N-glycolneuraminic acid (Neu5Gc) is a sialic acid mainly found in mammalian species. It is absent in humans. This is due to an irreversible mutation of the CMP-Neu5Ac hydroxylase (CMAH) enzyme in humans, rendering them unable to synthesize Neu5Gc. The human body identifies Neu5Gc as “foreign”; and anti-Neu5Gc antibodies are produced by the human body in response to any metabolically incorporated, diet-derived Neu5Gc, as found in ingested red meats and dairy products.Varying quantities of Neu5Gc is found in some approved biotherapeutics used for the treatment of numerous medical conditions. This leads to the debate of potential risks and/or benefits of Neu5Gc in humans. The effects of the interaction between anti-Neu5Gc antibodies and antigenic Neu5Gc-containing biotherapeutics in humans are largely unknown and there are many discrepancies in terms of scientific evidence. This article reviews and discusses the current knowledge in the understanding of Neu5Gc in the human body and its potential significance.

Keywords: N-glycolneuraminic acid, Neu5Gc, anti-Neu5Gc antibodies,sialic acid,CMAH

Sialic acids are a family of nine-carbon sugar acids. They are monosaccharides  displayed on the non-reducing termini of cell surface glycans in all vertebrates.¹ These acidic 9-carbon saccharides cover the ends of millions of glycan chains per cell. In mammalian species and other vertebrates, the sialic acids N-acetylneuraminic acid (Neu5Ac) and its derivative N-glycolylneuraminic acid (Neu5Gc) are the two most common forms, each a family of molecules with various modifications of the canonical, 9-carbon monosaccharide. They are hydrophilic, with a negative charge; and are involved in many biological, molecular and cellular interactions.^2,3 Catalysis of the Neu5Ac precursor by the enzyme CMP-Neu5Ac hydroxylase (CMAH) produces Neu5Gc by the addition of a hydroxyl group.⁴ However, the human species is genetically unable to produce Neu5Gc, due to the irreversibly mutated CMAH gene.⁵ This absence of Neu5Gc in humans possibly affects the recognition process of endogenous and exogenous sialic-acid binding lectins.⁶

Besides the human species, absence of the CMAH homologs has been reported in several species of birds, reptiles, New World monkeys, and sperm whales. These species are unable to produce Neu5Gc.⁷ Nonhuman Neu5Gc was first discovered in humans when Hanganutziu and Deicher independently observed heterophilic antibody in sera of patients who had received therapeutic injections of horse serum-based anti-toxoid.^8,9 These nonhuman Neu5Gc have since been referred to as Hanganutziu–Deicher (H–D) antibodies or serum sickness antibodies. When Higashi et al. (1977) and Merrick et al. (1978) studied the characterisation of the antigenic determinants of the H-D antibodies, it was found that some of the major epitopes recognized were gangliosides containing Neu5Gc.^10,11

In spite of the inability of the human species to produce Neu5Gc, accumulations of xenoglycan Neu5Gc have been reported in humans; the amounts of Neu5Gc are highly variable between individuals, and between tissues within individuals. Neu5Gc has been detected in smaller quantities on certain human cell types, in particular epithelial and endothelial cells.⁵ Tangvoranuntakul et al. (2003) had reported that small quantities of Neu5Gc were being metabolically incorporated into newly synthesized glycoproteins in humans after the ingestion of porcine submaxillary mucin sialic acids (95% of Neu5Gc, 5% Neu5Ac).¹² How ingested Neu5Gc becomes incorporated into the human body is still unclear. It seems that Neu5Gc is first converted to GalNGc and then incorporated into the glycosaminoglycan chondroitin sulfate, an important component of extracellular matrices and skeletal bone.¹³

Normal healthy humans would have circulating antibodies against Neu5Gc, but the levels of IgMs and IgGs anti-Neu5Gc antibodies may be variable.^12,14 Unfortunately, since anti-Neu5Gc antibodies recognise multiple Neu5Gc epitopes, it is difficult to determine the overall level of anti-Neu5Gc antibodies in individual human samples.¹⁵ It is believed that anti-Neu5Gc antibodies begin to be generated in humans during infancy, specifically in relation to weaning and dietary exposure.¹⁶

Many of the red meats we consume contain high amounts of Neu5Gc.⁴Among the red meats, the highest levels of Neu5Gc are found in beef, followed by pork and lamb.¹⁷ Minute traces of Neu5Gc, less than 0.7% of total sialic acid fraction, are also found in chicken.¹⁸ Long term consumption and high intake of red meats have been associated with various types of cancer,¹⁹ and anti-Neu5Gc antibodies are alleged to be involved in these and many other diseases. However, none of these mechanisms is conclusively proven. This article attempts to assess the possible deleterious and/or therapeutic roles of this xeno-sialic acid Neu5Gc and its associated antibodies.

Cancer progression and anti-tumour activities of Neu5Gc

The human body is unable to produce Neu5Gc, and regard these as foreign. When exposed to Neu5Gc molecules, humoral immunity is activated, and this initiates the production of anti-Neu5Gc antibodies.^16,20 Studies have shown that the metabolic incorporation of dietary Neu5Gc into human tissues as xeno-autoantigens, induces xenosialitis, caused by the reaction with circulating anti-Neu5Gc antibodies in human tissues.^14,21 This inflammatory process, xenosialitis, has been associated with cancer progression, cardiovascular diseases and autoimmune diseases.^5,14

There are concerns that the incorporation of dietary Neu5Gc could elicit an immune response which may result in chronic inflammation and cancer.²² However, a large cohort study of over 200,000 kidney transplant patients including 522 patients with colon cancer did not support the hypothesis that long term over-exposure to anti-Neu5Gc antibodies triggers malignancies in the colon.²³ In the study, there was no estimation regarding red meat intake, as patients with renal failure are typically advised to reduce meat intake. In addition, some of these patients were also under immunosuppression, which would alter the outcomes. Pearce et al. (2014) have shown that anti-Neu5Gc antibodies promote liver tumour progression by enhancing inflammation in Neu5Gc-deficient CMAHnull mice.²⁴ The same group of researchers also reported that tumour growth was stimulated at low anti-Neu5Gc antibody concentrations; however, tumour growth was inhibited at higher anti-Neu5Gc concentrations in Neu5Gc deficient mice.²⁴ Rodríguez‐Zhurbenko et al. (2013) found that low levels of anti-Neu5Gc antibodies are found in non-small cell lung cancer patients, while a higher level of the antibodies was found in healthy volunteers, suggesting that anti-Neu5Gc antibodies might possess antitumor immune surveillance function.²⁵The study led by De villliers and ZurHausen’s group demonstrate another potential carcinogenic mechanism arising from red meat that revealed by the isolation of a number of small DNAs derived from specific plasmids of Acinetobacter bacteria from commercially available cow milk.^26–28 Possible host infections from replicating plasmids are potential risk factors for human colon and breast cancers,²⁹ that incorporated Neu5Gc from dietary sources could present receptors for the viruses, and antibodies against these viral proteins orchestrate together Neu5Gc-induced xenosialitis.Further research is warranted to further investigate the exact mechanism of how Neu5Gc promotes or inhibits the progression of tumour.

Significance of elevated anti-Neu5Gc antibodies in allergies and asthma

Frei et al. (2018) investigated the role of exposure to Neu5Gc in children of farmers and non-farmers. The findings were that higher levels of anti-Neu5Gc antibodies were detected in these farmers' children when compared to nonfarmers’ children. Their results showed that the higher anti-Neu5Gc IgG levels were correlated with less asthma and wheezing.³⁰ It has been shown that mice sensitized with ovalbumin and house dust mites resulted in reduced airway hyperresponsiveness and reduced inflammatory cell recruitment to the lungs.³⁰ This suggests that the exposure to Neu5Gc in children living in rural areas induced an elevated anti-Neu5Gc antibodies, which reduces their response to allergens, thus providing a reduced incidence of allergies.²⁰

Properties of anti-Neu5Gc antibodies, and its association to red meats

The presence of anti-Neu5Gc antibodies has been reported to protect against reactive airways, inflammation, and colitis.³⁰However, there is inadequate evidence to support the association of red meats consumption with increased inflammations. Hodgson et al. (2007) revealed that the intake of red meats did not increase oxidative stress and inflammation in humans.³¹ In contrast, increased lean meats consumption at the expense of partial carbohydrate-rich food, leads to a reduction of some inflammatory markers.³¹

Causal link between serum sickness and anti-Neu5Gc antibodies

Couvrat-Desvergnes et al. (2015) suggested a causal link between anti-Neu5Gc antibody levels and long-term graft survival, although this causal link was not confirmed.³² The author noted that serum sickness disease is a major contributing factor to late graft loss, and that the anti-Neu5Gc antibodies levels are increased in patients with serum sickness disease. The author made no mention of the dietary habits of these patients. Any plausible link between anti-Neu5Gc antibody levels and long-term graft survival cannot be confirmed from this clinical study.

Neu5Gc and xeno-derived biotherapeutics

In recent decades, pharmaceutical products that have animal-based and animal-derived components, have significantly diversified. Many different animal sources were investigated as technology advances and purification techniques progress; this ranged from pufferfish in the 1960s to, most recently, genetically engineered animals.³³ These biotherapeutic products involve non-human mammalian cells (such as cow, horse, hamsters and pig); which contain Neu5Gc.¹² The levels of Neu5Gc in these biotherapeutics vary according to the production systems.³⁴ Examples of some of these pharmaceutical products that incorporate animal-derived components are as shown in Table 1.^33,35,36

Neu5Gc is found in many biotherapeutic agents such as Alemtuzumab (Campath®, Mabcampath®), Bevacizumab (Avastin®), Cetuximab (Erbitux®), Daclizumab (Zenapax®), Erythropoietin (Procrit®), Rituximab (Rituxan®, Mabthera®) and Trastuzumab (Herceptin®). Among these therapeutic agents, Cetuximab has the highest Neu5Gc content (1.77 mol Neu5Gc/mol antibody), followed by Daclizumab (0.081 mol Neu5Gc/mol antibody) and Erythropoietin (0.014 mol Neu5Gc/mol antibody).³⁷ Cetuximab has been approved by the US FDA on February 2004 and is used to treat patients with advanced colorectal cancer. In in-vivo studies using mouse models, Cetuximab’s efficiency was noted to be reduced by Neu5Gc/anti-Neu5Gc antibodies.³⁷ However, there is no clear evidence of their effects on Cetuximab’s efficiency in humans.

Breakthrough advances in medical science have been in the fields of transplantations, stem cells therapy, and use of xeno-derived biotherapeutics. Porcine pancreatic islet transplantation has been used to successfully treat type 1 diabetes patients, even without the use of any immunosuppressant.³⁸ Xeno-derived fetal stem cells, that have low expression of MHC class I, and no expression of MHC class II, do not evoke an immune response during transplantation when compared to their adult counterpart.³⁹ This may be link to the absence, low levels, or lack of expression of Neu5Gc. Research has shown that the absence or decreased number of dendritic cells linked to a reduced immunogenicity of the progenitor stem cell makes these cross-species transplantations less susceptible to rejection and are better tolerated.^40–42 The significance of the roles and effects of Neu5Gc in these transplanted xeno-derived biotherapeutics are yet to be determined.

Neu5Gc is a ‘foreign entity’ to the human body. It is antigenic and provokes the production of anti-Neu5Gc antibodies. Its entry into the body is via ingestion of animal proteins, via xeno-derived pharmaceuticals, via xeno-transplantations of tissues and organs, and via injections of xeno-derived progenitor stem cells and peptides. There are many discrepancies in terms of scientific evidences to support or refute the potential benefits or harm of Neu5Gcand anti-Neu5Gc antibodies in humans. Current literature and research suggest that Neu5Gc and anti-Neu5Gc antibodies have shown both a therapeutic and deleterious role. Further studies in the field of Neu5Gc will allow us to gain a better understanding of the roles and mechanisms of actions of this unique sialic acid. Studies on the anti-Neu5Gc antibodies will allow an understanding of the possible modulating effects to the immune system of the human body. For instance, the details of particular Neu5Gc metabolism processes might provide a variety of potential targets for new drug development as clinicians use a huge range of products derived from non-human mammalian cells and organs and it is very important which of them produce significant amounts of antigen-antibodies reactions especially to those in critical condition with compromised immune system.

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