Influenza D virus utilizes both 9-O-acetylated N-acetylneuraminic and 9-O-acetylated N-glycolylneuraminic acids as functional entry receptors.

Influenza D virus (IDV) utilizes bovines as a primary reservoir with periodical spillover to other hosts. We have previously demonstrated that IDV binds both 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac). Bovines produce both Neu5,9Ac2 and Neu5Gc9Ac, while humans are genetically unable to synthesize Neu5Gc9Ac. 9-O-Acetylation of sialic acids is catalyzed by CASD1 via a covalent acetyl-enzyme intermediate. To characterize the role of Neu5,9Ac2 and Neu5Gc9Ac in IDV infection and determine which form of 9-O-acetylated sialic acids drives IDV entry, we took advantage of a CASD1 knockout (KO) MDCK cell line and carried out feeding experiments using synthetic 9-O-acetyl sialic acids in combination with the single-round and multi-round IDV infection assays. The data from our studies show that (i) CASD1 KO cells are resistant to IDV infection and lack of IDV binding to the cell surface is responsible for the failure of IDV replication; (ii) feeding CASD1 KO cells with Neu5,9Ac2 or Neu5Gc9Ac resulted in a dose-dependent rescue of IDV infectivity; and (iii) diverse IDVs replicated robustly in CASD1 KO cells fed with either Neu5,9Ac2 or Neu5Gc9Ac at a level similar to that in wild-type cells with a functional CASD1. These data demonstrate that IDV can utilize Neu5,9Ac2- or non-human Neu5Gc9Ac-containing glycan receptor for infection. Our findings provide evidence that IDV has acquired the ability to infect and transmit among agricultural animals that are enriched in Neu5Gc9Ac, in addition to posing a zoonotic risk to humans expressing only Neu5,9Ac2.IMPORTANCEInfluenza D virus (IDV) has emerged as a multiple-species-infecting pathogen with bovines as a primary reservoir. Little is known about the functional receptor that drives IDV entry and promotes its cross-species spillover potential among different hosts. Here, we demonstrated that IDV binds exclusively to 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and non-human 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac) and utilizes both for entry and infection. This ability in effective engagement of both 9-O-acetylated sialic acids as functional receptors for infection provides an evolutionary advantage to IDV for expanding its host range. This finding also indicates that IDV has the potential to emerge in humans because Neu5,9Ac2 is ubiquitously expressed in human tissues, including lung. Thus, results of our study highlight a need for continued surveillance of IDV in humans, as well as for further investigation of its biology and cross-species transmission mechanism.

Single Particle Analysis of H3N2 Influenza Entry Differentiates the Impact of the Sialic Acids (Neu5Ac and Neu5Gc) on Virus Binding and Membrane Fusion.

Entry of influenza A viruses (IAVs) into host cells is initiated by binding to sialic acids (Sias), their primary host cell receptor, followed by endocytosis and membrane fusion to release the viral genome into the cytoplasm of the host cell. Host tropism is affected by these entry processes, with a primary factor being receptor specificity. Sias exist in several different chemical forms, including the hydroxylated N-glycolylneuraminic acid (Neu5Gc), which is found in many hosts; however, it has not been clear how modified Sias affect viral binding and entry. Neu5Gc is commonly found in many natural influenza hosts, including pigs and horses, but not in humans or ferrets. Here, we engineered HEK293 cells to express the hydoxylase gene (CMAH) that converts Neu5Ac to Neu5Gc, or knocked out the Sia-CMP transport gene (SLC35A1), resulting in cells that express 95% Neu5Gc or minimal level of Sias, respectively. H3N2 (X-31) showed significantly reduced infectivity in Neu5Gc-rich cells compared to wild-type HEK293 (>95% Neu5Ac). To determine the effects on binding and fusion, we generated supported lipid bilayers (SLBs) derived from the plasma membranes of these cells and carried out single particle microscopy. H3N2 (X-31) exhibited decreased binding to Neu5Gc-containing SLBs, but no significant difference in H3N2 (X-31)'s fusion kinetics to either SLB type, suggesting that reduced receptor binding does not affect subsequent membrane fusion. This finding suggests that for this virus to adapt to host cells rich in Neu5Gc, only receptor affinity changes are required without further adaptation of virus fusion machinery. IMPORTANCE Influenza A virus (IAV) infections continue to threaten human health, causing over 300,000 deaths yearly. IAV infection is initiated by the binding of influenza glycoprotein hemagglutinin (HA) to host cell sialic acids (Sias) and the subsequent viral-host membrane fusion. Generally, human IAVs preferentially bind to the Sia N-acetylneuraminic acid (Neu5Ac). Yet, other mammalian hosts, including pigs, express diverse nonhuman Sias, including N-glycolylneuraminic acid (Neu5Gc). The role of Neu5Gc in human IAV infections in those hosts is not well-understood, and the variant form may play a role in incidents of cross-species transmission and emergence of new epidemic variants. Therefore, it is important to investigate how human IAVs interact with Neu5Ac and Neu5Gc. Here, we use membrane platforms that mimic the host cell surface to examine receptor binding and membrane fusion events of human IAV H3N2. Our findings improve the understanding of viral entry mechanisms that can affect host tropism and virus evolution.

Sialic acid in human milk and infant formulas in China: concentration, distribution and type.

This study compared the concentrations, types and distributions of sialic acid (SA) in human milk at different stages of the postnatal period with those in a range of infant formulas. Breast milk from mothers of healthy, full-term and exclusively breastfed infants was collected on the 2nd (n 246), 7th (n 135), 30th (n 85) and 90th (n 48) day after birth. The SA profiles of human milk, including their distribution, were analysed and compared with twenty-four different infant formulas. Outcome of this observational study was the result of natural exposure. Only SA of type Neu5Ac was detected in human milk. Total SA concentrations were highest in colostrum and reduced significantly over the next 3 months. Approximately 68·7­76·1 % of all SA in human milk were bound to oligosaccharides. Two types of SA, Neu5Ac and Neu5Gc, have been detected in infant formulas. Most SA was present in infant formulas combined with protein. Breastfed infants could receive more SA than formula-fed infants with the same energy intake. Overall, human milk is a preferable source of SA than infant formulas in terms of total SA content, dynamics, distribution and type. These SA profiles in the natural state are worth to be considered by the production of formulas because they may have a great effect on infant nutrition and development.

Tick bite induced α-gal syndrome highlights anticancer effect of allergy.

Tick bite induced α-gal syndrome (AGS) following consumption of mammalian meat is a recently described intriguing disease occurring worldwide. Here we argue that AGS and delayed allergy in general is an adaptive defence method against cancer. Our hypothesis synthesizes two lines of supporting evidence. First, allergy has been shown to have direct anti-cancer effects with unknown mechanism. Second, eating processed meat was shown to be linked to developing cancer. Humans lost their genes encoding molecules α-gal 30 MYA and Neu5Gc 2 MYA, the latter co-occurring with the start of using fire. These molecules are acquired from external sources, as tick bite for α-gal and mammalian meat for Neu5Gc, the latter accumulating in tumors. The resulting specific delayed allergic response is a molecular adaptation to fight cancer. By further testing and applying our hypothesis, new avenues in cancer research and therapy will open that might save lives and decrease human suffering.

Characterization of an Aptamer Targeting Neu5Gc, as an Endogenous Pathogenic Factor Derived from Red Meat.

N-glycolylneuraminic acid (Neu5Gc), a sialic acid predominantly found in the non-neurohumoral fluids of hind-mouthed animals, is incapable of synthesizing Neu5Gc due to a deletion in the CMAH exon of the gene encoding human CMP-Neu5Gc hydroxylase. But consumption of animal-derived foods that contain Neu5Gc, such as red meat, can instigate an immune response in humans, as Neu5Gc is recognized as a foreign substance by the human immune system. This recognition leads to the production of anti-Neu5Gc antibodies, subsequently resulting in chronic inflammation. When Neu5Gc is consumed excessively or frequently, it may contribute to the development of heart disease and cancer. This makes Neu5Gc, an endogenous pathogenic factor derived from red meat, a new hot topic in red meat safety research. In this study, aptamers obtained by the magnetic bead SELEX technique were subjected to homology and secondary structure prediction analysis as well as affinity determination. The result indicated that the aptamer 2B.N2A9 exhibited a robust binding affinity, with an affinity constant (Ka) of 1.87 × 108 L/mol. This aptamer demonstrated optimal binding specificity within a pH range of 5.4 to 7.4. Molecular docking analysis further revealed that aptamer 2B.N2A9 formed stable binding interactions with the target Neu5Gc at specific sites, namely G-14, C-15, G-13, G-58, G-60, and C-59. An Enzyme-Linked Oligonucleotide Sorbent Assay (ELOSA) methodology was established to detect the endogenous pathogenic factor Neu5Gc present in red meat. This method demonstrated a limit of detection (LOD) of 0.71 ng/mL, along with an average recovery rate of 92.23%. The aptamer obtained in this study exhibited favorable binding properties to Neu5Gc. The assay was relatively convenient and demonstrated good sensitivity. Further investigation into the distribution of Neu5Gc in various red meats is of public health significance and scientific potential. A practical detection method should be provided to guide red meat diets and ensure the nutrition and safety of meat products.

N-glycolylneuraminic acid in red meat and processed meat is a health concern: A review on the formation, health risk, and reduction.

One of the most consistent epidemiological associations between diet and human disease risk is the impact of consuming red meat and processed meat products. In recent years, the health concerns surrounding red meat and processed meat have gained worldwide attention. The fact that humans have lost the ability to synthesize N-glycolylneuraminic acid (Neu5Gc) makes red meat and processed meat products the most important source of exogenous Neu5Gc for humans. As our research of Neu5Gc has increased, it has been discovered that Neu5Gc in red meat and processed meat is a key factor in many major diseases. Given the objective evidence of the harmful risk caused by Neu5Gc in red meat and processed meat to human health, there is a need for heightened attention in the field of food. This updated review has several Neu5Gc aspects given including biosynthetic pathway of Neu5Gc and its accumulation in the human body, the distribution of Neu5Gc in food, the methods for detecting Neu5Gc, and most importantly, a systematic review of the existing methods for reducing the content of Neu5Gc in red meat and processed meat. It also provides some insights into the current status and future directions in this area.

Paleo-immunology of human anti-carbohydrate antibodies preventing primate extinctions.

Two human natural anti-carbohydrate antibodies appeared in critical evolutionary events that brought primates and hominins to brink of extinction. The first is the anti-Gal antibody, produced in Old-World monkeys (OWM), apes and humans. It binds the carbohydrate-antigen 'α-gal epitope' (Galα1-3Galß1-4GlcNAc-R) on carbohydrate-chains (glycans) synthesized by non-primate mammals, lemurs and New-World monkeys (NWM). The second is anti-N-glycolylneuraminic-acid (anti-Neu5Gc) antibody binding Neu5Gc on glycans synthesized by OWM, apes and most non-primate mammals. Ancestral OWM and apes synthesized α-gal epitopes and were eliminated ~20-30 million-years-ago (mya). Only few accidentally mutated offspring lacking α-gal epitopes, produced anti-Gal and survived. Hominin-populations living ~3 mya synthesized Neu5Gc and were eliminated, but few mutated offspring that accidently lost their ability to synthesize Neu5Gc, produced natural anti-Neu5Gc antibody. These hominins survived and ultimately evolved into present-day humans. It is argued that these two near-extinction events were likely to be the result of epidemics caused by highly virulent and lethal enveloped viruses that killed parental-populations. These viruses presented α-gal epitopes or Neu5Gc synthesized in host-cells of the parental-populations. Mutated offspring survived the epidemics because they were protected from the lethal virus by the natural anti-Gal or anti-Neu5Gc antibodies they produced due to loss of immune-tolerance to α-gal epitopes or to Neu5Gc, respectively.

