Cloning, Expression, and Substrate Specificity of MeCPA, a Zinc Carboxypeptidase That Is Secreted into Infected Tissues by the Fungal Entomopathogen Metarhizium anisopliae

Abstract

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.The vertebrate pancreas synthesizes and secretes a subset of zinc carboxypeptidases that hydrolyze alimentary proteins and peptides from their COOH-terminal ends. These are traditionally classified into the A types (with a preference for apolar COOH-terminal residues) and the B types (with a preference for basic COOH-terminal residues) (1, 2). In contrast, actinomycetes (bacteria) produce carboxypeptidases with a dual A ϩ B specificity toward both neutral and basic substrates and the distinct vertebrate A and B types presumably arose from such a precursor (3, 4). Why or when in the course of evolution a single carboxypeptidase with both A and B specificities was abandoned in favor of multiple enzymes with more limited specificities is not known. The classification of carboxypeptidases into the A and B forms has been further expanded with the identification of the A1 and A2 isoforms in rat and humans. Carboxypeptidase A1 preferentially catalyzes aliphatic COOHterminal residues of peptide substrates, while the A2-type selectively acts on the bulkier aromatic COOH-terminal residues, being the only isoform that shows specificity toward tryptophan (2, 5, 6). Carboxypeptidase A2 is apparently absent from bovine pancreas, so in contrast the single carboxypeptidase gene has a relatively broad substrate specificity (2). A number of non-digestive zinc carboxypeptidases involved in hormone and neuropeptide processing, bioactive peptide activation or inactivation, or functional modulation of regulatory proteins have also been reported in the literature expanding the field of interest in metallocarboxypeptidases (1). ...

Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts

“…Total RNA was extracted from frozen fungus using TRI Reagent as described by Joshi & St Leger (1999). The cDNA libraries were constructed in the unidirectional lZAP II vector (Stratagene) exploiting the EcoRI and XhoI restriction sites.…”

“…acridum was intended to mimic conditions occurring during infection and colonization of the host insect and increase the probability of cloning transcripts involved in pathogenicity. The success of this strategy was supported by expression of several EST sequences, including proteases (Pr1a, chymotrypsin, trypsins, carboxypeptidase), chitinases and a cAMP-dependent protein kinase catalytic subunit (AJ273794) that are produced in vivo and have been implicated in the events leading to formation of infection structures and pathogenicity (Joshi & St Leger, 1999;St Leger et al, 1996b, c).…”

Applications of Differential-Display Reverse Transcription-PCR to Molecular Pathogenesis and Medical Mycology

“…Genes have been identified in plant and insect studies which may be relevant to medically important reactions. For instance, a subtilisin-like protease (PrlB) and a metallocarboxypeptidase (MeCPA) were induced in Metarhizium anisopliae after contact with the host (cockroach) cuticle (65,66). Both proteases were secreted into the host cuticle during invasion, although subtilisin is the major secreted protein.…”

“…The subtilisin-like protease rapidly degrades cuticle into peptides. The peptides can then be further degraded by MeCPA to amino acids as a source of nutrition for the fungus (65,66). Other proteases also are differentially expressed in fungal pathogen interactions with plant or insect hosts.…”

IDENTIFICATION OF POTENTIAL ENTOMOPATHOGENIC FUNGI OF Tetranychus kanzawai Kishida (Tetranychidae: Acarina) USING ITS-5.8s rDNA REGION AS MOLECULAR MARKER

“…Obviously, taxonomic procedures are becoming more complex, and it is generally accepted that some forms of molecular identification techniques are needed in addition to the traditional morphological characteristics formally used to classify fungi species (Bridge and Arora, 1998). Different molecular techniques are used for various applications and on different entomopathogenic and mycoparasitic fungi, including identification of fungi isolates based on DNA polymorphism using RAPD-PCR technique (Joshi and St Leger, 1999). The RAPD (Random Amplified Polymorphic DNA) technique was introduced in 1990 (Samšiňáková et al, 1983).…”