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. 1984 Sep;81(17):5320–5324. doi: 10.1073/pnas.81.17.5320

Sulfation of lutropin oligosaccharides with a cell-free system.

E D Green, J Gruenebaum, M Bielinska, J U Baenziger, I Boime
PMCID: PMC391695  PMID: 6591193

Abstract

Sulfate is covalently linked to the oligosaccharides on the alpha and beta subunits of bovine lutropin (luteinizing hormone; LH) but not to those on human chorionic gonadotropin (hCG). Since the amino acid sequences of the pituitary and placental alpha subunits are homologous, comparison of their asparagine-linked sugars can provide information regarding tissue specificity of oligosaccharide maturation. To characterize this post-translational modification, we have developed a reconstituted cell-free sulfation system. Sulfate is incorporated into exogenously added glycoproteins by sulfotransferases from Triton X-100-lysed Golgi membranes in the presence of 3'-phosphoadenosine 5'-phospho[35S]sulfate, which is generated from [35S]sulfate by a ribosome-free supernate from Krebs ascites tumor cells. LH is sulfated by pituitary and liver membranes but not by those from placenta. Desialylated hCG (AshCG) is sulfated by membranes from placenta and pituitary, but not liver, while hCG is not sulfated by any of these membranes. Endoglycosidase F releases all the incorporated sulfate from LH in the form of a heterogeneous mixture of mono- and disulfated oligosaccharides. In contrast, the sulfate added to AshCG is apparently attached to peptide rather than oligosaccharide. As found with the cell-free system, sulfate metabolically incorporated into LH by pituitary cells is present on a heterogeneous population of mono- and disulfated oligosaccharides. Thus the cell-free sulfation system accurately duplicates the in vivo process.

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Selected References

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

  1. Baenziger J. U., Fiete D. Structural determinants of concanavalin A specificity for oligosaccharides. J Biol Chem. 1979 Apr 10;254(7):2400–2407. [PubMed] [Google Scholar]
  2. Baenziger J. U., Natowicz M. Rapid separation of anionic oligosaccharide species by high performance liquid chromatography. Anal Biochem. 1981 Apr;112(2):357–361. doi: 10.1016/0003-2697(81)90305-5. [DOI] [PubMed] [Google Scholar]
  3. Bedi G. S., French W. C., Bahl O. P. Structure of carbohydrate units of ovine luteinizing hormone. J Biol Chem. 1982 Apr 25;257(8):4345–4355. [PubMed] [Google Scholar]
  4. Elder J. H., Alexander S. endo-beta-N-acetylglucosaminidase F: endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4540–4544. doi: 10.1073/pnas.79.15.4540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Heifetz A., Watson C., Johnson A. R., Roberts M. K. Sulfated glycoproteins secreted by human vascular endothelial cells. J Biol Chem. 1982 Nov 25;257(22):13581–13586. [PubMed] [Google Scholar]
  6. Hogan B. L., Taylor A., Kurkinen M., Couchman J. R. Synthesis and localization of two sulphated glycoproteins associated with basement membranes and the extracellular matrix. J Cell Biol. 1982 Oct;95(1):197–204. doi: 10.1083/jcb.95.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hortin G., Natowicz M., Pierce J., Baenziger J., Parsons T., Boime I. Metabolic labeling of lutropin with [35S]sulfate. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7468–7472. doi: 10.1073/pnas.78.12.7468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hoshina H., Boime I. Combination of rat lutropin subunits occurs early in the secretory pathway. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7649–7653. doi: 10.1073/pnas.79.24.7649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hoshina H., Hortin G., Boime I. Rat pro-opiomelanocortin contains sulfate. Science. 1982 Jul 2;217(4554):63–64. doi: 10.1126/science.6283633. [DOI] [PubMed] [Google Scholar]
  10. Hsu C. H., Kingsbury D. W. Contribution of oligosaccharide sulfation to the charge heterogeneity of a viral glycoprotein. J Biol Chem. 1982 Aug 10;257(15):9035–9038. [PubMed] [Google Scholar]
  11. Hubbard S. C., Ivatt R. J. Synthesis and processing of asparagine-linked oligosaccharides. Annu Rev Biochem. 1981;50:555–583. doi: 10.1146/annurev.bi.50.070181.003011. [DOI] [PubMed] [Google Scholar]
  12. Kessler M. J., Reddy M. S., Shah R. H., Bahl O. P. Structures of N-glycosidic carbohydrate units of human chorionic gonadotropin. J Biol Chem. 1979 Aug 25;254(16):7901–7908. [PubMed] [Google Scholar]
  13. Lee R. W., Huttner W. B. Tyrosine-O-sulfated proteins of PC12 pheochromocytoma cells and their sulfation by a tyrosylprotein sulfotransferase. J Biol Chem. 1983 Sep 25;258(18):11326–11334. [PubMed] [Google Scholar]
  14. Liau Y. H., Horowitz M. I. Incorporation in vitro of [3H]glucosamine or [3H]glucose and [35S]SO42- into rat gastric mucosa. Presence of N-acetylhexosamine mono- and disulfates and galactose monosulfate in glycoprotein. J Biol Chem. 1982 May 10;257(9):4709–4718. [PubMed] [Google Scholar]
  15. Mellis S. J., Baenziger J. U. Size fractionation of anionic oligosaccharides and glycopeptides by high-performance liquid chromatography. Anal Biochem. 1983 Oct 15;134(2):442–449. doi: 10.1016/0003-2697(83)90320-2. [DOI] [PubMed] [Google Scholar]
  16. Miller R. R., Waechter C. J. Enzymatic sulfation of a large molecular weight glycoprotein associated with pig brain membranes. Arch Biochem Biophys. 1979 Nov;198(1):31–41. doi: 10.1016/0003-9861(79)90392-8. [DOI] [PubMed] [Google Scholar]
  17. Mizuochi T., Kobata A. Different asparagine-linked sugar chains on the two polypeptide chains of human chorionic gonadotropin. Biochem Biophys Res Commun. 1980 Nov 28;97(2):772–778. doi: 10.1016/0006-291x(80)90331-9. [DOI] [PubMed] [Google Scholar]
  18. Moore H. P., Gumbiner B., Kelly R. B. A subclass of proteins and sulfated macromolecules secreted by AtT-20 (mouse pituitary tumor) cells is sorted with adrenocorticotropin into dense secretory granules. J Cell Biol. 1983 Sep;97(3):810–817. doi: 10.1083/jcb.97.3.810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nakamura K., Compans R. W. Effects of glucosamine, 2-deoxyglucose, and tunicamycin on glycosylation, sulfation, and assembly of influenza viral proteins. Virology. 1978 Feb;84(2):303–319. doi: 10.1016/0042-6822(78)90250-7. [DOI] [PubMed] [Google Scholar]
  20. Papkoff H., Gan J. Bovine interstitial cell-stimulating hormone: purification and properties. Arch Biochem Biophys. 1970 Feb;136(2):522–528. doi: 10.1016/0003-9861(70)90224-9. [DOI] [PubMed] [Google Scholar]
  21. Parsons T. F., Bloomfield G. A., Pierce J. G. Purification of an alternate form of the alpha subunit of the glycoprotein hormones from bovine pituitaries and identification of its O-linked oligosaccharide. J Biol Chem. 1983 Jan 10;258(1):240–244. [PubMed] [Google Scholar]
  22. Parsons T. F., Pierce J. G. Oligosaccharide moieties of glycoprotein hormones: bovine lutropin resists enzymatic deglycosylation because of terminal O-sulfated N-acetylhexosamines. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7089–7093. doi: 10.1073/pnas.77.12.7089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pierce J. G., Parsons T. F. Glycoprotein hormones: structure and function. Annu Rev Biochem. 1981;50:465–495. doi: 10.1146/annurev.bi.50.070181.002341. [DOI] [PubMed] [Google Scholar]
  24. Prehm P., Scheid A., Choppin P. W. The carbohydrate structure of the glycoproteins of the paramyxovirus SV5 grown in bovine kidney cells. J Biol Chem. 1979 Oct 10;254(19):9669–9677. [PubMed] [Google Scholar]
  25. Reitman M. L., Kornfeld S. Lysosomal enzyme targeting. N-Acetylglucosaminylphosphotransferase selectively phosphorylates native lysosomal enzymes. J Biol Chem. 1981 Dec 10;256(23):11977–11980. [PubMed] [Google Scholar]
  26. Rogers G., Gruenebaum J., Boime I. Reconstitution of a tandem Co- and post-translational processing pathway with rat liver subcellular fractions. J Biol Chem. 1982 Apr 25;257(8):4179–4186. [PubMed] [Google Scholar]
  27. Spik G., Bayard B., Fournet B., Strecker G., Bouquelet S., Montreuil J. Studies on glycoconjugates. LXIV. Complete structure of two carbohydrate units of human serotransferrin. FEBS Lett. 1975 Feb 15;50(3):296–299. doi: 10.1016/0014-5793(75)80513-8. [DOI] [PubMed] [Google Scholar]
  28. Stadler J., Gerisch G., Bauer G., Suchanek C., Huttner W. B. In vivo sulfation of the contact site A glycoprotein of Dictyostelium discoideum. EMBO J. 1983;2(7):1137–1143. doi: 10.1002/j.1460-2075.1983.tb01558.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Szczesna E., Boime I. mRNA-dependent synthesis of authentic precursor to human placental lactogen: conversion to its mature hormone form in ascites cell-free extracts. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1179–1183. doi: 10.1073/pnas.73.4.1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Takasaki S., Mizuochi T., Kobata A. Hydrazinolysis of asparagine-linked sugar chains to produce free oligosaccharides. Methods Enzymol. 1982;83:263–268. doi: 10.1016/0076-6879(82)83019-x. [DOI] [PubMed] [Google Scholar]
  31. Weintraub B. D., Krauth G., Rosen S. W., Babson A. S. Differences between purified ectopic and normal alpha subnits of human glycoprotein hormones. J Clin Invest. 1975 Oct;56(4):1043–1052. doi: 10.1172/JCI108151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yamashita K., Ueda I., Kobata A. Sulfated asparagine-linked sugar chains of hen egg albumin. J Biol Chem. 1983 Dec 10;258(23):14144–14147. [PubMed] [Google Scholar]
  33. Yonekura H., Oguri K., Nakazawa K., Shimizu S., Nakanishi Y., Okayama M. Isolation and partial characterization of sulfated glycoproteins synthesized by corneal epithelium. J Biol Chem. 1982 Sep 25;257(18):11166–11175. [PubMed] [Google Scholar]

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