Glycoengineering with neuraminic acid analogs to label lipooligosaccharides and detect native sialyltransferase activity in gram-negative bacteria.

Lipooligosaccharides are the most abundant cell surface glycoconjugates on the outer membrane of Gram-negative bacteria. They play important roles in host-microbe interactions. Certain Gram-negative pathogenic bacteria cap their lipooligosaccharides with the sialic acid, N-acetylneuraminic acid (Neu5Ac), to mimic host glycans that among others protects these bacteria from recognition by the hosts immune system. This process of molecular mimicry is not fully understood and remains under investigated. To explore the functional role of sialic acid-capped lipooligosaccharides at the molecular level, it is important to have tools readily available for the detection and manipulation of both Neu5Ac on glycoconjugates and the involved sialyltransferases, preferably in live bacteria. We and others have shown that the native sialyltransferases of some Gram-negative bacteria can incorporate extracellular unnatural sialic acid nucleotides onto their lipooligosaccharides. We here report on the expanded use of native bacterial sialyltransferases to incorporate neuraminic acids analogs with a reporter group into the lipooligosaccharides of a variety of Gram-negative bacteria. We show that this approach offers a quick strategy to screen bacteria for the expression of functional sialyltransferases and the ability to use exogenous CMP-Neu5Ac to decorate their glycoconjugates. For selected bacteria we also show this strategy complements two other glycoengineering techniques, Metabolic Oligosaccharide Engineering and Selective Exo-Enzymatic Labeling, and that together they provide tools to modify, label, detect and visualize sialylation of bacterial lipooligosaccharides.

Bioisosteres at C9 of 2-Deoxy-2,3-didehydro-N-acetyl Neuraminic Acid Identify Selective Inhibitors of NEU3.

Human neuraminidases play critical roles in many physiological and pathological processes. Humans have four isoenzymes of NEU, making selective inhibitors important tools to investigate the function of individual isoenzymes. A typical scaffold for NEU inhibitors is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) where C9 modifications can be critical for potency and selectivity against human NEU. To design improved DANA analogues, we generated a library of compounds with either a short alkyl chain or a biphenyl substituent linked to the C9 position through one of six amide bioisosteres. Bioisostere linkers included triazole, urea, thiourea, carbamate, thiocarbamate, and sulfonamide groups. Within this library, we identified a C9 biphenyl carbamate derivative (963) that showed high selectivity and potency for NEU3 (Ki = 0.12 ± 0.01 µM). In contrast, NEU1 and NEU4 isoenzymes preferred amide and triazole linkers, respectively. Finally, analogues with urea, sulfonamide, and amide linkers showed enhanced inhibitory activity for a bacterial NEU, NanI from Clostridium perfringens.

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.

N-Glycolylneuraminic Acid Binding of Avian and Equine H7 Influenza A Viruses.

Influenza A viruses (IAV) initiate infection by binding to glycans with terminal sialic acids on the cell surface. Hosts of IAV variably express two major forms of sialic acid, N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). NeuGc is produced in most mammals, including horses and pigs, but is absent in humans, ferrets, and birds. The only known naturally occurring IAV that exclusively bind NeuGc are extinct highly pathogenic equine H7N7 viruses. We determined the crystal structure of a representative equine H7 hemagglutinin (HA) in complex with NeuGc and observed high similarity in the receptor-binding domain with an avian H7 HA. To determine the molecular basis for NeuAc and NeuGc specificity, we performed systematic mutational analyses, based on the structural insights, on two distant avian H7 HAs and an H15 HA. We found that the A135E mutation is key for binding α2,3-linked NeuGc but does not abolish NeuAc binding. The additional mutations S128T, I130V, T189A, and K193R converted the specificity from NeuAc to NeuGc. We investigated the residues at positions 128, 130, 135, 189, and 193 in a phylogenetic analysis of avian and equine H7 HAs. This analysis revealed a clear distinction between equine and avian residues. The highest variability was observed at key position 135, of which only the equine glutamic acid led to NeuGc binding. These results demonstrate that genetically distinct H7 and H15 HAs can be switched from NeuAc to NeuGc binding and vice versa after the introduction of several mutations, providing insights into the adaptation of H7 viruses to NeuGc receptors. IMPORTANCE Influenza A viruses cause millions of cases of severe illness and deaths annually. To initiate infection and replicate, the virus first needs to bind to a structure on the cell surface, like a key fitting in a lock. For influenza A viruses, these "keys" (receptors) on the cell surface are chains of sugar molecules (glycans). The terminal sugar on these glycans is often either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc). Most influenza A viruses bind NeuAc, but a small minority bind NeuGc. NeuGc is present in species like horses, pigs, and mice but not in humans, ferrets, and birds. Here, we investigated the molecular determinants of NeuGc specificity and the origin of viruses that bind NeuGc.

N-Glycomic Analysis of the Cell Shows Specific Effects of Glycosyl Transferase Inhibitors.

Glycomic profiling methods were used to determine the effect of metabolic inhibitors on glycan production. These inhibitors are commonly used to alter the cell surface glycosylation. However, structural analysis of the released glycans has been limited. In this research, the cell membranes were enriched and the glycans were released to obtain the N-glycans of the glycocalyx. Glycomic analysis using liquid chromatography-mass spectrometry (LC-MS) with a PGC chip column was used to profile the structures in the cell membrane. Glycans of untreated cells were compared to glycans of cells treated with inhibitors, including kifunensine, which inhibits the formation of complex- and hybrid-type structures, 2,4,7,8,9-Penta-O-acetyl-N-acetyl-3-fluoro-b-d-neuraminic acid methyl ester for sialylated glycans, 2-deoxy-2-fluorofucose, and 6-alkynyl fucose for fucosylated glycans. Kifunensine was the most effective, converting nearly 95% of glycans to high mannose types. The compound 6-alkynyl fucose inhibited some fucosylation but also incorporated into the glycan structure. Proteomic analysis of the enriched membrane for the four inhibitors showed only small changes in the proteome accompanied by large changes in the N-glycome for Caco-2. Future works may use these inhibitors to study the cellular behavior associated with the alteration of glycosylation in various biological systems, e.g., viral and bacterial infection, drug binding, and cell-cell interactions.

Chemoenzymatic Synthesis of 9NHAc-GD2 Antigen to Overcome the Hydrolytic Instability of O-Acetylated-GD2 for Anticancer Conjugate Vaccine Development.

Ganglioside GD2 is an attractive tumor-associated carbohydrate antigen for anti-cancer vaccine development. However, its low immunogenicity and the significant side effects observed with anti-GD2 antibodies present significant obstacles for vaccines. To overcome these, a new GD2 derivative bearing an N-acetamide (NHAc) at its non-reducing end neuraminic acid (9NHAc-GD2) has been designed to mimic the 9-O-acetylated-GD2 (9OAc-GD2), a GD2 based antigen with a restricted expression on tumor cells. 9NHAc-GD2 was synthesized efficiently via a chemoenzymatic method and subsequently conjugated with a powerful carrier bacteriophage Qß. Mouse immunization with the Qß-9NHAc-GD2 conjugate elicited strong and long-lasting IgG antibodies, which were highly selective toward 9NHAc-GD2 with little cross-recognition of GD2. Immunization of canines with Qß-9NHAc-GD2 showed the construct was immunogenic in canines with little adverse effects, paving the way for future clinical translation to humans.

Current views on N-glycolylneuraminic acid in therapeutic recombinant proteins.

The incorporation of the non-human N-glycolylneuraminic acid (Neu5Gc) in therapeutic recombinant proteins raises clinical concerns due to its immunogenic potential and the high prevalence of pre-existing anti-Neu5Gc antibodies in humans. The scientific literature is ambiguous regarding the actual impact of Neu5Gc-containing biotherapeutics as no severe adverse clinical manifestations were unequivocally attributed to Neu5Gc for currently marketed biotherapeutics. This review discusses structural and functional considerations of Neu5Gc-containing glycans regarding the potential impact on drug clearance, their recognition by pre-existing antibodies, and recent hypotheses regarding the tolerance to low Neu5Gc levels. Furthermore, it provides recommendations regarding the standardization of analysis and reporting, analytical aspects relevant for assessing risks associated with Neu5Gc-containing biotherapeutics, and approaches to minimize Neu5Gc incorporation in recombinant protein manufacturing.

N-Glycolylneuraminic Acid in Animal Models for Human Influenza A Virus.

The first step in influenza virus infection is the binding of hemagglutinin to sialic acid-containing glycans present on the cell surface. Over 50 different sialic acid modifications are known, of which N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the two main species. Animal models with α2,6 linked Neu5Ac in the upper respiratory tract, similar to humans, are preferred to enable and mimic infection with unadapted human influenza A viruses. Animal models that are currently most often used to study human influenza are mice and ferrets. Additionally, guinea pigs, cotton rats, Syrian hamsters, tree shrews, domestic swine, and non-human primates (macaques and marmosets) are discussed. The presence of NeuGc and the distribution of sialic acid linkages in the most commonly used models is summarized and experimentally determined. We also evaluated the role of Neu5Gc in infection using Neu5Gc binding viruses and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH)-/- knockout mice, which lack Neu5Gc and concluded that Neu5Gc is unlikely to be a decoy receptor. This article provides a base for choosing an appropriate animal model. Although mice are one of the most favored models, they are hardly naturally susceptible to infection with human influenza viruses, possibly because they express mainly α2,3 linked sialic acids with both Neu5Ac and Neu5Gc modifications. We suggest using ferrets, which resemble humans closely in the sialic acid content, both in the linkages and the lack of Neu5Gc, lung organization, susceptibility, and disease pathogenesis.

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.

Glycoside Hydrolases Restrict the Side Chain Conformation of Their Substrates To Gain Additional Transition State Stabilization.

Carbohydrate side chain conformation confers a significant influence on reactivity during glycosylation and anomeric bond hydrolysis due to stabilization of the oxocarbenium-like transition state. By analysis of 513 pyranoside-bound glycoside hydrolase (GH) crystal structures, we determine that most glucosidases and ß-mannosidases preferentially bind their substrates in the most reactive gauche,gauche (gg) conformation, thereby maximizing stabilization of the corresponding oxocarbenium ion-like transition state during hydrolysis. α-Galactoside hydrolases mostly show a preference for the second most activating gauche,trans (gt) conformation to avoid the energy penalty that would arise from imposing the gg conformation on galacto-configured ligands. These preferences stand in stark contrast to the side chain populations observed for these sugars both in free solution and bound to nonhydrolytic proteins, where for the most part a much greater diversity of side chain conformations is observed. Analysis of sequences of GH-ligand complexes reveals that side chain restriction begins with the enzyme-substrate complex and persists through the transition state until release of the hydrolysis product, despite changes in ring conformation along the reaction coordinate. This work will inform the design of new generations of glycosidase inhibitors with restricted side chains that confer higher selectivity and/or affinity.

Influence of sulfonated and diet-derived human milk oligosaccharides on the infant microbiome and immune markers.

Human milk oligosaccharides (HMOs) promote the development of the neonatal intestinal, immune, and nervous systems and has recently received considerable attention. Here we investigated how the maternal diet affects HMO biosynthesis and how any diet-induced HMO alterations influence the infant gut microbiome and immunity. Using capillary electrophoresis and MS-based analyses, we extracted and measured HMOs from breast milk samples and then correlated their levels with results from validated 24-h diet recall surveys and breast milk fatty acids. We found that fruit intake and unsaturated fatty acids in breast milk were positively correlated with an increased absolute abundance of numerous HMOs, including 16 sulfonated HMOs we identified here in humans for the first time. The diet-derived monosaccharide 5-N-glycolyl-neuraminic acid (Neu5Gc) was unambiguously detected in all samples. To gain insights into the potential impact of Neu5Gc on the infant microbiome, we used a constrained ordination approach and identified correlations between Neu5Gc levels and Bacteroides spp. in infant stool. However, Neu5Gc was not associated with marked changes in infant immune markers, in contrast with sulfonated HMOs, whose expression correlated with suppression of two major Th2 cytokines, IL-10 and IL-13. The findings of our work highlight the importance of maternal diet for HMO biosynthesis and provide as yet unexplored targets for future studies investigating interactions between HMOs and the intestinal microbiome and immunity in infants.

Are there specific antibodies against Neu5Gc epitopes in the blood of healthy individuals?

Strong discrepancies in published data on the levels and epitope specificities of antibodies against the xenogenic N-glycolyl forms of sialoglycans (Hanganutziu-Deicher Neu5Gcɑ2-3Galß1-4Glc and related antigens) in healthy donors prompted us to carry out a systematic study in this area using the printed glycan array and other methods. This article summarizes and discusses our published and previously unpublished data, as well as publicly available data from the Consortium for Functional Glycomics. As a result, we conclude that (1) the level of antibodies referred to as anti-Neu5Gc in healthy individuals is low; (2) there are antibodies that seem to interact with Neu5Gc-containing epitopes, but in fact they recognize internal fragments of Neu5Gc-containing glycans (without sialic acids), which served as antigens in the assays used and; (3) a population capable of interacting specifically with Neu5Gc (it does not bind the corresponding NAc analogs) does exist, but it binds the monosaccharide Neu5Gc better than the entire glycans containing it. In other words, in healthy donors, there are populations of antibodies capable of binding the Neu5Gc monosaccharide or the inner core -Galß1-4Glc, but very few true anti-Neu5Gcɑ2-3Galß1-4Glc antibodies, i.e., antibodies capable of specifically recognizing the entire trisaccharide.

Distinct glycosylation in membrane proteins within neonatal versus adult myocardial tissue.

Mammalian hearts have regenerative potential restricted to early neonatal stage and lost within seven days after birth. Carbohydrates exclusive to cardiac neonatal tissue may be key regulators of regenerative potential. Although cell surface and extracellular matrix glycosylation are known modulators of tissue and cellular function and development, variation in cardiac glycosylation from neonatal tissue to maturation has not been fully examined. In this study, glycosylation of the adult rat cardiac ventricle showed no variability between the two strains analysed, nor were there any differences between the glycosylation of the right or left ventricle using lectin histochemistry and microarray profiling. However, in the Sprague-Dawley strain, neonatal cardiac glycosylation in the left ventricle differed from adult tissues using mass spectrometric analysis, showing a higher expression of high mannose structures and lower expression of complex N-linked glycans in the three-day-old neonatal tissue. Man6GlcNAc2 was identified as the main high mannose N-linked structure that was decreased in adult while higher expression of sialylated N-linked glycans and lower core fucosylation for complex structures were associated with ageing. The occurrence of mucin core type 2 O-linked glycans was reduced in adult and one sulfated core type 2 O-linked structure was identified in neonatal tissue. Interestingly, O-linked glycans from mature tissue contained both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), while all sialylated N-linked glycans detected contained only Neu5Ac. As glycans are associated with intracellular communication, the specific neonatal structures found may indicate a role for glycosylation in the neonatal associated regenerative capacity of the mammalian heart. New strategies targeting tissue glycosylation could be a key contributor to achieve an effective regeneration of the mammalian heart in pathological scenarios such as myocardial infarction.

Analysis of cetuximab N-Glycosylation using multiple fractionation methods and capillary electrophoresis mass spectrometry.

Site-specific glycosylation of Cetuximab was characterized in this study using multiple fractionation methods and capillary electrophoresis coupled to mass spectrometry (CE-MS) based glycomics. IdeS digested Cetuximab with subsequent reduction was fractionated using reversed-phase chromatography resulting in 3 fragments; Fd, Lc and Fc/2. Glycan release of the different fragments was performed in 18O enriched water providing the possible quantification of site occupancy. 2-AA labelled glycan structures were annotated by CE-MS profiling in combination with exoglycosidase sequencing, revealing potential structures with terminal α-galactose and N-glycolyl-neuraminic acid (NGNA) mainly originating from the Fd fragment. Glycosylation analysis was also performed on different charge variants of Cetuximab that were separated using pH gradient cation-exchange chromatography to investigate the impact of glycosylation on the net charge of the protein.

Effects of sialic acid linkage on antibody-fragment crystallizable receptor binding and antibody dependent cytotoxicity depend on levels of fucosylation/bisecting.

Aim: Fragment crystallizable (Fc) glycosylation of immunoglobulin G-type monoclonal antibodies applied to therapeutic applications is regarded a critical quality attribute and can influence bioactivity, pharmacokinetics and/or immunogenicity/safety. Investigating the impact of certain Fc N-glycans is therefore of importance to assess its criticality for a therapeutic product. This has been done for N-glycan types like fucosylation, galactosylation or sialylation. There were contradictory results reported for functionality especially with regard to sialylation. Material & methods: We elucidated the effect of terminal sialic acid residues on Fcγ receptor binding and antibody dependent cytotoxicity activity of two immunoglobulin G1 antibodies with different levels of fucosylation/bi-secting. Conclusion: We found the impact to be specific to the sialylation linkage type, in other words, α2,3- versus α2,6-linked sialic acid attached to the terminal galactose residues.

Understanding the Effects of Lactose Hydrolysis Modeling on the Main Oligosaccharides in Goat Milk Whey Permeate.

