Correlation of glycosylation forms with position in amino acid sequence.

We surveyed published reports on about 50 glycoproteins whose amino acid sequence, glycosylation sites, and type of glycosylation at a particular site have been established. We note that high-mannose substances were rarely found at the N-terminal side of a previously glycosylated complex site. There was a very definite distribution of complex sites about the N-terminal region. Furthermore, secreted glycoproteins usually contained only complex oligosaccharides whereas membrane proteins contained both types. We suggest that the position of the glycosylation site with respect to the N-terminus affects the extent of oligosaccharide processing and subsequent presentation of complex or high-mannose structures in the mature glycoprotein. This review relates glycosylation type to its position in the known sequence of given proteins and discusses these observations in light of known glycosylation processing reactions.

Primary sequence domains required for the retention of rotavirus VP7 in the endoplasmic reticulum.

Rotavirus VP7 is a membrane-associated protein of the endoplasmic reticulum (ER). It is the product of rotavirus gene 9 which potentially encodes a protein of 326 amino acids that contains two amino terminal hydrophobic domains, h1 and h2, each preceded by an initiation codon. Comparison of the size of products derived from altered genes containing coding sequences for both h1 and h2 with those lacking the h1 sequence ('dhl' mutants), indicates that initiation takes place at M30 immediately preceding h2 (residues F32 to L48) and that h2 is cleaved, confirming the studies of others (Stirzaker, S.C., P.L. Whitfeld, D.L. Christie, A.R. Bellamy, and G.W. Both. 1987. J. Cell Biol. 105:2897-2903). Our previous work had shown that deletions in the carboxy end of h2, extending to amino acid 61 in the open reading frame, resulted in secretion of VP7. The region from amino acid number 51-61, present in wild-type VP7 but missing in the secreted mutant delta 47-61, was thus implicated to have a role in ER retention. To test this, a series of chimeric genes were constructed by fusing the first 63 codons of wild-type VP7, delta 1-14 or delta 51-61/dhl, to the mouse salivary alpha-amylase gene, a secretory protein, such that the fusion junction was located at the exact mature terminus of amylase. The chimeric proteins VP7(63)/amylase, delta 1-14(63)/amylase and delta 51-61(63)/dhl/amylase were secreted when expressed in cells and the h2 domain was cleaved when mRNA was translated in vitro. These results imply that the sequence 51-61 is necessary but not sufficient for ER retention. When a second series of VP7/amylase chimera were constructed extending the VP7 contribution to amino acid 111, the product expressed by delta 1-14(111)/amylase was not secreted whereas that of delta 47-61(111)/amylase was. Significantly, the intracellular delta 1-14(111)/amylase product exhibited an amylase enzymatic specific activity that was similar to that of the wild-type amylase product. We conclude that two regions of VP7 mediate its retention in the ER, the first lies within the sequence 51-61 and the second within the sequence 62-111, which contains the glycosylation site for VP7. Both regions are necessary for retention, though neither is sufficient alone.

Co-translational excision of alpha-glucose and alpha-mannose in nascent vesicular stomatitis virus G protein.

Membrane bound polysomes were prepared from HeLa cells infected with vesicular stomatitis virus (VSV), after pulse labeling with [3H]mannose for various times from 15 to 90 min. Oligosaccharides on nascent chains were released from peptides by treatment with endoglycosidase H and sized by high resolution Biogel P4 chromatography. Processing on some nascent chains proceeded to the removal of all three types of alpha-linked glucose and one alpha-1,2-mannose from the Glc3Man9GlcNAc precursor showing that the enzymes responsible were not only active on nascent chains but were present in the rough endoplasmic reticulum (RER). Incubation of cells for various times in cycloheximide, where chain elongation had ceased, made no difference to the profile of oligosaccharides on the nascent chains, and trimming proceeded no further than Man8GlcNAc2Asn . Carbonyl cyanide m-chlorophenylhydrazone (CCCP), an energy inhibitor reportedly able to block the transfer of glycoproteins from the RER, increases the amount of Man8-oligosaccharides on the nascent chains and also the amount of Glc3Man9GlcNAc precursor. On completed G protein in the RER fraction from which membrane bound polysomes were prepared, processing occurred to Man6 - but not to Man5GlcNAc sized oligosaccharides in the CCCP-treated cells. By contrast, processing to Man5GlcNAc oligosaccharides was observed in unfractionated control cells.

Processing of the rough endoplasmic reticulum membrane glycoproteins of rotavirus SA11.

The synthesis and oligosaccharide processing of the glycoproteins of SA11 rotavirus in infected Ma104 cells was examined. Rotavirus assembles in the rough endoplasmic reticulum (RER) and encodes two glycoproteins: VP7, a component of the outer viral capsid, and NCVP5, a nonstructural protein. A variety of evidence suggests the molecules are limited to the ER, a location consistent with the high mannose N-linked oligosaccharides modifying these proteins. VP7 and NCVP5 were shown to be integral membrane proteins. In an in vitro translation system supplemented with dog pancreas microsomes, they remained membrane associated after high salt treatment and sodium carbonate-mediated release of microsomal contents. In infected cells, the oligosaccharide processing of these molecules proceeded in a time-dependent manner. For VP7, Man8GlcNAc2 and Man6GlcNAc2 were the predominant intracellular species after a 5-min pulse with [3H]mannose and a 90 min chase, while in contrast, trimming of NCVP5 halted at Man8GlcNAc2. VP7 on mature virus was processed to Man5GlcNAc2. It is suggested that the alpha-mannosidase activities responsible for the formation of these structures reside in the ER. In the presence of the energy inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), processing of VP7 and the vesicular stomatitis virus G protein was blocked at Man8GlcNAc2. After a 20-min chase of [3H]mannose-labeled molecules followed by addition of CCCP, trimming of VP7 could continue while processing of G protein remained blocked. Thus, an energy-sensitive translocation step within the ER may mark the divergence of the processing pathways of these glycoproteins.

Calcium depletion blocks the maturation of rotavirus by altering the oligomerization of virus-encoded proteins in the ER.

Maturation of rotavirus occurs in the ER. The virus transiently acquires an ER-derived membrane surrounding the virus particle before the eventual formation of double-shelled particles. The maturation process includes the retention and selective loss of specific viral protein(s) as well as the ER-derived membrane during formation of the outer capsid of the mature virus. When infected cells were depleted of Ca++ by use of the ionophore A23187 in calcium-free medium, membrane-enveloped intermediates were seen to accumulate. When Mn++, an efficient Ca++ competitor, was used to replace Ca++ in the medium, the accumulation of the enveloped intermediate was again observed, pointing to an absolute requirement of Ca++ in the maturation process. It was previously demonstrated in this laboratory that a hetero-oligomeric complex of NS28, VP7, and VP4 exists which may participate in the budding of the single-shelled particle into the ER (Maass, D. R., and P. H. Atkinson, 1990. J. Virol. 64:2632-2641). The present study demonstrates that either in the absence of Ca++ or in the presence of tunicamycin, a glycosylation inhibitor, VP7 is excluded from these hetero-oligomers. In the presence of Mn++, VP4 was blocked in forming a hetero-oligomeric complex with NS28 and VP7. The electrophoretic mobility of the viral glycoproteins synthesized in the presence of the ionophore were found to be altered. This size difference was attributed to altered N-linked glycosylation and carbohydrate processing of the viral glycoproteins. These results imply a major role for calcium and the state of glycosylation of NS28 in the assembly and acquisition of specific viral protein conformations necessary for the correct association of proteins during virus maturation in the ER.

Further characterization of HeLa S3 plasma membrane ghosts.

A plasma membrane fraction of HeLa S3 cells, consisting of ghosts, is characterized more fully. A simple procedure is described which permits light and electron microscope study of the plasma membrane fraction through the entire depth of the final product pellet and through large areas parallel to the surface. Contamination by nuclei is 0.14%, too little for DNA detection by the diphenylamine reaction. Contamination by rough endoplasmic reticulum and ribosomes is small, a single ghost containing about 3% of the RNA in a single cell. Mitochondria were not encountered. Electron microscopy also shows (a) small vesicles associated with the outer surface of the ghosts, and (b) a filamentous web at the inner face of the ghost membrane. Sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis shows that of the many Coomassie Blue-stained bands two were prominent. One, 43,000 daltons, co-migrated with purified rabbit muscle actin and constituted about 7.5% of the plasma membrane protein. The other major band, 34,000 daltons, was concentrated in the plasma membrane fraction. Two major glycoproteins detected by autoradiography of [14C]fucose-labeled glycoproteins on the gels, had apparent molecular weights of 35,000 daltons and 32,000 daltons. These major bands did not stain with Coomassie Blue. There were many other minor glycoprotein bands in the 200,000- to 80,000-dalton range. Ouabain-sensitive, Na+, K+-adenosine triphosphatase (ATPase) activity of the ghost fraction is purified 9.1 (+/- 2.2) times over the homogenate; recover of the activity is 12.0 (+/- 3.8%) of the homogenate. Enrichment and recovery of fucosylglycoprotein parallel those for ouabain-sensitive Na+, K+-ATPase activity. Fucosyl glycoprotein is recovered more than the enzyme activity in a smooth membrane vesicle fraction probably containing the bulk of plasma membrane not recovered as ghosts.

Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein.

Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon. A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N-acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N-acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal.

Biosynthesis and processing of ribophorins in the endoplasmic reticulum.

Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s). Direct analysis of the oligosaccharides of ribophorin l showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from Man8GlcNAc to Man5GlcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus.

Studies on mannose-containing glycopeptides from a normal and an SV40 transformed human cell.

The oligosaccharide moiety of cell-surface mannose-labelled glycopeptides from a normal (WI38) and an SV40 transformed cell (W118Va) have been investigated using specific glycosidases. Partially purified mannose-containing glycopeptides were separated into acidic and neutral species by high voltage paper electrophoresis. Endo-beta-N-acetylgucosaminidase D, in the presence of three exoglycosidases, released from the acidic glycopeptides of non-growing cells a product completely absent in growing cells. However, the acidic species from growing WI18 Va and WI38 were found to be similar in the products released by enzyme digestion. The neutral species from growing normal cells contained a proportion of the glycopeptides resistant to endoglycosidase D while those from the non-growing cells were almost free of these resistant species. The SV40 transformed cells were further enriched, when compared to normal cells (WI38), in these neutral resistant species. We suggest that the oligomannosyl core of the majority of the susceptible species contains three mannose residues while that of the resistant species contains between six and eight.

Fractionation of ovalbumin glycopeptide AC-C by high-pressure liquid chromatography. Determination of structure by 1H-NMR spectroscopy.

We describe for the first time a method for the separation of intact ovalbumin glycopeptides by high-pressure liquid chromatography (HPLC). The separation was achieved using two reverse-phase columns connected in series and eluting with an isocratic solvent system at acid pH containing 1-hexane sulfonate. Ovalbumin glycopeptide fraction AC-C has been separated into at least four distinct glycopeptides. High resolution 1H-NMR spectroscopy has confirmed the reported structure of the two major species. We also extend our structural studies to the two other glycopeptides and establish the structure of a previously unreported ovalbumin glycopeptide, Man3GalGlcNAcAsn.

Determination of the structure of ovalbumin glycopeptide AC-B by 1H nuclear magnetic resonance spectroscopy at 500 MHz.

Three unique, unmodified ovalbumin glycopeptides were separated to homogeneity by high-pressure liquid chromatography. The nuclear magnetic resonance data, at 500 MHz, confirmed the structure of two of the three species and for the first time established the presence of a Man8GlcNAc2Asn glycopeptide in ovalbumin. This compound was a single homogeneous isomeric form out of three possible compounds expected as processing intermediates.

Rhodopsin's protein and carbohydrate structure: selected aspects.

A topographic model for rhodopsin has been constructed based upon evaluation of rhodopsin's sequence by a secondary structure prediction algorithm as well as chemical and enzymatic modification of rhodopsin in the membrane [Hargrave et al. (1983) Biophys. Struct. Mech. 9, 235-244]. The non-uniform distribution of several amino acids in the primary structure and within the topographic model is discussed. The seven predicted helices were evaluated and each helix was found to have one surface which is much more hydrophobic than the other. Stereoscopic views of a three dimensional model with a functional color-coding scheme incorporating these features are presented. The amino acid sequence of rhodopsin has been compared to other proteins in the Dayhoff Protein Data Bank. No obvious relationship to any other protein sequenced was found. High resolution proton magnetic resonance spectroscopy was used to reinvestigate the structure and relative proportions of rhodopsin's major and minor oligosaccharide chains. One major (Man3GlcNAc3) and two minor (Man4GlcNAc3 and Man5GlcNAc3) were observed.

Chemical and spectroscopic characterisation of the phosphatidylinositol manno-oligosaccharides from Mycobacterium bovis AN5 and WAg201 and Mycobacterium smegmatis mc2 155.

Two classes of phosphatidylinositol manno-oligosaccharides (PIMs) were isolated from each of Mycobacterium bovis AN5 and WAg201 and Mycobacterium smegmatis mc2 155. The deacylated PIMs (dPIMs), were identified as hexasaccharide (dPIM-6) and disaccharide (dPIM-2) species composed of mannose, myo-inositol, glycerol and phosphate residues in the proportions of 6:1:1:1 and 2:1:1:1, respectively. Structural analysis, employing a combination of microanalytical methods, nuclear magnetic resonance spectroscopy, and mass spectrometric techniques established that the sequence of residues within dPIM-6, as shown below, was identical in the three mycobacterial strains investigated.

Glycosylation of prion strains in transmissible spongiform encephalopathies.

This is a review of prion replication in the context of the cell biology of membrane proteins especially folding quality control in the endoplasmic reticulum (ER). Transmissible spongiform encephalopathies, such as scrapie and BSE, are infectious lethal diseases of mammalian neurons characterised by conversion of the normal membrane protein PrPC to the disease-associated conformational isomer called PrPSc. PrPSc, apparently responsible for infectivity, forms a number of different conformations and specific N-glycosylation site occupancies that correlate with TSE strain differences. Dimerisation and specific binding of PrPc and PrPSc seems critical in PrPSc biosynthesis and is influenced by N-glycosylation and disulfide bond formation. PrPsc can be amplified in vitro but new glycosylation cannot occur in cell free environments without the special conditions of microsome mediated in vitro translation, thus strain specific glycosylation of PrPSc formed in vitro in the absence of these conditions must take place by imprintation of PrPc from existing glycosylation site-occupancies. PrPSc formed in cell free homogenates is not infectious pointing to events necessary for infectivity that only occur in intact cells. Such events may include glycosylation site occupancy and ER folding chaperone activity. In the biosynthetic pathway of PrPSc, early acquisition of sensitivity of the GPI anchor to phospholipase C can be distinguished from the later acquisition of protease resistance and detergent insolubility. By analogy to the co-translational formation of the MHC I loading complex, it is postulated that PrPSc or its specific peptides could imprint nascent PrPc chains thereby ensuring its own folds and the observed glycosylation site occupancy ratios of strains.

Synthesis and assembly of HeLa cell plasma membrane glycoproteins and proteins.

At least 60 percent of the fucose residues in HeLa cell glycoprotein are nonreducing, terminal, and closely proximal to the protein carbohydrate linkage. As determined by pulse-labeling with (3H) fucose and sizing glycopeptides in Sephadex chromatography these residues are added near the time of completion of oligosaccharide chains. Glycoproteins, the large bulk if not the only macromolecules labeled with radioactive fucose in HeLa cells, were not soluble in ethanol or chloroform-methanol, 2:1, but were substanially solubilized by chloroform-methanol-water, 10:10:3. Folch extraction of labeled cells and analysis of the upper phase revealed little if any (3H) fucose-labeled glycosphingolipids. Studies on the distribution of radioactively labeled glycoprotein in various cell fractions show that in uniform labeling conditions fucosylated glycoproteins accumulate in the plasma membrane specifically. Pulse-chase and protein synthesis inhibitor studies show that there is an internal pool of completed fucosylated glycoprotein, taking not less than 12 min to deplete. From this pool newly synthesized glycoprotein moves to the plasma membrane with a transit time of 12 min and little was found soluble in the cell. By contrast, a pool of protein labeled with 14C-aminoacids and precursor to plasma membrane protein is small and depleted almost immediately. From this pool newly synthesized protein molecules move to the plasma membrane with a transit time of less than 2 min. It would appear that these two distinct molecular components of plasma membranes may be assembled into membranes sequentially or into the plasma membrane independently.