Quantitative mapping of the in vivo O-GalNAc glycoproteome in mouse tissues identifies GalNAc-T2 O-glycosites in metabolic disorder.

The family of GalNAc-Ts (GalNAcpolypeptide:N-Acetylgalactosaminyl transferases) catalyzes the first committed step in the synthesis of O-glycans, which is an abundant and biologically important protein modification. Abnormalities in the activity of individual GalNAc-Ts can result in congenital disorders of O-glycosylation (CDG) and influence a broad array of biological functions. How site-specific O-glycans regulate biology is unclear. Compiling in vivo O-glycosites would be an invaluable step in determining the function of site-specific O-glycans. We integrated chemical and enzymatic conditions that cleave O-glycosites, a higher-energy dissociation product ions-triggered electron-transfer/higher-energy collision dissociation mass spectrometry (MS) workflow and software to study nine mouse tissues and whole blood. We identified 2,154 O-glycosites from 595 glycoproteins. The O-glycosites and glycoproteins displayed consensus motifs and shared functions as classified by Gene Ontology terms. Limited overlap of O-glycosites was observed with protein O-GlcNAcylation and phosphorylation sites. Quantitative glycoproteomics and proteomics revealed a tissue-specific regulation of O-glycosites that the differential expression of Galnt isoenzymes in tissues partly contributes to. We examined the Galnt2-null mouse model, which phenocopies congenital disorder of glycosylation involving GALNT2 and revealed a network of glycoproteins that lack GalNAc-T2-specific O-glycans. The known direct and indirect functions of these glycoproteins appear consistent with the complex metabolic phenotypes observed in the Galnt2-null animals. Through this study and interrogation of databases and the literature, we have compiled an atlas of experimentally identified mouse O-glycosites consisting of 2,925 O-glycosites from 758 glycoproteins.

Characterization of Circulating Tumor Cells Using Imaging Flow Cytometry in Liver Disease Patients.

Background: Hepatocellular carcinoma (HCC) is asymptomatic at an early stage which delays its timely diagnosis and treatment. Circulating tumor cells (CTCs), derived from a primary or secondary tumor, may help in the management of HCC. Here, we evaluate and characterize CTCs in liver disease patients. Methods: In total, 65 patients, categorized into liver cirrhosis (LC) (n = 30) and HCC (n = 35), were enrolled. Using ImagestreamX MkII imaging flow cytometer, CTCs were detected and characterized using biomarker expression of EpCAM, CK, AFP, CD45, and DRAQ5 in LC and HCC patients. Results: CTCs were detected in 33/35 (94%) HCC patients and in 28/30 (93%) LC patients. In the HCC group, the number of biomarker-positive CTCs was higher in BCLC stage D when compared with others. EpCAM + CK was the most expressed biomarker on CTCs in LC versus HCC (83.3% vs. 77.14%), followed by AFP (80% vs. 65.71%), EpCAM (30% vs. 28.57%), and CK (16.6% vs. 14.28%). The EpCAM cell area was significantly associated (P value = 0.031) with the CTC-positive status. The combination biomarker expression of CTCs cell area (EpCAM, CK, and AFP) performed well with the area under the curve of 0.92, high sensitivity, and specificity in detecting early-stage and AFP-negative HCC as well as in AFP-negative LC cases. Conclusion: Enumeration and cell area of CTCs may be used as a biomarker for early detection of HCC and guiding treatment.

A novel role for GalNAc-T2 dependent glycosylation in energy homeostasis.

OBJECTIVE: GALNT2, encoding polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2), was initially discovered as a regulator of high-density lipoprotein metabolism. GalNAc-T2 is known to exert these effects through post-translational modification, i.e., O-linked glycosylation of secreted proteins with established roles in plasma lipid metabolism. It has recently become clear that loss of GALNT2 in rodents, cattle, nonhuman primates, and humans should be regarded as a novel congenital disorder of glycosylation that affects development and body weight. The role of GALNT2 in metabolic abnormalities other than plasma lipids, including insulin sensitivity and energy homeostasis, is poorly understood. METHODS: GWAS data from the UK Biobank was used to study variation in the GALNT2 locus beyond changes in high-density lipoprotein metabolism. Experimental data were obtained through studies in Galnt2-/- mice and wild-type littermates on both control and high-fat diet. RESULTS: First, we uncovered associations between GALNT2 gene variation, adiposity, and body mass index in humans. In mice, we identify the insulin receptor as a novel substrate of GalNAc-T2 and demonstrate that Galnt2-/- mice exhibit decreased adiposity, alterations in insulin signaling and a shift in energy substrate utilization in the inactive phase. CONCLUSIONS: This study identifies a novel role for GALNT2 in energy homeostasis, and our findings suggest that the local effects of GalNAc-T2 are mediated through posttranslational modification of the insulin receptor.

Differentially expressed serum host proteins in hepatitis B and C viral infections.

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infection often lead to hepatocellular carcinoma (HCC), which is mostly detected in advanced stage. Hence, its early detection is of paramount importance using a biomarker having sensitivity and specificity both. The present study highlights differentially expressed host proteins in response to HBV/HCV infection at different stages. Comparative proteomic study was done by two-dimensional gel electrophoresis followed by mass spectrometry. Sera from each of chronically infected, liver cirrhosis and HCC in HBV or HCV infection along with controls were selected. Analysis of functional association between differentially expressed proteins with viral hepatitis was extensively carried out. Forty-three differentially expressed spots (≥ 1.5 fold; P < 0.05) on two-dimensional gel electrophoresis were corresponded to 28 proteins by mass spectrometry in variable liver diseases. Haptoglobin protein levels were decreased upon disease progression to HCC due to HBV infection. The other proteins expressed differentially are ceruloplasmin, serum paraoxonase 1, retinol binding protein and leucine rich alpha 2 proteins in plasma maybe associated to HBV HCC. Whereas, upregulation of C4a/C4b showed it as a reliable marker in patients with end stage liver disease related to HCV infection. ApolipoproteinA1 levels in liver diseases in both HBV and HCV infection corresponding to healthy controls may be a common marker for early diagnosis and disease monitoring. Protein interaction studies by extensive pathway analysis using bioinformatics tools such as EnrichNet application and STRING revealed significant associations with specific infections.