Addition of truncated oligosaccharides to influenza virus hemagglutinin results in its temperature-conditional cell-surface expression.

Abstract

we described the isolation and initial characterization of seven Chinese hamster ovary cell lines that are temperature conditional for the cell-surface expression of influenza virus hemagglutinin (HA) and other integral membrane glycoproteins. Two of these cell lines appeared to be defective for the synthesis and/or addition of mannose-rich oligosaccharide chains to nascent glycoproteins. In this paper we show that at both 32 and 39°C the two mutant cell lines accumulate a truncated version, MansGlcNAc2, of the normal lipid-linked precursor oligosaccharide, Glc3MangGlcNAc2. This is possibly due to a defect in the synthesis of dolichol phosphate because in vitro assays indicate that the mutant cells are not deficient in mannosylphosphoryldolichol synthase at either temperature. A mixture of truncated and complete oligosaccharide chains was transferred to newly synthesized glycoproteins at both the permissive and restrictive temperatures. Both mutant cell lines exhibited altered sensitivity to cytotoxic plant lectins when grown at 32°C, indicating that cellular glycoproteins bearing abnormal oligosaccharide chains were transported to the cell surface at the permissive temperature. Although glycosylation was defective at both 32 and 39°C, the cell lines were temperature conditional for growth, suggesting that cellular glycoproteins were adversely affected by the glycosylation defect at the elevated temperature. The temperature-conditional expression of HA on the cell surface was shown to be due to impairment at 39°C of the folding, trimerization, and stability of HA molecules containing truncated oligosaccharide chains. W'E have isolated a series of mutant Chinese hamster ovary (CHO) l cell lines that express the hemagglutinin (HA) of influenza virus on the cell surface in a temperature-conditional fashion (see accompanying paper, Hearing et al., 1989). Two of these cell lines, clones 4B and 4J, differed from the others in that they synthesize abnormally modified glycoproteins. Our initial experiments suggested that the apparent defect in the addition of asparaginelinked oligosaccharide chains to nascent glycoproteins was independent of temperature. This observation prompted us to investigate further the defect in glycosylation in these mutant cell lines and to determine how the attachment of aberrant carbohydrate chains influenced the movement of HA through the secretory pathway, resulting in the temperature-

Retention of secretory proteins in an intermediate compartment and disappearance of the Golgi complex in an END4 mutant of Chinese hamster ovary cells

“…However, this does not explain the defect in End4 mutants, because HA folded and trimerized at the same rate in V.24.1 and wild-type cells at the restrictive temperature. The failure to secrete could also be related to unusual protein glycosylation, since Hearing et al (19,20) reported that addition of truncated N-linked oligosaccharides to HA impaired secretion in a temperaturesensitive mutant of CHO cells. However, in the present work, and in previous studies (39,56), we have not observed any unusual electrophoretic migration of newly synthesized glycoproteins in SDS-polyacrylamide gels that would suggest aberrant or heterogeneous oligosaccharides were present on glycoproteins.…”

How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum.

“…Numerous cell lines in yeast and mammalian cells have been isolated with defects in synthesis, transfer, and trimming of N-linked core carbohydrates (Reitman et al, 1982;Stanley, 1984;Hearing et al, 1989a). Of these, many show a temperature-sensitive growth phenotype consistent with protein folding defects.…”

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

“…Cells infected with influenza virus were infected and labeled, and the immunoprecipitates were treated with endoglycosidase H (endo H; ICN) as described elsewhere (24). All immunoprecipitations were performed as previously described (17).…”

“…While glycoprotein modification in the Golgi apparatus of Saccharomyces cerevisiae differs from that occurring in mammalian cells, the biochemical or enzymatic modifications and processing that occur in the ER are believed to be essentially identical in yeast and mammalian cells (25,30). In all eucaryotic organisms, the initial N-linked glycosylation and subsequent processing of secretory proteins by various glycosyltransferases and glycosidases are part of a complex enzymatic pathway that is not only essential for the correct folding and stability of many proteins (4,17,21,24,36,37) but is in fact coupled with and necessary for the correct routing of some of these proteins to their final destinations (16,26).…”