Photoidentification of mannosyltransferases of dolichol cycle in the mammary gland. Purification and characterization of GDP-Man:Man beta 1-->4GlcNAc beta 1-->4GlcNAc-P-P-dolichol mannosyltransferase.
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-[beta-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 Man beta 1-->4GlcNAc beta 1-->4GlcNAc-P-P-Dol in alpha 1,3-linkage to give [14C]Man alpha 1-->3Man beta 1-->4GlcNAc beta 1-->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, Km approximately 4 microM for GDP-Man, approximately 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.
Mudgapalli
A
A
Department of Animal Sciences, University of Maryland, College Park 20742.
Roy
S K
SK
Holmes
E H
EH
Vijay
I K
IK
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004286:Dolichols / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D005260:Female; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D007700:Kinetics; D007774:Lactation; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008364:Mannosyltransferases / Q000302:isolation & purification* / Q000378:metabolism* / Q000528:radiation effects; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D004286:Dolichols; D009844:Oligosaccharides; D006155:Guanosine Diphosphate Mannose; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase