One of the major obstacles in xenotransplantation is tissue expression of the non-human mammalian carbohydrate N-glycolylneuraminic acid (Neu5Gc). This 9-carbon backbone acidic sugar is the ...hydroxylated form of N-acetylneuraminic acid (Neu5Ac), and both constitute the two most common sialic acid types in mammals. Loss of CMP-Neu5Ac hydroxylase encoding gene in humans dictates the immunogenic nature of Neu5Gc-containing xenografts. Here we describe an immunohistochemistry method for the detection of Neu5Gc in mammalian-derived tissues using affinity-purified chicken anti-Neu5Gc IgY. Specificity is further demonstrated by competitive inhibition with free Neu5Gc or Neu5Gc-glycoproteins, but not with Neu5Ac or Neu5Ac-glycoproteins. This method can be used to evaluate potential Neu5Gc-immunogenicity of xenografts.
•Aberrant glycosylation is a well-known hallmark of human tumors, and one of the forms of aberrant glycosylation in human tumors is the expression of Neu5Gc.•Neu5Gc-containing antigens as a class of ...cancer biomarkers have been under-explored for cancer diagnosis, monitoring and prognosis.•The mechanism and source of Neu5Gc expressed by human tumors remains to be clarified.•The emerging hypothesis of cancer cell-generated Neu5Gc provides a rationale for the use of Neu5Gc as a universal cancer biomarker.
One of the forms of aberrant glycosylation in human tumors is the expression of N-glycolylneuraminic acid (Neu5Gc). The only known enzyme to biosynthesize Neu5Gc in mammals, cytidine-5′-monophosphate-N-acetylneuraminic acid (CMAH), appears to be genetically inactivated in humans. Regardless, low levels of Neu5Gc have been detected in healthy humans. Therefore, it is proposed that the presence of Neu5Gc in humans is from dietary acquisition, such as red meat. Notably, detection of elevated Neu5Gc levels has been repeatedly found in cancer tissues, cells and serum samples, thereby Neu5Gc-containing antigens may be exploited as a class of cancer biomarkers. Here we review the findings to date on using Neu5Gc-containing tumor glycoconjugates as a class of cancer biomarkers for cancer detection, surveillance, prognosis and therapeutic targets. We review the evidence that supports an emerging hypothesis of de novo Neu5Gc biosynthesis in human cancer cells as a source of Neu5Gc in human tumors, generated under certain metabolic conditions.
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Cardiovascular disease (CVD) events due to atherosclerosis cause one-third of worldwide deaths and risk factors include physical inactivity, age, dyslipidemia, hypertension, diabetes, obesity, ...smoking, and red meat consumption. However, ∼15% of first-time events occur without such factors. In contrast, coronary events are extremely rare even in closely related chimpanzees in captivity, despite human-like CVD–risk-prone blood lipid profiles, hypertension, and mild atherosclerosis. Similarly, red meat-associated enhancement of CVD event risk does not seem to occur in other carnivorous mammals. Thus, heightened CVD risk may be intrinsic to humans, and genetic changes during our evolution need consideration. Humans exhibit a species-specific deficiency of the sialic acid N-glycolylneuraminic acid (Neu5Gc), due to pseudogenization of cytidine monophosphate-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH), which occurred in hominin ancestors ∼2 to 3 Mya. Ldlr
−/− mice with human-like Cmah deficiency fed a sialic acids (Sias)-free high-fat diet (HFD) showed ∼1.9-fold increased atherogenesis over Cmah wild-type Ldlr
−/− mice, associated with elevated macrophage cytokine expression and enhanced hyperglycemia. Human consumption of Neu5Gc (from red meat) acts as a “xeno-autoantigen” via metabolic incorporation into endogenous glycoconjugates, as interactions with circulating anti-Neu5Gc “xeno-autoantibodies” potentiate chronic inflammation (“xenosialitis”). Cmah
−/−
Ldlr
−/− mice immunized with Neu5Gc-bearing antigens to generate human-like anti-Neu5Gc antibodies suffered a ∼2.4-fold increased atherosclerosis on a Neu5Gc-rich HFD, compared with Neu5Ac-rich or Sias-free HFD. Lesions in Neu5Gc-immunized and Neu5Gc-rich HFD-fed Cmah
−/−
Ldlr
−/− mice were more advanced but unexplained by lipoprotein or glucose changes. Human evolutionary loss of CMAH likely contributes to atherosclerosis predisposition via multiple intrinsic and extrinsic mechanisms, and future studies could consider this more human-like model.
