Single N-acetylglucosamine Residue Research Articles

An in vivo approach for the identification of acceptor sites for O-glycosyltransferases: motifs for the addition of O-GlcNAc in Dictyostelium discoideum.

To identify and analyze acceptor sequences for O-glycosylation, we have developed an in vivo system expressing short peptides as glutathione S-transferase fusion proteins in the eukaryotic host Dictyostelium discoideum. Using this approach, we show that a short peptide motif (PTVTPT), present in the D. discoideum cell-surface glycoprotein PsA, is sufficient as a signal for O-glycosylation, even when fused to a heterologous protein. Monosaccharide analysis and solid-phase protein sequencing showed that the modification is a single N-acetylglucosamine attached to threonine residues. This was further confirmed by electrospray-mass spectrometry. The O-linked glycosylation of both this peptide and authentic PsA presents the modB-dependent carbohydrate-specific epitope identified by the monoclonal antibody MUD50. Substitution of threonine by serine residues in this peptide also yields a glycosylated fusion protein which is modified with single N-acetylglucosamine residues, but not all of the serines are glycosylated.

Oligomannosides or oligosaccharide-lipids as potential substrates for rat liver cytosolic alpha-D-mannosidase.

We have previously reported the substrate specificity of the cytosolic alpha-D-mannosidase purified from rat liver using Man9GlcNAc, i.e. Man alpha 1-2Man alpha 1-3(Man alpha 1-2Man alpha 1-6)Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3) Man beta 1-4G1cNAc, as substrate [Grard, Saint-Pol, Haeuw, Alonso, Wieruszeski, Strecker and Michalski (1994) Eur. J. Biochem. 223, 99-106]. Man9 G1cNAc is hydrolysed giving Man5GlcNAc, i.e. Man alpha 1-2 Man alpha 1-2Man alpha 1-3(Man alpha 1-6)Man beta 1-4GlcNAc, possessing the same structure as the oligosaccharide of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. We study here the activity of the purified cytosolic alpha-D-mannosidase towards the oligosaccharide-diphosphodolichol intermediates formed during the biosynthesis of N-glycans, and also towards soluble oligosaccharides released from the endoplasmic reticulum which are glucosylated or not and possessing at their reducing end either a single N-acetylglucosamine residue or a di-N-acetylchitobiose sequence. We demonstrate that (1) dolichol pyrophosphate oligosaccharide substrates are poorly hydrolysed by the cytosolic alpha-D-mannosidase; (2) oligosaccharides with a terminal reducing di-N-acetylchitobiose sequence are not hydrolysed at all; (3) soluble oligosaccharides bearing a single reducing N-acetylglucosamine are the real substrates for the enzyme. These results suggest a role for alpha-D-mannosidase in the catabolism of glycans released from the endoplasmic reticulum rather than in the regulation of the biosynthesis of asparagine-linked oligosaccharides.

Recombinant prespore-specific antigen from Dictyostelium discoideum is a beta-sheet glycoprotein with a spacer peptide modified by O-linked N-acetylglucosamine.

Prespore-specific antigen (PsA) is a putative cell-adhesion molecule of the cellular slime mould Dictyostelium discoideum, which has a similar molecular architecture to several mammalian cell-surface proteins. It has an N-terminal globular domain presented to the extracellular environment on an O-glycosylated stem (glycopeptide) that is attached to the cell membrane through a glycosyl-PtdIns anchor. The sequence of PsA suggests that PsA may belong to a new family of cell-surface molecules and here we present information on the structure of the N-terminal globular domain and determine the reducing-terminal linkage of the O-glycosylation. To obtain a sufficient amount of pure protein, a secreted recombinant form of PsA (rPsA), was expressed in D. discoideum and characterised. 1H-NMR spectra of rPsA contained features consistent with a high degree of beta-sheet in the N-terminal globular domain, a feature commonly observed in cell-adhesion proteins. Solid-phase Edman degradation of the glycopeptide of rPsA indicated that 14 of the 15 threonines and serines in the spacer region were glycosylated. The chemical structures of the O-glycosylations were determined to be single N-acetylglucosamine residues.

Ligand binding by the immunoglobulin superfamily recognition molecule CD2 is glycosylation-independent.

The evolutionary success of the immunoglobulin superfamily (IgSF) is thought to reflect the ability of IgSF protein domains to form stable structural units. The role of glycosylation in stabilizing these domains is controversial, however. In this study a systematic analysis of the effect of glycosylation on the ligand-binding properties of the cell-cell recognition molecule CD2, which consists of two IgSF domains, was undertaken. A form of human soluble CD2 (hsCD2) with single N-acetylglucosamine residues at each glycosylation site was produced by inhibiting glucosidase I with N-butyldeoxynojirimycin during expression in Chinese hamster ovary cells and digesting the expressed hsCD2 with endoglycosidase H. The ligand and antibody binding properties of this form of hsCD2 were indistinguishable from those of fully glycosylated hsCD2 as determined by surface plasmon resonance analyses. The protein also formed diffraction quality crystals and analysis of the 2.5-A resolution crystal structure indicated that the single N-acetylglucosamine residue present on domain 1 is unlikely to stabilize the ligand binding face of hsCD2. A second, fully deglycosylated form of hsCD2 also bound the ligand and antibodies although this form of the protein tended to aggregate. In contrast to the results of previous studies, the current data indicate that the structural integrity and ligand binding function of human CD2 are glycosylation-independent.

Crystal structure of the extracellular region of the human cell adhesion molecule CD2 at 2.5 A resolution.

The T-lymphocyte antigen CD2 is an adhesion molecule implicated in immune responses in vivo. The extracellular regions of the human and rat homologues of CD2 share only 45% sequence identity and bind different protein ligands. Comparison of the human and rat soluble CD2 (sCD2) structures should provide insights into the structural basis of cell surface recognition. We therefore determined the crystal structure of a form of human sCD2 with single N-acetylglucosamine residues at each glycosylation site to 2.5 A resolution with an R-factor of 19.3%. It is composed of two immunoglobulin superfamily domains similar to those of rat sCD2, but the relative orientation of the domains in the two homologues differs by up to 20 degrees. An interaction involving the flat, highly charged, ligand binding GFCC'C" faces of crystallographically related human sCD2 molecules duplicates, in a different lattice, that observed in the rat sCD2 crystals. Intramolecular flexibility appears to be a conserved feature of CD2. The head-to-head interaction between molecules represents a general model for interactions between adhesion molecules of this structural class. Ligand specificity may be influenced by the distribution of charged residues on the binding face.

Intracellular compartmentalization and degradation of free polymannose oligosaccharides released during glycoprotein biosynthesis.

The intracellular site for the degradation of free polymannose oligosaccharides released during glycoprotein biosynthesis has been studied by permeabilizing the plasma membrane of metabolically radiolabeled HepG2 cells with streptolysin O. This pore-forming agent permitted us to examine the breakdown in both the cytosolic and vesicular compartments of the previously recognized (Anumula, K. R., and Spiro, R. G. (1983) J. Biol. Chem. 258, 15274-15282) polymannose components terminating in a di-N-acetylchitobiose sequence (OS-Glc-NAc2) or a single N-acetylglucosamine residue (OS-Glc-NAc1) residue. Pulse-chase studies indicated that although the OS-GlcNAc2 saccharides were about equally distributed between vesicles and cytosol and rapidly disappeared after reaching the Man8 stage, the OS-GlcNAc1 species were found predominantly in the extravesicular compartment and there underwent a distinctive demannosylation sequence resulting in the formation of a Man5GlcNAc isomer (Man alpha 1-->2Man alpha 1-->2Man alpha 1-->3(Man alpha 1-->6)Man beta 1-->4GlcNAc) which was different from the product of Golgi processing enzymes. Further trimming of this cytosolic limit product required its translocation into a vesicular compartment, believed to be lysosomes, in which Man2-4GlcNAc components appeared as the metabolic chase progressed. The accumulation of Glc1Man5GlcNAc in the cytosol during the chase suggested that glucose interferes with the cytosolic-vesicular transfer and this became even more evident by the pronounced pile-up of extravesicular Glc3Man5GlcNAc when the cells were incubated in the presence of castanospermine. Although the biological significance and mechanism of free polymannose oligosaccharide entry into the cytosol is not yet known, the possibility that it may reflect an endoplasmic reticulum-situated degradative process of glycoproteins merits consideration.

Mitotic arrest-associated enhancement of O-linked glycosylation and phosphorylation of human keratins 8 and 18.

Arrest of the human colonic cell line HT29 at the G2/M phase of the cell cycle resulted in changes in keratin assembly that were coupled with a significant increase in the O-linked glycosylation and serine phosphorylation of keratin polypeptides 8 and 18 (K8/18). With mitotic arrest, enhanced keratin phosphorylation occurred preferentially on K8, whereas K18 showed a higher glycosylation level than K8. Removal of the arresting agent allowed cells to proceed through the cell cycle with a concomitant decrease in K8/18 glycosylation. In contrast, keratins isolated from S phase-enriched cells, obtained after synchronization with aphidicolin, did not show enhanced glycosylation. Tryptic peptide analysis of keratins in G2/M-arrested cells showed changes in the glycopeptide pattern of K8 and in the phosphopeptide patterns of K8 and K18. Labeling of K8/18 immunoprecipitates, isolated from G2/M-arrested cells, with [3H]galactose followed by beta-elimination showed that K8/18 glycosylation consisted of single N-acetylglucosamine residues. Threonine was identified as the site of glycosylation after comparing acid hydrolysis products of beta-eliminated and non-beta-eliminated K8 and K18. Specific cleavage at tryptophan residues indicated that K18 glycosylation and phosphorylation were restricted to the head and proximal rod domains, whereas K8 did not show the same restriction. Our results show a unique association of the single O-linked N-acetylglucosamine type of modification of keratins with mitotic arrest in HT29 cells. There was no reciprocal relationship between K8/18 glycosylation and phosphorylation, and each keratin showed a preferential G2/M cell cycle-associated increase in either serine phosphorylation or threonine glycosylation.

O-linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins.

Glycoproteins bearing a single N-acetylglucosamine (GlcNAc) residue attached by an O-glycosidic linkage to the polypeptide chain (Holt, G. D., and Hart, G. W. (1986) J. Biol. Chem. 261, 8049-8057) have been found to be enriched in the nuclear and soluble fractions of rat liver. Our goal was to determine the localization and membrane topography of proteins bearing O-linked GlcNAc using galactosyltransferase and wheat germ agglutinin (WGA) as membrane-impermeant probes. Latency of the enzyme mannose-6-phosphatase was used to quantitatively confirm the intactness of the nuclear envelope during incubations with galactosyltransferase or WGA. The O-linked GlcNAc residues of nuclei were fully accessible to modification by galactosyltransferase under conditions where the nuclear envelope mannose-6-phosphatase was 70% latent. Addition of detergent destroyed the permeability barrier but did not increase galactosylation of the O-linked GlcnAc. The major polypeptides bearing O-linked GlcNAc residues on nuclei were peripheral rather than integral membrane proteins with apparent molecular masses ranging from 210 to 54 kDa. The proteins were also detected on sealed nuclei using conjugates of WGA. WGA-rhodamine labeled intact nuclei when examined by immunofluorescence; WGA-peroxidase was used to identify the nuclear glycoproteins after transfer to nitrocellulose. WGA-ferritin selectively labels the cytoplasmic and nucleoplasmic faces of the nuclear pore complex when examined by electron microscopy. Taken together, these data strongly suggest that proteins bearing cytoplasmically oriented O-linked GlcNAc are components of the nuclear pore complex, thereby raising the possibility that cytoplasmic and nucleoplasmic glycoproteins are involved in the assembly or functioning of the nuclear pore.

Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine.

A novel form of protein-saccharide linkage consisting of single N-acetylglucosamine (GlcNAc) residues attached in O-linkages directly to the polypeptide backbone has been described (Holt, G. D., and G. W. Hart, 1986, J. Biol. Chem., 261:8049-8057). This modification was found on proteins distributed throughout the cell, although proteins bearing O-linked GlcNAc moieties were particularly abundant in the cytosolic and nuclear envelope fractions of rat liver. In the accompanying article (Snow, C. M., A. Senior, and L. Gerace, 1987, J. Cell. Biol., 104: 1143-1156), the authors describe monoclonal antibodies directed against eight proteins localized to the nuclear pore complex. These proteins occur on the cytoplasmic and nucleoplasmic (but not lumenal) sides of nuclear membranes. In this report, we demonstrate that all members of this group of pore complex proteins bear multiple O-linked GlcNAc residues. Further, we show that the O-linked GlcNAc moieties are linked via serine (and possibly threonine) side chains to these proteins. Perturbing the O-linked GlcNAc residues either by covalently attaching galactose to them or by releasing them with beta-N-acetylglucosaminidase strongly diminishes the immunoreactivity of the proteins with all of the monoclonal antibodies. However, the O-linked GlcNAc moieties are only part of the epitopes recognized, since O-GlcNAc-containing limit pronase fragments of nuclear pore complex proteins cannot be immunoprecipitated by these antibodies. These findings, taken together with those in the accompanying article, are a direct demonstration that proteins of the cytoplasm and nucleoplasm bear O-linked GlcNAc residues.

The subcellular distribution of terminal N-acetylglucosamine moieties. Localization of a novel protein-saccharide linkage, O-linked GlcNAc.

Previously our laboratory reported the discovery of a novel protein-saccharide linkage in which single N-acetylglucosamine (GlcNAc) residues are attached in O-linkages to protein (Torres, C. R., and Hart, G. W. (1984) J. Biol. Chem. 259, 3308-3317). This linkage was first found on plasma membrane proteins of living cells by galactosylation with bovine milk galactosyltransferase. Here we report the distribution of O-linked GlcNAc in highly enriched rat liver subcellular organelles. Nonidet P-40 solubilized organelles were labeled by galactosyltransferase with UDP-[3H]galactose, and the amount of radiolabel occurring on GlcNAc residues in O-linkages was assessed by its sensitivity to beta-elimination and by its resistance to deglycosylation with endo-beta-N-acetylglucosaminidase F. The presence of galactose-labeled O-linked GlcNAc residues was confirmed by high voltage paper electrophoresis. There is a 17-fold range per mg of protein in the amount of galactosylatable terminal GlcNAc residues found in the various organelles, as well as a wide range in the organelles' apparent content of O-linked GlcNAc residues. Nuclei and the soluble fraction of rat liver cells are particularly enriched with proteins bearing O-linked GlcNAc residues, although these residues are demonstrable in virtually all organelles tested. Furthermore, examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that many different organelle-specific proteins are glycosylated with O-linked GlcNAc residues. Because of the wide occurrence of this unique linkage, these data suggest that glycosylation with O-linked GlcNAc residues is not an exclusive marker for a particular organelle. In addition, we have surveyed the organelles for their content of glycoproteins bearing GlcNAc-terminated N-linked oligosaccharides. Our data demonstrate that there are significant amounts of these oligosaccharides in rough and stripped microsomes, nuclei, and nuclear envelopes. In light of evidence that terminal GlcNAc transferases are localized to the Golgi complex, these data suggest that there are glycoproteins which enter into the Golgi for processing and then are transported back into the rough endoplasmic reticulum, and possibly the nucleus.

Structural characterization of novel complex oligosaccharides accumulated in the caprine beta-mannosidosis kidney. Occurrence of tetra- and pentasaccharides containing a beta-linked mannose residue at the nonreducing terminus.

Four oligosaccharide fractions were isolated and purified from the kidney of goats affected with beta-mannosidosis by repeating Bio-Gel P-2 column chromatography. The structural characterization of the purified oligosaccharide fractions (oligosaccharides A, B, C1,2, and D) included sugar composition analysis by gas chromatography, sugar sequence analysis by mass spectrometry of their permethylated alditols, and by methylation analysis as well as anomeric configuration studies by exoglycosidase digestions. Oligosaccharides A and B were the major oligosaccharides accumulating in the kidney and were elucidated as Man beta 1-4GlcNAc and Man beta 1-4GlcNAc beta 1-4GlcNAc, respectively (Matsuura, F., Laine, R. A., and Jones, M. Z. (1981) Arch. Biochem. Biophys. 211, 485-493). Oligosaccharide C1,2 was a mixture of two tetrasaccharides and oligosaccharide D was a pentasaccharide. The proposed structures are: oligosaccharide C1, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc; oligosaccharide C2, Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc; oligosaccharide D, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc beta 1-4GlcNAc. Tetrasaccharide C1 and pentasaccharide D are heretofore undiscovered oligosaccharides. There is no precedent for these structures in glycoproteins or other glycoconjugates. One possibility which accounts for the presence of oligosaccharide C1 and D is that a bisecting N-acetylglucosamine (the beta-N-acetylglucosamine residue linked at the C-4 position of the beta-mannosyl residue of the trimannosyl core of the asparagine-linked sugar chains) is linked by a beta-mannosyl residue. Moreover, the detection of oligosaccharides containing two N-acetylglucosamine residues at the reducing terminus, together with those containing a single N-acetylglucosamine residue, is further corroboration of species-specific differences in glycoprotein catabolic pathways (Hancock, L. W., and Dawson, G. (1984) Fed. Proc. 43, 1552) or in glycoprotein structures.

Novel carbohydrate structures of cathepsin B from porcine spleen.

Two 13-residue glycopeptides were isolated from the digestion of purified porcine spleen cathepsin B by Staphylococcus aureus protease using high performance liquid chromatography. The major peptide, which is about 73% of the total, had the amino acid sequence His-His-Val-Asn(CH2O)-Gly-Ser-Arg-Pro-Pro-Cys-Thr-Gly-Glu. This peptide contains only a single N-acetylglucosamine residue linked to asparagine at the fourth residue by a beta-linkage. The minor peptide had a single amino acid replacement in a sequence otherwise identical to that of the major peptide. A serine was found at residue 10 instead of a half-cystine. The minor peptide also contains different carbohydrates, which were determined using proton NMR to be Man alpha 1----6 Man beta 1----4 GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAc beta 1----n Asn. These results suggest that the cathepsin B carbohydrates are processed in vivo by enzymic systems specific to each isozyme.

Demonstration and partial characterization of endo- N-acetyl-β-d-glucosaminidase in human tissues

It has been postulated that the biological degradation of the oligosaccharide part of the serum type glycoproteins would be initiated by the splitting of the di-N-acetyl chitobiose moiety by an endo-Nacetyl-/3-D-glucosaminidase (EC 3.2.1.96 endoglucosaminidase) [ 1,2]. This hypothesis is sustained by the finding of oligosaccharide chains of various lengths and compositions in urine and tissues of patients, suffering from lysosomal storage disorders as in sialidosis 131. These oligosaccharides share a single N-acetylglucosamine residue at the reducing terminus. At the other end of the chain the sugar is present for which the hydrolysis is blocked due to the enzyme deficiency. Endoglucosaminidase activity towards oligomannosidic glycans was reported in rat and pig tissues [2]. This finding was confirmed [4], localizing this type of enzymatic activity in the cytosolic fraction of rat liver and kidney, with an oligomannosidic substrate derived from ovalbumin. In the same tissue activity was also shown towards a glycopeptide isolated from asialotransferrin, a substrate of the N-acetyl-lactosaminic type [5]. To obtain support for the hypothesis [ 1,2], it was of interest to see whether endoglucosaminidase activity could be detected in human tis-