O-glycosylation pathway for mucin-type glycoproteins

O-glycosylation is the post-translational process whereby carbohydrate is added to hydroxylated amino acids of proteins. The major O-glycosylation pathway in animal cells is involved in the synthesis of oligosaccharides linked by N-acetylgalactosamine to serine or threonine residues in 'mucin-type' proteins or their analogs. In this review, we discuss the evidence for the cellular localization of the biosynthetic steps in this pathway and propose a simplified, consensus version. We also propose variations of the simple pathway to account for its heterogeneity and variability in different cell types and differentiation states.     

Cell surface transport, oligomerization, and endocytosis of chimeric type II glycoproteins: role of cytoplasmic and anchor domains.

We investigated the role of cytoplasmic and anchor domains of type II glycoproteins in intracellular transport, oligomerization, and endocytosis by expressing the wild-type and chimeric genes in mammalian cells. Chimeric genes were constructed by exchanging the DNA segments that encode the cytoplasmic and anchor domains between the human influenza virus (A/WSN/33) neuraminidase (NA) and transferrin receptor (TR). The chimeric proteins in which domains were exchanged precisely were productively targeted to the cell surface. However, the proteins appeared to assemble differently in the intracellular compartment. For example, while TR existed predominantly as a dimer, NATR delta 90, containing the cytoplasmic and signal-anchor domains of NA and the ectodomain of TR, was present as a tetramer, a dimer, and a monomer. Similarly, the influenza virus NA existed predominantly as a tetramer but TRNA delta 35, in which the cytoplasmic and signal-anchor domains of TR were joined to the ectodomain of NA, existed predominantly as a dimer, suggesting that the cytoplasmic and anchor domains of type II glycoproteins affect the subunit assembly of heterologous ectodomains. In addition, we analyzed the role of the cytoplasmic domain in endocytosis. NA and NATR delta 90 did not undergo endocytosis, whereas both TR and TRNA delta 35 were internalized efficiently, demonstrating that the NH2 cytoplasmic domain of TR was capable of internalizing a heterologous ectodomain (NA) from the cell surface.     

Typing of core and backbone domains of mucin-type oligosaccharides from human ovarian-cyst glycoproteins by 500-MHz 1H-NMR spectroscopy

Human blood-group A active glycoproteins from ovarian-cyst fluid were subjected to Smith degradation and subsequent beta-elimination. The resulting oligosaccharide-alditols represent the core and backbone domains of the O-linked carbohydrate chains. Nine of these, ranging in size from disaccharides to hexasaccharides, were investigated by 1H-NMR spectroscopy. Their primary structures could be adequately characterized. In particular, the core types, i.e. the substitution patterns of N-acetylgalactosaminitol (GalNAc-ol) as well as the types of backbone, i.e. the linkage types of alternating Gal-GlcNAc sequences, were unambiguously identified. The core type GlcNAc beta(1-3)GalNAc-ol is described for the first time as occurring in ovarian-cyst glycoprotein.     

Recent advances in the chemical synthesis of mucin-like glycoproteins

This purpose of this mini review is to familiarizereaders with the tools currently available for the synthesis of mucin-typeglycoproteins. The article will highlight recent approaches to thesynthesis of glycopeptide fragments bearing complex O-linkedglycans, as well as new strategies for the generation of full-lengthglycoproteins.     

Cell surface expression of v-fms-coded glycoproteins is required for transformation.

The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane.     

Oviductins possess chitinase- and mucin-like domains: A lead in the search for the biological function of these oviduct-specific ZP-associating glycoproteins

Over the last 10 years considerable progress has been made in the immunological and biochemical characterization of oviduct-specific glycoproteins. It is now well established that a subclass of these secretory products, designated as oviductins, associate with the zona pellucida of the ovulated oocyte and with the early embryo. Recent reports on the cloning of cDNAs of oviductins from various species, including that of golden hamster (Mesocricetus auratus) oviductin by our laboratory, allowed us to compare their deduced amino acid sequences with those of other proteins. Optimal alignment analysis showed that oviductins contain regions of significant similarity with catalytically inactive mammalian members of the bacterial and microfilarial chitinase protein family. Most importantly, a close examination of the hamster and human deduced amino acid sequences revealed that both glycoproteins possess contiguous Ser/Thr rich repeated units, clustered in their carboxy-terminal portions. These mucin-type motifs are similar in the hamster and human glycoprotein, although hamster oviductin contains more of these complete units. This striking feature might indicate that these molecules play a similar role to mucin-type glycoproteins, e.g., in protecting the oocyte and early embryo against attacks from their environment. We propose a model whereby oviductins are targeted to the oocyte via the interaction of their chitinase-like domains with specific oligosaccharide moieties of the zona pellucida. Once localized to this structure, oviductin molecules would act as a protective shield around the oocyte and early embryo by virtue of their densely glycosylated mucin-type domains. © 1995 Wiley-Liss, Inc.     

Tumour-associated and differentiation antigens on the carbohydrate moieties of mucin-type glycoproteins

In this report the carbohydrate antigens expressed on the three oligosaccharide domains, core, backbone and peripheral, of mucin-type glycoproteins are briefly reviewed in the light of recent observations with monoclonal antibodies. These have revealed that a number of cell-surface antigens which behave as tumour-associated and differentiation antigens of man or mouse are abundantly expressed on the carbohydrate chains of a variety of secreted mucins of human and animal origins and they belong to an antigen system which also includes the major blood group antigens. Examples are given of the use of well-characterized anti-carbohydrate antibodies to derive structural information on (a) mucin-type glycoproteins of human B lymphocyte membranes, (b) the high molecular weight glycoproteins of the normal human gastric and distal-colon mucosae and (c) tumour-derived glycoproteins from these two organs. Major differences between the antigenicities of the normal stomach and distal-colon, and between their tumour-derived glycoproteins, and the important effect of the secretor status in the expression of these antigens are described. These observations have enabled a better understanding of the individual and tissue differences in the expression of tumour-associated antigens. The possibility is raised that these carbohydrate structures (many of which also occur on certain N-linked oligosaccharides and glycolipids) are components of receptor systems for endogenous ligands. More tangible evidence is cited for the role of certain structures in this family of saccharides as receptors for infective agents.     

Role of the cytoplasmic domains of viral glycoproteins in antibody-induced cell surface mobility.

We have investigated the role of the cytoplasmic domains of the influenza virus hemagglutinin (HA) and the parainfluenza virus type 3 (PI3) fusion (F) glycoproteins as a determinant of their ability to undergo antibody-induced redistribution on plasma membranes. The viral envelope genes were truncated in their cytoplasmic domains by using oligonucleotide-directed mutagenesis and expressed by using recombinant vaccinia viruses. In HeLa cells, the truncated HA (HAt), like the full-length HA, did not cap in response to specific antibody. In CV-1 cells, HAt showed patchy surface immunofluorescence with few caps, whereas full-length HA exhibited capping in many cells in response to bivalent antibody. Quantitation of cap formation indicated a sevenfold decrease in the frequency of capping of HAt in comparison with full-length HA. Similarly, truncated F also exhibited a significant decrease in cap formation in comparison with full-length F. These results indicate that the ability of influenza virus HA and PI3 F to undergo redistribution in response to bivalent antibody has been altered by truncation of the viral glycoproteins and suggest that capping may involve interactions between the cytoplasmic domain of the viral glycoproteins and host cell components.     

Fluorometric determination of mucin-type glycoproteins by the galactose oxidase-peroxidase method

We developed a convenient and specific method for the determination of mucin-type glycoproteins using galactose oxidase and horseradish peroxidase on the basis of the contents of galactosyl and N-acetylgalactosaminyl residues in glycoproteins. Galactose and galactosamine residues released from glycoproteins after hydrolysis were oxidized with galactose oxidase and subsequently the resultant hydrogen peroxide was determined by a combination of horseradish peroxidase and 3-(p-hydroxyphenyl) propionic acid as a fluorogenic substrate. The contents of galactose/galactosamine residues in N- and O-glycans, as determined by the galactose oxidase-peroxidase method, were in good agreement with those described in the previous reports. We applied the present method to determine mucin-type glycoproteins secreted from rat gastric mucosa by stimulation with misoprostol, a prostaglandin E(1) analogue in vivo. Thus, the galactose oxidase-peroxidase method is useful for the determination of mucin-type glycoproteins in biological materials.     

Macrophage signaling by glycosylphosphatidylinositol-anchored mucin-like glycoproteins derived from Trypanosoma cruzi trypomastigotes

Activation of cells from the innate immune system has an important role in host resistance to early infection with the intracellular protozoan parasite, Trypanosoma cruzi. Here we review the studies that have identified and structurally characterized the glycosylphosphatidylinositol (GPI) anchors, as parasite molecules responsible for the activation of cells from the macrophage lineage. We also cover the studies that have identified the receptor, signaling pathways as well as the array of genes expressed in macrophages that are activated by these glycoconjugates. We discuss the possible implications of such response on the host resistance to T. cruzi infection and the pathogenesis of Chagas disease.     

mu-Protocadherin, a novel developmentally regulated protocadherin with mucin-like domains

Branching morphogenesis is a central event during the development of kidneys, lungs, and other organs. We previously generated a monoclonal antibody, 3D2-E9, that inhibited branching morphogenesis and caused widespread apoptosis. We now report the purification of its antigen and cloning of its full-length cDNA. Its cDNA encodes an integral membrane protein that contains four cadherin-like ectodomains and a thrice tandemly repeated region enriched in threonine, serine, and proline, similar to those of mucins. We thus term this protein mu-protocadherin, reflecting the hybrid nature of its extracellular region. mu-Protocadherin is expressed in two forms that are developmentally regulated, with the shorter isoform lacking the mucin-like repeats. Expression of the long isoform in heterologous cells results in adhesion of the expressing cells, suggesting that it is a new cell adhesion molecule. mu-Protocadherin contains both N and O glycosylations. It is expressed at lateral and basal surfaces of epithelia during kidney and lung development and is located in coated pits. Colocalization of mu-protocadherin with beta-catenin was noted primarily at the junction of the lateral and basal membrane. The cytoplasmic domain contains four proline-rich regions, similar to SH3 binding regions. Thus, it is likely that adhesive interactions mediated by mu-protocadherin induce signaling events that regulate branching morphogenesis.     

Bacterial surface layer glycoproteins.

Crystalline cell surface layers (S-layers) are ubiquitously present in bacterial species from almost all phylogenetic branches. Recent investigations have shown that the S-layer proteins of many archaebacteria and eubacteria contain covalently linked carbohydrate chains. This evidence clearly shows that the ability for protein glycosylation is present as a common biosynthetic pathway in prokaryotic organisms.     

N-acetyl-beta-D-glucosaminyltransferases related to the synthesis of mucin-type glycoproteins in human ovarian tissue

The presence of N-acetyl-beta-D-glucosaminyltransferases in microsome preparations from human ovarian tissues was investigated with UDP-GlcNAc and several synthetic oligosaccharides as acceptors. The products were identified by paper chromatography and the linkage of the 2-acetamido-2-deoxy-beta-D-glucopyranosyl group incorporated into oligosaccharides was determined by exoglycosidase digestions, 1H-n.m.r. spectroscopy, and methylation analysis. These results showed that ovarian microsome preparations contain both beta-(1----3)- and beta-(1----6)-N-acetyl-D-glucosaminyltransferase activities which might be involved in the synthesis of mucin-type glycoproteins. Substrate competition tests suggested that both UDP-GlcNAc:-Bn glycoside of beta-D-GlcpNAc-(1----6)-alpha-D-GalpNAc [GlcNAc to GalNAc] and -Bn glycoside of beta-D-Galp-(1----3)-[beta-D-GlcNAc-(1----6)]-alpha-D-GalpNAc [GlcNAc to Gal] beta-(1----3)-N-acetyl-D-glucosaminyltransferase activities reside in a single enzyme species.     

Cell surface glycoproteins of hepatocytes and hepatoma cells identified by monoclonal antibodies

Eight hybridoma cell lines secreting monoclonal antibodies (MABs) directed to cell surface components of rat hepatocytes were isolated. The antigens of seven MABs were identified as glycosylated plasma membrane proteins. The presence of these glycoproteins on normal hepatocytes and hepatocellular carcinoma cells was analyzed. A semi-quantitative enzyme-linked immunosorbent assay revealed that only two MABs (Be 8.7, Ne 11.3) recognized proteins which were expressed not only in normal liver but also in chemically induced transplantable Morris hepatomas and hepatoma-derived cell lines. The expression of six antigens was found to be sensitive to transformation. The domain specificity of the MABs was determined by indirect immunofluorescence on sections of liver tissue containing neoplastic nodules. Three MABs (Be 8.4, Ne 11.1, Ne 11.3) specifically bound to the sinusoidal domain and two MABs (Be 9.2, De 13.4) to the bile canalicular domain. These five antigens were transformation-sensitive except for the glycoprotein recognized by the MAB Ne 11.3. Three MABs (Be 8.7, Be 9.1, De 13.2) also showed intracellular immunofluorescence. Two of the antigens (Be 9.1, De 13.2) were not present in hepatomas. The relative molar masses (Mr) of the glycoproteins were determined after protein immunoblotting and immunoprecipitation. Four MABs (Be 8.7, Be 9.1, Be 9.2, De 13.4) recognized antigens with a Mr of 110 000 but did not mutually cross-react. The antigen recognized by MAB De 13.4 was identified as the ectoenzyme dipeptidyl peptidase IV (EC 3.4.14.-).