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Assessing liver fibrosis is important for predicting the efficacy of antiviral therapy and patient prognosis. Liver biopsy is the gold standard for diagnosing liver fibrosis, despite its invasiveness and problematic diagnostic accuracy. Although noninvasive techniques to assess liver fibrosis are becoming important, reliable serum surrogate markers are not available. A glycoproteomics study aimed at identifying such markers discovered Mac 2-Binding Protein Gylcan Isomer (M2BPGi), which is a reliable marker for assessing liver fibrosis in patients with viral hepatitis and other fibrotic liver diseases such as primary biliary cholangitis, biliary atresia, autoimmune hepatitis, and nonalcoholic fatty liver disease. M2BPGi predicts the development of hepatocellular carcinoma (HCC) in patients infected with hepatitis B and C as well as the prognosis of liver cirrhosis in those with HCC after therapy. The unique features of M2BPGi are as follows: (1) cut-off values differ for the same stages of fibrosis according to the cause of fibrosis; and (2) M2BPGi levels rapidly decrease after patients achieve a sustained antiviral response to hepatitis C virus. These observations cannot be explained if M2BPGi levels reflect the amount of fibrotic tissue. Hepatic stellate cells (HSCs) secrete M2BPGi, which may serve as a messenger between HSCs and Kupffer cells via Mac-2 (galectin 3) that is expressed in Kupffer cells during fibrosis progression. Here we show that M2BPGi is a surrogate marker for assessing HSC activation. These findings may reveal the roles of HSCs in extrahepatic fibrotic disease progression.

Background Recently, a novel marker, hyperglycosylated Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA + -M2BP), was developed for liver fibrosis using the glycan “sugar chain”-based immunoassay; however, the feasibility of WFA + -M2BP for assessing liver fibrosis has not been proven with clinical samples of hepatitis. Methods Serum WFA + -M2BP values were evaluated in 200 patients with chronic liver disease who underwent histological examination of liver fibrosis. The diagnostic accuracy of WFA + -M2BP values was compared with various fibrosis markers, such as ultrasound based-virtual touch tissue quantification (VTTQ), magnetic resonance imaging based-liver-to-major psoas muscle intensity ratio (LMR), and serum markers, including hyaluronic acid, type 4 collagen, and aspartate transaminase to platelet ratio index (APRI). Results Serum WFA + -M2BP levels in patients with fibrosis grades F0, F1, F2, F3, and F4 had cutoff indices 1.62, 1.82, 3.02, 3.32, and 3.67, respectively, and there were significant differences between fibrosis stages F1 and F2, and between F2 and F3 ( P  < 0.01). The area under the receiver operating characteristic curves for the diagnosis of fibrosis ( F  ≥ 3) using serum WFA + -M2BP values (0.812) was almost comparable to that using VTTQ examination (0.814), but was superior to the other surrogate markers, including LMR index (0.766), APRI (0.694), hyaluronic acid (0.683), and type 4 collagen (0.625) ( P  < 0.01 each). Conclusions Serum WFA + -M2BP values based on a glycan-based immunoassay is an accurate, reliable, and reproducible method for the assessment of liver fibrosis. This approach could be clinically feasible for evaluation of beneficial therapy through the quantification of liver fibrosis in hepatitis patients if this measurement application is commercially realized.

Background Accurately evaluating liver fibrosis in patients with non-alcoholic fatty liver disease (NAFLD) is important for identifying those who may develop complications. The aims of this study were (1) to measure serum Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA + -M2BP) using the glycan sugar chain-based immunoassay and (2) to compare the results with clinical assessments of fibrosis. Methods Serum WFA + -M2BP values were retrospectively evaluated in 289 patients with NAFLD who had undergone liver biopsy. Histological findings were evaluated by three blinded, experienced liver-specific pathologists. Results For stages 0 ( n  = 35), 1 ( n  = 113), 2 ( n  = 49), 3 ( n  = 41), and 4 ( n  = 51) of liver fibrosis, the serum WFA + -M2BP cutoff indexes were 0.57, 0.70, 1.02, 1.57, and 2.96, respectively. Multivariate regression analysis showed that serum WFA + -M2BP values were associated with the stage of fibrosis (≥stage 2). The areas under the receiver operating characteristic curve (AUROC), sensitivity, and specificity of serum WFA + -M2BP were 0.876, 85.9, and 74.6 %, respectively, for severe fibrosis (≥stage 3) and were 0.879, 74.6, and 87.0 %, respectively, for cirrhosis. When compared with six non-invasive conventional markers, serum WFA + -M2BP had the greatest AUROC for diagnosing severe fibrosis and cirrhosis. Conclusions Serum WFA + -M2BP values are useful for assessing the stage of liver fibrosis in patients with NAFLD.

Recent advancements in proteomics technology have stimulated the widespread research and development in the area of biomarker discovery using mass spectrometry (MS). The final goal of biomarker discovery and development is to establish clinically useful and reliable diagnostic methods for various diseases. Specific alterations in the nature and composition of glycans attached to proteins are seen during the development and progression of a number of diseases and disorders. Therefore, development of glyco-biomarkers, which detect disease-specific glycoproteins and changes in glycoforms, is gaining much attention. The combined use of multiple technologies, not solely MS, is the key to the discovery of clinically significant and reliable biomarkers. We have employed the combination of quantitative real-time polymerase chain reaction (PCR), lectin microarray, liquid chromatography/mass spectrometry-based technique with isotope-coded glycosylation site-specific tagging (IGOT-LC/MS), and bioinformatics to successfully develop a novel diagnostic kit for the quantitative evaluation of liver fibrosis. Efforts to develop highly effective glyco-biomarkers for other diseases are also currently underway.

The mucin‐type sialoglycoprotein podoplanin (aggrus) is involved in tumor cell‐induced platelet aggregation and tumor metastasis. C‐type lectin‐like receptor‐2 (CLEC‐2) was recently identified as an endogenous receptor of podoplanin on platelets. However, the pathophysiological importance and function of CLEC‐2 have not been elucidated. Here we clarified the pathophysiological interaction between podoplanin and CLEC‐2 in vitro and in vivo. Using several deletion mutants of CLEC‐2 expressed as Fc chimeras, we first identified an important podoplanin‐recognition domain in CLEC‐2. Furthermore, the podoplanin–CLEC‐2 interaction was confirmed using several deletion mutants of podoplanin expressed as Fc chimeras. Not only the disialyl‐core1‐attached glycopeptide but also the stereostructure of the podoplanin protein was found to be critical for the CLEC‐2‐binding activity of podoplanin. We next synthesized various glycopeptides of podoplanin that included both the platelet aggregation‐stimulating domain and O‐glycan on Thr52. Interestingly, a disialyl‐core1‐attached glycopeptide was recognized specifically by CLEC‐2. Moreover, the anti‐podoplanin monoclonal antibody NZ‐1 suppressed both the podoplanin–CLEC‐2 interaction and podoplanin‐induced pulmonary metastasis, suggesting that CLEC‐2 is the first pathophysiological receptor of podoplanin to be identified. In summary, we clarified the molecular interaction in vitro and in vivo between a platelet aggregation‐inducing factor, podoplanin, and its specific pathophysiological receptor on platelets, CLEC‐2. Podoplanin and CLEC‐2 might represent promising therapeutic targets in cancer metastasis. (Cancer Sci 2008; 99: 54–61)

Mucin-type O -glycans are involved in cancer initiation and progression, although details of their biological and clinicopathological roles remain unclear. The aim of this study was to investigate the clinicopathological significance of β1,3- N -acetylglucosaminyltransferase 6 (β3Gn-T6), an essential enzyme for the synthesis of core 3 O -glycan and several other O -glycans in pancreatic ductal adenocarcinoma (PDAC). We performed immunohistochemical and lectin-histochemical analyses to detect the expression of β3Gn-T6 and several O -glycans in 156 cases of PDAC with pancreatic intraepithelial neoplasias (PanINs) and corresponding normal tissue samples. The T antigen, Tn antigen, sialyl Lewis X (sLeX) antigen, and sLeX on core 2 O -glycan were more highly expressed in PDAC cells than in normal pancreatic duct epithelial cells (NPDEs). Conversely, the expression of 6-sulfo N -acetyllactosamine on extended core 1 O -glycan was found in NPDEs and was low in PDAC cells. These glycan expression levels were not associated with patient outcomes. β3Gn-T6 was expressed in ~20% of PDAC cases and 30–40% of PanINs but not in NPDEs. Higher expression of β3Gn-T6 was found in PDAC cells in more differentiated adenocarcinoma cases showing significantly longer disease-free survival in both univariate and multivariate analyses. In addition, the expression of β3Gn-T6 in PDAC cells and PanINs significantly correlated with the expression of MUC5AC in these cells, suggesting that β3Gn-T6 expression is related to cellular differentiation status of the gastric foveolar phenotype. Thus, it is likely that β3Gn-T6 expression in PDAC cells is a favorable prognostic factor in PDAC patients, and that the expression of β3Gn-T6 correlates with the gastric foveolar phenotype in pancreatic carcinogenesis.

The poor prognosis of hepatocellular carcinoma (HCC) is mainly because of its high rate of metastasis. Thus, elucidation of the molecular mechanisms underlying HCC metastasis is of great significance. Glycosylation is an important post-translational modification that is closely associated with tumor progression. Altered glycosylation including the altered sialylation resulting from aberrant expression of β-galactoside α2,6 sialyltransferase 1 (ST6GAL1) has long been considered as an important feature of cancer cells. However, there is limited information on the roles of ST6GAL1 and α2,6 sialylation in HCC metastasis. Here, we found that ST6GAL1 and α2,6 sialylation were negatively correlated with the metastatic potentials of HCC cells. Moreover, ST6GAL1 overexpression inhibited migration and invasion of HCC cells in vitro and suppressed HCC metastasis in vivo. Using a metabolic labeling-based glycoproteomic strategy, we identified a list of sialylated proteins that may be regulated by ST6GAL1. In particular, an increase in α2,6 sialylation of melanoma cell adhesion molecule (MCAM) inhibited its interaction with galectin-3 and decreased its expression on cell surface. In vitro and in vivo analysis showed that ST6GAL1 exerted its function in HCC metastasis by regulating MCAM expression. Finally, we found the relative intensity of sialylated MCAM was negatively correlated with tumor malignancy in HCC patients. Taken together, these results demonstrate that ST6GAL1 may be an HCC metastasis suppressor by affecting sialylation of MCAM on cell surface, which provides a novel insight into the roles of ST6GAL1 in HCC progression and supports the functional complexity of ST6GAL1 in a cancer type- and tissue type-specific manner.

Background Wisteria floribunda agglutinin positive Mac-2-binding protein (WFA + -M2BP) is a novel serum marker of liver fibrosis identified in glycoproteomic biomarker screening studies, and its clinicopathological characteristics have yet to be elucidated sufficiently for clinical utilization. Methods We retrospectively analyzed the clinicopathology data and serum WFA + -M2BP levels in 376 hepatocellular carcinoma patients undergoing liver surgery. WFA + -M2BP was quantified in frozen serum samples collected at the time of surgery using the FastLec-Hepa method. Results Significant independent determinants of serum WFA + -M2BP levels included pathological diagnosis of cirrhosis, female gender, hepatitis C virus (HCV) infection, and liver dysfunction characteristics, such as abnormal indocyanine green retention rate at 15 min, platelet counts, albumin levels, alanine aminotransferase levels, and total bilirubin levels. Serum WFA + -M2BP levels increased with the pathological fibrosis stage and liver dysfunction severity. HCV infection significantly affected serum WFA + -M2BP levels throughout the pathological and functional progression of liver fibrosis, and the effect of gender was significant only in F4 stage patients with severe liver dysfunction. The diagnostic thresholds for cutoff index values for cirrhosis were 1.435 and 4.615 in HCV-negative and HCV-positive patients, respectively. Serum WFA + -M2BP levels at the time of operation were a significant predictor of hepatocellular carcinoma recurrence and overall survival in both HCV-negative and HCV-positive patients. Conclusions Serum WFA + -M2BP levels reflected both the pathological and functional progression of liver fibrosis comprehensively and continuously. Elevated WFA + -M2BP levels were a significant risk factor for tumor recurrence and decreased overall survival after liver surgery independent of HCV infection.

Background Serum hepatitis B surface antigen (HBsAg) is a component of both hepatitis B virus (HBV) virions and non-infectious subviral particles (SVPs). Recently, O -glycosylation of the PreS2 domain of middle HBsAg protein has been identified as a distinct characteristic of genotype C HBV virions versus SVPs. This study aimed to evaluate serum O -glycosylated HBsAg levels in patients with chronic hepatitis B (CHB) treated with nucleos(t)ide analogs (NAs). Methods Forty-seven treatment-naïve patients with genotype C CHB were retrospectively enrolled. Serum O -glycosylated HBsAg levels at baseline and after 48 weeks of NA therapy were quantified by immunoassay using a monoclonal antibody against the O -glycosylated PreS2 domain of middle HBsAg, and their correlations with conventional HBV marker levels were analyzed. Results At baseline, the serum O -glycosylated HBsAg levels were significantly correlated with the HBV DNA ( P  = 0.004), HBsAg ( P  = 0.005), and hepatitis B-core related antigen (HBcrAg, P  = 0.001) levels. Both HBV DNA and O -glycosylated HBsAg levels were decreased after 48 weeks of NA therapy. The significant correlation of the O -glycosylated HBsAg level with the HBsAg or HBcrAg level was lost in patients who achieved undetectable HBV DNA (HBsAg, P  = 0.429; HBcrAg, P  = 0.065). Immunoprecipitation assays demonstrated that HBV DNA and RNA were detected in the O -glycosylated HBsAg-binding serum fraction, and the proportion of HBV RNA increased during NA therapy ( P  = 0.048). Conclusion Serum O -glycosylated HBsAg levels change during NA therapy and may reflect combined levels of serum HBV DNA and RNA virions. An O -glycosylated HBsAg-based immunoassay may provide a novel means to monitor viral kinetics during NA therapy.