Complex N-glycans are important for interspecies transmission of H7 influenza A viruses.

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N-glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N-acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N-glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection.

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.

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.

Biosynthesis of UDP-α-N-Acetyl-d-mannosaminuronic Acid and CMP-ß-N-Acetyl-d-neuraminic Acid for the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni is a human pathogen and a leading cause of food poisoning in North America and Europe. The exterior surface of the bacterial cell wall is attached to a polymeric coat of sugar molecules known as the capsular polysaccharide (CPS) that helps protect the organism from the host immune response. The CPS is composed of a repeating sequence of common and unusual sugar residues. In the HS:11 serotype of C. jejuni, we identified two enzymes in the gene cluster for CPS formation that are utilized for the biosynthesis of UDP-α-N-acetyl-d-mannosaminuronic acid (UDP-ManNAcA). In the first step, UDP-α-N-acetyl-d-glucosamine (UDP-GlcNAc) is epimerized at C2 to form UDP-α-N-acetyl-d-mannosamine (UDP-ManNAc). This product is then oxidized by a NAD+-dependent C6-dehydrogenase to form UDP-ManNAcA. In the HS:6 serotype (C. jejuni strain 81116), we identified three enzymes that are required for the biosynthesis of CMP-ß-N-acetyl-d-neuraminic acid (CMP-Neu5Ac). In the first step, UDP-GlcNAc is epimerized at C2 and subsequently hydrolyzed to form N-acetyl-d-mannosamine (ManNAc) with the release of UDP. This product is then condensed with PEP by N-acetyl-d-neuraminate synthase to form N-acetyl-d-neuraminic acid (Neu5Ac). In the final step, CMP-N-acetyl-d-neuraminic acid synthase utilizes CTP to convert this product into CMP-Neu5Ac. A bioinformatic analysis of these five enzymes from C. jejuni serotypes HS:11 and HS:6 identified other bacterial species that can produce UDP-ManNAcA or CMP-Neu5Ac for CPS formation.

Facile synthesis of C5-azido derivatives of thiosialosides and 2,3-dehydro-5-N-acetylneuraminic acid (DANA).

Neuraminic acid (Neu5Ac, also known as sialic acid) is an important monosaccharide found in glycoproteins and glycolipids which plays a vital role in regulation of physiological functions and pathological conditions. The study of sialoglycans has benefitted from the development of glycomimetic probes and inhibitors for proteins and enzymes that interact with and modify neuraminic acid in glycan chains. Methods to access sialoside intermediates with high yield are needed to facilitate the design of new targets. Here, we report the synthesis of C5-azido thiosialosides using a mild method to deprotect the C5-acetamido functional group followed by the use of a diazo-transfer reagent. We examined two diazo-transfer strategies and compared their yields and tolerance of acetate protecting groups. The same methods and comparisons were also performed for the 2,3-dehydro-5-N-acetylneuraminic acid (DANA) scaffold which is commonly used to generate inhibitors of neuraminidase (sialidase) enzymes. We found that C5-azido derivatives of both thiosialosides and DANA could be produced in five or six steps with yields up to 76 % and 83 %, respectively. Diazo-transfer reagents compared in this study were trifluoromethanesulfonyl azide (TfN3) and imidazole-1-sulfonyl azide (ImzSO2N3). We found that both reagents were compatible with this method and showed comparable yields. Finally, we show that C5-azido derivatives can help to avoid O, N-acyl protecting group migration which was observed in C5-NHAc analogs.

Detection of N-glycolyl-neuraminic acid-containing glycolipids in human skin.

Humans lack the enzyme that produces the sialic acid N-glycolyl neuraminic acid (Neu5Gc), but several lines of evidence have shown that Neu5Gc can be taken up by mammalian food sources and replace the common human sialic acid N-acetyl neuraminic acid (Neu5Ac) in glycans. Cancer tissue has been shown to have increased the presence of Neu5Gc and Neu5Gc-containing glycolipids such as the ganglioside GM3, which have been proposed as tumor-specific antigens for antibody treatment. Here, we show that a previously described antibody against Neu5Gc-GM3 is binding to Neu5GC-containing gangliosides and is strongly staining different cancer tissues. However, we also found a strong intracellular staining of keratinocytes of healthy skin. We confirmed this staining on freshly isolated keratinocytes by flow cytometry and detected Neu5Gc by mass spectrometry. This finding implicates that non-human Neu5Gc can be incorporated into gangliosides in human skin, and this should be taken into consideration when targeting Neu5Gc-containing gangliosides for cancer immunotherapy.

Amphiphilic Sialic Acid Derivatives as Potential Dual-Specific Inhibitors of Influenza Hemagglutinin and Neuraminidase.

In the shadow of SARS-CoV-2, influenza seems to be an innocent virus, although new zoonotic influenza viruses evolved by mutations may lead to severe pandemics. According to WHO, there is an urgent need for better antiviral drugs. Blocking viral hemagglutinin with multivalent N-acetylneuraminic acid derivatives is a promising approach to prevent influenza infection. Moreover, dual inhibition of both hemagglutinin and neuraminidase may result in a more powerful effect. Since both viral glycoproteins can bind to neuraminic acid, we have prepared three series of amphiphilic self-assembling 2-thio-neuraminic acid derivatives constituting aggregates in aqueous medium to take advantage of their multivalent effect. One of the series was prepared by the azide-alkyne click reaction, and the other two by the thio-click reaction to yield neuraminic acid derivatives containing lipophilic tails of different sizes and an enzymatically stable thioglycosidic bond. Two of the three bis-octyl derivatives produced proved to be active against influenza viruses, while all three octyl derivatives bound to hemagglutinin and neuraminidase from H1N1 and H3N2 influenza types.

A platform for qualitative and quantitative characterization of α-Gal and NeuGc at the oligosaccharide level.

Glycosylation modification serves as a pivotal quality attribute in glycoprotein-based therapeutics, emphasizing its role in drug safety and efficacy. Prior studies have underscored the potential immunogenic risks posed by the presence of galactose-α-1,3-galactose (α-Gal) and N-glycolylneuraminic acid (NeuGc) in glycoprotein formulations. This accentuates the imperative for developing robust qualitative and quantitative analytical methods to monitor these immunogenic epitopes, thereby ensuring drug safety. In the present investigation, α-Gal and NeuGc were accurately quantified using UPLC-FLR-MS/MS at the oligosaccharide level. Remarkably, α-Gal could be identified when the ion intensity ratio or the mass-to-charge ratio (m/z) of 528.19 to 366.14 exceeded 1. Concurrently, NeuGc and N-acetylneuraminic acid (NeuAc) could be unambiguously identified through their respective fragment ions at m/z 673.23 and m/z 657.23. Furthermore, relative quantification of α-Gal and NeuGc was achieved using fluorescence signals. This study introduces a sensitive and reliable analytical approach for the qualitative and quantitative assessment of α-Gal and NeuGc in glycoprotein pharmaceuticals. The methodology offers significant potential for application in process control and optimization of glycoprotein production, aimed at minimizing immunogenicity and enhancing product quality.

Heterophile carbohydrate antigen N-glycolylneuraminic acid as a potential biomarker in patients with hepatocellular carcinoma.

BACKGROUND AND OBJECTIVES: Hepatocellular carcinoma (HCC) has a high recurrence rate even after radical hepatectomy. More optimal biomarkers may help improve recurrence and prognosis. METHODS: We investigated whether the oncological properties of N-glycolylneuraminic acid (NeuGc) can participate in the prognosis of HCC. We evaluated the NeuGc antigen (Ag) expression in the HCC tissues and measured the preoperative anti-NeuGc IgG antibodies (Abs) in the sera of the patients with HCC. We compared the clinical characteristics and survival rate in the hepatectomized patients (initial; n = 66, recurrent; n = 34) with and without the NeuGc Ag or Abs. RESULTS: Multivariate analyses showed positive expression of NeuGc Ag in HCC tissues (Odds ratio; initial = 6.3, recurrent = 14.0) and higher titers of preoperative anti-NeuGc Ab (Odds ratio; initial = 4.9; recurrent = 3.8), which could be the predictive factors related to early recurrence. Both the NeuGc Ag-positive and Ab-positive groups in the initial hepatectomized patients exhibited significantly shorter recurrent free survival compared to those in the negative groups. CONCLUSIONS: Our findings suggested that anti-NeuGc Ab titers and NeuGc Ag expression in the HCC tissues can be used as the predictive factors for the postoperative recurrence and prognosis of HCC.

The generation of 5-N-glycolylneuraminic acid as a consequence of high levels of reactive oxygen species.

The presence of N-glycolylneuraminic acid (Neu5Gc), a non-human sialic acid in cancer patients, is currently attributed to the consumption of red meat. Excess dietary red meat has been considered a risk factor causing chronic inflammation and for the development of cancers. However, it remains unknown whether Neu5Gc can be generated via a chemical reaction rather than via a metabolic pathway in the presence of high levels of reactive oxygen species (ROS) found in the inflammatory and tumor environments. In this study, the conversion of N-acetylneuraminic acid (Neu5Ac) to Neu5Gc has been assessed in vitro under conditions mimicking the hydroxyl radical-rich humoral environment found in inflammatory and cancerous tissues. As a result, Neu5Gc has been detected via liquid chromatography-multiple reaction monitoring mass spectrometry. Furthermore, this conversion has also been found to take place in serum biomatrix containing ROS and in cancer cell cultures with induced ROS production.

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.

Immune disguise: the mechanisms of Neu5Gc inducing autoimmune and transplant rejection.

Organ (stem cell) transplantation is the most effective treatment for advanced organ failure. Neu5Gc (N-hydroxyacetylneuraminic acid) is a pathogenic non-human sialic acid, which is very similar to the molecular structure of Neu5Ac (N-acetylneuraminic acid) in human body. Neu5Gc has the function of "immune disguise", which is the main obstacle to transplantation. Gene knockout such as cytidine monophosphate-N-acetylneuraminidase (CMAH) reduces donor antigenicity, making xenotransplantation from fiction to reality. Exploring the immune disguise event in this emerging field has become a hot topic in the research of transplantation immune tolerance mechanism.

B-cell-specific ablation of ß-1,4-galactosyltransferase 1 prevents aging-related IgG glycans changes and improves aging phenotype in mice.

IgG N-glycans levels change with advancing age, making it a potential biomarker of aging. ß-1,4-galactosyltransferase (B4GALT) gene expression levels also increase with aging. Ultra performance liquid chromatography (UPLC) was used to examine changes inserum IgG N-glycans at six time points during the aging process. Most serum IgG N-glycans changed with aging in WT but not in CD19-cre B4GALT1 floxed mice. The relative abundance of fucosylated biantennary glycans with or without Neu5Gc structures changed with aging in heterozygous B4GALT1 floxed mice but not in homozygous B4GALT1 floxed mice. Additionally, the aging phenotype was more apparent in WT mice than in B4GALT1 floxed mice. These results demonstrate that fucosylated biantennary glycans and fucosylated biantennary glycans containing N-glycolylneuraminic acid (Neu5Gc)-linked N-acetyllactosamine (LacNAc) were highly associated with aging and were affected by the B4GALT1 floxed mouse genotype. The changing levels of fucosylated monoantennary glycans observed with aging in WT mice was reversed in B4GALT1 floxed mice and was not sex specific. In summary, B-cell-specific ablation of B4GALT1 from a glycoproteomic perspective prevented age-related changes in IgG N-glycans in mice. SIGNIFICANCE: In this study, serum IgG glycoproteomic data in wild-type (WT) and B-cell-specific ablation of ß-1,4-galactosyltransferase 1 mice (B4GALT) were analyzed. Results showed that fucosylated biantennary glycans with or without N-glycolylneuraminic acid (Neu5Gc)-linked N-acetyllactosamine (LacNAc) were highly associated with aging and were also affected by the B4GALT1 floxed mouse genotype. In terms of gender-specific information, the trend towards elevated fucosylated monoantennary glycans in WT mice was not seen in CD19-cre B4GALT1 floxed mice in either sex. B-cell-specific ablation of B4GALT1 plays an important role in age-related glycan changes; its specific functions and mechanisms are worthy of in-depth study. Our data suggest that investigating the relationship between galactosylation and aging may help advance the field of glycoproteomics and aging research.

Lectin Staining to Detect Human and Avian Influenza Virus Receptors in the Airway of Nonhuman Primates.

Hemagglutinin (HA) on the surface of influenza viruses binds to sialic acids, mainly N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid. Neu5Ac and N-glycolylneuraminic acid lie at the terminal end of sugar chains on the cell surface. Human influenza viruses preferentially bind to sialic acids bound to galactose by the alpha2-6 linkage (Neu5Acα2-6Gal), abundant in the human airway. In contrast, avian influenza viruses preferentially bind to Neu5Acα2-3Gal, abundant in the intestine of ducks. Sambucus nigra lectin (SNA) and Maackia amurensis lectin (MAA) bind to Neu5Acα2-6Gal and Neu5Acα2-3Gal, respectively. These two lectins have therefore been applied to detect sialic acids on the airway epithelium of animals.

N-glycolylated carbohydrates in nature.

N-glycolylated carbohydrates are amino sugars with an N-glycolyl amide group. These glycans have not been well studied due to their surprising rarity in nature in comparison with N-acetylated carbohydrates. Recently, however, there has been increasing interest in N-glycolylated sugars because the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc), apparently the only source of all N-glycolylated sugars in deuterostomes, appears to be involved in xenosialitis (inflammation associated with consumption of Neu5Gc-rich red meats). Xenosialitis has been implicated in cancers as well as other diseases including atherosclerosis. Furthermore, metabolites of Neu5Gc have been shown to be incorporated into glycosaminoglycans (GAGs), resulting in N-glycolylated GAGs. These N-glycolylated GAGs have important potential applications, such as dating the loss of the Neu5Gc-generating CMAH gene in humans and being explored as a xenosialitis biomarker and/or estimate of the body burden of diet-derived Neu5Gc, to understand the risks associated with the consumption of red meats. This review explores N-glycolylated carbohydrates, how they are metabolized to N-glycolylglucosamine and N-glycolylgalactosamine, and how these metabolites can be incorporated into N-glycolylated GAGs in human tissues. We also discuss other sources of N-glycolylated sugars, such as recombinant production from microorganisms using metabolic engineering as well as chemical synthesis.

A novel C-domain-dependent inhibition of the rainbow trout CMP-sialic acid synthetase activity by CMP-deaminoneuraminic acid.

The CMP-sialic acid synthetase (CSS) activates free sialic acid (Sia) to CMP-Sia using CTP, and is prerequisite for the sialylation of cell surface glycoconjugates. The vertebrate CSS consists of two domains, a catalytic N-domain and a non-catalytic C-domain. Although the C-domain is not required for the CSS enzyme to synthesize CMP-Sia, its involvement in the catalytic activity remains unknown. First, the real-time monitoring of CSS-catalyzed reaction was performed by 31P NMR using the rainbow trout CSS (rtCSS). While a rtCSS lacking the C-domain (rtCSS-N) similarly activated both deaminoneuraminic acid (Kdn) and N-acetylneuraminic acid (Neu5Ac), the full-length rtCSS (rtCSS-FL) did not activate Kdn as efficiently as Neu5Ac. These results suggest that the C-domain of rtCSS affects the enzymatic activity, when Kdn was used as a substrate. Second, the enzymatic activity of rtCSS-FL and rtCSS-N was measured under various concentrations of CMP-Kdn. Inhibition by CMP-Kdn was observed only for rtCSS-FL, but not for rtCSS-N, suggesting that the inhibition was C-domain-dependent. Third, the inhibitory effect of CMP-Kdn was also investigated using the mouse CSS (mCSS). However, no inhibition was observed with mCSS even at high concentrations of CMP-Kdn. Taken together, the data demonstrated that the C-domain is involved in the CMP-Kdn-dependent inhibition of rtCSS, which is a novel regulation of the Sia metabolism in rainbow trout.

Wild and domestic animals variably display Neu5Ac and Neu5Gc sialic acids.

Sialic acids are used as a receptor by several viruses and variations in the linkage type or C-5 modifications affect the binding properties. A species barrier for multiple viruses is present due to α2,3- or α2,6-linked sialic acids. The C-5 position of the sialic acid can be modified to form N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), which acts as a determinant for host susceptibility for pathogens such as influenza A virus, rotavirus, and transmissible gastroenteritis coronavirus. Neu5Gc is present in most mammals such as pigs and horses but is absent in humans, ferrets, and dogs. However, little is known about C-5 content in wildlife species or how many C-5 modified sialic acids are present on N-linked glycans or glycolipids. Using our previously developed tissue microarray system, we investigated how 2 different lectins specific for Neu5Gc can result in varying detection levels of Neu5Gc glycans. We used these lectins to map Neu5Gc content in wild Suidae, Cervidae, tigers, and European hedgehogs. We show that Neu5Gc content is highly variable among different species. Furthermore, the removal of N-linked glycans reduces the binding of both Neu5Gc lectins while retention of glycolipids by omitting methanol treatment of tissues increases lectin binding. These findings highlight the importance of using multiple Neu5Gc lectins as the rich variety in which Neu5Gc is displayed can hardly be detected by a single lectin.

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.

Synthetic O-Acetyl-N-glycolylneuraminic Acid Oligosaccharides Reveal Host-Associated Binding Patterns of Coronaviral Glycoproteins.

A panel of O-acetylated N-glycolylneuraminic acid oligosaccharides has been prepared by diversification of common synthetic precursors by regioselective de-O-acetylation by coronaviral hemagglutinin-esterase (HE) combined with C7-to-C9 acetyl ester migration. The resulting compound library was printed on streptavidin-coated glass slides to give a microarray to investigate receptor binding specificities of viral envelope glycoproteins, including spike proteins and HEs from animal and human coronaviruses. It was found that the binding patterns of the viral proteins for N-glycolylated sialosides differ considerable from those of the previously synthesized N-acetylated counterparts. Generally, the spike proteins tolerate N-glycolyl modification, but selectivities differ among viruses targeting different hosts. On the other hand, the lectin domain of the corresponding HEs showed a substantial decrease or loss of binding of N-glycolylated sialosides. MD simulations indicate that glycolyl recognition by HE is mediated by polar residues in a loop region (109-119) that interacts with the 5-N-glycolyl moiety. Collectively, the results indicate that coronaviruses have adjusted their receptor fine specificities to adapt to the sialoglycome of their host species.

Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid.

Background: Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells. Methods: Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed. Results: The C3 deposition level in GTKO pPBMCs or pECs in GVB++ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs. Conclusion: TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.