symptomatic cryptosporidiosis in 51 children from a birth cohort study in an area in South India where Cryptosporidium is endemic and a major cause of parasitic diarrhea. IgG levels to gp15 and to homotypic and heterotypic gp40 antigens were measured in pre- and postdiarrheal sera by enzyme-linked immunosorbent assay (ELISA). There was a significant IgG response to gp15 (P < 0.001) following the first episode of cryptosporidial diarrhea. Using a general additive model, we determined the estimated time of the peak IgG response to gp15 to be 9.3 weeks (confidence interval, 5.2 to 13.4) following the diarrheal episode. In a subset of 30 children infected with Cryptosporidium hominis subtype Ia, there was a significant difference in IgG responses to homotypic C. hominis Ia and to heterotypic Cryptosporidium parvum II gp40 antigens (P = 0.035). However, there was also a significant correlation (P = 0.001) in the responses to both antigens in individual children, suggesting that while responses are in part subtype specific, there is significant cross-reactivity to both antigens. This is the first report of the characterization of immune responses to cryptosporidiosis in Indian children and the first study to investigate human immune responses to the polymorphic gp40 antigen. However, further studies are needed to determine whether immune responses to these antigens are protective against subsequent infections.