Cloning and characterisation of angiotensin-converting enzyme from the dipteran species, Haematobia irritans exigua, and its expression in the maturing male reproductive system.
The angiotensin-converting enzymes (ACE) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. A carboxydipeptidase similar to mammalian ACE has now been identified in the adult stage of the haematophagous fly, Haematobia irritans exigua (buffalo fly), a close relative of the horn fly of North America. The enzyme was purified by lectin-affinity chromatography and ion-exchange chromatography and migrated as a doublet of 70 kDa upon reducing SDS/PAGE. Unlike mammalian ACE, the fly carboxydipeptidase (HieACE) is not membrane bound. The amino acid sequence of an internal peptide from HieACE and a conserved amino acid region present in all mammalian ACE were used to design degenerate oligonucleotide primers suitable for PCR. A DNA fragment amplified from adult buffalo fly cDNA was used to identify a cDNA clone that encoded the enzyme. The cDNA sequence encodes a carboxydipeptidase with 41-42% amino acid identity to the mammalian testicular ACE. The active-site regions of mammalian ACE are conserved in the deduced amino acid sequence of HieACE. Enzymatically, HieACE is very similar to its mammalian counterparts, with comparable Km and V(max) values for the synthetic substrate, benzoylglycylglycylglycine, and similar patterns of inhibition by EDTA, ACE inhibitor peptide and captopril. HieACE also specifically activates angiotensin I to angiotensin II and degrades other mammalian ACE substrates such as bradykinin, substance P and cholecystokinin-8. In the adult fly, HieACE is expressed in the compound ganglion and in the posterior region of the midgut. Similar to the mammalian system, expression of this enzyme is induced in the maturing male reproductive system, which suggests conservation of ACE function in these species.
Wijffels
G
G
CSIRO Division of Tropical Animal Production, Queensland, Australia.
Fitzgerald
C
C
Gough
J
J
Riding
G
G
Elvin
C
C
Kemp
D
D
Willadsen
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D017344:Genes, Insect; D005837:Genitalia, Male / Q000201:enzymology / Q000254:growth & development; D007700:Kinetics; D008297:Male; D008322:Mammals; D008969:Molecular Sequence Data; D009117:Muscidae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D009842:Oligopeptides; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D009842:Oligopeptides; C029275:hippuryl-glycyl-glycine; D007703:Peptidyl-Dipeptidase A