|Budget Amount *help
¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 1990 : ¥300,000 (Direct Cost : ¥300,000)
Fiscal Year 1989 : ¥1,300,000 (Direct Cost : ¥1,300,000)
I have isolated a temperatureーsensitive (ts) Asnーlinked glycosylation mutant, designated G258, from a mouse mammary carcinoma cell line FM3A by [2-^3H] mannose suicide selection. G258 is not only ts for Asnーlinked glycosylation, but also ts for cell growth. The biochemical defect resides in the formation of oligosaccharidelipid (OL). At 33^ﾟC, G258 cells synthesize Glc_3Man_9GlcNAc_2-PP-Dol, the fully assembled OL, but at 39^ﾟC, G258 cells are able to synthesize merely the mediumーsized OL (up to Man_3GlcNAc_2-PP-Dol), but are unable to synthesize the larger OL [J. Cell. Physiol. 119 : 260-266 (1984) ]. Judging from the current model of the topography of OL synthesis in the rER membrane, medium-sized OLs such as Man_3GlcNAc_2-PP-Dol would not be recognized by oligosaccharyltransferase. Consequently, at 39ﾟC, VSV-infected G258 cells could not perform Asn-linked glycosylation of G (glyco) protein (submitted paper). Moreover, syntheses of GRP78 (Bip) and GRP94 were induced at 39^ﾟC in G258
cells (submitted paper). The GRP78 (Bip) is known to associate with malfolded proteins. This result implies that aberrant foldings and aberrant assemblies of (glyco) proteins occur at 39ﾟC in G258 cells.
Spontaneous revertants for cell growth were isolated from G258 cells. All growth-revertants also showed reversions on the Asn-linked glycosylation and on the synthesis of OL [Biochim. Biophys. Acta 1091 : 135-140 (1991) ]. These results imply that both temperature-sensitivities of G258 cells were derived from a single gene mutation. This characteristic will enable me to clone human cDNA on the OL synthesis by screening its activity to let G258 cells recover from their temperature sensitivity for cell growth.
A possible mechanism of the coupling between impaired OL synthesis and growth arrest in G258 cells at 39^ﾟC is as follows : At 39^ﾟC, G258 cells are unable to synthesize OLs beyond Man_3GlcNAc_2-PP-Dol. Consequently G258 cells are defective in Asn-linked glycosylation. Resultant aglycoproteins fold abnormally, and accumulate in the rER, forming complex with GRP78 (Bip). Thus the functional expression of essential (glyco) proteins for cell growth [perhaps receptors for growth factors, and/or receptors for nutrients] may be impaired. This may lead G258 cells to stop their growth at 39^ﾟC. Less