Human glycogene cloning: focus on beta 3-glycosyltransferase and beta 4-glycosyltransferase families

Glycogenes encode proteins involved in glycan synthesis, such as glycosyltransferases, sulfotransferases and sugar-nucleotide transporters. The comprehensive identification and functional analysis of human glycogenes has been ongoing for some time. During the past 20 years, 183 human glycogenes have been cloned and their substrate specificities analyzed. All current information on these human glycogenes and their links with orthologous genes in other species is summarized in the GlycoGene database. In recent years, two glycogene families, beta3-glycosyltransferases and beta4-glycosyltransferases, have been identified and analyzed in particular detail.     

Bioinformatics for comprehensive finding and analysis of glycosyltransferases

Bioinformatics is a very powerful tool in the field of glycoproteomics as well as genomics and proteomics. As a part of the Glycogene Project (GG project), we have developed a novel bioinformatics system for the comprehensive identification and in silico cloning of human glycogenes. Using our system, a total of 105 candidate human glycogenes were identified and then engineered for heterologous expression. Of these candidates, 38 recombinant proteins were successfully identified for their enzyme activity and substrate specificity. We also classified 47 out of 60 carbohydrate-active enzyme glycosyltransferase families into 4 superfamilies using the profile Hidden Markov Model method. On the basis of our classification and the relationship between glycosylation pathways and superfamilies, we propose the evolution of glycosyltransferases.     

Custom microarray for glycobiologists: considerations for glycosyltransferase gene expression profiling

The development of microarray technology offers the unprecedented possibility of studying the expression of thousands of genes in one experiment. Its exploitation in the glycobiology field will eventually allow the parallel investigation of the expression of many glycosyltransferases, which will ultimately lead to an understanding of the regulation of glycoconjugate synthesis. While numerous gene arrays are available on the market, e.g. the Affymetrix GeneChip arrays, glycosyltransferases are not adequately represented, which makes comprehensive surveys of their gene expression difficult. This chapter describes the main issues related to the establishment of a custom glycogenes array.     

Cloning and nucleotide sequences of the homoserine dehydrogenase genes (hom) and the threonine synthase genes (thrC) of the gram-negative obligate methylotroph Methylobacillus glycogenes.

We have cloned the homoserine dehydrogenase genes (hom) from the gram-negative obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and ATCC 21371 by complementation of an Escherichia coli homoserine dehydrogenase-deficient mutant. The 4.15-kb DNA fragment cloned from M. glycogenes ATCC 21371 also complemented an E. coli threonine synthase-deficient mutant, suggesting the DNA fragment contained the thrC gene in addition to the hom gene. The homoserine dehydrogenases expressed in the E. coli recombinants were hardly inhibited by L-threonine, L-phenylalanine, or L-methionine. However, they became sensitive to the amino acids after storage at 4 degrees C for 4 days as in M. glycogenes. The structures of the homoserine dehydrogenases overexpressed in E. coli were thought to be different from those in M. glycogenes, probably in subunit numbers of the enzyme, and were thought to have converted to the correct structures during the storage. The nucleotide sequences of the hom and thrC genes were determined. The hom genes of M. glycogenes ATCC 21276 and ATCC 21371 encode peptides with M(r)s of 48,225 and 44,815, respectively. The thrC genes were located 50 bp downstream of the hom genes. The thrC gene of ATCC 21371 encodes a peptide with an M(r) of 52,111, and the gene product of ATCC 21276 was truncated. Northern (RNA) blot analysis suggests that the hom and thrC genes are organized in an operon. Significant homology between the predicted amino acid sequences of the hom and thrC genes and those from other microorganisms was found.     

Activation of human endothelial cells by tumor necrosis factor-alpha results in profound changes in the expression of glycosylation-related genes

The endothelium plays a central role in the logistics of the immune system by allowing the selective transmigration of leukocytes, as well as the maintenance of the circulation and coagulation homeostasis. Evidence is increasing that the carbohydrate composition of the endothelial cell surface is critical for the cells to exert their physiological function. The major aim of this study is to unravel the mechanisms underlying the expression of carbohydrate structures by endothelial cells, which are involved in leukocyte adhesion and migration. Using quantitative real-time PCR, the expression profile of a selected group of 74 glycosylation-related genes has been determined in human umbilical vein endothelial cells (HUVEC) and human foreskin microvascular endothelial cells (FMVEC) under control and TNFalpha-induced conditions. The set of genes comprised 59 glycosyltransferases, 6 mannosidases and 9 sulfotransferases. In parallel, the overall cell surface glycan profile has been assessed by the use of glycan-specific lectins and monoclonal antibodies. The results demonstrate that HUVEC and FMVEC differ substantially in the expression of glycosylation-related genes and, accordingly, also in the presence of different glycan epitopes on the cell membrane. Induction of an inflamed phenotype of the cells by treatment with TNFalpha differentially modulates a set of these genes in HUVEC and FMVEC resulting in a change in the cell membrane associated glycans that are of importance in inflammation-related endothelial cell-surface processes.     

Insights into miRNA regulation of the human glycome

Glycosylation is an intricate process requiring the coordinated action of multiple proteins, including glycosyltransferases, glycosidases, sugar nucleotide transporters and trafficking proteins. Work by several groups points to a role for microRNA (miRNA) in controlling the levels of specific glycosyltransferases involved in cancer, neural migration and osteoblast formation. Recent work in our laboratory suggests that miRNA are a principal regulator of the glycome, translating genomic information into the glycocode through tuning of enzyme levels. Herein we overlay predicted miRNA regulation of glycosylation related genes (glycogenes) onto maps of the common N-linked and O-linked glycan biosynthetic pathways to identify key regulatory nodes of the glycome. Our analysis provides insights into glycan regulation and suggests that at the regulatory level, glycogenes are non-redundant.     

Essential roles of carbohydrate signals in development, immune response and tissue functions, as revealed by gene targeting

Knockout mice lacking glycosyltransferases or sulfotransferases provide unequivocal evidence that the carbohydrate moieties of glycoproteins, glycolipids, and proteoglycans play essential roles in various biological phenomena such as development, the immune response, and tissue functions. Examples of abnormalities of null mutants include arrest of embryogenesis due to deletion of N-acetylglucosaminyltransferase I or glucosylceramide synthase, failure of kidney formation in heparan sulfate 2-O-sulfotransferase deficiency, suppressed antibody production in alpha-2, 6-sialyltransferase deficiency, male sterility in GM2/GD2 synthase deficiency, and abnormalities in the function and stability of myelin in galactosylceramide deficiency.     

Peptide-displaying phage technology in glycobiology

Phage display technology is an emerging drug discovery tool. Using that approach, short peptides that mimic part of a carbohydrate's conformation are selected by screening a peptide-displaying phage library with anti-carbohydrate antibodies. Chemically synthesized peptides with an identified sequence have been used as an alternative ligand to carbohydrate-binding proteins. These peptides represent research tools useful to assay the activities of glycosyltransferases and/or sulfotransferases or to inhibit the carbohydrate-dependent binding of proteins in vitro and in vivo. Peptides can also serve as immunogens to raise anti-carbohydrate antibodies in vivo in animals. Phage display has also been used in single-chain antibody technology by inserting an immunoglobulin's variable region sequence into the phage. A single-chain antibody library can then be screened with a carbohydrate antigen as the target, resulting in a recombinant anti-carbohydrate antibody with high affinity to the antigen. This review provides examples of successful applications of peptide-displaying phage technology to glycobiology. Such an approach should benefit translational research by supplying carbohydrate-mimetic peptides and carbohydrate-binding polypeptides.     

Characterization and mutagenesis of Gal/GlcNAc-6-O-sulfotransferases

The installation of sulfate groups on the carbohydrate residues of glycoproteins, glycolipids, and glycosaminoglycans is a critical posttranslational modification that occurs in all higher eukaryotes. The Gal/GalNAc/GlcNAc-6-O-sulfotransferases (GSTs) are a recently discovered family of carbohydrate sulfotransferases that share significant sequence homology at the amino acid level and mediate a number of different biological processes such as leukocyte adhesion at sites of chronic inflammation. Structural and mechanistic studies of this family of sulfotransferases have been hindered by the lack of a productive recombinant protein expression system. We developed a baculovirus expression system for five of the seven cloned GSTs and determined their kinetic parameters using both thin-layer chromatography and a recently developed polymer dot-blot assay. We used these tools to perform the first site-directed mutagenesis study of a member of this sulfotransferase family, GST2. Using sequence alignments with other carbohydrate and cytosolic sulfotransferases, we selected residues within the putative binding regions for 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the carbohydrate substrate for mutagenesis. Kinetic analysis of the mutants identified residues that are essential for catalytic activity. These results should facilitate mechanistic studies and the development of small molecule inhibitors of this enzyme family to ameliorate chronic inflammatory diseases.     

Expression of human glycosyltransferase genes in yeast as a tool for enzymatic synthesis of sugar chain

We planned the production of human glycosyltransferases in yeast for the enzymatic synthesis of various sugar chains. More than 160 genes encoding various glycosyltransferases were prepared as N-terminal transmembrane region truncated forms by PCR and were inserted into the entry vector of Invitrogen Ltd's Gateway system. About fifty glycosyltransferases were chosen for the synthesis of human type oligosaccharides, and expressed as two different forms in yeast. One is a soluble form, which is secreted into the culture medium by methylotrophic yeast, and the other is an immobilized form, which is displayed at the budding yeast cell wall as a fusion protein with Pir protein. To date, in both systems, some sialyltranferases and fucosyltransferases have been produced as active forms, indicating the potential usefulness of these systems for the enzymatic synthesis of various types of human sugar chains attached to proteins and lipids.     

Enzymatic synthesis in vitro of the disulfated disaccharide unit of corneal keratan sulfate

Among the enzymes of the carbohydrate sulfotransferase family, human corneal GlcNAc 6-O-sulfotransferase (hCGn6ST, also known as human GlcNAc6ST-5/GST4beta) and human intestinal GlcNAc 6-O-sulfotransferase (hIGn6ST or human GlcNAc6ST-3/GST4alpha) are highly homologous. In the mouse, intestinal GlcNAc 6-O-sulfotransferase (mIGn6ST or mouse GlcNAc6ST-3/GST4) is the only orthologue of hCGn6ST and hIGn6ST. In the previous study, we found that hCGn6ST and mIGn6ST, but not hIGn6ST, have sulfotransferase activity to produce keratan sulfate (Akama, T. O., Nakayama, J., Nishida, K., Hiraoka, N., Suzuki, M., McAuliffe, J., Hindsgaul, O., Fukuda, M., and Fukuda, M. N. (2001) J. Biol. Chem. 276, 16271-16278). In this study, we analyzed the substrate specificities of these sulfotransferases in vitro using synthetic carbohydrate substrates. We found that all three sulfotransferases can transfer sulfate to the nonreducing terminal GlcNAc of short carbohydrate substrates. Both hCGn6ST and mIGn6ST, but not hIGn6ST, transfer sulfate to longer carbohydrate substrates that have poly-N-acetyllactosamine structures, suggesting the involvement of hCGn6ST and mIGn6ST in production of keratan sulfate. To clarify further the involvement of hCGn6ST in biosynthesis of keratan sulfate, we reconstituted the biosynthetic pathway in vitro by sequential enzymatic treatment of a synthetic carbohydrate substrate. Using four enzymes, beta1,4-galactosyltransferase-I, beta1,3-N-acetylglucosaminyltransferase-2, hCGn6ST, and keratan sulfate Gal 6-O-sulfotransferase, we were able to synthesize in vitro a product that conformed to the basic structural unit of keratan sulfate. Based on these results, we propose a biosynthetic pathway for N-linked keratan sulfate on corneal proteoglycans.     

A focused microarray approach to functional glycomics: transcriptional regulation of the glycome

Glycosylation is the most common posttranslational modification of proteins, yet genes relevant to the synthesis of glycan structures and function are incompletely represented and poorly annotated on the commercially available arrays. To fill the need for expression analysis of such genes, we employed the Affymetrix technology to develop a focused and highly annotated glycogene-chip representing human and murine glycogenes, including glycosyltransferases, nucleotide sugar transporters, glycosidases, proteoglycans, and glycan-binding proteins. In this report, the array has been used to generate glycogene-expression profiles of nine murine tissues. Global analysis with a hierarchical clustering algorithm reveals that expression profiles in immune tissues (thymus [THY], spleen [SPL], lymph node, and bone marrow [BM]) are more closely related, relative to those of nonimmune tissues (kidney [KID], liver [LIV], brain [BRN], and testes [TES]). Of the biosynthetic enzymes, those responsible for synthesis of the core regions of N- and O-linked oligosaccharides are ubiquitously expressed, whereas glycosyltransferases that elaborate terminal structures are expressed in a highly tissue-specific manner, accounting for tissue and ultimately cell-type-specific glycosylation. Comparison of gene expression profiles with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) profiling of N-linked oligosaccharides suggested that the alpha1-3 fucosyltransferase 9, Fut9, is the enzyme responsible for terminal fucosylation in KID and BRN, a finding validated by analysis of Fut9 knockout mice. Two families of glycan-binding proteins, C-type lectins and Siglecs, are predominately expressed in the immune tissues, consistent with their emerging functions in both innate and acquired immunity. The glycogene chip reported in this study is available to the scientific community through the Consortium for Functional Glycomics (CFG) (http://www.functionalglycomics.org).     

Functional expression of enterobacterial O-polysaccharide biosynthesis enzymes in Bacillus subtilis

The expression of heterologous bacterial glycosyltransferases is of interest for potential application in the emerging field of carbohydrate engineering in gram-positive organisms. To assess the feasibility of using enzymes from gram-negative bacteria, the functional expression of the genes wbaP (formerly rfbP), wecA (formerly rfe), and wbbO (formerly rfbF) from enterobacterial lipopolysaccharide O-polysaccharide biosynthesis pathways was examined in Bacillus subtilis. WbaP and WecA are initiation enzymes for O-polysaccharide formation, catalyzing the transfer of galactosyl 1-phosphate from UDP-galactose and N-acetylglucosaminyl 1-phosphate from UDP-N-acetylglucosamine, respectively, to undecaprenylphosphate. The WecA product (undecaprenylpyrophosphoryl GlcNAc) is used as an acceptor to which the bifunctional wbbO gene product sequentially adds a galactopyranose and a galactofuranose residue from the corresponding UDP sugars to form a lipid-linked trisaccharide. Genes were cloned into the shuttle vectors pRB374 and pAW10. In B. subtilis hosts, the genes were effectively transcribed under the vegII promoter control of pRB374, but the plasmids were susceptible to rearrangements and deletion. In contrast, pAW10-based constructs, in which genes were cloned downstream of the tet resistance cassette, were stable but yielded lower levels of enzyme activity. In vitro glycosyltransferase assays were performed in Escherichia coli and B. subtilis, using membrane preparations as sources of enzymes and endogenous undecaprenylphosphate as an acceptor. Incorporation of radioactivity from UDP-alpha-D-(14)C-sugar into reaction products verified the functionality of WbaP, WecA, and WbbO in either host. Enzyme activities in B. subtilis varied between 20 and 75% of those measured in E. coli.     

Regulation of expression of carbohydrate blood group antigens

The carbohydrate antigens associated with the human ABO and Lewis blood group systems are excellent models for the study of the genetic regulation of glycoconjugate biosynthesis because their expression on erythrocytes and in saliva has been thoroughly investigated in terms of classical genetics and the chemical structures and pathways for the formation of the antigens are now well understood. The primary protein products of the blood group genes are believed to be the glycosyltransferase enzymes that complete the biosynthesis of the determinants. The important controlling factors still to be elucidated are the genetic and environmental influences leading to the tissue specific expression of these antigens. The 3 types of regulation mechanisms discussed in this review are those arising: 1) from the specificity requirements of the glycosyltransferases encoded by the blood group genes; 2) from the competition or co-operation of glycosyltransferases encoded by genes at the same or independent loci; and 3) from the existence and tissue distribution of glycosyltransferases with related, but not identical, substrate specificities.     

Engineered N-acetylhexosamine-active enzymes in glycoscience

BackgroundIn recent years, enzymes modifying N-acetylhexosamine substrates have emerged in numerous theoretical studies as well as practical applications from biology, biomedicine, and biotechnology. Advanced enzyme engineering techniques converted them into potent synthetic instruments affording a variety of valuable glycosides.Scope of reviewThis review presents the diversity of engineered enzymes active with N-acetylhexosamine carbohydrates: from popular glycoside hydrolases and glycosyltransferases to less known oxidases, epimerases, kinases, sulfotransferases, and acetylases. Though hydrolases in natura, engineered chitinases, β-N-acetylhexosaminidases, and endo-β-N-acetylglucosaminidases were successfully employed in the synthesis of defined natural and derivatized chitooligomers and in the remodeling of N-glycosylation patterns of therapeutic antibodies. The genes of various N-acetylhexosaminyltransferases were cloned into metabolically engineered microorganisms for producing human milk oligosaccharides, Lewis X structures, and human-like glycoproteins. Moreover, mutant N-acetylhexosamine-active glycosyltransferases were applied, e.g., in the construction of glycomimetics and complex glycostructures, industrial production of low-lactose milk, and metabolic labeling of glycans. In the synthesis of biotechnologically important compounds, several innovative glycoengineered systems are presented for an efficient bioproduction of GlcNAc, UDP-GlcNAc, N-acetylneuraminic acid, and of defined glycosaminoglycans.Major conclusionsThe above examples demonstrate that engineering of N-acetylhexosamine-active enzymes was able to solve complex issues such as synthesis of tailored human-like glycoproteins or industrial-scale production of desired oligosaccharides. Due to the specific catalytic mechanism, mutagenesis of these catalysts was often realized through rational solutions.General significanceSpecific N-acetylhexosamine glycosylation is crucial in biological, biomedical and biotechnological applications and a good understanding of its details opens new possibilities in this fast developing area of glycoscience.     

Construction of a library of human glycosyltransferases immobilized in the cell wall of Saccharomyces cerevisiae

Fifty-one human glycosyltransferases were expressed in Saccharomyces cerevisiae as immobilized enzymes and were assayed for enzymatic activities. The stem and catalytic regions of sialyl-, fucosyl-, galactosyl-, N-acetylgalactosaminyl-, and N-acetylglucosaminyltransferases were fused with yeast cell wall Pir proteins, which anchor glycosyltransferases at the yeast cell wall glucan. More than 75% of expressed recombinant glycosyltransferases retained their enzymatic activities in the yeast cell wall fraction and will be used as a human glycosyltransferase library. In increasing the enzymatic activities of immobilized glycosyltransferases, several approaches were found to be effective. Additional expression of yeast protein disulfide isomerase increased the expression levels and activities of polypeptide N-acetylgalactosaminyltransferases and other glycosyltransferases. PIR3 and/or PIR4 was more effective than PIR1 as a cell wall anchor when the Pir-glycosyltransferase fusions were expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Oligosaccharides such as Lewis x, Lewis y, and H antigen were successfully synthesized using this immobilized glycosyltransferase library, indicating that the Pir-fused glycosyltransferases are useful for the production of various human oligosaccharides.