Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis.

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.

Core2 O-glycan structure is essential for the cell surface expression of sucrase isomaltase and dipeptidyl peptidase-IV during intestinal cell differentiation.

Alterations in glycosylation play an important role during intestinal cell differentiation. Here, we compared expression of mucin-type O-glycan synthases from proliferating and differentiated HT-29 and Caco-2 cells. Mucin-type O-glycan structures were analyzed at both stages by mass spectrometry. Core2 ß1,6-N-acetylglucosaminyltransferase-2 (C2GnT-2) was markedly increased in differentiated HT-29 and Caco-2 cells, but the core3 structure was hardly detectable. To determine whether such differential expression of mucin-type O-glycan structures has physiological significance in intestinal cell differentiation, expression of sucrase isomaltase (SI) and dipeptidyl-peptidase IV (DPP-IV), two well known intestinal differentiation markers, was examined. Interestingly, the fully glycosylated mature form of SI was decreased in C2GnT-2 knock-out mice but not in core2 N-acetylglucosaminyltransferase-3 (C2GnT-3) nulls. In addition, expression of SI and DPP-IV was dramatically reduced in C2GnT-1-3 triple knock-out mice. These patterns were confirmed by RNAi analysis; C2GnT-2 knockdown significantly reduced cell surface expression of SI and DPP-IV in Caco-2 cells. Similarly, overexpression of the core3 structure in HT-29 cells attenuated cell surface expression of both enzymes. These findings indicate that core3 O-glycan structure regulates cell surface expression of SI and DPP-IV and that core2 O-glycan is presumably an essential mucin-type O-glycan structure found in both molecules in vivo. Finally, goblet cells in the upper part of the crypt showed impaired maturation in the core2 O-glycan-deficient mice. These studies are the first to clearly identify functional mucin-type O-glycan structures modulating cell surface expression of SI and DPP-IV during the intestinal cell differentiation.

Core3 O-glycan synthase suppresses tumor formation and metastasis of prostate carcinoma PC3 and LNCaP cells through down-regulation of alpha2beta1 integrin complex.

Although there are numerous reports of carbohydrates enriched in cancer cells, very few studies have addressed the functions of carbohydrates present in normal cells that decrease in cancer cells. It has been reported that core3 O-glycans are synthesized in normal gastrointestinal cells but are down-regulated in cancer cells. To determine the roles of core3 O-glycans, we transfected PC3 and LNCaP prostate cancer cells with beta3-N-acetylglucosaminyltransferase-6 (core3 synthase) required to synthesize core3 O-glycans. Both engineered cell lines exhibited reduced migration and invasion through extracellular matrix components compared with mock-transfected cells. Moreover we found that alpha2beta1 integrin acquired core3 O-glycans in cells expressing core3 synthase with decreased maturation of beta1 integrin, leading to decreased levels of the alpha2beta1 integrin complex, decreased activation of focal adhesion kinase, and reduced lamellipodia formation. Upon inoculation into the prostate of nude mice, PC3 cells expressing core3 O-glycans produced much smaller tumors without metastasis to the surrounding lymph nodes in contrast to robust tumor formation and metastasis seen in mock-transfected PC3 cells. Similarly LNCaP cells expressing core3 O-glycans barely produced subcutaneous tumors in contrast to robust tumor formation by mock-transfected LNCaP cells. These findings indicate that addition of core3 O-glycans to beta1 and alpha2 integrin subunits in prostate cancer cells suppresses tumor formation and tumor metastasis.