Serum Neu5Gc biomarkers are elevated in primary cutaneous melanoma.

Cutaneous melanoma is one of the most aggressive and deadly types of skin cancer and rates of disease are continuing to increase worldwide. Currently, no serum biomarkers exist for the early detection of cutaneous melanoma. Normal human cells cannot make the sialic acid sugar, Neu5Gc, yet human tumor cells express Neu5Gc and Neu5Gc-containing glycoconjugates have been proposed as tumor biomarkers. We engineered a Neu5Gc-specific lectin based on the pentameric B-subunit of the Shiga toxigenic Escherichia coli subtilase cytotoxin, termed SubB2M. We have detected elevated Neu5Gc-containing biomarkers in the sera of ovarian and breast cancer patients in a highly sensitive surface plasmon resonance (SPR)-based assay using our SubB2M lectin. Here, we used the SubB2M-SPR assay to investigate Neu5Gc-containing glycoconjugates in the serum of cutaneous melanoma patients. We found elevated total serum Neu5Gc levels in primary (n = 24) and metastatic (n = 38) patients compared to cancer-free controls (n = 34). Serum Neu5Gc levels detected with SubB2M can distinguish cutaneous melanoma patients from cancer-free controls with high sensitivity and specificity as determined by ROC curve analysis. These data indicate that serum Neu5Gc-containing glycoconjugates are a novel class of biomarkers for cutaneous melanoma, particularly for primary melanoma, and have the potential to contribute to the early diagnosis of this disease.

Lamprey immunity protein enables detection for bladder cancer through recognizing N-hydroxyacetylneuraminic acid (Neu5Gc)-modified as a diagnostic marker and exploration of its production mechanism.

Previous studies have demonstrated that Neu5Gc is highly expressed in breast, ovarian, prostate, colon and lung cancers, but not in normal human cells. The presence of Neu5Gc is important for prognosis and is associated with aggressiveness, metastasis, and tumor grade. However, increased Neu5Gc in bladder cancer remains unclear. LIP from lamprey binds the carbohydrate receptor of N-glycolylneuraminic acid (Neu5Gc). The combination of Neu5Gc and LIP suggested that it might be used as a diagnostic tool for the detection of Neu5Gc tumor antigen. Here, the classical animal model of bladder cancer was successfully induced by MNU bladder perfusion. The ELISA results showed that the expression level of Neu5Gc in the urine of normal rats was 94.96 ± 21.01ng/mg, and that of bladder cancer rats was 158.28 ± 34.86 ng/mg. In addition, the results of SNA and LIP immunohistochemistry demonstrated the high expression of Neu5Gc in bladder cancer. After the addition of Neu5Gc to BIU-87 and SV-HUC-1 cells, transcriptomic sequencing and real-time quantitative PCR analysis demonstrated that the gene expression of Neu5Gc synthesis pathway was significantly increased. These data suggest that LIP provides a new tool for the detection of biological samples, especially urine from patients with bladder cancer or suspected cancer, and that revealing the mechanism of abnormal glycosylation can provide theoretical basis for clinical studies.

How bacteria utilize sialic acid during interactions with the host: snip, snatch, dispatch, match and attach.

N -glycolylneuraminic acid (Neu5Gc), and its precursor N-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.

N-glycolylneuraminic acid serum biomarker levels are elevated in breast cancer patients at all stages of disease.

BACKGROUND: Normal human tissues do not express glycans terminating with the sialic acid N-glycolylneuraminic acid (Neu5Gc), yet Neu5Gc-containing glycans have been consistently found in human tumor tissues, cells and secretions and have been proposed as a cancer biomarker. We engineered a Neu5Gc-specific lectin called SubB2M, and previously reported elevated Neu5Gc biomarkers in serum from ovarian cancer patients using a Surface Plasmon Resonance (SPR)-based assay. Here we report an optimized SubB2M SPR-based assay and use this new assay to analyse sera from breast cancer patients for Neu5Gc levels. METHODS: To enhance specificity of our SPR-based assay, we included a non-sialic acid binding version of SubB, SubBA12, to control for any non-specific binding to SubB2M, which improved discrimination of cancer-free controls from early-stage ovarian cancer. We analysed 96 serum samples from breast cancer patients at all stages of disease compared to 22 cancer-free controls using our optimized SubB2M-A12-SPR assay. We also analysed a collection of serum samples collected at 6 monthly intervals from breast cancer patients at high risk for disease recurrence or spread. RESULTS: Analysis of sera from breast cancer cases revealed significantly elevated levels of Neu5Gc biomarkers at all stages of breast cancer. We show that Neu5Gc serum biomarker levels can discriminate breast cancer patients from cancer-free individuals with 98.96% sensitivity and 100% specificity. Analysis of serum collected prospectively, post-diagnosis, from breast cancer patients at high risk for disease recurrence showed a trend for a decrease in Neu5Gc levels immediately following treatment for those in remission. CONCLUSIONS: Neu5Gc serum biomarkers are a promising new tool for early detection and disease monitoring for breast cancer that may complement current imaging- and biopsy-based approaches.

Implication of N-glycolylneuraminic acid in regulation of cell adhesiveness of C2C12 myoblast cells during differentiation into myotube cells.

A transition of sialic acid (Sia) species on GM3 ganglioside from N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc) takes place in mouse C2C12 myoblast cells during their differentiation into myotube cells. However, the meaning of this Sia transition remains unclear. This study thus aims to gain a functional insight into this phenomenon. The following lines of evidence show that the increased de novo synthesis of Neu5Gc residues in differentiating myoblast cells promotes adhesiveness of the cells, which is beneficial for promotion of differentiation. First, the Sia transition occurred even in the C2C12 cells cultured in serum-free medium, indicating that it happens through de novo synthesis of Neu5Gc. Second, GM3(Neu5Gc) was localized in myoblast cells, but not in myotube cells, and related to expression of the CMP-Neu5Ac hydroxylase (CMAH) gene. Notably, expression of CMAH precedes myotube formation not only in differentiating C2C12 cells, but also in mouse developing embryos. Since the myoblast cells were attached on the dish surface more strongly than the myotube cells, expression of GM3(Neu5Gc) may be related to the surface attachment of the myoblast cells. Third, exogenous Neu5Gc, but not Neu5Ac, promoted differentiation of C2C12 cells, thus increasing the number of cells committed to fuse with each other. Fourth, the CMAH-transfected C2C12 cells were attached on the gelatin-coated surface much more rapidly than the mock-cells, suggesting that the expression of CMAH promotes cell adhesiveness through the expression of Neu5Gc.

Knock-out of N-glycolylneuraminic acid attenuates antibody-mediated rejection in xenogenically perfused porcine lungs.

BACKGROUND: Antibody-mediated rejection has long been known to be one of the major organ failure mechanisms in xenotransplantation. In addition to the porcine α1,3-galactose (α1,3Gal) epitope, N-Glycolylneuraminic acid (Neu5Gc), a sialic acid, has been identified as an important porcine antigen against which most humans have pre-formed antibodies. Here we evaluate GalTKO.hCD46 lungs with an additional cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene knock-out (Neu5GcKO) in a xenogeneic ex vivo perfusion model METHODS: Eleven GalTKO.hCD46.Neu5GcKO pig lungs were perfused for up to 6 h with fresh heparinized human blood. Six of them were treated with histamine (H) blocker famotidine and 1-thromboxane synthase inhibitor Benzylimidazole (BIA) and five were left untreated. GalTKO.hCD46 lungs without Neu5GcKO (n = 18: eight untreated and 10 BIA+H treated) served as a reference. Functional parameters, blood, and tissue samples were collected at pre-defined time points throughout the perfusion RESULTS: All but one Neu5GcKO organs maintained adequate blood oxygenation and "survived" until elective termination at 6 h whereas two reference lungs failed before elective termination at 4 h. Human anti-Neu5Gc antibody serum levels decreased during the perfusion of GalTKO.hCD46 lungs by flow cytometry (∼40% IgM, 60% IgG), whereas antibody levels in Neu5GcKO lung perfusions did not fall (IgM p = .007; IgG p < .001). Thromboxane elaboration, thrombin generation, and histamine levels were significantly reduced with Neu5GcKO lungs compared to reference in the untreated groups (p = .007, .005, and .037, respectively); treatment with BIA+H masked these changes. Activation of platelets, measured as CD62P expression on circulating platelets, was lower in Neu5GcKO experiments compared to reference lungs (p = .023), whereas complement activation (as C3a rise in plasma) was not altered. MCP-1 and lactotransferin level elevations were blunted in Neu5GcKO lung perfusions (p = .007 and .032, respectively). Pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in untreated GalTKO.hCD46.Neu5GcKO lungs in comparison to the untreated GalTKO.hCD46 lungs (p = .003) CONCLUSION: Additional Neu5GcKO in GalTKO.hCD46 lungs significantly reduces parameters associated with antibody-mediated inflammation and activation of the coagulation cascade. Knock-out of the Neu5Gc sialic acid should be beneficial to reduce innate immune antigenicity of porcine lungs in future human recipients.

Human species-specific loss of CMP-N-acetylneuraminic acid hydroxylase enhances atherosclerosis via intrinsic and extrinsic mechanisms.

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.

Determination of N-acetylneuraminic and N-glycolylneuraminic acids in unprocessed milk of four cattle breeds.

This research communication reports concentrations of two sialic acids (SA), N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic (Neu5Gc), in fresh milk from different cow breeds throughout lactation. According to published studies, the two SA types found in animal-derived products have diverse and conflicting effects on human health, but SA content is not routinely analysed in individual milk cows samples. We measured the content of Neu5Ac and Neu5Gc in milk from Holstein Friesian (HO), Simmental (SM), Simmental × Holstein crossbred (SM×HO), and Podolica (POD) cows at 60 and 120 d following calving. HO, SM and SM×HO were reared in an intensive production while POD were raised in an extensive system. Results showed that total Neu5Ac was overall thirty times more abundant than Neu5Gc, and their concentrations were higher at 120 d than at 60 d (P < 0.001). Neu5Gc values were greater in HO, SM, and SM × HO than in POD (P < 0.001), while HO had a higher Neu5Ac value than the other three breeds (P < 0.001). These findings shed light on the differences in SA content among cow breeds and lay the groundwork for future research to select animals that produce milk with desirable characteristics for human health.

Comparative Assessment of the Structural Features of Originator Recombinant Human Follitropin Alfa Versus Recombinant Human Follitropin Alfa Biosimilar Preparations Approved in Non-European Regions.

Although the full primary structures of the alfa and beta subunits of reference r-hFSH-alfa and its biosimilars are identical, cell context-dependent differences in the expressing cell lines and manufacturing process can lead to variations in glycosylation profiles. In the present study, we compared the structural features of reference r-hFSH-alfa with those of five biosimilar preparations approved in different global regions outside Europe (Primapur®, Jin Sai Heng®, Follitrope®, Folisurge®, and Corneumon®) with respect to glycosylation, macro- and microheterogeneity, and other post-translational modifications and higher order structure. The mean proportion of N-glycosylation-site occupancy was highest in reference r-hFSH-alfa, decreasing sequentially in Primapur, Jin Sai Heng, Corneumon, Follisurge and Follitrope, respectively. The level of antennarity showed slightly higher complexity in Corneumon, Primapur and Follitrope versus reference r-hFSH-alfa, whereas Jin Sai Heng and Folisurge were aligned with reference r-hFSH-alfa across all N-glycosylation sites. Sialylation level was higher in Corneumon and Follitrope, but small differences were detected in other biosimilar preparations compared with reference r-hFSH-alfa. Jin Sai Heng showed higher levels of N-glyconeuramic acid than the other preparations. Minor differences in oxidation levels were seen among the different products. Therefore, in summary, we identified var ious differences in N-glycosylation occupancy, antennarity, sialylation and oxidation between reference r-hFSH-alfa and the biosimilar preparations analyzed.

Identification of chicken-derived scFv against N-glycolylneuraminic acid retrieved from an immune library by phage display.

N- glycolylneuraminic acid (Neu5Gc) is a type of sialic acid, it can be synthesized by a range of mammals except chickens and healthy human. After entering human body, Neu5Gc in foods such as red meat and milk can cause chronic inflammation, thus promoting the development of cancer and related diseases. In this study, we identified a gene sequence of Neu5Gc-specific single-chain variable fragment (ScFv) by phage display from a primary chicken antibodies library. Then the gene sequence was used to express a 29 kDa anti-Neu5Gc ScFv protein as detection probe in competitive inhibition ELISA (IC-ELISA). The linear regression equation of the IC-ELISA was y = 23.12x+33.19 (R = 0.980), and the half-maximal inhibitory concentration (IC50) and the limit of detection (LOD) was 5.333 and 0.66 µg/mL. The mean recovery of the spiked samples was 83.04%, and the intra-assay and inter-assay coefficients of variation (CVs) were both 5.59%. The results suggested that the specific anti-Neu5Gc ScFv is a promising probe for the development of IC-ELISA and test strip in order to detect the presence of Neu5Gc in red meat, milk, and tumor tissues.

Histopathology of pig kidney grafts with/without expression of the carbohydrate Neu5Gc in immunosuppressed baboons.

INTRODUCTION: Pigs deficient in three glycosyltransferase enzymes (triple-knockout [TKO] pigs, that is, not expressing the three known carbohydrate xenoantigens) and expressing 'protective' human transgenes are considered a likely source of organs for transplantation into human recipients. Some human sera have no or minimal natural antibody binding to red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) from TKO pigs. However, all Old World monkeys exhibit natural antibody binding to TKO pig cells. The xenoantigen targets of Old World monkey natural antibodies are postulated to be carbohydrate moieties exposed when the expression of the carbohydrate N-glycolylneuraminic acid (Neu5Gc) is deleted. The aim of this study was to compare the survival in baboons and histopathology of renal grafts from pigs that either (a) expressed Neu5Gc (GTKO pigs; Group 1) or (b) did not express Neu5Gc (GTKO/CMAHKO [DKO] or TKO pigs; Group 2). METHODS: Life-supporting renal transplants were carried out using GTKO (n = 5) or DKO/TKO (n = 5) pig kidneys under an anti-CD40mAb-based immunosuppressive regimen. RESULTS: Group 1 baboons survived longer than Group 2 baboons (median 237 vs. 35 days; mean 196 vs. 57 days; p < 0.07) and exhibited histopathological features of antibody-mediated rejection in only two kidneys. Group 2 exhibited histopathological features of antibody-mediated rejection in all five grafts, with IgM and IgG binding to renal interstitial arteries and peritubular capillaries. Rejection-free survival was significantly longer in Group 1 (p < 0.05). CONCLUSIONS: The absence of expression of Neu5Gc on pig kidney grafts is associated with increased binding of baboon antibodies to pig endothelium and reduced graft survival.

In-depth characterization of non-human sialic acid (Neu5Gc) in human serum using label-free ZIC-HILIC/MRM-MS.

Sialic acid Neu5Gc, a non-human glycan, is recognized as a new harmful substance that can cause vascular disease and cancer. Humans are unable to synthesize Neu5Gc due to a genetic defect that converts Neu5Ac to Neu5Gc, but Neu5Gc is often observed in human biological samples. Therefore, the demand for accurately measuring the amount of Neu5Gc present in human blood or tissues is rapidly increasing, but there is still no method to reliably quantify trace amounts of a non-human sugar. In particular, selective isolation and detection of Neu5Gc from human serum is analytically challenging due to the presence of excess sialic acid Neu5Ac, which has physicochemical properties very similar to Neu5Gc. Herein, we developed the label-free approach based on ZIC-HILIC/MRM-MS that can enrich sialic acids released from human serum and simultaneously monitor Neu5Ac and Neu5Gc. The combination of complete separation of Neu5Gc from abundant Neu5Ac by hydrophilic and electrostatic interactions with selective monitoring of structure-specific cross-ring cleavage ions generated by negative CID-MS/MS was remarkably effective for quantification of Neu5Ac and Neu5Gc at the femtomole level. Indeed, we were able to successfully determine the absolute quantitation of Neu5Gc from 30 healthy donors in the range of 3.336 ± 1.252 pg/µL (mean ± SD), 10,000 times lower than Neu5Ac. In particular, analysis of sialic acids in protein-free serum revealed that both Neu5Ac and Neu5G are mostly bound to proteins and/or lipids, but not in free form. In addition, the correlation between expression level of Neu5Gc and biological factors such as BMI, age, and sex was investigated. This method can be widely used in studies requiring sialic acid-related measurements such as disease diagnosis or prediction of immunogenicity in biopharmaceuticals as it is both fast and highly sensitive.

Host Synthesized Carbohydrate Antigens on Viral Glycoproteins as "Achilles' Heel" of Viruses Contributing to Anti-Viral Immune Protection.

The glycans on enveloped viruses are synthesized by host-cell machinery. Some of these glycans on zoonotic viruses of mammalian reservoirs are recognized by human natural antibodies that may protect against such viruses. These antibodies are produced mostly against carbohydrate antigens on gastrointestinal bacteria and fortuitously, they bind to carbohydrate antigens synthesized in other mammals, neutralize and destroy viruses presenting these antigens. Two such antibodies are: anti-Gal binding to α-gal epitopes synthesized in non-primate mammals, lemurs, and New World monkeys, and anti-N-glycolyl neuraminic acid (anti-Neu5Gc) binding to N-glycolyl-neuraminic acid (Neu5Gc) synthesized in apes, Old World monkeys, and many non-primate mammals. Anti-Gal appeared in Old World primates following accidental inactivation of the α1,3galactosyltransferase gene 20-30 million years ago. Anti-Neu5Gc appeared in hominins following the inactivation of the cytidine-monophosphate-N-acetyl-neuraminic acid hydroxylase gene, which led to the loss of Neu5Gc <6 million-years-ago. It is suggested that an epidemic of a lethal virus eliminated ancestral Old World-primates synthesizing α-gal epitopes, whereas few mutated offspring lacking α-gal epitopes and producing anti-Gal survived because anti-Gal destroyed viruses presenting α-gal epitopes, following replication in parental populations. Similarly, anti-Neu5Gc protected few mutated hominins lacking Neu5Gc in lethal virus epidemics that eliminated parental hominins synthesizing Neu5Gc. Since α-gal epitopes are presented on many zoonotic viruses it is suggested that vaccines elevating anti-Gal titers may be of protective significance in areas endemic for such zoonotic viruses. This protection would be during the non-primate mammal to human virus transmission, but not in subsequent human to human transmission where the virus presents human glycans. In addition, production of viral vaccines presenting multiple α-gal epitopes increases their immunogenicity because of effective anti-Gal-mediated targeting of vaccines to antigen presenting cells for extensive uptake of the vaccine by these cells.