CLONING AND CHARACTERIZATION OF THE CDNAS AND GENES (MEP20) ENCODING HOMOLOGOUS METALLOPROTEINASES FROM ASPERGILLUS-FLAVUS AND A-FUMIGATUS

Citation
Mv. Ramesh et al., CLONING AND CHARACTERIZATION OF THE CDNAS AND GENES (MEP20) ENCODING HOMOLOGOUS METALLOPROTEINASES FROM ASPERGILLUS-FLAVUS AND A-FUMIGATUS, Gene, 165(1), 1995, pp. 121-125
Citations number
21
Categorie Soggetti
Genetics & Heredity
Journal title
GeneACNP
ISSN journal
03781119
Volume
165
Issue
1
Year of publication
1995
Pages
121 - 125
Database
ISI
SICI code
0378-1119(1995)165:1<121:CACOTC>2.0.ZU;2-V
Abstract
Aspergillus fumigatus (Afu) and A. flavus (Afl), two causative agents of invasive aspergillosis, produce highly homologous serine proteinase s. In addition, the former produces a 42-kDa metalloproteinase (MEP), whereas the latter produces a 23-kDa MEP. The cDNA and the gene encodi ng the 42-kDa MEP were cloned and sequenced. Here, we report the cloni ng of the cDNA and the gene encoding the 23-kDa MEP from Afl and a hom ologous gene from the Afu. Using degenerate primers based on the amino acid (aa) sequence of A. oryzae (Ao) MEP and thermolysin-like protein ases, a 282-bp fragment of the 23-kDa MEP-encoding gene of Afl was clo ned by PCR. A 6.5-kb KpnI fragment of Afl genomic DNA containing the c omplete gene was cloned. The open reading frame (ORF) in this gene enc odes a protein of 381 aa. Since the mature enzyme from this and other aspergilli would have a theoretical molecular mass of about 20 kDa, th is MEP-encoding gene is designated mep20. A Western blot of the protei n in the culture filtrate of Afl with polyclonal antibodies prepared a gainst 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 signa l 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 nu cleotide (nt) sequences of the cDNAs cloned by RT-PCR with their respe ctive genes showed that there are no introns in the ORF of mep20 in Af l, 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 t he 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 sim ilar MEP.