Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation.

Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophilic influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5'-monophosphate-N-acetylneuraminic acid, which is scavenged from the host using LOS sialyltransferase (Lst) since Gc cannot make its sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or Δlst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress the oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea. IMPORTANCE: Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subverts protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampens neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

Pancreatic cancer-associated fibroblasts modulate macrophage differentiation via sialic acid-Siglec interactions.

Despite recent advances in cancer immunotherapy, pancreatic ductal adenocarcinoma (PDAC) remains unresponsive due to an immunosuppressive tumor microenvironment, which is characterized by the abundance of cancer-associated fibroblasts (CAFs). Once identified, CAF-mediated immune inhibitory mechanisms could be exploited for cancer immunotherapy. Siglec receptors are increasingly recognized as immune checkpoints, and their ligands, sialic acids, are known to be overexpressed by cancer cells. Here, we unveil a previously unrecognized role of sialic acid-containing glycans on PDAC CAFs as crucial modulators of myeloid cells. Using multiplex immunohistochemistry and transcriptomics, we show that PDAC stroma is enriched in sialic acid-containing glycans compared to tumor cells and normal fibroblasts, and characterized by ST3GAL4 expression. We demonstrate that sialic acids on CAF cell lines serve as ligands for Siglec-7, -9, -10 and -15, distinct from the ligands on tumor cells, and that these receptors are found on myeloid cells in the stroma of PDAC biopsies. Furthermore, we show that CAFs drive the differentiation of monocytes to immunosuppressive tumor-associated macrophages in vitro, and that CAF sialylation plays a dominant role in this process compared to tumor cell sialylation. Collectively, our findings unravel sialic acids as a mechanism of CAF-mediated immunomodulation, which may provide targets for immunotherapy in PDAC.

Maackia amurensis seed lectin (MASL) and soluble human podoplanin (shPDPN) sequence analysis and effects on human oral squamous cell carcinoma (OSCC) cell migration and viability.

Maackia amurensis lectins serve as research and botanical agents that bind to sialic residues on proteins. For example, M. amurensis seed lectin (MASL) targets the sialic acid modified podoplanin (PDPN) receptor to suppress arthritic chondrocyte inflammation, and inhibit tumor cell growth and motility. However, M. amurensis lectin nomenclature and composition are not clearly defined. Here, we sought to definitively characterize MASL and its effects on tumor cell behavior. We utilized SDS-PAGE and LC-MS/MS to find that M. amurensis lectins can be divided into two groups. MASL is a member of one group which is composed of subunits that form dimers, evidently mediated by a cysteine residue in the carboxy region of the protein. In contrast to MASL, members of the other group do not dimerize under nonreducing conditions. These data also indicate that MASL is composed of 4 isoforms with an identical amino acid sequence, but unique glycosylation sites. We also produced a novel recombinant soluble human PDPN receptor (shPDPN) with 17 threonine residues glycosylated with sialic acid moieties with potential to act as a ligand trap that inhibits OSCC cell growth and motility. In addition, we report here that MASL targets PDPN with very strong binding kinetics in the nanomolar range. Moreover, we confirm that MASL can inhibit the growth and motility of human oral squamous cell carcinoma (OSCC) cells that express the PDPN receptor. Taken together, these data characterize M. amurensis lectins into two major groups based on their intrinsic properties, clarify the composition of MASL and its subunit isoform sequence and glycosylation sites, define sialic acid modifications on the PDPN receptor and its ability to act as a ligand trap, quantitate MASL binding to PDPN with KD in the nanomolar range, and verify the ability of MASL to serve as a potential anticancer agent.

Sialylation on vesicular integrin ß1 determined endocytic entry of small extracellular vesicles into recipient cells.

BACKGROUND: Small extracellular vesicles (sEV) are closely associated with the development and metastasis of many types of mammalian cancer. Glycoconjugates are highly expressed on sEV and play important roles in sEV biogenesis and their interaction with other cells. However, the study on vesicular glycoconjugates are far behind proteins and nucleic acids. Especially, the functions of sialic acids which are the terminal components of glycoconjugates, are poorly understood in sEV. METHODS: Sialic acid levels on sEV from plasma and bladder cancer cells were determined by ELISA and lectin blotting. Effects of sialylation on sEV uptake were determined by flow cytometry. Vesicular glycoproteins bearing sialic acids responsible for sEV uptake was identified by proteomics and density gradient centrifugation, and their site-specific sialylation functions were assayed by N-glycosylation site mutation. Effects of integrin ß1 bearing sialic acids on the pro-metastatic function of sEV in vivo were explored using Balb/c nu/nu mice. RESULTS: (1) Increased sialic acid levels were observed in sEV from malignant bladder cancer cells. (2) Elimination of sialic acids on sEV impaired sEV uptake by recipient cells. (3) Vesicular integrin ß1 bearing sialic acids was identified to play a key role in sEV uptake. (4) Desialylation of the hybrid domain of vesicular integrin ß1 inhibited its binding to matrix fibronectin, and reduced sEV entry into recipient cells. (5) Sialylation on integrin ß1 affected pro-metastatic function of sEV in Balb/c nu/nu mice. CONCLUSIONS: Taken together, our findings indicate important functional roles of sialic acids in sEV uptake and reprogramming plasticity of surrounding normal epithelial cells.

Potential small effector molecules restoring cellular defects due to sialic acid biosynthetic enzyme deficiency: Pathological relevance to GNE myopathy.

GNEM (GNE Myopathy) is a rare neuromuscular disease caused due to biallelic mutations in sialic acid biosynthetic GNE enzyme (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine Kinase). Recently direct or indirect role of GNE in other cellular functions have been elucidated. Hyposialylation of IGF-1R leads to apoptosis due to mitochondrial dysfunction while hyposialylation of ß1 integrin receptor leads to altered F-actin assembly, disrupted cytoskeletal organization and slow cell migration. Other cellular defects in presence of GNE mutation include altered ER redox state and chaperone expression such as HSP70 or PrdxIV. Currently, there is no cure to treat GNEM. Possible therapeutic trials focus on supplementation with sialic acid, ManNAc, sialyllactose and gene therapy that slows the disease progression. In the present study, we analyzed the effect of small molecules like BGP-15 (HSP70 modulator), IGF-1 (IGF-1R ligand) and CGA (cofilin activator) on cellular phenotypes of GNE heterozygous knock out L6 rat skeletal muscle cell line (SKM­GNEHz). Treatment with BGP-15 improved GNE epimerase activity by 40 % and reduced ER stress by 45 % for SKM­GNEHz. Treatment with IGF-1 improved epimerase activity by 37.5 %, F-actin assembly by 100 %, cell migration upto 36 % (36 h) and atrophy by 0.44-fold for SKM­GNEHz. Treatment with CGA recovered epimerase activity by 49 %, F-actin assembly by 132 % and cell migration upto 41 % (24 h) in SKM­GNEHz. Our study shows that treatment with these small effector molecules reduces the detrimental phenotype observed in SKM­GNEHz, thereby, providing insights into potential therapeutic targets for GNEM.

Regulation of sialic acid metabolism in cancer.

Sialic acid, the terminal structure of cell surface glycans, has essential functions in regulating immune response, cell-to-cell communication, and cell adhesion. More importantly, an increased level of sialic acid, termed hypersialylation, has emerged as a commonly observed phenotype in cancer. Therefore, targeting sialic acid ligands (sialoglycans) and their receptors (Siglecs) may provide a new therapeutic approach for cancer immunotherapy. We highlight the complexity of the sialic acid metabolism and its involvement in malignant transformation within individual cancer subtypes. In this review, we focus on the dysregulation of sialylation, the intricate nature of sialic acid synthesis, and clinical perspective. We aim to provide a brief insight into the mechanism of hypersialylation and how our understanding of these processes can be leveraged for the development of novel therapeutics.

Functional dissection of the spike glycoprotein S1 subunit and identification of cellular cofactors for regulation of swine acute diarrhea syndrome coronavirus entry.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus, and the broad interspecies infection of SADS-CoV poses a potential threat to human health. This study provides experimental evidence to dissect the roles of distinct domains within the SADS-CoV spike S1 subunit in cellular entry. Specifically, we expressed the S1 and its subdomains, S1A and S1B. Cell binding and invasion inhibition assays revealed a preference for the S1B subdomain in binding to the receptors on the cell surface, and this unknown receptor is not utilized by the porcine epidemic diarrhea virus. Nanoparticle display demonstrated hemagglutination of erythrocytes from pigs, humans, and mice, linking the S1A subdomain to the binding of sialic acid (Sia) involved in virus attachment. We successfully rescued GFP-labeled SADS-CoV (rSADS-GFP) from a recombinant cDNA clone to track viral infection. Antisera raised against S1, S1A, or S1B contained highly potent neutralizing antibodies, with anti-S1B showing better efficiency in neutralizing rSADS-GFP infection compared to anti-S1A. Furthermore, depletion of heparan sulfate (HS) by heparinase treatment or pre-incubation of rSADS-GFP with HS or constituent monosaccharides could inhibit SADS-CoV entry. Finally, we demonstrated that active furin cleavage of S glycoprotein and the presence of type II transmembrane serine protease (TMPRSS2) are essential for SADS-CoV infection. These combined observations suggest that the wide cell tropism of SADS-CoV may be related to the distribution of Sia or HS on the cell surface, whereas the S1B contains the main protein receptor binding site. Specific host proteases also play important roles in facilitating SADS-CoV entry.IMPORTANCESwine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel pathogen infecting piglet, and its unique genetic evolution characteristics and broad species tropism suggest the potential for cross-species transmission. The virus enters cells through its spike (S) glycoprotein. In this study, we identify the receptor binding domain on the C-terminal part of the S1 subunit (S1B) of SADS-CoV, whereas the sugar-binding domain located at the S1 N-terminal part of S1 (S1A). Sialic acid, heparan sulfate, and specific host proteases play essential roles in viral attachment and entry. The dissection of SADS-CoV S1 subunit's functional domains and identification of cellular entry cofactors will help to explore the receptors used by SADS-CoV, which may contribute to exploring the mechanisms behind cross-species transmission and host tropism.

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.

In Situ Visualization of RNA-Specific Sialylation on Living Cell Membranes to Explore N-Glycosylation Sites.

The small RNAs on living cell membranes were recently found to be N-glycosylated and terminated with sialic acids, although the glycosylation sites and potential functions remain unclear. Herein, we designed a second-generation hierarchical coding strategy (HieCo 2) for in situ visualization of cell surface RNA-specific sialylation. After covalently binding DNA codes to sialic acids and then binding a DNA code to a target RNA via sequence specificity, cascade decoding processes were performed with subsequent signal amplification that enabled sensitive in situ visualization of low-abundance Y5 RNA-specific sialic acids on living cell membranes. The proposed strategy unveils the number of glycosylation sites on a single RNA and reveals the binding preference of glycosylated RNAs to different sialic acid binding-immunoglobulin lectin-type receptors, demonstrating a new route for exploration of the glycosylated RNA-related biological and pathological processes.

Bacterial-derived sialidases inhibit porcine rotavirus OSU replication by interfering with the early steps of infection.

Rotavirus infections in suckling and weaning piglets cause severe dehydration and death, resulting in significant economic losses in the pig breeding industry. With the continuous emergence of porcine rotavirus (PoRV) variants and poor vaccine cross-protection among various genotypes, there is an urgent need to develop alternative strategies such as seeking effective antiviral products from nature, microbial metabolites and virus-host protein interaction. Sialidases play a crucial role in various physiopathological processes and offer a promising target for developing antivirus drugs. However, the effect of bacterial-derived sialidases on the infection of PoRVs remains largely unknown. Herein, we investigated the impact of bacterial-derived sialidases (sialidase Cp and Vc) on PoRV strain OSU(Group A) infection, using differentiated epithelial monkey kidney cells (MA104) as a model. Our results indicated that the pretreatment of MA104 with exogenous sialidases effectively suppressed PoRV OSU in a concentration-dependent manner. Notably, even at a concentration of 0.01 µU/mL, sialidases significantly inhibited the virus (MOI = 0.01). Meanwhile, we found that sialidase Vc pretreatment sharply reduced the binding rate of PoRV OSU. Last, we demonstrated that PoRV OSU might recognize α-2,3-linked sialic acid as the primary attachment factor in MA104. Our findings provide new insights into the underlying mechanism of PoRV OSU infections, shedding lights on the development of alternative antivirus approaches based on bacteria-virus interaction.

Sialylated glycoproteins and sialyltransferases in digestive cancers: Mechanisms, diagnostic biomarkers, and therapeutic targets.

Sialic acid (SA), as the ultimate epitope of polysaccharides, can act as a cap at the end of polysaccharide chains to prevent their overextension. Sialylation is the enzymatic process of transferring SA residues onto polysaccharides and is catalyzed by a group of enzymes known as sialyltransferases (SiaTs). It is noteworthy that the sialylation level of glycoproteins is significantly altered when digestive cancer occurs. And this alteration exhibits a close correlation with the progression of these cancers. In this review, from the perspective of altered SiaTs expression levels and changed glycoprotein sialylation patterns, we summarize the pathogenesis of gastric cancer (GC), colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), and hepatocellular carcinoma (HCC). Furthermore, we propose potential early diagnostic biomarkers and prognostic indicators for different digestive cancers. Finally, we summarize the therapeutic value of sialylation in digestive system cancers.

Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2.

The overexpression of sialic acids on glycans, called hypersialylation, is a common alteration found in cancer cells. Sialylated glycans can enhance immune evasion by interacting with sialic acid-binding immunoglobulin-like lectin (Siglec) receptors on tumor-infiltrating immune cells. Here, we investigated the effect of sialylated glycans and their interaction with Siglec receptors on myeloid-derived suppressor cells (MDSCs). We found that MDSCs derived from the blood of lung cancer patients and tumor-bearing mice strongly express inhibitory Siglec receptors and are highly sialylated. In murine cancer models of emergency myelopoiesis, Siglec-E knockout in myeloid cells resulted in prolonged survival and increased tumor infiltration of activated T cells. Targeting suppressive myeloid cells by blocking Siglec receptors or desialylation strongly reduced their suppressive potential. We further identified CCL2 as a mediator involved in T-cell suppression upon interaction between sialoglycans and Siglec receptors on MDSCs. Our results demonstrated that sialylated glycans inhibit anticancer immunity by modulating CCL2 expression.

Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter.

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the 'elevator-with-an-operator' mechanism of TRAP transporters.

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.

Sialic acids cleavage induced by elastin-derived peptides impairs the interaction between insulin and its receptor in adipocytes 3T3-L1.

The insulin receptor (IR) plays an important role in insulin signal transduction, the defect of which is believed to be the root cause of type 2 diabetes. In 3T3-L1 adipocytes as in other cell types, the mature IR is a heterotetrameric cell surface glycoprotein composed of two α subunits and two ß subunits. Our objective in our study, is to understand how the desialylation of N-glycan chains, induced by elastin-derived peptides, plays a major role in the function of the IR. Using the 3T3-L1 adipocyte line, we show that removal of the sialic acid from N-glycan chains (N893 and N908), induced by the elastin receptor complex (ERC) and elastin derived-peptides (EDPs), leads to a decrease in the autophosphorylation activity of the insulin receptor. We demonstrate by molecular dynamics approaches that the absence of sialic acids on one of these two sites is sufficient to generate local and general modifications of the structure of the IR. Biochemical approaches highlight a decrease in the interaction between insulin and its receptor when ERC sialidase activity is induced by EDPs. Therefore, desialylation by EDPs is synonymous with a decrease of IR sensitivity in adipocytes and could thus be a potential source of insulin resistance associated with diabetic conditions.

Strain-specific requirements of susceptibility to rhesus enteric calicivirus infection.

Recently, we identified the coxsackie and adenovirus receptor (CAR) as the entry receptor for rhesus enteric calicivirus (ReCV) isolate FT285 and demonstrated that co-expression of the CAR and the type B histo-blood group antigen (HBGA) is required to convert the resistant CHO cell line susceptible to infection. To address whether the CAR is also the functional entry receptor for other ReCV isolates and the requirement for specific HBGAs or other glycans, here we used a panel of recombinant CHO cell lines expressing the CAR and the type A, B, or H HBGAs alone or in combination. Infection studies with three diverse ReCV strains, the prototype GI.1 Tulane virus (TV), GI.2 ReCV-FT285, and GI.3 ReCV-FT7, identified that cell surface expression of the CAR is an absolute requirement for all three strains to promote susceptibility to infection, while the requirement for HBGAs varies among the strains. In addition to the CAR, ReCV-FT285 and TV require type A or B HBGAs for infection. In the absence of HBGAs, TV, but not Re-CV FT285, can also utilize sialic acids, while ReCV-FT7 infection is HBGA-independent and relies on CAR and sialic acid expression. In summary, we demonstrated strain-specific diversity of susceptibility requirements for ReCV infections and that CAR, type A and B HBGA, and sialic acid expression control susceptibility to infection with the three ReCV isolates studied. Our study also indicates that the correlation between in vitro HBGA binding and HBGAs required for infection is relatively high, but not absolute. This has direct implications for human noroviruses.IMPORTANCEHuman noroviruses (HuNoVs) are important enteric pathogens. The lack of a robust HuNoV cell culture system is a bottleneck for HuNoV cell culture-based studies. Often, cell culture-adapted caliciviruses that rapidly replicate in conventional cell lines and recapitulate biological features of HuNoVs are utilized as surrogates. Particularly, rhesus enteric caliciviruses (ReCVs) display remarkable similarities, including the primate host, clinical manifestation of gastroenteritis, genetic/antigenic diversity, and reliance on histo-blood group antigens (HBGAs) for attachment. While the HuNoV entry receptor(s) is unknown, the coxsackie and adenovirus receptor (CAR) has recently been identified as the ReCV entry receptor. Here, we identified the CAR, the type A and B HBGAs, and sialic acids as critical cell surface molecules controlling susceptibility to ReCV infections. The CAR is required for all ReCV isolates studied. However, the requirement for the different carbohydrate molecules varies among different ReCV strains. Our findings have direct implications for HuNoVs.

Understanding pathophysiology of GNE myopathy and current progress towards drug development.

GNE myopathy is a rare genetic neuromuscular disease that is caused due to mutations in the GNE gene responsible for sialic acid biosynthesis. Foot drop is the most common initial symptom observed in GNE myopathy patients. There is slow progressive muscle weakness in the lower and upper extremities while the quadriceps muscles are usually spared. The exact pathophysiology of the disease is unknown. Besides sialic acid biosynthesis, recent studies suggest either direct or indirect involvement of GNE in other cellular functions such as protein aggregation, apoptosis, ER stress, cell migration, HSP70 chaperone activity, autophagy, muscle atrophy, and myogenesis. Both animal and in vitro cell-based model systems are generated to elucidate the mechanism of GNE myopathy and evaluate the efficacy of therapies. The many therapeutic avenues explored include supplementation with sialic acid derivatives or precursors and gene therapy. Recent studies suggest other therapeutic options such as modulators of HSP70 chaperone (BGP-15), cofilin activator (CGA), and ligands like IGF-1 that may help to rescue cellular defects due to GNE dysfunction. This review provides an overview of the pathophysiology associated with GNE function in the cell and promising therapeutic leads to be explored for future drug development.

Bacterial Enzyme Assay for Mucin Glycan Degradation.

Bacterial sialidase and sulfoglycosidase may act on the acidic modifications of mucin O-glycans, producing sialic acid and 6-sulfated N-acetylglucosamine, respectively. Assays for these enzymes, using mucin as a substrate, are enabled by the detection and/or quantification of the free monosaccharides that are released by these enzymes. This chapter describes enzyme reactions with mucin, detection by thin-layer chromatography of sialic acid, and quantification of 6-sulfated N-acetylglucosamine by liquid chromatography-tandem mass spectrometry.

The artificial amino acid change in the sialic acid-binding domain of the hemagglutinin neuraminidase of newcastle disease virus increases its specificity to HCT 116 colorectal cancer cells and tumor suppression effect.

BACKGROUND: Oncolytic viruses are being studied and developed as novel cancer treatments. Using directed evolution technology, structural modification of the viral surface protein increases the specificity of the oncolytic virus for a particular cancer cell. Newcastle disease virus (NDV) does not show specificity for certain types of cancer cells during infection; therefore, it has low cancer cell specificity. Hemagglutinin is an NDV receptor-binding protein on the cell surface that determines host cell tropism. NDV selectivity for specific cancer cells can be increased by artificial amino acid changes in hemagglutinin neuraminidase HN proteins via directed evolution, leading to improved therapeutic effects. METHODS: Sialic acid-binding sites (H domains) of the HN protein mutant library were generated using error-prone PCR. Variants of the H domain protein were screened by enzyme-linked immunosorbent assay using HCT 116 cancer cell surface molecules. The mutant S519G H domain protein showed the highest affinity for the surface protein of HCT 116 cells compared to that of different types of cancer cells. This showed that the S519G mutant H domain protein gene replaced the same part of the original HN protein gene, and S519G mutant recombinant NDV (rNDV) was constructed and recovered. S519G rNDV cancer cell killing effects were tested using the MTT assay with various cancer cell types, and the tumor suppression effect of the S519G mutant rNDV was tested in a xenograft mouse model implanted with cancer cells, including HCT 116 cells. RESULTS: S519G rNDV showed increased specificity and enhanced killing ability of HCT 116 cells among various cancer cells and a stronger suppressive effect on tumor growth than the original recombinant NDV. Directed evolution using an artificial amino acid change in the NDV HN (S519G mutant) protein increased its specificity and oncolytic effect in colorectal cancer without changing its virulence. CONCLUSION: These results provide a new methodology for the use of directed evolution technology for more effective oncolytic virus development.

GNE deficiency impairs Myogenesis in C2C12 cells and cannot be rescued by ManNAc supplementation.

GNE myopathy (GNEM) is a late-onset muscle atrophy, caused by mutations in the gene for the key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). With an incidence of one to nine cases per million it is an ultra-rare, so far untreatable, autosomal recessive disease. Several attempts have been made to treat GNEM patients by oral supplementation with sialic acid precursors (e.g. N-acetylmannosamine, ManNAc) to restore sarcolemmal sialylation and muscle strength. In most studies, however, no significant improvement was observed. The lack of a suitable mouse model makes it difficult to understand the exact pathomechanism of GNEM and many years of research have failed to identify the role of GNE in skeletal muscle due to the lack of appropriate tools. We established a CRISPR/Cas9-mediated Gne-knockout cell line using murine C2C12 cells to gain insight into the actual role of the GNE enzyme and sialylation in a muscular context. The main aspect of this study was to evaluate the therapeutic potential of ManNAc and N-acetylneuraminic acid (Neu5Ac). Treatment of Gne-deficient C2C12 cells with Neu5Ac, but not with ManNAc, showed a restoration of the sialylation level back to wild type levels-albeit only with long-term treatment, which could explain the rather low therapeutic potential. We furthermore highlight the importance of sialic acids on myogenesis, for C2C12 Gne-knockout myoblasts lack the ability to differentiate into mature myotubes.