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. 1993 Sep 15;294(Pt 3):923–927. doi: 10.1042/bj2940923

Demonstration by electrospray mass spectrometry that the peptidyldipeptidase activity of cathepsin B is capable of rat cathepsin B C-terminal processing.

A D Rowan 1, R Feng 1, Y Konishi 1, J S Mort 1
PMCID: PMC1134550  PMID: 8379946

Abstract

Electrospray mass spectrometric techniques were used to demonstrate that mature (single-chain) recombinant rat cathepsin B is capable of sequentially removing the three dipeptides which comprise the C-terminal extension of the proenzyme. A pepsin-cleaved form of a non-active mutant recombinant rat procathepsin B (Cys-29-Ser) was used as a 'substrate' to study C-terminal processing by mature cathepsin B. The results indicate that the first two residues (Arg-Phe) are removed efficiently, while the remaining four (Gln-Tyr-Trp-Gly), particularly the final two, are much more resistant to proteolysis. These cleavages were pronounced at pH 5.0 compared with pH 6.0, in agreement with the lower pH optimum for cathepsin B exopeptidase activity reported previously. From this example of the peptidyldipeptidase activity of cathepsin B we conclude that removal of the C-terminal extension may occur in any intracellular compartment where active cathepsin B is found.

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Selected References

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  1. Aronson N. N., Jr, Barrett A. J. The specificity of cathepsin B. Hydrolysis of glucagon at the C-terminus by a peptidyldipeptidase mechanism. Biochem J. 1978 Jun 1;171(3):759–765. doi: 10.1042/bj1710759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barrett A. J. Human cathepsin B1. Purification and some properties of the enzyme. Biochem J. 1973 Apr;131(4):809–822. doi: 10.1042/bj1310809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barrett A. J., Kirschke H. Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol. 1981;80(Pt 100):535–561. doi: 10.1016/s0076-6879(81)80043-2. [DOI] [PubMed] [Google Scholar]
  4. Baudys M., Meloun B., Gan-Erdene T., Pohl J., Kostka V. Disulfide bridges of bovine spleen cathepsin B. Biol Chem Hoppe Seyler. 1990 Jun;371(6):485–491. doi: 10.1515/bchm3.1990.371.1.485. [DOI] [PubMed] [Google Scholar]
  5. Bond J. S., Barrett A. J. Degradation of fructose-1,6-bisphosphate aldolase by cathepsin B. Biochem J. 1980 Jul 1;189(1):17–25. doi: 10.1042/bj1890017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Béchet D. M., Ferrara M. J., Mordier S. B., Roux M. P., Deval C. D., Obled A. Expression of lysosomal cathepsin B during calf myoblast-myotube differentiation. Characterization of a cDNA encoding bovine cathepsin B. J Biol Chem. 1991 Jul 25;266(21):14104–14112. [PubMed] [Google Scholar]
  7. Chan S. J., San Segundo B., McCormick M. B., Steiner D. F. Nucleotide and predicted amino acid sequences of cloned human and mouse preprocathepsin B cDNAs. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7721–7725. doi: 10.1073/pnas.83.20.7721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Covey T. R., Bonner R. F., Shushan B. I., Henion J. The determination of protein, oligonucleotide and peptide molecular weights by ion-spray mass spectrometry. Rapid Commun Mass Spectrom. 1988 Nov;2(11):249–256. doi: 10.1002/rcm.1290021111. [DOI] [PubMed] [Google Scholar]
  9. Erickson A. H., Blobel G. Carboxyl-terminal proteolytic processing during biosynthesis of the lysosomal enzymes beta-glucuronidase and cathepsin D. Biochemistry. 1983 Oct 25;22(22):5201–5205. doi: 10.1021/bi00291a021. [DOI] [PubMed] [Google Scholar]
  10. Fenn J. B., Mann M., Meng C. K., Wong S. F., Whitehouse C. M. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989 Oct 6;246(4926):64–71. doi: 10.1126/science.2675315. [DOI] [PubMed] [Google Scholar]
  11. Hasnain S., Hirama T., Tam A., Mort J. S. Characterization of recombinant rat cathepsin B and nonglycosylated mutants expressed in yeast. New insights into the pH dependence of cathepsin B-catalyzed hydrolyses. J Biol Chem. 1992 Mar 5;267(7):4713–4721. [PubMed] [Google Scholar]
  12. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. King J., Laemmli U. K. Polypeptides of the tail fibres of bacteriophage T4. J Mol Biol. 1971 Dec 28;62(3):465–477. doi: 10.1016/0022-2836(71)90148-3. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Lee X., Ahmed F. R., Hirama T., Huber C. P., Rose D. R., To R., Hasnain S., Tam A., Mort J. S. Crystallization of recombinant rat cathepsin B. J Biol Chem. 1990 Apr 15;265(11):5950–5951. [PubMed] [Google Scholar]
  16. Loo J. A., Udseth H. R., Smith R. D. Peptide and protein analysis by electrospray ionization-mass spectrometry and capillary electrophoresis-mass spectrometry. Anal Biochem. 1989 Jun;179(2):404–412. doi: 10.1016/0003-2697(89)90153-x. [DOI] [PubMed] [Google Scholar]
  17. Mason R. W. Interaction of lysosomal cysteine proteinases with alpha 2-macroglobulin: conclusive evidence for the endopeptidase activities of cathepsins B and H. Arch Biochem Biophys. 1989 Sep;273(2):367–374. doi: 10.1016/0003-9861(89)90495-5. [DOI] [PubMed] [Google Scholar]
  18. Musil D., Zucic D., Turk D., Engh R. A., Mayr I., Huber R., Popovic T., Turk V., Towatari T., Katunuma N. The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity. EMBO J. 1991 Sep;10(9):2321–2330. doi: 10.1002/j.1460-2075.1991.tb07771.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pohl J., Davinic S., Bláha I., Strop P., Kostka V. Chromophoric and fluorophoric peptide substrates cleaved through the dipeptidyl carboxypeptidase activity of cathepsin B. Anal Biochem. 1987 Aug 15;165(1):96–101. doi: 10.1016/0003-2697(87)90205-3. [DOI] [PubMed] [Google Scholar]
  20. Ritonja A., Popovic T., Turk V., Wiedenmann K., Machleidt W. Amino acid sequence of human liver cathepsin B. FEBS Lett. 1985 Feb 11;181(1):169–172. doi: 10.1016/0014-5793(85)81136-4. [DOI] [PubMed] [Google Scholar]
  21. Rowan A. D., Mason P., Mach L., Mort J. S. Rat procathepsin B. Proteolytic processing to the mature form in vitro. J Biol Chem. 1992 Aug 5;267(22):15993–15999. [PubMed] [Google Scholar]
  22. San Segundo B., Chan S. J., Steiner D. F. Identification of cDNA clones encoding a precursor of rat liver cathepsin B. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2320–2324. doi: 10.1073/pnas.82.8.2320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  24. Smith R. D., Loo J. A., Edmonds C. G., Barinaga C. J., Udseth H. R. New developments in biochemical mass spectrometry: electrospray ionization. Anal Chem. 1990 May 1;62(9):882–899. doi: 10.1021/ac00208a002. [DOI] [PubMed] [Google Scholar]
  25. Takahashi T., Dehdarani A. H., Yonezawa S., Tang J. Porcine spleen cathepsin B is an exopeptidase. J Biol Chem. 1986 Jul 15;261(20):9375–9381. [PubMed] [Google Scholar]
  26. Takahashi T., Yonezawa S., Dehdarani A. H., Tang J. Comparative studies of two cathepsin B isozymes from porcine spleen. Isolation, polypeptide chain arrangements, and enzyme specificity. J Biol Chem. 1986 Jul 15;261(20):9368–9374. [PubMed] [Google Scholar]
  27. Takio K., Towatari T., Katunuma N., Teller D. C., Titani K. Homology of amino acid sequences of rat liver cathepsins B and H with that of papain. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3666–3670. doi: 10.1073/pnas.80.12.3666. [DOI] [PMC free article] [PubMed] [Google Scholar]

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