Enzymatic hydrolysis of lactose is a crucial step to improve the efficiency and selectivity of membrane-based separations toward the recovery of milk oligosaccharides free from simple sugars. Response surface methodology was used to investigate the effects temperature (25.9 to 54.1 °C) and amount of enzyme (0.17 to 0.32% w/w) at 1, 2, and 4 h of reaction on the efficiency of lactose hydrolysis by Aspergillus oryzae ß-galactosidase, preservation of major goat whey oligosaccharides, and on the de-novo formation of oligosaccharides. Lactose hydrolysis above 99% was achieved at 1, 2, and 4 h, not being significantly affected by temperature and amount of enzyme within the tested conditions. Formation of 4 Hexose (Hex) and 4 Hex 1 Hex and an increased de-novo formation of 2 Hex 1 N-Acetyl-Neuraminic Acid (NeuAc) and 2 Hex 1 N-Glycolylneuraminic acid (NeuGc) was observed in all treatments. Overall, processing conditions using temperatures ≤40 °C and enzyme concentration ≤0.25% resulted in higher preservation/formation of goat whey oligosaccharides.

N-Glycolylneuraminic Acid as a Receptor for Influenza A Viruses.

A species barrier for the influenza A virus is the differential expression of sialic acid, which can either be α2,3-linked for avians or α2,6-linked for human viruses. The influenza A virus hosts also express other species-specific sialic acid derivatives. One major modification at C-5 is N-glycolyl (NeuGc), instead of N-acetyl (NeuAc). N-glycolyl is mammalian specific and expressed in pigs and horses, but not in humans, ferrets, seals, or dogs. Hemagglutinin (HA) adaptation to either N-acetyl or N-glycolyl is analyzed on a sialoside microarray containing both α2,3- and α2,6-linkage modifications on biologically relevant N-glycans. Binding studies reveal that avian, human, and equine HAs bind either N-glycolyl or N-acetyl. Structural data on N-glycolyl binding HA proteins of both H5 and H7 origin describe this specificity. Neuraminidases can cleave N-glycolyl efficiently, and tissue-binding studies reveal strict species specificity. The exclusive manner in which influenza A viruses differentiate between N-glycolyl and N-acetyl is indicative of selection.

Profiling and Structural Characterization of High Neu5Gc or Sulfate-containing O-glycans from Hyla Rabbit Intestinal Mucin.

Intestinal mucins constitute the major component of the mucus covering the epithelium of the gastrointestinal tract, thereby forming a barrier against microbial colonization. Rabbits are bred in large numbers worldwide, with little known about intestinal O-glycosylation despite this insight being crucial to the understanding of host-pathogen interactions. In the present study, a major mucin-type glycopeptide (RIF6) of hyla rabbit intestine was isolated and the O-glycans were extensively characterized based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with bioinformatics approaches. Thirty-three O-glycans were identified, and most of them were sulfated or sialylated glycans. It was worth noting that Neu5Gc-containing structures within sialylated O-glycans accounted for 91%, which were extremely different from that of other species including humans, mice, chickens, etc. Sulfated glycans accounted for 58%, unique disufated and sulfated-sialylated glycans were also detected in rabbit intestinal mucin. These structural characterization reflected species diversity and may provide deeper insights into explaining the adaptability of hyla rabbit to the environment.

Intestine specific regulation of pig cytidine-5'-monophospho-N-acetylneuraminic acid hydroxylase gene for N-glycolylneuraminic acid biosynthesis.

N-glycolylneuraminic acid (Neu5Gc), a generic form of sialic acid, is enzymatically synthesized by cytidine-5'-monophospho-N-acetylneuraminic acid hydroxylase (CMAH). Although expression of pig CMAH gene pcmah encoding CMAH has been reported to be regulated by pathogenic infection and developmental processes, little is known about the mechanisms underlying the regulation of pcmah gene expression. The objective of this study was to determine mechanism(s) involved in intestine specific regulation of pcmah gene by identifying several cis-acting elements and nuclear transcription factors that could directly interact with these cis-acting elements. We identified intestine specific promoter region (Pi) of pcmah gene located at upstream regions of the 5'flanking region of exon 1a and found that the promoter region is responsible for the transcriptional regulation of 5'pcmah-1. Based on reporter assays using serially constructed luciferase genes with each deleted promoter, we demonstrated that the Pi promoter activity was more active in intestinal IPI-2I cells than that in kidney PK15 cells, corresponding to both mRNA expression patterns in the two cell lines. In addition, we found that Sp1 transcription factor was necessary for basal activity of Pi promoter and that Ets-1 contributed to intestine-specific activity of Pi promoter. This study helps us understand transcriptional regulation of pcmah in the intestine of pig tissues. It also allows us to consider potential roles of Neu5Gc in interaction with environmental factors present in the intestinal tissue during pathogenic infection and developmental process.