O-glycosylated HBsAg peptide can induce specific antibody neutralizing HBV infection.

BACKGROUND: Hepatitis B virus (HBV), which causes hepatitis, liver cirrhosis, and hepatocellular carcinoma, is a global human health problem. HBV contains three envelope proteins, S-, M-, and L-hepatitis B surface antigen (HBsAg). We recently found that O-glycosylated M-HBsAg, reactive with jacalin lectin, is one of the primary components of HBV DNA-containing virus particles. Thus, we aimed to analyze and target the glycosylation of HBsAg. METHODS: HBsAg prepared from the serum of Japanese patients with HBV were analyzed using mass spectrometry. The glycopeptide modified with O-glycan was generated and used for immunization. The specificity of the generated antibody and the HBV infection inhibition activity was examined. RESULTS: Mass spectrometry analysis revealed that T37 and/or T38 on M-HBsAg of genotype C were modulated by ±NeuAc(α2,3)Gal(ß1,3)GalNAc. Chemically and enzymatically synthesized O-glycosylated peptide (Glyco-PS2) induced antibodies that recognize mainly PreS2 in M-HBsAg not in L-HBsAg, whereas the non-glycosylated peptide (PS2) induced antisera recognizing L-HBsAg but not O-glycosylated M-HBsAg. The removal of O-glycan from M-HBsAg partly decreased the reactivity of the Glyco-PS2 antibody, suggesting that peptide part was also recognized by the antibody. The antibody further demonstrated the inhibition of HBV infection in human hepatic cells in vitro. CONCLUSIONS: Glycosylation of HBsAg occurs differently in different HBsAgs in a site-specific manner. The new Glyco-PS2 antibody, recognizing O-glycosylated M-HBsAg of genotype C, could inhibit HBV infection. GENERAL SIGNIFICANCE: The detailed analysis of HBsAg identified different glycosylations of HBV surface. The glycosylated peptide based on mass spectrometry analysis showed higher potential to induce functional antibody against HBV.

Products of Chemoenzymatic Synthesis Representing MUC1 Tandem Repeat Unit with T-, ST- or STn-antigen Revealed Distinct Specificities of Anti-MUC1 Antibodies.

Anti-mucin1 (MUC1) antibodies have long been used clinically in cancer diagnosis and therapy and specific bindings of some of them are known to be dependent on the differential glycosylation of MUC1. However, a systematic comparison of the binding specificities of anti-MUC1 antibodies was not previously conducted. Here, a total of 20 glycopeptides including the tandem repeat unit of MUC1, APPAHGVTSAPDTRPAPGSTAPPAHGV with GalNAc (Tn-antigen), Galß1-3GalNAc (T-antigen), NeuAcα2-3Galß1-3GalNAc (sialyl-T-antigen), or NeuAcα2-6GalNAc (sialyl-Tn-antigen) at each threonine or serine residue were prepared by a combination of chemical glycopeptide synthesis and enzymatic extension of carbohydrate chains. These glycopeptides were tested by the enzyme-linked immunosorbent assay (ELISA) for their capacity to bind 13 monoclonal antibodies (mAbs) known to be specific for MUC1. The results indicated that anti-MUC1 mAbs have diverse specificities but can be classified into a few characteristic groups based on their binding pattern toward glycopeptides in some cases having a specific glycan at unique glycosylation sites. Because the clinical significance of some of these antibodies was already established, the structural features identified by these antibodies as revealed in the present study should provide useful information relevant to their further clinical use and the biological understanding of MUC1.

Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover.

The collagenase subfamily of matrix metalloproteinases (MMPs) have important roles in the remodeling of collagenous matrices. The proteinase-activated receptor (PAR) family has a unique mechanism of activation requiring proteolysis of an extracellular domain forming a neo-N terminus that acts as a tethered ligand, a process that has been associated with the development of arthritis. Canonical PAR2 activation typically occurs via a serine proteinase at Arg36-Ser37, but other proteinases can cleave PARs downstream of the tethered ligand and "disarm" the receptor. To identify additional cleavage sites within PAR2, we synthesized a 42-amino-acid peptide corresponding to the extracellular region. We observed that all three soluble MMP collagenases, MMP-1, MMP-8, and MMP-13, cleave PAR2 and discovered a novel cleavage site (Ser37-Leu38). Metalloproteinases from resorbing bovine nasal cartilage and recombinant human collagenases could cleave a quenched fluorescent peptide mimicking the canonical PAR2 activation region, and kinetic constants were determined. In PAR2-overexpressing SW1353 chondrocytes, we demonstrated that the activator peptide SLIGKV-NH2 induces rapid calcium flux, inflammatory gene expression (including MMP1 and MMP13), and the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase. The corresponding MMP cleavage-derived peptide (LIGKVD-NH2) exhibited no canonical activation; however, we observed phosphorylation of ERK, providing evidence of biased agonism. Importantly, we demonstrated that preincubation with active MMP-1 reduced downstream PAR2 activation by a canonical activator, matriptase, but not SLIGKV-NH2 These results support a role for collagenases as proteinases capable of disarming PAR2, revealing a mechanism that suppresses PAR2-mediated inflammatory responses.

Structural basis for specific recognition of core fucosylation in N-glycans by Pholiota squarrosa lectin (PhoSL).

Fucosylation of the N-glycan core via the α1-6 linkage (core fucosylation) is detected in specific types of cancers and related diseases, and thereby serves for a relevant biomarker. The lectin from a mushroom Pholiota squarrosa (PhoSL) shows a clear specificity to core fucosylation, without recognizing those with other types of fucosylation, such as the H type via the α1-2 linkage or the Lewis type via the α1-3 or α1-4 linkage. Here we determined the crystal structure of the PhoSL trimer in complex with a disaccharide fucose(α1-6)N-acetylglucosamine (GlcNAc). In the three sugar-binding pockets of PhoSL, extensive hydrophobic and hydrogen-bonding contacts were formed with the fucose moiety. In contrast, the GlcNAc moiety showed only a few hydrophobic and hydrogen-bonding contacts. To elucidate the mechanism for the specificity, we performed molecular dynamics simulations on this disaccharide and a trisaccharide fucose(α1-6)[GlcNAc(ß1-4)]GlcNAc in complex with PhoSL. It was observed that the GlcNAc corresponding to the outer one of the N-glycan core entered the sugar-binding pocket with the N-acetyl group placed stably at the bottom, forming extensive hydrophobic and hydrogen-bonding interactions. In addition, these glycans adopted unstressed favorable conformations when bound to PhoSL. In contrast, H- and Lewis-types of fucosylated trisaccharides adopting favorable conformations caused inevitable steric hindrance with the steep edge of the binding pocket, when docked with PhoSL. Therefore, the specificity to core fucosylation of PhoSL was achieved by a combination of these preferential and exclusive mechanisms.

p62/SQSTM1 plays a protective role in oxidative injury of steatotic liver in a mouse hepatectomy model.

AIMS: Liver injury and regeneration involve complicated processes and are affected by various physio-pathological factors. We investigated the mechanisms of steatosis-associated liver injury and delayed regeneration in a mouse model of partial hepatectomy. RESULTS: Initial regeneration of the steatotic liver was significantly delayed after hepatectomy. Although hepatocyte proliferation was not significantly suppressed, severe liver injury with oxidative stress (OS) occurred immediately after hepatectomy in the steatotic liver. Fas-ligand (FasL)/Fas expression was upregulated in the steatotic liver, whereas the expression of antioxidant and anti-apoptotic molecules (catalase/MnSOD/Ref-1 and Bcl-2/Bcl-xL/FLIP, respectively) and p62/SQSTM1, a steatosis-associated protein, was downregulated. Interestingly, pro-survival Akt was not activated in response to hepatectomy, although it was sufficiently expressed even before hepatectomy. Suppression of p62/SQSTM1 increased FasL/Fas expression and reduced nuclear factor erythroid 2-related factor-2 (Nrf-2)-dependent antioxidant response elements activity and antioxidant responses in steatotic and nonsteatotic hepatocytes. Exogenously added FasL induced severe cellular OS and necrosis/apoptosis in steatotic hepatocytes, with only the necrosis being inhibited by pretreatment with antioxidants, suggesting that FasL/Fas-induced OS mainly leads to necrosis. Furthermore, p62/SQSTM1 re-expression in the steatotic liver markedly reduced liver injury and improved liver regeneration. INNOVATION: This study is the first which demonstrates that reduced expression of p62/SQSTM1 plays a crucial role in posthepatectomy acute injury and delayed regeneration of steatotic liver, mainly via redox-dependent mechanisms. CONCLUSION: In the steatotic liver, reduced expression of p62/SQSTM1 induced FasL/Fas overexpression and suppressed antioxidant genes, mainly through Nrf-2 inactivation, which, along with the hypo-responsiveness of Akt, caused posthepatectomy necrotic/apoptotic liver injury and delayed regeneration, both mainly via a redox-dependent mechanism.