Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1992 Jul;1(7):902–909. doi: 10.1002/pro.5560010708

Complete enzymatic deglycosylation of native sex steroid-binding protein (SBP or SHBG) of human and rabbit plasma: effect on the steroid-binding activity.

P H Petra 1, P R Griffin 1, J R Yates 3rd 1, K Moore 1, W Zhang 1
PMCID: PMC2142156  PMID: 1304375

Abstract

An enzymatic procedure for the complete removal of the N-linked and O-linked oligosaccharide side chains of the sex steroid-binding proteins (SBP or SHBG) of human and rabbit plasma under native conditions is described. Deglycosylation was catalyzed by N-glycanase, neuraminidase, and O-glycanase and was monitored by SDS-PAGE, lectin blotting, and molecular weight analyses by electrospray mass spectrometry. Digestion of rabbit SBP with N-glycanase generated a major 39,777-Da protein and two minor ones of 39,389 and 39,545 Da. The molecular weight of the major protein agrees with the molecular weight calculated from the sequence of the sugar-free polypeptide monomer (39,769 Da: Griffin, P.R., Kumar, S., Shabanowitz, J., Charbonneau, H., Namkung, P.C., Walsh, K.A., Hunt, D.F., & Petra, P.H., 1989, J. Biol. Chem. 264, 19066-19075), whereas the other two are deglycosylated proteolytic cleavage products lacking the TQR and TQ sequences at the amino-terminus. The N- and O-linked side chains of human SBP were removed by sequential digestion with N-glycanase and neuraminidase/O-glycanase. A 38,771-Da protein was generated, which agrees well with the molecular weight of the sugar-free polypeptide monomer (Walsh, K.A., Titani, K., Kumar, S., Hayes, R., & Petra, P.H., 1986, Biochemistry 25, 7584-7590). N-deglycosylation of human and rabbit SBP has no effect on the steroid-binding activity, but removal of the O-linked side chains of N-deglycosylated human SBP results in an apparent 50% loss of steroid-binding activity and an increase in the Kd for the binding of 5 alpha-dihydrotestosterone from 0.3 mM to 0.9 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Full Text

The Full Text of this article is available as a PDF (2.8 MB).

Selected References

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

  1. Bardin C. W., Lipsett M. B. Testosterone and androstenedione blood production rates in normal women and women with idiopathic hirsutism or polycystic ovaries. J Clin Invest. 1967 May;46(5):891–902. doi: 10.1172/JCI105588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bocchinfuso W. P., Warmels-Rodenhiser S., Hammond G. L. Expression and differential glycosylation of human sex hormone-binding globulin by mammalian cell lines. Mol Endocrinol. 1991 Nov;5(11):1723–1729. doi: 10.1210/mend-5-11-1723. [DOI] [PubMed] [Google Scholar]
  3. Bordin S., Torres R., Petra P. H. An enzyme-immunoassay (ELISA) for the sex steroid-binding protein (SBP) of human serum. J Steroid Biochem. 1982 Oct;17(4):453–457. doi: 10.1016/0022-4731(82)90641-0. [DOI] [PubMed] [Google Scholar]
  4. Cheng C. Y., Musto N. A., Gunsalus G. L., Bardin C. W. The role of the carbohydrate moiety on the size heterogeneity and immunologic determinants of human testosterone-estradiol-binding globulin. J Steroid Biochem. 1985 Jan;22(1):127–134. doi: 10.1016/0022-4731(85)90152-9. [DOI] [PubMed] [Google Scholar]
  5. Danzo B. J., Bell B. W., Black J. H. Human testosterone-binding globulin is a dimer composed of two identical protomers that are differentially glycosylated. Endocrinology. 1989 Jun;124(6):2809–2817. doi: 10.1210/endo-124-6-2809. [DOI] [PubMed] [Google Scholar]
  6. Gershagen S., Henningsson K., Fernlund P. Subunits of human sex hormone binding globulin. Interindividual variation in size. J Biol Chem. 1987 Jun 15;262(17):8430–8437. [PubMed] [Google Scholar]
  7. Gershagen S., Lundwall A., Fernlund P. Characterization of the human sex hormone binding globulin (SHBG) gene and demonstration of two transcripts in both liver and testis. Nucleic Acids Res. 1989 Nov 25;17(22):9245–9258. doi: 10.1093/nar/17.22.9245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Grenot C., de Montard A., Blachère T., Mappus E., Cuilleron C. Y. Identification d'un site de photomarquage de la protéine plasmatique de liaison de la testostérone et de l'oestradiol (SBP) par l'hydroxy-17 beta oxo-3 androstadiène-4,6 tritié. C R Acad Sci III. 1988;307(7):391–396. [PubMed] [Google Scholar]
  9. Griffin P. R., Kumar S., Shabanowitz J., Charbonneau H., Namkung P. C., Walsh K. A., Hunt D. F., Petra P. H. The amino acid sequence of the sex steroid-binding protein of rabbit serum. J Biol Chem. 1989 Nov 15;264(32):19066–19075. [PubMed] [Google Scholar]
  10. Hammond G. L., Robinson P. A., Sugino H., Ward D. N., Finne J. Physicochemical characteristics of human sex hormone binding globulin: evidence for two identical subunits. J Steroid Biochem. 1986 Apr;24(4):815–824. doi: 10.1016/0022-4731(86)90442-5. [DOI] [PubMed] [Google Scholar]
  11. Hammond G. L., Underhill D. A., Rykse H. M., Smith C. L. The human sex hormone-binding globulin gene contains exons for androgen-binding protein and two other testicular messenger RNAs. Mol Endocrinol. 1989 Nov;3(11):1869–1876. doi: 10.1210/mend-3-11-1869. [DOI] [PubMed] [Google Scholar]
  12. Kotite N. J., Musto N. A. Subunit structure of rabbit testosterone estradiol-binding globulin. J Biol Chem. 1982 May 10;257(9):5118–5124. [PubMed] [Google Scholar]
  13. Mahoudeau J. A., Corvol P. Rabbit testosterone-binding-globulin. I. Physico-chemical properties. Endocrinology. 1973 Apr;92(4):1113–1119. doi: 10.1210/endo-92-4-1113. [DOI] [PubMed] [Google Scholar]
  14. Mickelson K. E., Petra P. H. A filter assay for the sex steroid binding protein (SBP) of human serum. FEBS Lett. 1974 Aug 15;44(1):34–38. doi: 10.1016/0014-5793(74)80300-5. [DOI] [PubMed] [Google Scholar]
  15. Namkung P. C., Kumar S., Walsh K. A., Petra P. H. Identification of lysine 134 in the steroid-binding site of the sex steroid-binding protein of human plasma. J Biol Chem. 1990 Oct 25;265(30):18345–18350. [PubMed] [Google Scholar]
  16. Namkung P. C., Stanczyk F. Z., Cook M. J., Novy M. J., Petra P. H. Half-life of plasma sex steroid-binding protein (SBP) in the primate. J Steroid Biochem. 1989 May;32(5):675–680. doi: 10.1016/0022-4731(89)90511-6. [DOI] [PubMed] [Google Scholar]
  17. Petra P. H., Namkung P. C., Senear D. F., McCrae D. A., Rousslang K. W., Teller D. C., Ross J. B. Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins. J Steroid Biochem. 1986 Aug;25(2):191–200. doi: 10.1016/0022-4731(86)90416-4. [DOI] [PubMed] [Google Scholar]
  18. Petra P. H., Stanczyk F. Z., Senear D. F., Namkung P. C., Novy M. J., Ross J. B., Turner E., Brown J. A. Current status of the molecular structure and function of the plasma sex steroid-binding protein (SBP). J Steroid Biochem. 1983 Jul;19(1B):699–706. doi: 10.1016/0022-4731(83)90238-8. [DOI] [PubMed] [Google Scholar]
  19. Petra P. H. The plasma sex steroid binding protein (SBP or SHBG). A critical review of recent developments on the structure, molecular biology and function. J Steroid Biochem Mol Biol. 1991;40(4-6):735–753. doi: 10.1016/0960-0760(91)90299-k. [DOI] [PubMed] [Google Scholar]
  20. Plymate S. R., Namkung P. C., Metej L. A., Petra P. H. Direct effect of plasma sex hormone binding globulin (SHBG) on the metabolic clearance rate of 17 beta-estradiol in the primate. J Steroid Biochem. 1990 Jul 4;36(4):311–317. doi: 10.1016/0022-4731(90)90223-f. [DOI] [PubMed] [Google Scholar]
  21. Pétra P. H., Lewis J. Modification in the purification of the sex steroid-binding protein of human serum by affinity chromatography. Anal Biochem. 1980 Jun;105(1):165–169. doi: 10.1016/0003-2697(80)90440-6. [DOI] [PubMed] [Google Scholar]
  22. Pétra P. H., Stanczyk F. Z., Namkung P. C., Fritz M. A., Novy M. J. Direct effect of sex steroid-binding protein (SBP) of plasma on the metabolic clearance rate of testosterone in the rhesus macaque. J Steroid Biochem. 1985 Jun;22(6):739–746. doi: 10.1016/0022-4731(85)90280-8. [DOI] [PubMed] [Google Scholar]
  23. Renoir J. M., Mercier-Bodard C., Baulieu E. E. Hormonal and immunological aspects of the phylogeny of sex steroid binding plasma protein. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4578–4582. doi: 10.1073/pnas.77.8.4578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rosner W., Darmstadt R. A. Demonstration and partial characterization of a rabbit serum protein which binds testosterone and dihydrotestosterone. Endocrinology. 1973 Jun;92(6):1700–1707. doi: 10.1210/endo-92-6-1700. [DOI] [PubMed] [Google Scholar]
  25. Schiller H. S., Pétra P. H. A filter assay for the corticosteroid binding globulin of human serum. J Steroid Biochem. 1976 Jan;7(1):55–59. doi: 10.1016/0022-4731(76)90165-5. [DOI] [PubMed] [Google Scholar]
  26. Shanbhag V. P., Södergård R. The temperature dependence of the binding of 5 alpha-dihydrotestosterone, testosterone and estradiol to the sex hormone globulin (SHBG) of human plasma. J Steroid Biochem. 1986 Feb;24(2):549–555. doi: 10.1016/0022-4731(86)90118-4. [DOI] [PubMed] [Google Scholar]
  27. Strel'chyonok O. A., Avvakumov G. V. Specific steroid-binding glycoproteins of human blood plasma: novel data on their structure and function. J Steroid Biochem. 1990 Apr;35(5):519–534. doi: 10.1016/0022-4731(90)90195-x. [DOI] [PubMed] [Google Scholar]
  28. Tarentino A. L., Gómez C. M., Plummer T. H., Jr Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F. Biochemistry. 1985 Aug 13;24(17):4665–4671. doi: 10.1021/bi00338a028. [DOI] [PubMed] [Google Scholar]
  29. Umemoto J., Bhavanandan V. P., Davidson E. A. Purification and properties of an endo-alpha-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae. J Biol Chem. 1977 Dec 10;252(23):8609–8614. [PubMed] [Google Scholar]
  30. Van Baelen H., Heyns W., De Moor P. Microheterogeneity of the testosterone binding globulin of human pregnancy serum demonstrated by isoelectric focusing. Ann Endocrinol (Paris) 1969;30(Suppl):199–203. [PubMed] [Google Scholar]
  31. Walsh K. A., Titani K., Takio K., Kumar S., Hayes R., Petra P. H. Amino acid sequence of the sex steroid binding protein of human blood plasma. Biochemistry. 1986 Nov 18;25(23):7584–7590. doi: 10.1021/bi00371a048. [DOI] [PubMed] [Google Scholar]
  32. Whitehouse C. M., Dreyer R. N., Yamashita M., Fenn J. B. Electrospray interface for liquid chromatographs and mass spectrometers. Anal Chem. 1985 Mar;57(3):675–679. doi: 10.1021/ac00280a023. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES