Mannosylation of a synthetic gpi precursor by hamster membranes requires prior palmttoyl-CoA dependent acylation

W. T. Doerrier, J. Ye, J R Falck, Mark A Lehrman

Research output: Contribution to journalArticle

Abstract

GlcN-PI(CS) (di-octanoyl ghicosamine-α-phosphatidylinositol), a short chain analog of an intermediate in the synthesis of glycosylphosphatidylinositol (GPI) anchois was used in an in vitro assay with Chinese hamster ovary cell membranes and pHJ-Mannose-PDolichol (generated with exogenous GDP-[3H]-Mannose) to investigate conditions required for its mannosylation. Mannosylation of GicN-Pl(C8) required palnritoyl-CoA either added exogenousry or synthesized in vitro by adding ATP and free CoA. Nitrous acid, HF, a-mannosidase, GPIPLD and PI-PLC treatments and Bio-Gel P-2 chromatography showed that the structure of major mannosylated product was consistent with Man-G)cN-(acyl)-PI(C8). Pataritoyl-CoA was an acyl donor as different chain length acyl-CoA's gave products with different mobilities on TLC. At higher concentrations, GlcN-PI(C8) was an inhibitor of its own mannosylation as well as mannosylation of endogenous GlcN(acyl)-PI. This suggests that palmitoyl-CoA dependent acylation converted GlcNPI(C8) from an inhibitor into an acceptor of the mannosyltransferase.

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number6
StatePublished - 1996

Fingerprint

Acylation
Acyl Coenzyme A
acylation
Coenzyme A
mannose
hamsters
Cricetinae
Mannosyltransferases
Guanosine Diphosphate Mannose
Mannosidases
mannosidases
Nitrous Acid
Palmitoyl Coenzyme A
Mannose
Membranes
Glycosylphosphatidylinositols
phosphatidylinositols
Chinese hamsters
Phosphatidylinositols
Cricetulus

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Mannosylation of a synthetic gpi precursor by hamster membranes requires prior palmttoyl-CoA dependent acylation. / Doerrier, W. T.; Ye, J.; Falck, J R; Lehrman, Mark A.

In: FASEB Journal, Vol. 10, No. 6, 1996.

Research output: Contribution to journalArticle

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PY - 1996

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AB - GlcN-PI(CS) (di-octanoyl ghicosamine-α-phosphatidylinositol), a short chain analog of an intermediate in the synthesis of glycosylphosphatidylinositol (GPI) anchois was used in an in vitro assay with Chinese hamster ovary cell membranes and pHJ-Mannose-PDolichol (generated with exogenous GDP-[3H]-Mannose) to investigate conditions required for its mannosylation. Mannosylation of GicN-Pl(C8) required palnritoyl-CoA either added exogenousry or synthesized in vitro by adding ATP and free CoA. Nitrous acid, HF, a-mannosidase, GPIPLD and PI-PLC treatments and Bio-Gel P-2 chromatography showed that the structure of major mannosylated product was consistent with Man-G)cN-(acyl)-PI(C8). Pataritoyl-CoA was an acyl donor as different chain length acyl-CoA's gave products with different mobilities on TLC. At higher concentrations, GlcN-PI(C8) was an inhibitor of its own mannosylation as well as mannosylation of endogenous GlcN(acyl)-PI. This suggests that palmitoyl-CoA dependent acylation converted GlcNPI(C8) from an inhibitor into an acceptor of the mannosyltransferase.

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