Cloning and characterization of angiotensin converting enzyme related dipeptidylcarboxypeptidase from Leishmania donovani.
We report the first identification, gene cloning, recombinant expression and biochemical characterization of an angiotensin converting enzyme (ACE) related dipeptidylcarboxypeptidase (DCP) in a protozoan parasite. The mammalian counterpart of this enzyme, peptidyl dipeptidase A (a carboxyl dipeptidase) also known as ACE leads to the cleavage of angiotensin I to produce a potent vasopressor. The catalytic enzyme activity of its Escherichia coli DCP counter part can be inhibited by the antihypertensive drug captopril, suggesting that this class of enzymes constitutes a novel target for drugs and vaccines. By utilizing a DNA microarray expression profiling approach, we identified a gene encoding a DCP enzyme for the kinetoplast protozoan Leishmania donovani (LdDCP) that was differentially expressed in promastigote and amastigote stages of the parasite life cycle. Both RNA and protein levels of LdDCP are higher in axenic amastigotes compared to promastigotes. Immuno-fluorescence analysis revealed the cytosolic expression of the protein. Primary structure analysis of LdDCP revealed the presence of an active Zn binding site. When expressed in E. coli, the recombinant enzyme showed carboxy-dipeptidase activity with synthetic substrates. Replacement of two histidine and one glutamic acid at positions 466, 470 and 467, respectively, with alanine residues in its active site resulted in loss of enzyme activity. Captopril, an ACE specific inhibitor was able both to reduce significantly LdDCP enzyme activity and to inhibit promastigote growth. Both its cytosolic location and close homology to DCPs from bacterial species suggests a role in parasite nutrition. Further, identification of LdDCP now provides an opportunity to investigate Leishmania peptidases for their potential as drug and vaccine targets.
Goyal
Neena
N
Division of Biochemistry, Central Drug Research Institute, Lucknow 226001, Uttar Pradesh, India. neenacdri@yahoo.com
Duncan
Robert
R
Selvapandiyan
Angamuthu
A
Debrabant
Alain
A
Baig
Mirza S
MS
Nakhasi
Hira L
HL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002216:Captopril / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D003600:Cytosol / Q000737:chemistry; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D007150:Immunohistochemistry; D007893:Leishmania donovani / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D007703:Peptidyl-Dipeptidase A / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D016054:DNA, Protozoan; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C017800:zinc-binding protein; D002216:Captopril; D010450:Endopeptidases; C020071:dipeptidyl carboxypeptidase; D007703:Peptidyl-Dipeptidase A