Molecular cloning, expression and characterization of an Ascaris inhibitor for pepsin and cathepsin E
- PMID: 9654082
- DOI: 10.1046/j.1432-1327.1998.2530804.x
Molecular cloning, expression and characterization of an Ascaris inhibitor for pepsin and cathepsin E
Abstract
Molecular cloning of a cDNA for a pepsin inhibitor in the round worm, Ascaris suum, was achieved. The ORF was found to encode a 20-residue potential signal peptide and a 149-residue inhibitor moiety. Northern analysis showed the mRNA for the inhibitor to be expressed in the body wall and not in the viscera. To obtain the active inhibitor, we constructed a yeast expression vector, pYES2API, containing the inducible galactosidase promoter and a DNA fragment encoding a fusion protein of the yeast alpha-factor leader and the Ascaris pepsin inhibitor. The active inhibitor was secreted in the culture medium, the yield being approximately 3 mg x l(-1) x day(-1), and purified by a two-step procedure that included HPLC. The inhibitor inactivated pepsin A and cathepsin E almost completely at amounts equimolar with the enzymes, but was 100-fold less effective against pepsin C and did not act on cathepsin D and renin. Ki values for the inhibition of pepsin A and cathepsin E were in the nanomolar range below pH 5. Since the inhibitor activity was lost by modification of specific Lys residues, including Lys110, an electrostatic interaction between these Lys residues and Asp/Glu residues of pepsin A or cathepsin E was thought to be essential for the inhibition.
Similar articles
-
The disulphide bond arrangement in the major pepsin inhibitor PI-3 of Ascaris suum.FEBS Lett. 2000 Jun 2;474(2-3):253-4. doi: 10.1016/s0014-5793(00)01589-1. FEBS Lett. 2000. PMID: 10896483 No abstract available.
-
Structural basis for the inhibition of porcine pepsin by Ascaris pepsin inhibitor-3.Nat Struct Biol. 2000 Aug;7(8):653-7. doi: 10.1038/77950. Nat Struct Biol. 2000. PMID: 10932249
-
Human cathepsin F. Molecular cloning, functional expression, tissue localization, and enzymatic characterization.J Biol Chem. 1998 Nov 27;273(48):32000-8. doi: 10.1074/jbc.273.48.32000. J Biol Chem. 1998. PMID: 9822672
-
Cathepsin E and cathepsin D: biosynthesis, processing and subcellular location.Adv Exp Med Biol. 1995;362:223-9. doi: 10.1007/978-1-4615-1871-6_26. Adv Exp Med Biol. 1995. PMID: 8540322 Review. No abstract available.
-
The specificity and mechanism of pepsin action.Adv Enzymol Relat Areas Mol Biol. 1970;33:401-43. doi: 10.1002/9780470122785.ch9. Adv Enzymol Relat Areas Mol Biol. 1970. PMID: 4916858 Review. No abstract available.
Cited by
-
Immune responses to recombinant Brugia malayi pepsin inhibitor homolog (Bm-33) in patients with human lymphatic filariaisis.Parasitol Res. 2011 Feb;108(2):407-15. doi: 10.1007/s00436-010-2081-x. Epub 2010 Oct 7. Parasitol Res. 2011. PMID: 20927633 Free PMC article. Clinical Trial.
-
Characterization of a novel aspartyl protease inhibitor from Haemonchus contortus.Parasit Vectors. 2017 Apr 19;10(1):191. doi: 10.1186/s13071-017-2137-1. Parasit Vectors. 2017. PMID: 28420411 Free PMC article.
-
Mammalian metallopeptidase inhibition at the defense barrier of Ascaris parasite.Proc Natl Acad Sci U S A. 2009 Feb 10;106(6):1743-7. doi: 10.1073/pnas.0812623106. Epub 2009 Jan 28. Proc Natl Acad Sci U S A. 2009. PMID: 19179285 Free PMC article.
-
Recombinant Brugia malayi pepsin inhibitor (rBm33) induced monocyte function and absence of apoptotic cell death: an in vitro study.Microb Pathog. 2012 Jul;53(1):19-27. doi: 10.1016/j.micpath.2012.03.009. Epub 2012 Mar 30. Microb Pathog. 2012. PMID: 22484090 Free PMC article.
-
A structural biology approach to understand human lymphatic filarial infection.PLoS Negl Trop Dis. 2014 Feb 6;8(2):e2662. doi: 10.1371/journal.pntd.0002662. eCollection 2014 Feb. PLoS Negl Trop Dis. 2014. PMID: 24516678 Free PMC article.
Publication types
MeSH terms
Substances
Associated data
- Actions
LinkOut - more resources
Full Text Sources
