Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Jul 15;357(Pt 2):385–392. doi: 10.1042/0264-6021:3570385

Site-directed removal of N-glycosylation sites in BST-1/CD157: effects on molecular and functional heterogeneity.

S Yamamoto-Katayama 1, A Sato 1, M Ariyoshi 1, M Suyama 1, K Ishihara 1, T Hirano 1, H Nakamura 1, K Morikawa 1, H Jingami 1
PMCID: PMC1221964  PMID: 11439087

Abstract

Cyclic ADP ribose (cADPR) is a novel second messenger that releases calcium from intracellular calcium stores, but works independently of inositol 1,4,5-trisphosphate. In mammals ADP-ribosyl cyclase function is found in two membrane proteins, CD38 and bone marrow stromal cell antigen 1 (BST-1)/CD157. These enzymes are exposed extracellularly and also possess cADPR hydrolase activity, but an intracellular soluble ADP-ribosyl cyclase has been reported in human T-cells. Previously, a soluble form of BST-1/CD157 (sBST-1), which lacked the glycosylphosphatidylinositol-anchored portion, was expressed by a baculovirus-insect-cell system. In this study, we have purified the sBST-1, and it migrated as two major bands by SDS/PAGE, suggesting that it is post-translationally modified. BST-1 contains four putative N-glycosylation sites. Tunicamycin treatment reduced sBST-1 expression in the culture medium, indicating that N-glycosylation is essential for secretion. Site-directed mutagenesis was performed to generate sBST-1 mutants (N1-N4), each preserving a single N-glycosylation site. N1, N3 and N4 were well secreted into the medium, and were each detected as a single band. Although N3 and N4 retained the ADP-ribosyl cyclase activity, the cADPR-hydrolase activity was retained only in N4. We conclude that N-glycosylation of sBST-1 facilitates the folding of the nascent polypeptide chain into a conformation that is conductive for intracellular transport and enzymic activity. Furthermore a crystal has been obtained using the N4 mutant, but not the wild-type sBST-1. Thus the artificial engineering of N-glycosylation sites could be an effective method to generate homogeneous material for structural studies.

Full Text

The Full Text of this article is available as a PDF (319.3 KB).

Selected References

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

  1. Berthelier V., Tixier J. M., Muller-Steffner H., Schuber F., Deterre P. Human CD38 is an authentic NAD(P)+ glycohydrolase. Biochem J. 1998 Mar 15;330(Pt 3):1383–1390. doi: 10.1042/bj3301383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Chidambaram N., Chang C. F. Functional role of glycosylation on the recombinant CD38/ADP-ribosyl cyclase in CHO cells. Int J Biochem Cell Biol. 1998 Sep;30(9):1011–1018. doi: 10.1016/s1357-2725(98)00057-0. [DOI] [PubMed] [Google Scholar]
  4. Deaglio S., Morra M., Mallone R., Ausiello C. M., Prager E., Garbarino G., Dianzani U., Stockinger H., Malavasi F. Human CD38 (ADP-ribosyl cyclase) is a counter-receptor of CD31, an Ig superfamily member. J Immunol. 1998 Jan 1;160(1):395–402. [PubMed] [Google Scholar]
  5. Franco L., Guida L., Bruzzone S., Zocchi E., Usai C., De Flora A. The transmembrane glycoprotein CD38 is a catalytically active transporter responsible for generation and influx of the second messenger cyclic ADP-ribose across membranes. FASEB J. 1998 Nov;12(14):1507–1520. doi: 10.1096/fasebj.12.14.1507. [DOI] [PubMed] [Google Scholar]
  6. Funaro A., Reinis M., Trubiani O., Santi S., Di Primio R., Malavasi F. CD38 functions are regulated through an internalization step. J Immunol. 1998 Mar 1;160(5):2238–2247. [PubMed] [Google Scholar]
  7. Graeff R. M., Walseth T. F., Fryxell K., Branton W. D., Lee H. C. Enzymatic synthesis and characterizations of cyclic GDP-ribose. A procedure for distinguishing enzymes with ADP-ribosyl cyclase activity. J Biol Chem. 1994 Dec 2;269(48):30260–30267. [PubMed] [Google Scholar]
  8. Guse A. H., da Silva C. P., Berg I., Skapenko A. L., Weber K., Heyer P., Hohenegger M., Ashamu G. A., Schulze-Koops H., Potter B. V. Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose. Nature. 1999 Mar 4;398(6722):70–73. doi: 10.1038/18024. [DOI] [PubMed] [Google Scholar]
  9. Hart G. W., Brew K., Grant G. A., Bradshaw R. A., Lennarz W. J. Primary structural requirements for the enzymatic formation of the N-glycosidic bond in glycoproteins. Studies with natural and synthetic peptides. J Biol Chem. 1979 Oct 10;254(19):9747–9753. [PubMed] [Google Scholar]
  10. Hellmich M. R., Strumwasser F. Purification and characterization of a molluscan egg-specific NADase, a second-messenger enzyme. Cell Regul. 1991 Mar;2(3):193–202. doi: 10.1091/mbc.2.3.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hirata Y., Kimura N., Sato K., Ohsugi Y., Takasawa S., Okamoto H., Ishikawa J., Kaisho T., Ishihara K., Hirano T. ADP ribosyl cyclase activity of a novel bone marrow stromal cell surface molecule, BST-1. FEBS Lett. 1994 Dec 19;356(2-3):244–248. doi: 10.1016/0014-5793(94)01279-2. [DOI] [PubMed] [Google Scholar]
  12. Hussain A. M., Chang C. F. Novel kinetics, behaviour and cell-type specificity of CD157-mediated tyrosine kinase signalling. Cell Signal. 1999 Dec;11(12):891–897. doi: 10.1016/s0898-6568(99)00057-1. [DOI] [PubMed] [Google Scholar]
  13. Ishihara K., Hirano T. BST-1/CD157 regulates the humoral immune responses in vivo. Chem Immunol. 2000;75:235–255. doi: 10.1159/000058772. [DOI] [PubMed] [Google Scholar]
  14. Jackson D. G., Bell J. I. Isolation of a cDNA encoding the human CD38 (T10) molecule, a cell surface glycoprotein with an unusual discontinuous pattern of expression during lymphocyte differentiation. J Immunol. 1990 Apr 1;144(7):2811–2815. [PubMed] [Google Scholar]
  15. Kaisho T., Ishikawa J., Oritani K., Inazawa J., Tomizawa H., Muraoka O., Ochi T., Hirano T. BST-1, a surface molecule of bone marrow stromal cell lines that facilitates pre-B-cell growth. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5325–5329. doi: 10.1073/pnas.91.12.5325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Koshiyama H., Lee H. C., Tashjian A. H., Jr Novel mechanism of intracellular calcium release in pituitary cells. J Biol Chem. 1991 Sep 15;266(26):16985–16988. [PubMed] [Google Scholar]
  17. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  18. Lee B. O., Ishihara K., Denno K., Kobune Y., Itoh M., Muraoka O., Kaisho T., Sasaki T., Ochi T., Hirano T. Elevated levels of the soluble form of bone marrow stromal cell antigen 1 in the sera of patients with severe rheumatoid arthritis. Arthritis Rheum. 1996 Apr;39(4):629–637. doi: 10.1002/art.1780390414. [DOI] [PubMed] [Google Scholar]
  19. Lee H. C., Aarhus R. ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite. Cell Regul. 1991 Mar;2(3):203–209. doi: 10.1091/mbc.2.3.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lee H. C. Modulator and messenger functions of cyclic ADP-ribose in calcium signaling. Recent Prog Horm Res. 1996;51:355–389. [PubMed] [Google Scholar]
  21. Marshall R. D. Glycoproteins. Annu Rev Biochem. 1972;41:673–702. doi: 10.1146/annurev.bi.41.070172.003325. [DOI] [PubMed] [Google Scholar]
  22. Masuda W., Takenaka S., Tsuyama S., Tokunaga M., Yamaji R., Inui H., Miyatake K., Nakano Y. Inositol 1,4,5-trisphosphate and cyclic ADP-ribose mobilize Ca2+ in a protist, Euglena gracilis. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1997 Nov;118(3):279–283. doi: 10.1016/s0742-8413(97)00173-4. [DOI] [PubMed] [Google Scholar]
  23. Okuyama Y., Ishihara K., Kimura N., Hirata Y., Sato K., Itoh M., Ok L. B., Hirano T. Human BST-1 expressed on myeloid cells functions as a receptor molecule. Biochem Biophys Res Commun. 1996 Nov 21;228(3):838–845. doi: 10.1006/bbrc.1996.1741. [DOI] [PubMed] [Google Scholar]
  24. Peitsch M. C. ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling. Biochem Soc Trans. 1996 Feb;24(1):274–279. doi: 10.1042/bst0240274. [DOI] [PubMed] [Google Scholar]
  25. Prasad G. S., McRee D. E., Stura E. A., Levitt D. G., Lee H. C., Stout C. D. Crystal structure of Aplysia ADP ribosyl cyclase, a homologue of the bifunctional ectozyme CD38. Nat Struct Biol. 1996 Nov;3(11):957–964. doi: 10.1038/nsb1196-957. [DOI] [PubMed] [Google Scholar]
  26. Sato A., Yamamoto S., Ishihara K., Hirano T., Jingami H. Novel peptide inhibitor of ecto-ADP-ribosyl cyclase of bone marrow stromal cell antigen-1 (BST-1/CD157). Biochem J. 1999 Feb 1;337(Pt 3):491–496. [PMC free article] [PubMed] [Google Scholar]
  27. Sato A., Yamamoto S., Kajimura N., Oda M., Usukura J., Jingami H. Inhibitor peptide SNP-1 binds to a soluble form of BST-1/CD157 at a 2:2 stoichiometry. Eur J Biochem. 1999 Sep;264(2):439–445. doi: 10.1046/j.1432-1327.1999.00632.x. [DOI] [PubMed] [Google Scholar]
  28. Sauve A. A., Munshi C., Lee H. C., Schramm V. L. The reaction mechanism for CD38. A single intermediate is responsible for cyclization, hydrolysis, and base-exchange chemistries. Biochemistry. 1998 Sep 22;37(38):13239–13249. doi: 10.1021/bi981248s. [DOI] [PubMed] [Google Scholar]
  29. Wu Y., Kuzma J., Maréchal E., Graeff R., Lee H. C., Foster R., Chua N. H. Abscisic acid signaling through cyclic ADP-ribose in plants. Science. 1997 Dec 19;278(5346):2126–2130. doi: 10.1126/science.278.5346.2126. [DOI] [PubMed] [Google Scholar]
  30. Zhang Y., Dahms N. M. Site-directed removal of N-glycosylation sites in the bovine cation-dependent mannose 6-phosphate receptor: effects on ligand binding, intracellular targetting and association with binding immunoglobulin protein. Biochem J. 1993 Nov 1;295(Pt 3):841–848. doi: 10.1042/bj2950841. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES