title: Cloning and characterization of the cDNAs and genes (mep20) encoding homologous metalloproteinases from Aspergillus flavus and A. fumigatus.
abstract: Aspergillus fumigatus (Afu) and A. flavus (Afl), two causative agents of invasive aspergillosis, produce highly homologous serine proteinases. In addition, the former produces a 42-kDa metalloproteinase (MEP), whereas the latter produces a 23-kDa MEP. The cDNA and the gene encoding the 42-kDa MEP were cloned and sequenced. Here, we report the cloning of the cDNA and the gene encoding the 23-kDa MEP from Afl and a homologous gene from the Afu. Using degenerate primers based on the amino acid (aa) sequence of A. oryzae (Ao) MEP and thermolysin-like proteinases, a 282-bp fragment of the 23-kDa MEP-encoding gene of Afl was cloned by PCR. A 6.5-kb KpnI fragment of Afl genomic DNA containing the complete gene was cloned. The open reading frame (ORF) in this gene encodes a protein of 381 aa. Since the mature enzyme from this and other aspergilli would have a theoretical molecular mass of about 20 kDa, this MEP-encoding gene is designated mep20. A Western blot of the protein in the culture filtrate of Afl with polyclonal antibodies prepared against the MEP showed a single band at 23 kDa. The N-terminal sequence of the extracellular MEP20, TKVAS, was found at aa 194-198 within the ORF. Thus, the primary translation product has a putative 19-aa signal and a pro region of 174 aa. A homologous gene cloned from a genomic DNA library of Afu showed an ORF encoding 365 aa. Comparison of the nucleotide (nt) sequences of the cDNAs cloned by RT-PCR with their respective genes showed that there are no introns in the ORF of mep20 in Afl, but there is a 59-bp intron in the gene from Afu. The MEP20 of Afl and Afu have 68% identity and show weak immunological cross reactivity. MEP20 from both these fungi share about 60% sequence identity with the penicillolysin of Penicillium citrinum and the neutral protease II of Ao. MEP20 of Afl and Afu show only the conserved sequence, HEFTHA, but not the two other conserved sequences seen in thermolysins and similar MEP.
mesh_terms: D000595:Amino Acid Sequence; D001231:Aspergillus flavus / Q000201:enzymology* / Q000235:genetics; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D005800:Genes, Fungal; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D008666:Metalloendopeptidases