Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1995 May 15;308(Pt 1):321–325. doi: 10.1042/bj3080321

Endoproteolytic processing of recombinant proalbumin variants by the yeast Kex2 protease.

E C Ledgerwood 1, P M George 1, R J Peach 1, S O Brennan 1
PMCID: PMC1136879  PMID: 7755581

Abstract

The yeast Kex2 protease is regarded as the prototype of the eukaryotic family of subtilisin-like serine proteases involved in processing after dibasic amino acid sequences. Here we investigate the specificity of Kex2 using recombinant human proalbumin variants. Proalbumins with the processing site sequences Arg-Arg and Lys-Arg were cleaved after the dibasic sequence at approximately the same rate by Kex2 in vitro, and yeast expressing either of these sequences secreted mature albumin into the culture medium. As expected, the Arg-Gly-Val-Phe-His-Arg-albumin (proalbumin Lille) was not a substrate for Kex2 and neither was the Arg-Gly-Arg-Phe-His-Arg-albumin. In contrast to the mammalian endoproteases furin and the hepatic proalbumin convertase, the Kex2 protease was adversely affected by a P4 arginine. There was an 85% decrease in the cleavage of Arg-Gly-Arg-Phe-Arg-Arg-albumin compared with normal; also chicken proalbumin with an Arg-Phe-Ala-Arg processing site sequence was not a substrate for Kex2. A P1' arginine had a marked negative effect on processing and N-terminal sequence analysis confirmed that cleavage was occurring at the P1-P1' bond. The sequence context surrounding the classical dibasic site is critical in determining susceptibility to cleavage by the Kex2 protease.

Full text

PDF
321

Images in this article

323Figure 2
on p.323
323Figure 3
on p.323
324Figure 4
on p.324

Selected References

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

  1. Azaryan A. V., Wong M., Friedman T. C., Cawley N. X., Estivariz F. E., Chen H. C., Loh Y. P. Purification and characterization of a paired basic residue-specific yeast aspartic protease encoded by the YAP3 gene. Similarity to the mammalian pro-opiomelanocortin-converting enzyme. J Biol Chem. 1993 Jun 5;268(16):11968–11975. [PubMed] [Google Scholar]
  2. Bathurst I. C., Brennan S. O., Carrell R. W., Cousens L. S., Brake A. J., Barr P. J. Yeast KEX2 protease has the properties of a human proalbumin converting enzyme. Science. 1987 Jan 16;235(4786):348–350. doi: 10.1126/science.3541206. [DOI] [PubMed] [Google Scholar]
  3. Bourbonnais Y., Germain D., Latchinian-Sadek L., Boileau G., Thomas D. Y. Prohormone processing by yeast proteases. Enzyme. 1991;45(5-6):244–256. doi: 10.1159/000468899. [DOI] [PubMed] [Google Scholar]
  4. Brennan S. O., Nakayama K. Cleavage of proalbumin peptides by furin reveals unexpected restrictions at the P2 and P'1 sites. FEBS Lett. 1994 Jun 20;347(1):80–84. doi: 10.1016/0014-5793(94)00511-7. [DOI] [PubMed] [Google Scholar]
  5. Brennan S. O., Owen M. C., Boswell D. R., Lewis J. H., Carrell R. W. Circulating proalbumin associated with a variant proteinase inhibitor. Biochim Biophys Acta. 1984 Nov 6;802(1):24–28. doi: 10.1016/0304-4165(84)90029-1. [DOI] [PubMed] [Google Scholar]
  6. Brennan S. O., Peach R. J. Calcium-dependent KEX2-like protease found in hepatic secretory vesicles converts proalbumin to albumin. FEBS Lett. 1988 Feb 29;229(1):167–170. doi: 10.1016/0014-5793(88)80819-6. [DOI] [PubMed] [Google Scholar]
  7. Brennan S. O., Peach R. J. The processing of human proinsulin and chicken proalbumin by rat hepatic vesicles suggests a convertase specific for X-Y-Arg-Arg or Arg-X-Y-Arg sequences. J Biol Chem. 1991 Nov 15;266(32):21504–21508. [PubMed] [Google Scholar]
  8. Brenner C., Fuller R. S. Structural and enzymatic characterization of a purified prohormone-processing enzyme: secreted, soluble Kex2 protease. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):922–926. doi: 10.1073/pnas.89.3.922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Creemers J. W., Siezen R. J., Roebroek A. J., Ayoubi T. A., Huylebroeck D., Van de Ven W. J. Modulation of furin-mediated proprotein processing activity by site-directed mutagenesis. J Biol Chem. 1993 Oct 15;268(29):21826–21834. [PubMed] [Google Scholar]
  10. Elble R. A simple and efficient procedure for transformation of yeasts. Biotechniques. 1992 Jul;13(1):18–20. [PubMed] [Google Scholar]
  11. Foster D. C., Holly R. D., Sprecher C. A., Walker K. M., Kumar A. A. Endoproteolytic processing of the human protein C precursor by the yeast Kex2 endopeptidase coexpressed in mammalian cells. Biochemistry. 1991 Jan 15;30(2):367–372. doi: 10.1021/bi00216a009. [DOI] [PubMed] [Google Scholar]
  12. Fuller R. S., Sterne R. E., Thorner J. Enzymes required for yeast prohormone processing. Annu Rev Physiol. 1988;50:345–362. doi: 10.1146/annurev.ph.50.030188.002021. [DOI] [PubMed] [Google Scholar]
  13. Gluschankof P., Fuller R. S. A C-terminal domain conserved in precursor processing proteases is required for intramolecular N-terminal maturation of pro-Kex2 protease. EMBO J. 1994 May 15;13(10):2280–2288. doi: 10.1002/j.1460-2075.1994.tb06510.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hosaka M., Nagahama M., Kim W. S., Watanabe T., Hatsuzawa K., Ikemizu J., Murakami K., Nakayama K. Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway. J Biol Chem. 1991 Jul 5;266(19):12127–12130. [PubMed] [Google Scholar]
  15. Hunter W. M. Radioimmunoassay and saturation analysis. Preparation and assessment of radioactive tracers. Br Med Bull. 1974 Jan;30(1):18–23. doi: 10.1093/oxfordjournals.bmb.a071161. [DOI] [PubMed] [Google Scholar]
  16. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mizuno K., Nakamura T., Ohshima T., Tanaka S., Matsuo H. Yeast KEX2 genes encodes an endopeptidase homologous to subtilisin-like serine proteases. Biochem Biophys Res Commun. 1988 Oct 14;156(1):246–254. doi: 10.1016/s0006-291x(88)80832-5. [DOI] [PubMed] [Google Scholar]
  18. Ortmann D., Ohuchi M., Angliker H., Shaw E., Garten W., Klenk H. D. Proteolytic cleavage of wild type and mutants of the F protein of human parainfluenza virus type 3 by two subtilisin-like endoproteases, furin and Kex2. J Virol. 1994 Apr;68(4):2772–2776. doi: 10.1128/jvi.68.4.2772-2776.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Peach R. J., Brennan S. O. Identification of a calcium-dependent microsomal proteinase responsible for monobasic cleavage of chicken proalbumin. Biochim Biophys Acta. 1989 Mar 24;990(3):276–279. doi: 10.1016/s0304-4165(89)80045-5. [DOI] [PubMed] [Google Scholar]
  20. Rehemtulla A., Kaufman R. J. Preferred sequence requirements for cleavage of pro-von Willebrand factor by propeptide-processing enzymes. Blood. 1992 May 1;79(9):2349–2355. [PubMed] [Google Scholar]
  21. Schild D., Brake A. J., Kiefer M. C., Young D., Barr P. J. Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations. Proc Natl Acad Sci U S A. 1990 Apr;87(8):2916–2920. doi: 10.1073/pnas.87.8.2916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Seidah N. G., Day R., Chrétien M. The family of pro-hormone and pro-protein convertases. Biochem Soc Trans. 1993 Aug;21(3):685–691. doi: 10.1042/bst0210685. [DOI] [PubMed] [Google Scholar]
  23. Siezen R. J., Creemers J. W., Van de Ven W. J. Homology modelling of the catalytic domain of human furin. A model for the eukaryotic subtilisin-like proprotein convertases. Eur J Biochem. 1994 Jun 1;222(2):255–266. doi: 10.1111/j.1432-1033.1994.tb18864.x. [DOI] [PubMed] [Google Scholar]
  24. Smeekens S. P., Montag A. G., Thomas G., Albiges-Rizo C., Carroll R., Benig M., Phillips L. A., Martin S., Ohagi S., Gardner P. Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8822–8826. doi: 10.1073/pnas.89.18.8822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Smeekens S. P. Processing of protein precursors by a novel family of subtilisin-related mammalian endoproteases. Biotechnology (N Y) 1993 Feb;11(2):182–186. doi: 10.1038/nbt0293-182. [DOI] [PubMed] [Google Scholar]
  26. Thim L., Hansen M. T., Norris K., Hoegh I., Boel E., Forstrom J., Ammerer G., Fiil N. P. Secretion and processing of insulin precursors in yeast. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6766–6770. doi: 10.1073/pnas.83.18.6766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Thomas G., Thorne B. A., Thomas L., Allen R. G., Hruby D. E., Fuller R., Thorner J. Yeast KEX2 endopeptidase correctly cleaves a neuroendocrine prohormone in mammalian cells. Science. 1988 Jul 8;241(4862):226–230. doi: 10.1126/science.3291117. [DOI] [PubMed] [Google Scholar]
  28. Walker J. A., Molloy S. S., Thomas G., Sakaguchi T., Yoshida T., Chambers T. M., Kawaoka Y. Sequence specificity of furin, a proprotein-processing endoprotease, for the hemagglutinin of a virulent avian influenza virus. J Virol. 1994 Feb;68(2):1213–1218. doi: 10.1128/jvi.68.2.1213-1218.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES