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

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

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6 Citations (Scopus)

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 - Jan 1 1994

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Mannosyltransferases
Dolichol
Human Mammary Glands
Purification
Peptides
Enzymes
Rats
Uridine Diphosphate
Detergents
Microsomes
Nucleotides
Chloromercuribenzoates
Breast
Glycosylation
Glucosyltransferases
Proteins
Ethylmaleimide
Antibodies
Cloning
Asparagine

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{3012ca2000534a1da9e5928cf19d11b1,
title = "Photoidentification of mannosyltransferases of dolichol cycle in the mammary gland. Purification and characterization of GDP-Man:Manβ1 → 4GlcNAcβ1 → 4GlcNAc-P-P-dolichol mannosyltransferase",
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.",
author = "Ashok Mudgapalli and Roy, {S. K.} and Holmes, {E. H.} and Vijay, {I. K.}",
year = "1994",
month = "1",
day = "1",
language = "English (US)",
volume = "269",
pages = "11327--11336",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "15",

}

TY - JOUR

T1 - Photoidentification of mannosyltransferases of dolichol cycle in the mammary gland. Purification and characterization of GDP-Man:Manβ1 → 4GlcNAcβ1 → 4GlcNAc-P-P-dolichol mannosyltransferase

AU - Mudgapalli, Ashok

AU - Roy, S. K.

AU - Holmes, E. H.

AU - Vijay, I. K.

PY - 1994/1/1

Y1 - 1994/1/1

N2 - 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.

AB - 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.

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SP - 11327

EP - 11336

JO - Journal of Biological Chemistry

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