Evaluation of SAT-1, SAT-2 and GalNAcT-1 mRNA in colon cancer by real-time PCR

By qualitative and quantitative PCR, we evaluated the expression of three messengers coding for SAT-1, SAT-2 and GalNAcT-1 in human samples of intestinal cancer and some cell lines (breast cancer and melanomas). Qualitative PCR demonstrated, in human tissues but not in the cell lines examined, the presence of an mRNA that lacks hexon 3; experiments performed on transfected SKMEL-28 excluded a regulative role of this noncanonical mRNA. Data from real-time PCR, statistically analysed by ANOVA indicated that the mRNA expression of all the considered glycosyltransferases (SAT-1, SAT-2 and GalNAcT-1) was significantly different in tumours versus their own control. The ganglioside patterns in the examined samples did not correlate with mRNA expression; this finding demonstrates that ganglioside expression is the result of a very complex balance between anabolic and catabolic enzyme activities. Although this study is still preliminary, it opens a new possibility for neoplastic prognosis finding potential molecular markers among the mRNAs that codify for glycosyltransferases.     

人体肠道菌群来源碳水化合物活性酶(CAZYmes)研究进展

肠道菌群是人体重要的代谢"器官",对人体的健康和疾病起着至关重要的作用.肠道菌群参与人体消化、免疫、神经系统调节机能的分子机理是特异性物质代谢通路在微生物与人体之间的协同耦合.酶是代谢通路中参与物质转化的基本功能单元,深入理解肠道菌群编码酶的分子催化机理将为以肠道菌群(或肠道酶)作为靶点的精准营养/医疗干预研究提供重要理论依据.特异性底物酶解研究表明,肠道菌群编码的酶系统不仅包含全部已知的碳水化合物活性酶(carbohydrateactive enzymes, CAZYmes)类,同时蕴含诸多潜在的新型CAZYmes.本文阐述CAZYmes的分类原则及催化机理,并主要从结构生物学方面综述人体肠道菌群来源的新型CAZYmes.     

Epigenetic Regulation of a Brain-specific Glycosyltransferase N-Acetylglucosaminyltransferase-IX (GnT-IX) by Specific Chromatin Modifiers

Expression of glycosyltransferase genes is essential for glycosylation. However, the detailed mechanisms of how glycosyltransferase gene expression is regulated in a specific tissue or during disease progression are poorly understood. In particular, epigenetic studies of glycosyltransferase genes are limited, although epigenetic mechanisms, such as histone and DNA modifications, are central to establish tissue-specific gene expression. We previously found that epigenetic histone activation is essential for brain-specific expression of N-acetylglucosaminyltransferase-IX (GnT-IX, also designated GnT-Vb), but the mechanism of brain-specific chromatin activation around GnT-IX gene (Mgat5b) has not been clarified. To reveal the mechanisms regulating the chromatin surrounding GnT-IX, we have investigated the epigenetic factors that are specifically involved with the mouse GnT-IX locus by comparing their involvement with other glycosyltransferase loci. We first found that a histone deacetylase (HDAC) inhibitor enhanced the expression of GnT-IX but not of other glycosyltransferases tested. By overexpression and knockdown of a series of HDACs, we found that HDAC11 silenced GnT-IX. We also identified the O-GlcNAc transferase (OGT) and ten-eleven translocation-3 (TET3) complex as a specific chromatin activator of GnT-IX gene. Moreover, chromatin immunoprecipitation (ChIP) analysis in combination with OGT or TET3 knockdown showed that this OGT-TET3 complex facilitates the binding of a potent transactivator, NeuroD1, to the GnT-IX promoter, suggesting that epigenetic chromatin activation by the OGT-TET3 complex is a prerequisite for the efficient binding of NeuroD1. These results reveal a new epigenetic mechanism of brain-specific GnT-IX expression regulated by defined chromatin modifiers, providing new insights into the tissue-specific expression of glycosyltransferases.     

Glycosyltransferase-specific Golgi-targeting Mechanisms

Glycosylation of secreted and membrane-bound mucins is carried out by glycosyltransferases localized to specific Golgi compartments according to the step in which each enzyme participates. However, the Golgi-targeting mechanisms of these enzymes are not clear. Herein, we investigate the Golgi-targeting mechanisms of core 1 β3 galactosyltransferase (C1GalT1) and core 2 β1,6-N-acetylglucosaminyltransferase-2 or mucus type (C2GnT-M), which participate in the early O-glycosylation steps. siRNAs, co-immunoprecipitation, and confocal fluorescence microscopy were employed to identify the golgins involved in the Golgi docking of vesicular complexes (VCs) that carry these two enzymes. We have found that these VCs use different golgins for docking: C2GnT-M-carrying VC (C2GnT-M-VC) utilizes Giantin, whereas C1GalT1-VC employs GM130-GRASP65 complex. However, in the absence of GRASP65, C1GalT1-VC utilizes GM130-Giantin complex. Also, we have found that these VCs are 1.1–1.2 μm in diameter, specific for each enzyme, and independent of coat protein complex II and I (COPII and COPI). These two fluorescently tagged enzymes exhibit different fluorescence recovery times in the Golgi after photobleaching. Thus, novel enzyme-specific Golgi-targeting mechanisms are employed by glycosyltransferases, and multiple Golgi docking strategies are utilized by C1GalT1.     

Family 6 Glycosyltransferases in Vertebrates and Bacteria: Inactivation and Horizontal Gene Transfer May Enhance Mutualism between Vertebrates and Bacteria

Glycosyltransferases (GTs) control the synthesis and structures of glycans. Inactivation and intense allelic variation in members of the GT6 family generate species-specific and individual variations in carbohydrate structures, including histo-blood group oligosaccharides, resulting in anti-glycan antibodies that target glycan-decorated pathogens. GT6 genes are ubiquitous in vertebrates but are otherwise rare, existing in a few bacteria, one protozoan, and cyanophages, suggesting lateral gene transfer. Prokaryotic GT6 genes correspond to one exon of vertebrate genes, yet their translated protein sequences are strikingly similar. Bacterial and phage GT6 genes influence the surface chemistry of bacteria, affecting their interactions, including those with vertebrate hosts.     

Aberrant expression of N-acetylglucosaminyltransferase-IVa and IVb (GnT-IVa and b) in pancreatic cancer

The goal of this study was to identify glycosyltransferases that are specifically expressed in pancreatic cancer. To investigate the gene expression of glycosyltransferases between pancreatic cancer and normal pancreatic tissues, we performed DNA-microarray (involving about 1000 oligosaccharide-related genes) using RNA mixtures of pancreatic cancer cells and normal pancreatic tissues. Eighty-six genes were up-regulated and thirty-two were down-regulated in pancreatic cancer, compared to normal pancreatic tissue. Among these changes, it is noteworthy that the expression of GnT-IVa was decreased and the expression of GnT-IVb was increased in pancreatic cancer, compared to normal pancreatic tissues. Although GnT-IVa and -IVb are involved in the same reaction as a glycosyltransferase, their chromosomal localization is different. When 5 cases of pancreatic cancer tissues were examined using the real-time RT-PCR method, the expression of GnT-IVb was dominant in tumor tissues and the expression of GnT-IVa was dominant in the surrounding normal tissues. The expression of GnT-IVa was increased in all 3 cell lines that had been treated with 5-aza-C and butyrate. These results suggest that the down-regulation of GnT-IVa in pancreatic cancer cells is due to an epigenetic abnormality in the gene.     

Golgi N-Glycosyltransferases Form Both Homo- and Heterodimeric Enzyme Complexes in Live Cells

Glycans (i.e. oligosaccharide chains attached to cellular proteins and lipids) are crucial for nearly all aspects of life, including the development of multicellular organisms. They come in multiple forms, and much of this diversity between molecules, cells, and tissues is generated by Golgi-resident glycosidases and glycosyltransferases. However, their exact mode of functioning in glycan processing is currently unclear. Here we investigate the supramolecular organization of the N-glycosylation pathway in live cells by utilizing the bimolecular fluorescence complementation approach. We show that all four N-glycosylation enzymes tested (β-1,2-N-acetylglucosaminyltransferase I, β-1,2-N-acetylglucosaminyltransferase II, 1,4-galactosyltransferase I, and α-2,6-sialyltransferase I) form Golgi-localized homodimers. Intriguingly, the same enzymes also formed two distinct and functionally relevant heterodimers between the medial Golgi enzymes β-1,2-N-acetylglucosaminyltransferase I and β-1,2-N-acetylglucosaminyltransferase II and the trans-Golgi enzymes 1,4-galactosyltransferase I and α-2,6-sialyltransferase I. Given their strict Golgi localization and sequential order of function, the two heterodimeric complexes are probably responsible for the processing and maturation of N-glycans in live cells.     

Galectin-1 as a fusion partner for the production of soluble and folded human β-1,4-galactosyltransferase-T7 in E. coli

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of a large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, β-1,4-galactosyltransferase-7 (β4Gal-T7), in E. coli. The enzyme β4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, β4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6× His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-β4Gal-T7 fusion protein, the unique protease cleavage site allows the protein β4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded β4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.     

Base-modified Donor Analogues Reveal Novel Dynamic Features of a Glycosyltransferase

Glycosyltransferases (GTs) are enzymes that are involved, as Nature''s “glycosylation reagents,” in many fundamental biological processes including cell adhesion and blood group biosynthesis. Although of similar importance to that of other large enzyme families such as protein kinases and proteases, the undisputed potential of GTs for chemical biology and drug discovery has remained largely unrealized to date. This is due, at least in part, to a relative lack of GT inhibitors and tool compounds for structural, mechanistic, and cellular studies. In this study, we have used a novel class of GT donor analogues to obtain new structural and enzymological information for a representative blood group GT. These analogues interfere with the folding of an internal loop and the C terminus, which are essential for catalysis. Our experiments have led to the discovery of an entirely new active site folding mode for this enzyme family, which can be targeted in inhibitor development, similar to the DFG motif in protein kinases. Taken together, our results provide new insights into substrate binding, dynamics, and utilization in this important enzyme family, which can very likely be harnessed for the rational development of new GT inhibitors and probes.