Cancer immunotherapy represents a promising approach to specifically target and treat cancer. The most common mechanisms by which monoclonal antibodies kill cells include antibody-dependent ...cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis, but also other mechanisms have been described. 14F7 is an antibody raised against the tumor-associated antigen NeuGc GM3, which was previously reported to kill cancer cells without inducing apoptotic pathways. The antibody was reported to induce giant membrane lesions in tumor cells, with apparent changes in the cytoskeleton. Here, we investigated the effect of humanized 14F7 on HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC) in combination with LC-MS and live cell imaging. 14F7 did not kill the HeLa cells, however, it caused altered protein expression (MS data are available
via
ProteomeXchange with identifier PXD024320). Several cytoskeletal and nucleic-acid binding proteins were found to be strongly down-regulated in response to antibody treatment, suggesting how 14F7 may induce membrane lesions in cells that contain higher amounts of NeuGc GM3. The altered expression profile identified in this study thus contributes to an improved understanding of the unusual killing mechanism of 14F7.
•Neu5Gc in skeletal muscle was highest in goat, but absent in kangaroo and dog.•Neu5Gc in organ meat was ~2–54% higher than muscle; >75% was in the conjugated form.•Bovine organs contained more ...Neu5Gc, but it was absent in female deer organs.•Nine commercial primal meat cuts in sheep contained similar levels of Neu5Gc.•Our study provides guidelines which meats to avoid sustaining a healthy diet.
Red meat-derived sialic acid (Sia), N-glycolylneuraminic acid (Neu5Gc), promotes the risk of carcinoma and inflammation. Expression in skeletal muscle and organs across animal species remains unknown. We measured Neu5Gc in skeletal muscle and organ tissues from nine species using UHPLC and found that: (1) neu5Gc concentration in skeletal muscle was highest in goats (166 ± 48.7 µg/g protein), followed by cattle, pig, sheep, horse, cat and deer: >75% was conjugated. No Neu5Gc was detected in kangaroo and dog muscles; (2) total Neu5Gc in organ meats was generally about 2–54% higher than in muscle. Surprisingly Neu5Gc was absent in seven organs of female deer; (3) nine commercial ovine meat cuts contained similar Neu5Gc levels. Thus, red meat Neu5Gc concentration is tissue and species-specific and absent in muscle and organ tissue of some species. Our study provides guidelines for animal meat preferences for consumers and sheds light on the functionality of Neu5Gc.
Human natural antibodies to mammalian carbohydrate antigens (MCA) bind to carbohydrate-antigens synthesized in other mammalian species and protect against zoonotic virus infections. Three such ...anti-MCA antibodies are: (1) anti-Gal, also produced in Old-World monkeys and apes, binds to α-gal epitopes synthesized in non-primate mammals, lemurs, and New-World monkeys; (2) anti-Neu5Gc binds to Neu5Gc (N-glycolyl-neuraminic acid) synthesized in apes, Old-World monkeys, and many non-primate mammals; and (3) anti-Forssman binds to Forssman-antigen synthesized in various mammals. Anti-viral protection by anti-MCA antibodies is feasible because carbohydrate chains of virus envelopes are synthesized by host glycosylation machinery and thus are similar to those of their mammalian hosts. Analysis of MCA glycosyltransferase genes suggests that anti-Gal appeared in ancestral Old-World primates following catastrophic selection processes in which parental populations synthesizing α-gal epitopes were eliminated in enveloped virus epidemics. However, few mutated offspring in which the α1,3galactosyltransferase gene was accidentally inactivated produced natural anti-Gal that destroyed viruses presenting α-gal epitopes, thereby preventing extinction of mutated offspring. Similarly, few mutated hominin offspring that ceased to synthesize Neu5Gc produced anti-Neu5Gc, which destroyed viruses presenting Neu5Gc synthesized in parental hominin populations. A present-day example for few humans having mutations that prevent synthesis of a common carbohydrate antigen (produced in >99.99% of humans) is blood-group Bombay individuals with mutations inactivating H-transferase; thus, they cannot synthesize blood-group O (H-antigen) but produce anti-H antibody. Anti-MCA antibodies prevented past extinctions mediated by enveloped virus epidemics, presently protect against zoonotic-viruses, and may protect in future epidemics. Travelers to regions with endemic zoonotic viruses may benefit from vaccinations elevating protective anti-MCA antibody titers.
-glycolylneuraminic acid (Neu5Gc), and its precursor
-acetylneuraminic acid (Neu5Ac), commonly referred to as sialic acids, are two of the most common glycans found in mammals. Humans carry a ...mutation in the enzyme that converts Neu5Ac into Neu5Gc, and as such, expression of Neu5Ac can be thought of as a 'human specific' trait. Bacteria can utilize sialic acids as a carbon and energy source and have evolved multiple ways to take up sialic acids. In order to generate free sialic acid, many bacteria produce sialidases that cleave sialic acid residues from complex glycan structures. In addition, sialidases allow escape from innate immune mechanisms, and can synergize with other virulence factors such as toxins. Human-adapted pathogens have evolved a preference for Neu5Ac, with many bacterial adhesins, and major classes of toxin, specifically recognizing Neu5Ac containing glycans as receptors. The preference of human-adapted pathogens for Neu5Ac also occurs during biosynthesis of surface structures such as lipo-oligosaccharide (LOS), lipo-polysaccharide (LPS) and polysaccharide capsules, subverting the human host immune system by mimicking the host. This review aims to provide an update on the advances made in understanding the role of sialic acid in bacteria-host interactions made in the last 5-10 years, and put these findings into context by highlighting key historical discoveries. We provide a particular focus on 'molecular mimicry' and incorporation of sialic acid onto the bacterial outer-surface, and the role of sialic acid as a receptor for bacterial adhesins and toxins.
•Neu5Gc is a dietary immunogenic sugar in humans.•It is expressed at low level on human tissues, accumulating mostly on carcinoma.•All humans have circulating anti-Neu5Gc antibodies.•Co-existence of ...Neu5Gc/anti-Neu5Gc spurs chronic inflammation mediated disease.•Concurrently, anti-Neu5Gc antibodies hold potential for novel targeted therapy.
Glycans at the forefront of cells facilitate immune recognition processes. Cancer cells commonly present altered cell surface glycosylation, especially manifested in the expression of sialic acid at the termini of glycolipids and glycoproteins. Although tumor-associated carbohydrate antigens (TACAs) result in expression of altered-self, most such carbohydrates do not elicit strong humoral responses. Various strategies had been devised to elicit increased immunogenicity of such TACA aiming for potent immunotherapeutic antibodies or cancer vaccines. However some carbohydrates are immunogenic in humans and hold potential for novel glycotherapies. N-Glycolylneuraminic acid (Neu5Gc) is a foreign immunogenic sugar in humans originating from the diet (e.g., red meat) and subsequently expressed on the cell surface, especially accumulating on carcinoma. Consequently, the human immune system detects this non-self carbohydrate generating a broad anti-Neu5Gc antibody response. The co-existence of Neu5Gc/anti-Neu5Gc within humans spurs chronic inflammation mediated disease, including cancer. Concurrently, anti-Neu5Gc antibodies hold potential for novel targeted therapy. αGal is another foreign immunogenic carbohydrate antigen in humans and all humans have circulating anti-Gal antibodies. This review aims to describe the immunogenicity of Neu5Gc and its implications for human diseases, highlighting differences and similarities with αGal and its potential for novel targeted theranostics.
N-glycolylneuraminic acid (Neu5Gc)-containing glycans are a prominent form of aberrant glycosylation found in human tumor cells and have been proposed as cancer biomarkers. The B subunit of the ...subtilase cytotoxin (SubB) produced by Shiga toxigenic Escherichia coli recognises Neu5Gc containing glycans. We have previously engineered this lectin, SubB2M, for greater specificity and enhanced recognition of Neu5Gc-containing glycans. Here we further explore the utility of SubB2M to detect Neu5Gc tumor biomarkers in sera from patients with ovarian cancer. Using surface plasmon resonance (SPR) we show that SubB2M can detect the established ovarian cancer biomarker, CA125, in a highly sensitive and specific fashion in the context of human serum. These studies established conditions for screening serum samples from patients with ovarian cancer for Neu5Gc glycans. We found that serum from patients with all stages of ovarian cancer had significantly elevated mean levels of Neu5Gc glycans compared to normal controls. Serum from patients with late stage disease (stages IIIC, IV) had uniformly elevated levels of Neu5Gc glycans. Detection of Neu5Gc-glycans using SubB2M has the potential to be used as a diagnostic ovarian cancer biomarker, as well as a tool for monitoring treatment and disease progression in late stage disease.
•N-glycolylneuraminic acid (Neu5Gc) has been proposed as cancer biomarker.•A Neu5Gc-specific lectin, SubB2M, was used to detect Neu5Gc in patient sera.•Serum from ovarian cancer patients has significantly elevated Neu5Gc levels.•Late stage ovarian cancer patients have uniformly elevated levels of Neu5Gc-glycans.
Gut microbiome composition depends heavily upon diet and has strong ties to human health. Dietary carbohydrates shape the gut microbiome by providing a potent nutrient source for particular microbes. ...This review explores how dietary carbohydrates in general, including individual monosaccharides and complex polysaccharides, influence the gut microbiome with subsequent effects on host health and disease. In particular, the effects of sialic acids, a prominent and influential class of monosaccharides, are discussed. Complex plant carbohydrates, such as dietary fiber, generally promote microbial production of compounds beneficial to the host while preventing degradation of host carbohydrates from colonic mucus. In contrast, simple and easily digestible sugars such as glucose are often associated with adverse effects on health and the microbiome. The monosaccharide class of sialic acids exerts a powerful but nuanced effect on gut microbiota. Sialic acid consumption (in monosaccharide form, or as part of human milk oligosaccharides or certain animal-based foods) drives the growth of organisms with sialic acid metabolism capabilities. Minor chemical modifications of Neu5Ac, the most common form of sialic acid, can alter these effects. All aspects of carbohydrate composition are therefore relevant to consider when designing dietary therapeutic strategies to alter the gut microbiome.
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