Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Apr 1;355(Pt 1):245–248. doi: 10.1042/0264-6021:3550245

The efficiency of N-linked glycosylation of bovine DNase I depends on the Asn-Xaa-Ser/Thr sequence and the tissue of origin.

A Nishikawa 1, S Mizuno 1
PMCID: PMC1221733  PMID: 11256970

Abstract

Bovine DNase I contains two potential N-linked glycosylation sites with the sequences Asn(18)-Ala-Thr and Asn(106)-Asp-Ser. A previous report established that pancreatic DNase I has only one sugar chain at Asn(18) [Liao, Salnikow, Moore and Stein (1973) J. Biol. Chem. 248, 1489-1495]. We found, however, that bovine DNase I expressed in COS-1 cells was glycosylated about 70% at Asn(106) in addition to being completely glycosylated at Asn(18). Glycosylation of Asn(106) increased to 97% when Asp(107) was mutated to Glu or when Ser(108) was mutated to Thr. Mutation of Asp(107) to Trp had no effect, whereas a substitution with Pro at this position abolished glycosylation of Asn(106). Analysis of the state of glycosylation of DNase I purified from a variety of bovine tissues revealed that DNase I from spleen, submaxillary gland, lung and adrenal had two sugar chains, whereas enzyme from pancreas and kidney had only one sugar chain. These findings demonstrate a major difference in the ability of various tissues to utilize N-linked glycosylation signals that contain suboptimal residues in the second and third positions.

Full Text

The Full Text of this article is available as a PDF (128.7 KB).

