The Multiple cpb Cysteine Proteinase Genes ofLeishmania mexicana Encode Isoenzymes That Differ in Their Stage Regulation and Substrate Preferences

doi:10.1074/jbc.272.22.14285

The Multiple cpb Cysteine Proteinase Genes ofLeishmania mexicana Encode Isoenzymes That Differ in Their Stage Regulation and Substrate Preferences*

From the ^‡Wellcome Unit of Molecular Parasitology, University of Glasgow, The Anderson College, Glasgow G11 6NU, and the ^¶Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Joseph Black Building, Glasgow G12 8QQ, Scotland, United Kingdom

Abstract

The cpb genes ofLeishmania mexicana encode stage-regulated, cathepsin L-like cysteine proteinases that are leishmanial virulence factors. Field inversion gel electrophoresis and genomic mapping indicate that there are 19 cpb genes arranged in a tandem array. Five genes from the array have been sequenced and their expression analyzed. The first two genes, cpb1 and cpb2, differ significantly from the remaining 17 copies (cpb3–cpb19) in that: 1) they are expressed predominantly in metacyclic promastigotes (the form in the insect vector which is infective to mammalian macrophages) rather than amastigotes (the form that parasitizes mammals); 2) they encode enzymes with a truncation in the COOH-terminal extension, an unusual feature of these cysteine proteinases of trypanosomatids. Transfection ofcpb1 into a cpb null mutant resulted in expression of an active enzyme that was shown by immunogold labeling with anti-CPB antibodies to be targeted to large lysosomes. This demonstrates that the 100-amino acid COOH-terminal extension is not essential for the activation or activity of the enzyme or for its correct intracellular trafficking. Transfection into thecpb null mutant of different copies of cpb and analysis of the phenotype of the lines showed that individual isoenzymes differ in their substrate preferences and ability to restore the loss of virulence associated with the null mutant. Comparison of the predicted amino acid sequences of the isoenzymes implicates five residues located in the mature domain (Asn¹⁸, Asp⁶⁰, Asn⁶¹, Ser⁶⁴, and Tyr⁸⁴) with differences in the activities of the encoded isoenzymes. The results suggest that the individual isoenzymes have distinct roles in the parasite’s interaction with its host. This complexity reflects the adaptation of cathepsin L-like cysteine proteinases to diverse functions in parasitic protozoa.

Footnotes

↵* This work was supported by the Medical Research Council, United Kingdom.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) Z49963 (cpb1), Z49965 (cpb19), and Y09958 (cpb18).
↵§ To whom correspondence should be addressed: Wellcome Unit of Molecular Parasitology, University of Glasgow, The Anderson College, 56 Dumbarton Rd, Glasgow G11 6NU, Scotland, U. K. Tel.: 44-141-339-8855 (ext. 2000); Fax: 44-1-41-330-5422; E-mail:j.mottram{at}udcf.gla.ac.uk.
↵‖ Present address: Max-Planck-Institut fur Biochemie, Am Klopferspitz 18a, D-82152, Martinsried, Munchen, Germany.
↵1 The abbreviations used are: kb, kilobase(s); bp, base pair(s); cpb1, gene 1 in the cpb tandem array; CPB1, protein encoded by cpb1; Δcpb,mutant cell line null for cpb; Δcpb[pTEXNE2], mutant cell line null for cpb re-expressing CPB1 from the pTEX episome; equivalent nomenclature is used for other genes in the array; CTE, COOH-terminal extension; FIGE, field inversion gel electrophoresis; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; BzFVR-NHMec,N-benzoyl-phenylalanyl-valinyl-arginyl-7-amido-4-methylcoumarin; SucLY-NHMec,N-succinyl-leucinyl-tyrosinyl-7-amido-4-methylcoumarin.
↵2 The single-letter code is used when referring to amino acid differences between CPB2.8 and other CPB isoenzymes. In the numbering system used, the NH₂ terminus of the mature CPB2.8 enzyme is designated as residue 1, and the pro-region is assigned negative numbers decreasing toward the NH₂terminus.
↵3 D. R. Brooks, G. H. Coombs, and J. C. Mottram, unpublished data.
- Received December 30, 1996.
- Revision received March 6, 1997.