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
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.