Oligosaccharide chains are trimmed during synthesis of the envelope glycoprotein of vesicular stomatitis virus.

Abstract

The biosynthesis and maturation of the oligosaccharide moieties of the envelope glycoprotein of vesicular stomatitis virus were investigated in virus-infected HeLa and BHK21 cells after pulse labeling with [2-3Hlmannose. Two major forms of the virus glycoprotein were detected by polyacrylamide gel electrophoresis, which appear to correspond to the viral glycoprotein with either "precursor" or "mature" oligosaccharide chains. The precursor chains in both HeLa and BHK21 cells infected with vesicular stomatitis virus obtained after a 30-min pulse were large oligomannose structures containing approximately 7-9 mannose residues as estimated by gel filtration analysis. The size of the oligomannose structures initially transferred to the protein may have been even larger. Mature, virus-size oligosaccharide chains, which could be detected after a 20-to 30-min delay, contained only three mannose residues and, in addition, contained branch structures terminating in sialic acid. A precursor-product relationship of these two forms of oligosaccharide chains was demonstrated by pulse-chase labeling of virus-infected HeLa cells. These studies indicated that the large oligomannosyl core structures initially added to the glycoprotein were being "trimmed" by the removal of mannose residues prior to (and/or during) the addition of the branch chains terminating in sialic acid. Vesicular stomatitis virus (VSV) is an enveloped, RNA-containing virus of the rhabdovirus group which matures by budding through host cell membranes that have been modified by replacement of host proteins with two virus-specified proteins, one of which is a glycoprotein (1). The VSV-infected animal cell is an excellent experimental system for elucidating the molecular and subcellular mechanisms involved in the biosynthesis and maturation of both cell membrane and virus envelope glycoproteins (2-8).The VSV G polypeptide contains two major glycosylation sites with complex oligosaccharides that are linked N-glycosidically to asparagine (9-11). As expected for the biosynthesis of a membrane glycoprotein, mRNA coding for VSV G is localized on membrane-bound polyribosomes in infected cells (12, 13), and newly synthesized G is found predominantly in rough endoplasmic reticulum membranes (14). The sugar residues in the oligomannosyl core [Mann(GlcNAc)2] are apparently added to the G polypeptide in the rough endoplasmic reticulum, whereas the branch sugars (NeuNAc-Gal-GlcNAc) and fucose are apparently added in smooth internal membranes prior to the appearance of the mature glycoprotein at plasma membranes (15). The initial glycosylation event probably occurs by the en bloc transfer of a preformed oligomannosyl core structure from a lipid intermediate (16).We have examined the product of the initial glycosylation of the VSV G glycoprotein and followed its conversion to mature, virus-size oligosaccharide chains in VSV-infected HeLa and BHK21 cells. Our results indicate that a large oligomannosyl core structure [Man>7(GlcNAc)2] is initially added to the glycopro...

Intercompartmental transport in the Golgi complex is a dissociative process: facile transfer of membrane protein between two Golgi populations.

“…Only the fractions containing glycopeptides are shown . The positions of VSV glycopeptides containing 0, 1, 2, and 3 sialic acid residues ($o, S,, S Z , and S3 , respectively) were located by reference to glycopeptides from VSV-infected baby hamster kidney cells (in which S3 is a major species [12,17]) and CHO 1021 cells (in which So is the only significant species due to the lack of sialic acid addition) . Note that the sialic acid-free species (So) chromatographs as a double peak .…”

Unusual Neutral Oligosaccharides in Mature Sindbis Virus Glycoproteins are Synthesized from Truncated Precursor Oligosaccharides in Chinese Hamster Ovary Cells

“…Pronase-digested glycopeptides were desalted on either a 120 × 1.5 cm or a 120 × 1.0 cm column of Sephadex G 15/50 (Pharmacia) before glycosidase digestion (Hunt & Summers, 1976 b). Digestion of glycopeptides and lipid-derived oligosaccharides with endo-fl-N-acetylglucosaminidase D and H (ENDO-D and ENDO-H) or a-mannosidase were performed as described previously (Etchison et al, 1977;Hunt et al, 1978). Purified ENDO-D from Diplococcus pneumoniae and ENDO-H from Streptomyces griseus were purchased from Miles Laboratories, and Canavalia ensiformis (jack bean) c~-mannosidase was purchased from Sigma.…”

“…Glycopeptides were analysed by gel filtration through columns (120 x 1.5 cm) of BioGel P-4 (minus 400 mesh; BioRad) along with unlabelled markers (blue dextran, stachyose, and mannose) and 14C_labelled gel filtration markers (Hunt & Summers, 1976 b;Hunt et al, 1978). The glycopeptides were eluted with 0.05 M-ammonium acetate, pH 6.…”

“…The initial step occurs in the rough endoplasmic reticulum with the en bloc transfer of a large glucose-containing oligomannosyl structure [Glc3(Man)9GlcNAc2; Fig. l a] from a lipid intermediate to the nascent polypeptide, followed by the rapid removal of glucose (Robbins et al, 1977;Hunt et al, 1978;. In the synthesis of complex acidic-type structures, the ~-1,2-1inked mannoses are removed in the Golgi membranes by c~-mannosidase J togive a (Man)sGlcNAcz structure ( Fig.…”

Processing of the env Gene Products of Moloney Murine Leukaemia Virus

“…This is not surprising as glueosamine residues are found not only in the first two sugar residues linked to the protein asparagine residue, but are also located in the terminal portions of the modified core oligosaccharide structure. Fucose incorporation is indicative of secondary oligosaccharide modification in that fucose addition occurs subsequent to addition and 'trimming' of the oligosaccharide core structure (Waechter & Lennarz, 1976_;Hunt et aL, 1978;Tabas et al, 1978;Turco & Robbins, 1979;Hubbard & Robbins, 1979 Electrophoresis was performed on a linear 6 to 12 % gradient slab gel as described in Methods.…”