Selected References

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

  1. Abe A., Liao T. H. The immunological and structural comparisons of deoxyribonucleases I. Glycosylation differences between bovine pancreatic and parotid deoxyribonucleases. J Biol Chem. 1983 Sep 10;258(17):10283–10288. [PubMed] [Google Scholar]
  2. Bause E., Hettkamp H. Primary structural requirements for N-glycosylation of peptides in rat liver. FEBS Lett. 1979 Dec 15;108(2):341–344. doi: 10.1016/0014-5793(79)80559-1. [DOI] [PubMed] [Google Scholar]
  3. Bause E., Legler G. The role of the hydroxy amino acid in the triplet sequence Asn-Xaa-Thr(Ser) for the N-glycosylation step during glycoprotein biosynthesis. Biochem J. 1981 Jun 1;195(3):639–644. doi: 10.1042/bj1950639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bause E. Studies on the acceptor specificity of asparagine-N-glycosyl-transferase from rat liver. FEBS Lett. 1979 Jul 15;103(2):296–299. doi: 10.1016/0014-5793(79)81348-4. [DOI] [PubMed] [Google Scholar]
  5. Carson D. D., Earles B. J., Lennarz W. J. Enhancement of protein glycosylation in tissue slices by dolichylphosphate. J Biol Chem. 1981 Nov 25;256(22):11552–11557. [PubMed] [Google Scholar]
  6. Gavel Y., von Heijne G. Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering. Protein Eng. 1990 Apr;3(5):433–442. doi: 10.1093/protein/3.5.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. KUNITZ M. Crystalline desoxyribonuclease; isolation and general properties; spectrophotometric method for the measurement of desoxyribonuclease activity. J Gen Physiol. 1950 Mar;33(4):349–362. doi: 10.1085/jgp.33.4.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kaplan H. A., Welply J. K., Lennarz W. J. Oligosaccharyl transferase: the central enzyme in the pathway of glycoprotein assembly. Biochim Biophys Acta. 1987 Jun 24;906(2):161–173. doi: 10.1016/0304-4157(87)90010-4. [DOI] [PubMed] [Google Scholar]
  10. Kasturi L., Chen H., Shakin-Eshleman S. H. Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors. Biochem J. 1997 Apr 15;323(Pt 2):415–419. doi: 10.1042/bj3230415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kasturi L., Eshleman J. R., Wunner W. H., Shakin-Eshleman S. H. The hydroxy amino acid in an Asn-X-Ser/Thr sequon can influence N-linked core glycosylation efficiency and the level of expression of a cell surface glycoprotein. J Biol Chem. 1995 Jun 16;270(24):14756–14761. doi: 10.1074/jbc.270.24.14756. [DOI] [PubMed] [Google Scholar]
  12. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  13. Liao T. H. Bovine pancreatic deoxyribonuclease D. J Biol Chem. 1974 Apr 25;249(8):2354–2356. [PubMed] [Google Scholar]
  14. Liao T. H., Salnikow J., Moore S., Stein W. H. Bovine pancreatic deoxyribonuclease A. Isolation of cyanogen bromide peptides; complete covalent structure of the polypeptide chain. J Biol Chem. 1973 Feb 25;248(4):1489–1495. [PubMed] [Google Scholar]
  15. Nadano D., Yasuda T., Kishi K. Measurement of deoxyribonuclease I activity in human tissues and body fluids by a single radial enzyme-diffusion method. Clin Chem. 1993 Mar;39(3):448–452. [PubMed] [Google Scholar]
  16. Nishikawa A., Gregory W., Frenz J., Cacia J., Kornfeld S. The phosphorylation of bovine DNase I Asn-linked oligosaccharides is dependent on specific lysine and arginine residues. J Biol Chem. 1997 Aug 1;272(31):19408–19412. doi: 10.1074/jbc.272.31.19408. [DOI] [PubMed] [Google Scholar]
  17. Nishikawa A., Nanda A., Gregory W., Frenz J., Kornfeld S. Identification of amino acids that modulate mannose phosphorylation of mouse DNase I, a secretory glycoprotein. J Biol Chem. 1999 Jul 2;274(27):19309–19315. doi: 10.1074/jbc.274.27.19309. [DOI] [PubMed] [Google Scholar]
  18. Rademacher T. W., Parekh R. B., Dwek R. A. Glycobiology. Annu Rev Biochem. 1988;57:785–838. doi: 10.1146/annurev.bi.57.070188.004033. [DOI] [PubMed] [Google Scholar]
  19. Ronin C., Bouchilloux S., Granier C., van Rietschoten J. Enzymatic N-glycosylation of synthetic Asn--X--Thr containing peptides. FEBS Lett. 1978 Dec 1;96(1):179–182. doi: 10.1016/0014-5793(78)81089-8. [DOI] [PubMed] [Google Scholar]
  20. Ronin C., Granier C., Caseti C., Bouchilloux S., Van Rietschoten J. Synthetic substrates for thyroid oligosaccharide transferase. Effects of peptide chain length and modifications in the Asn-Xaa-Thr-region. Eur J Biochem. 1981 Aug;118(1):159–164. doi: 10.1111/j.1432-1033.1981.tb05499.x. [DOI] [PubMed] [Google Scholar]
  21. Shakin-Eshleman S. H., Spitalnik S. L., Kasturi L. The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency. J Biol Chem. 1996 Mar 15;271(11):6363–6366. doi: 10.1074/jbc.271.11.6363. [DOI] [PubMed] [Google Scholar]
  22. Silberstein S., Gilmore R. Biochemistry, molecular biology, and genetics of the oligosaccharyltransferase. FASEB J. 1996 Jun;10(8):849–858. [PubMed] [Google Scholar]
  23. Suck D., Oefner C., Kabsch W. Three-dimensional structure of bovine pancreatic DNase I at 2.5 A resolution. EMBO J. 1984 Oct;3(10):2423–2430. doi: 10.1002/j.1460-2075.1984.tb02149.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Varki A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 1993 Apr;3(2):97–130. doi: 10.1093/glycob/3.2.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Williams A. F., Parekh R. B., Wing D. R., Willis A. C., Barclay A. N., Dalchau R., Fabre J. W., Dwek R. A., Rademacher T. W. Comparative analysis of the N-glycans of rat, mouse and human Thy-1. Site-specific oligosaccharide patterns of neural Thy-1, a member of the immunoglobulin superfamily. Glycobiology. 1993 Aug;3(4):339–348. doi: 10.1093/glycob/3.4.339. [DOI] [PubMed] [Google Scholar]
  26. Worrall A. F., Connolly B. A. The chemical synthesis of a gene coding for bovine pancreatic DNase I and its cloning and expression in Escherichia coli. J Biol Chem. 1990 Dec 15;265(35):21889–21895. [PubMed] [Google Scholar]
  27. Yamashita K., Hitoi A., Irie M., Kobata A. Fractionation by lectin affinity chromatography indicates that the glycosylation of most ribonucleases in human viscera and body fluids is organ specific. Arch Biochem Biophys. 1986 Oct;250(1):263–266. doi: 10.1016/0003-9861(86)90725-3. [DOI] [PubMed] [Google Scholar]
  28. Yamashita K., Hitoi A., Tateishi N., Higashi T., Sakamoto Y., Kobata A. Organ-specific difference in the sugar chains of gamma-glutamyltranspeptidase. Arch Biochem Biophys. 1983 Sep;225(2):993–996. doi: 10.1016/0003-9861(83)90116-9. [DOI] [PubMed] [Google Scholar]
  29. Yasuda T., Takeshita H., Nakajima T., Hosomi O., Nakashima Y., Kishi K. Rabbit DNase I: purification from urine, immunological and proteochemical characterization, nucleotide sequence, expression in tissues, relationships with other mammalian DNases I and phylogenetic analysis. Biochem J. 1997 Jul 15;325(Pt 2):465–473. doi: 10.1042/bj3250465. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES