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. 1974 Jun;71(6):2391–2395. doi: 10.1073/pnas.71.6.2391

The Role of a Dolichol-Oligosaccharide as an Intermediate in Glycoprotein Biosynthesis

An-Fei Hsu 1, John W Baynes 1,*, Edward C Heath 1,†
PMCID: PMC388461  PMID: 4526213

Abstract

Incubation of mouse myeloma microsomes with GDP-[14C]mannose results in the biosynthesis of [14C]mannose phosphoryl dolichol [Baynes, J. W., Hsu, A.-F. & Heath, E. C. (1973) J. Biol. Chem. 248, 5693-5704] and a [14C]mannose- and N-acetylglucosamine (GlcNAc)-containing oligosaccharide derivative of dolichol. Thus, [14C]mannose phosphoryl dolichol and [14C]mannose-labeled oligosaccharide pyrophosphoryl dolichol were isolated from incubation mixtures by solubilization in 2% (w/v) Triton X-100 and the lipids were separated from small molecules by gel filtration fractionation. After removal of radioactive protein from the preparation, the two lipid derivatives were separated quantitatively by fractionation on a concanavalin A-Sepharose column; [14C]mannose phosphoryl dolichol was not retained by the affinity resin but [14C]mannose-oligosaccharide pyrophosphoryl dolichol adsorbed to the gel and was eluted with α-methylmannoside.[14C]Mannose-oligosaccharide pyrophosphoryl dolichol appeared to be homogeneous when fractionated on DEAE-cellulose and in several thin-layer chromatographic systems. Treatment of [14C]mannose oligosaccharide pyrophosphoryl dolichol with 10% (w/v) NH4OH at 100° for 1 hr resulted in the formation of a water-soluble radioactive oligosaccharide phosphate which was isolated and characterized as [Man]5 → [GlcNAc → GlcNAc → P. Incubation of [14C]mannose-oligosaccharide pyrophosphoryl dolichol with myeloma microsomal preparations results in the transfer, presumably, of the entire oligosaccharide to endogenous protein. Kinetic studies indicate that the dolichol derivatives serve as intermediates in the glycosylation of protein as follows: [Formula: see text]

Keywords: myeloma proteins, mannosyl-dolichol, lipid intermediate

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arima T., Spiro R. G. Studies on the carbohydrate units of thyroglobulin. Structure of the mannose-N-acetylglucosamine unit (unit A) of the human and calf proteins. J Biol Chem. 1972 Mar 25;247(6):1836–1848. [PubMed] [Google Scholar]
  2. BURGOS J., HEMMING F. W., PENNOCK J. F., MORTON R. A. DOLICHOL: A NATURALLY-OCCURRING C100 ISOPRENOID ALCOHOL. Biochem J. 1963 Sep;88:470–482. [PMC free article] [PubMed] [Google Scholar]
  3. Baynes J. W., Hsu A. F., Heath E. C. The role of mannosyl-phosphoryl-dihydropolyisoprenol in the synthesis of mammalian glycoproteins. J Biol Chem. 1973 Aug 25;248(16):5693–5704. [PubMed] [Google Scholar]
  4. Behrens N. H., Carminatti H., Staneloni R. J., Leloir L. F., Cantarella A. I. Formation of lipid-bound oligosaccharides containing mannose. Their role in glycoprotein synthesis. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3390–3394. doi: 10.1073/pnas.70.12.3390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Behrens N. H., Parodi A. J., Leloir L. F. Glucose transfer from dolichol monophosphate glucose: the product formed with endogenous microsomal acceptor. Proc Natl Acad Sci U S A. 1971 Nov;68(11):2857–2860. doi: 10.1073/pnas.68.11.2857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hickman S., Kornfeld R., Osterland C. K., Kornfeld S. The structure of the glycopeptides of a human M-immunoglobulin. J Biol Chem. 1972 Apr 10;247(7):2156–2163. [PubMed] [Google Scholar]
  7. Lee C. Y., Scocca J. R. A common structural unit in asparagine-oligosaccharides of several glycoproteins from different sources. J Biol Chem. 1972 Sep 25;247(18):5753–5758. [PubMed] [Google Scholar]
  8. Leloir L. F., Staneloni R. J., Carminatti H., Behrens N. H. The biosynthesis of a N,N'-diacetylchitobiose containing lipid by liver microsomes. A probable dolichol pyrophosphate derivative. Biochem Biophys Res Commun. 1973 Jun 19;52(4):1285–1292. doi: 10.1016/0006-291x(73)90640-2. [DOI] [PubMed] [Google Scholar]
  9. Lennarz W. J., Scher M. G. Metabolism and function of polyisoprenol sugar intermediates in membrane-associated reactions. Biochim Biophys Acta. 1972 Aug 4;265(3):417–441. doi: 10.1016/0304-4157(72)90015-9. [DOI] [PubMed] [Google Scholar]
  10. Li Y. T., Lee Y. C. Pineapple - and -D-mannopyranosidases and their action on core glycopeptides. J Biol Chem. 1972 Jun 10;247(11):3677–3683. [PubMed] [Google Scholar]
  11. Melchers F. Biosynthesis of the carbohydrate portion of immunoglobulin. Radiochemical and chemical analysis of the carbohydrate moieties of two myeloma proteins purified from different subcellular fractions of plasma cells. Biochemistry. 1971 Feb 16;10(4):653–659. doi: 10.1021/bi00780a017. [DOI] [PubMed] [Google Scholar]
  12. Molnar J., Chao H., Ikehara Y. Phosphoryl-N-acetylglucosamine transfer to a lipid acceptor of liver microsomal preparations. Biochim Biophys Acta. 1971 Sep 1;239(3):401–410. doi: 10.1016/0005-2760(71)90033-6. [DOI] [PubMed] [Google Scholar]
  13. Richards J. B., Hemming F. W. The transfer of mannose from guanosine diphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticulum. Biochem J. 1972 Nov;130(1):77–93. doi: 10.1042/bj1300077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Waechter C. J., Lucas J. J., Lennarz W. J. Membrane glycoproteins. I. Enzymatic synthesis of mannosyl phosphoryl polyisoprenol and its role as a mannosyl donor in glycoprotein synthesis. J Biol Chem. 1973 Nov 10;248(21):7570–7579. [PubMed] [Google Scholar]

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