Photoidentification of mannosyltransferases of dolichol cycle in the mammary gland. Purification and characterization of GDP-Man:Manβ1 → 4GlcNAcβ1 → 4GlcNAc-P-P-dolichol mannosyltransferase

A. Mudgapalli, S. K. Roy, E. H. Holmes, I. K. Vijay

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6 Scopus citations

Abstract

Glc3Man9GlcNAc2-P-P-Dol serves as the major precursor for the biosynthesis of asparagine-linked glycoproteins in eukaryotes. The first 5 of the 9 mannosyl residues during the assembly of the oligosaccharide moiety within the dolichol cycle in the endoplasmic reticulum are incorporated directly by the action of GDP-Man-requiring mannosyltransferases while the remaining last 4 mannosyl residues are transferred by Man-P-Dol-requiring enzymes. In an earlier study (Shailubhai, K., Illeperuma, C., Tayal, M., and Vijay, I. K. (1990) J. Biol. Chem. 265, 14105-14108), we identified the enzyme UDP-Glc:Dol-P glucosyltransferase by photolabeling rat mammary microsomes with 5-N3-[β-32P]UDP-Glc. Applying a similar strategy, GDP- hexanolamine-125I-azidosalicylic acid, an analog of GDP-Man, was found to photolabel two polypeptides of 37 and 69 kDa among the microsomal proteins of the rat mammary gland. A differential ammonium sulfate saturation (60-80%) of the detergent-solubilized microsomal proteins enriched the 69-kDa polypeptide. Photolabeling of this polypeptide was specifically inhibited by guanine-containing nucleotides and nucleotide-sugars and was associated with a GDP-Man-requiring mannosyltransferase. The mannosyltransferase was purified nearly 16,000-fold and shown to contain the 69-kDa polypeptide. The purified enzyme catalyzes the transfer of [14C]Man from GDP-[14C]Man to Manβ1 → 4GlcNAcβ1 → 4GlcNAc-P-P-Dol in α1,3-linkage to give [14C]Manα1 → 3Manβ1 → 4GlcNAcβ1 → 4GlcNAc-P-P-Dol as the product. Antibodies raised against the 69-kDa polypeptide removed the enzymatic activity from the detergent extract of the rat mammary microsomes and reacted specifically with a polypeptide band of the same size on immunoblots. The purified enzyme showed a pH optima of 7.4-7.8, K(m) ~ 4 μM for GDP-Man, ~2-fold activation by phosphatidylcholine, and a strong inhibition by sulfhydryl-selective reagents, N-ethylmaleimide and p-chloromercuribenzoate. The availability of the highly purified enzyme and a monospecific antibody should allow its molecular cloning for investigating the regulation of the machinery for protein N-glycosylation upon hormonally modulated growth and differentiation of the mammary gland during its ontogeny.

Original languageEnglish (US)
Pages (from-to)11327-11336
Number of pages10
JournalJournal of Biological Chemistry
Volume269
Issue number15
StatePublished - 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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