Tilo Schwientek

Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions.

Enzymatic glycosylation of proteins and lipids is an abundant and important biological process. A great diversity of oligosaccharide structures and types of glycoconjugates is found in nature, and these are synthesized by a large number of glycosyltransferases. Glycosyltransferases have high donor and acceptor substrate specificities and are in general limited to catalysis of one unique glycosidic linkage. Emerging evidence indicates that formation of many glycosidic linkages is covered by large homologous glycosyltransferase gene families, and that the existence of multiple enzyme isoforms provides a degree of redundancy as well as a higher level of regulation of the glycoforms synthesized. Here, we discuss recent cloning strategies enabling the identification of these large glycosyltransferase gene families and exemplify the implication this has for our understanding of regulation of glycosylation by discussing two galactosyltransferase gene families.

O-glycosylation modulates proprotein convertase activation of angiopoietin-like protein 3: possible role of polypeptide GalNAc-transferase-2 in regulation of concentrations of plasma lipids.

The angiopoietin-like protein 3 (ANGPTL3) is an important inhibitor of the endothelial and lipoprotein lipases and a promising drug target. ANGPTL3 undergoes proprotein convertase processing (RAPR224↓TT) for activation, and the processing site contains two potential GalNAc O-glycosylation sites immediately C-terminal (TT226). We developed an in vivo model system in CHO ldlD cells that was used to show that O-glycosylation in the processing site blocked processing of ANGPTL3. Genome-wide SNP association studies have identified the polypeptide GalNAc-transferase gene, GALNT2, as a candidate gene for low HDL and high triglyceride blood levels. We hypothesized that the GalNAc-T2 transferase performed critical O-glycosylation of proteins involved in lipid metabolism. Screening of a panel of proteins known to affect lipid metabolism for potential sites glycosylated by GalNAc-T2 led to identification of Thr226 adjacent to the proprotein convertase processing site in ANGPTL3. We demonstrated that GalNAc-T2 glycosylation of Thr226 in a peptide with the RAPR224↓TT processing site blocks in vitro furin cleavage. The study demonstrates that ANGPTL3 activation is modulated by O-glycosylation and that this step is probably controlled by GalNAc-T2.

The Drosophila Gene brainiac Encodes a Glycosyltransferase Putatively Involved in Glycosphingolipid Synthesis

The Drosophila genesfringe and brainiac exhibit sequence similarities to glycosyltransferases. Drosophila and mammalian fringe homologs encode UDP-N-acetylglucosamine:fucose-O-Ser β1,3-N-acetylglucosaminyltransferases that modulate the function of Notch family receptors. The biological function of brainiac is less well understood. brainiac is a member of a large homologous mammalian β3-glycosyltransferase family with diverse functions. Eleven distinct mammalian homologs have been demonstrated to encode functional enzymes forming β1–3 glycosidic linkages with different UDP donor sugars and acceptor sugars. The putative mammalian homologs with highest sequence similarity tobrainiac encode UDP-N-acetylglucosamine:β1,3-N-acetylglucosaminyltransferases (β3GlcNAc-transferases), and in the present study we show thatbrainiac also encodes a β3GlcNAc-transferase that uses β-linked mannose as well as β-linked galactose as acceptor sugars. The inner disaccharide core structures of glycosphingolipids in mammals (Galβ1–4Glcβ1-Cer) and insects (Manβ1–4Glcβ1-Cer) are different. Both disaccharide glycolipids served as substrates for brainiac, but glycolipids of insect cells have so far only been found to be based on the GlcNAcβ1–3Manβ1–4Glcβ1-Cer core structure. Infection of High FiveTM cells with baculovirus containing full coding brainiac cDNA markedly increased the ratio of GlcNAcβ1–3Manβ1–4Glcβ1-Cer glycolipids compared with Galβ1–4Manβ1–4Glcβ1-Cer found in wild type cells. We suggest that brainiac exerts its biological functions by regulating biosynthesis of glycosphingolipids.

Drosophila egghead Encodes a β1,4-Mannosyltransferase Predicted to Form the Immediate Precursor Glycosphingolipid Substrate for brainiac

The neurogenic Drosophila genesbrainiac and egghead are essential for epithelial development in the embryo and in oogenesis. Analysis ofegghead and brainiac mutants has led to the suggestion that the two genes function in a common signaling pathway. Recently, brainiac was shown to encode a UDP-N-acetylglucosamine:βManβ1,3-N-acetylglucosaminyltransferase (β3GlcNAc-transferase) tentatively assigned a key role in biosynthesis of arthroseries glycosphingolipids and forming the trihexosylceramide, GlcNAcβ1–3Manβ1–4Glcβ1–1Cer. In the present study we demonstrate that egghead encodes a Golgi-located GDP-mannose:βGlcβ1,4-mannosyltransferase tentatively assigned a biosynthetic role to form the precursor arthroseries glycosphingolipid substrate for Brainiac, Manβ1–4Glcβ1–1Cer. Egghead is unique among eukaryotic gly- cosyltransferase genes in that homologous genes are limited to invertebrates, which correlates with the exclusive existence of arthroseries glycolipids in invertebrates. We propose that brainiac and egghead function in a common biosynthetic pathway and that inactivating mutations in either lead to sufficiently early termination of glycolipid biosynthesis to inactivate essential functions mediated by glycosphingolipids.

Glycoproteomic Analysis of Serum from Patients with Gastric Precancerous Lesions

Gastric cancer is preceded by a carcinogenesis pathway that includes gastritis caused by Helicobacter pylori infection, chronic atrophic gastritis that may progress to intestinal metaplasia (IM), dysplasia, and ultimately gastric carcinoma of the more common intestinal subtype. The identification of glycosylation changes in circulating serum proteins in patients with precursor lesions of gastric cancer is of high interest and represents a source of putative new biomarkers for early diagnosis and intervention. This study applies a glycoproteomic approach to identify altered glycoproteins expressing the simple mucin-type carbohydrate antigens T and STn in the serum of patients with gastritis, IM (complete and incomplete subtypes), and control healthy individuals. The immunohistochemistry analysis of the gastric mucosa of these patients showed expression of T and STn antigens in gastric lesions, with STn being expressed only in IM. The serum glycoproteomic analysis using 2D-gel electrophoresis, Western blot, and MALDI-TOF/TOF mass spectrometry led to the identification of circulating proteins carrying these altered glycans. One of the glycoproteins identified was plasminogen, a protein that has been reported to play a role in H. pylori chronic infection of the gastric mucosa and is involved in extracellular matrix modeling and degradation. Plasminogen was further characterized and showed to carry STn antigens in patients with gastritis and IM. These results provide evidence of serum proteins displaying abnormal O-glycosylation in patients with precursor lesions of gastric carcinoma and include a panel of putative targets for the non-invasive clinical diagnosis of individuals with gastritis and IM.

Initiation of Mammalian O-Mannosylation in Vivo Is Independent of a Consensus Sequence and Controlled by Peptide Regions within and Upstream of the α-Dystroglycan Mucin Domain

To reveal insight into the initiation of mammalian O-mannosylation in vivo, recombinant glycosylation probes containing sections of human α-dystroglycan (hDG) were expressed in epithelial cell lines. We demonstrate that O-mannosylation within the mucin domain of hDG occurs preferentially at Thr/Ser residues that are flanked by basic amino acids. Protein O-mannosylation is independent of a consensus sequence, but strictly dependent on a peptide region located upstream of the mucin domain. This peptide region cannot be replaced by other N-terminal peptides, however, it is not sufficient to induce O-mannosylation on a structurally distinct mucin domain in hybrid constructs. The presented in vivo evidence for a more complex regulation of mammalian O-mannosylation contrasts with a recent in vitro study of O-mannosylation in human α-dystroglycan peptides indicating the existence of an 18-meric consensus sequence. We demonstrate in vivo that the entire region p377–417 is necessary and sufficient for O-mannosylation initiation of hDG, but not of MUC1 tandem repeats. The feature of a doubly controlled initiation process distinguishes mammalian O-mannosylation from other types of O-glycosylation, which are largely controlled by structural properties of the substrate positions and their local peptide environment.

Cyclodextrin-assisted Glycan Chain Extension on a Protected Glycosyl Amino Acid

By the use of cyclodextrins, we have enhanced the solubility of the protected amino acid glycan Fmoc-Thr(GalNAcα1)-OtBu (1b) up to 100-fold. This improvement enabled us to carry out an enzymatic glycosylation employing a β-galactosidase in combination with an α2,3-sialyltransferase without the aid of organic cosolvents. After optimization of the one-pot reaction, the sialylated core 1 structure Fmoc-Thr[Neu5Ac(α2-3)Gal(β1-3)GalNAcα1]-OtBu (3b) could be obtained with 50% yield.

Delineation of the minimal catalytic domain of human Galβ1-3GalNAc α2,3-sialyltransferase (hST3Gal I)

The CMP-Neu5Ac:GalL1-3GalNAc K2,3-sialyltransferase (ST3Gal I, EC 2.4.99.4) is a Golgi membrane-bound type II glycoprotein that catalyses the transfer of sialic acid residues to GalL1-3GalNAc disaccharide structures found on O-glycans and glycolipids. In order to gain further insight into the structure/function of this sialyltransferase, we studied protein expression, N-glycan processing and enzymatic activity upon transient expression in the COS-7 cell line of various constructs deleted in the N-terminal portion of the protein sequence. The expressed soluble polypeptides were detected within the cell and in the cell culture media using a specific hST3Gal I monoclonal antibody. The soluble forms of the protein consisting of amino acids 26^340 (hST3-v25) and 57^340 (hST3-v56) were efficiently secreted and active. In contrast, further deletion of the N-terminal region leading to hST3-v76 and hST3-v105 gave also rise to various polypeptides that were not active within the transfected cells and not secreted in the cell culture media. The kinetic parameters of the active secreted forms were determined and shown to be in close agreement with those of the recombinant enzyme already described (H. Kitagawa, J.C. Paulson, J. Biol. Chem. 269 (1994)). In addition, the present study demonstrates that the recombinant hST3Gal I polypeptides transiently expressed in COS-7 cells are glycosylated with complex and high mannose type glycans on each of the five potential N-glycosylation sites. fl 2001 Elsevier Science B.V. All rights reserved.

Rescue of Drosophila Melanogaster l(2)35Aa lethality is only mediated by polypeptide GalNAc-transferase pgant35A, but not by the evolutionary conserved human ortholog GalNAc-transferase-T11

The Drosophila l(2)35Aa gene encodes a UDP-N-acetylgalactosamine: Polypeptide N-acetylgalactosaminyltransferase, essential for embryogenesis and development (J. Biol. Chem. 277, 22623–22638; J. Biol. Chem. 277, 22616–22). l(2)35Aa, also known as pgant35A, is a member of a large evolutionarily conserved family of genes encoding polypeptide GalNAc-transferases. Phylogenetic and functional analyses have proposed that subfamilies of orthologous GalNAc-transferase genes are conserved in species, suggesting that they serve distinct functions in vivo. Based on sequence alignments, pgant35A and human GALNT11 are thought to belong to a distinct subfamily. Recent in vitro studies have shown that pgant35A and pgant7, encoding enzymes from different subfamilies, prefer different acceptor substrates, whereas the orthologous pgant35A and human GALNT11 gene products possess, 1) conserved substrate preferences and 2) similar acceptor site preferences in vitro. In line with the in vitro pgant7 studies, we show that l(2)35Aa lethality is not rescued by ectopic pgant7 expression. Remarkably and in contrast to this observation, the human pgant35A ortholog, GALNT11, was shown not to support rescue of the l(2)35Aa lethality. By use of genetic “domain swapping” experiments we demonstrate, that lack of rescue was not caused by inappropriate sub-cellular targeting of functionally active GalNAc-T11. Collectively our results show, that fly embryogenesis specifically requires functional pgant35A, and that the presence of this gene product during fly embryogenesis is functionally distinct from other Drosophila GalNAc-transferase isoforms and from the proposed human ortholog GALNT11.

Core 2 β6-N- Acetylglucosaminyltransferase-I and -III

Core 2 β6-N-acetylglucosaminyltransferase (Core2GlcNAcT) is a glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to αGalNAc residues in core 1, Galβ1- 3GalNAcα1-Ser/Thr with β1,6-linkage, forming Galβ1-3(GlcNAcβ1-6)GalNAcα1- Ser/Thr. The formation of the core 2 branch is usually followed by galactosylation by β4-galactosyltransferase-IV, a member of the β4-galactosyltransferase gene family (Ujita et al. 1998), resulting in the formation of N-acetyllactosamines in O-glycans. Such N-acetyllactosamines can be modified to form functional oligosaccharides such as sialyl Lewis X (Fig. 1). Open image in new window Fig. 1 The proposed biosynthetic pathways of O-glycans (A) and poly-N-acetyllactosaminyl O-glycans (B). A It has been shown that the tetrasaccharide (bottom left) is formed by the sequential action of α2-3-sialyltransferase followed by α2-6-sialyltransferase. When β6 Nacetylglucosaminyltransferase, Core2GlcNAcT, is present, the branched hexasaccharide (bottom right) is formed. B Poly-N-acetyllactosaminyl chain can be extended from the GlcNAcβ1,6- linkage synthesized in core 2. Poly-N-acetyllactosaminyl extension can be further modified by α3 fucosyltransferase, forming sialyl Lex termini. (From Maemura and Fukuda 1992)