Cloning, expression, and substrate specificity of MeCPA, a zinc carboxypeptidase that is secreted into infected tissues by the fungal entomopathogen Metarhizium anisopliae.
To date zinc carboxypeptidases have only been found in animals and actinomycete bacteria. A cDNA clone (MeCPA) for a novel fungal (Metarhizium anisopliae) carboxypeptidase (MeCPA) was obtained by using reverse transcription differential display polymerase chain reaction to identify pathogenicity genes. MeCPA resembles pancreatic carboxypeptidases in being synthesized as a precursor species (418 amino acids) containing a large amino-terminal fragment (99 amino acids). The mature (secreted) form of MeCPA shows closest amino acid identity to human carboxypeptidases A1 (35%) and A2 (37%). MeCPA was expressed in an insect cell line yielding an enzyme with dual A1 + A2 specificity for branched aliphatic and aromatic COOH-terminal amino acids. However, in contrast to the very broad spectrum A + B-type bacterial enzymes, MeCPA lacks B-type activity against charged amino acids. This is predictable as key catalytic residues determining the specificity of MeCPA are conserved with those of mammalian A-type carboxypeptidases. Thus, in evolutionary terms the fungal enzyme is an intermediate between the divergence of A and B forms and the differentiation of the A form into A1 and A2 isoforms. Ultrastructural immunocytochemistry of infected host (Manduca sexta) cuticle demonstrated that MeCPA participates with the concurrently produced endoproteases in procuring nutrients; an equivalent function to digestive pancreatic enzymes.
Joshi
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Boyce Thompson Institute at Cornell University, Ithaca, New York 14853, USA.
St Leger
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D043422:Carboxypeptidases A; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004271:DNA, Fungal / Q000737:chemistry; D004792:Enzyme Precursors / Q000737:chemistry; D005656:Fungal Proteins; D006801:Humans; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009181:Mycoses / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D018076:DNA, Complementary; D004271:DNA, Fungal; D004792:Enzyme Precursors; D005656:Fungal Proteins; D002268:Carboxypeptidases; C118453:MCBP protein, Metarhizium anisopliae; D043422:Carboxypeptidases A