A secreted aspartic proteinase from Glomerella cingulata: purification of the enzyme and molecular cloning of the cDNA.
A secreted aspartic proteinase from Glomerella cingulata (GcSAP) was purified to homogeneity by ion exchange chromatography. The enzyme has an M, of 36000 as estimated by SDS-PAGE, optimal activity from pH 3.5 to pH 4.0 and is inhibited by pepstatin. The N-terminal sequence, 23 residues long, was used to design a gene-specific primer. This was used in 3' RACE (rapid amplification of cDNA ends) PCR to amplify a 1.2 kb fragment of the gcsap cDNA. A second gene-specific primer was designed and used in 5' RACE PCR to clone the 5' region. This yielded a 600 bp DNA fragment and completed the open reading frame. The gcsap open reading frame encodes a protein with a 78 residue prepro-sequence typical of other fungal secreted aspartic proteinases. Based on the deduced sequence, the mature enzyme contains 329 amino acids and shows approximately 40% identity to other fungal aspartic proteinases. Subsequent cloning and sequencing of gcsap fragments obtained from PCR with genomic DNA revealed a 73 bp intron beginning at nt 728. Southern analyses at medium and high stringency indicated that G. cingulata possesses one gene for the secreted aspartic proteinase, and Northern blots indicated that gene expression was induced by exogenous protein and repressed by ammonium salts. GcSAP is a putative pathogenicity factor of G. cingulata, and it will now be possible to create SAP-mutants and assess the role GcSAP plays in pathogenicity.
Clark
Sarah J
SJ
Biochemistry Department, University of Otago, PO Box 56, Dunedin, New Zealand.
Templeton
Matthew D
MD
Molecular Genetics Group, Horticulture and Food Research Institute of New Zealand Ltd, Mt Albert, Auckland, New Zealand.
Sullivan
Patrick A
PA
Biochemistry Department, Massey University, Private Bag 11222, Palmerston North, New Zealand.
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology / Q000235:genetics*; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D016681:Genome, Fungal; D007438:Introns; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D005656:Fungal Proteins; D016282:Aspartic Acid Endopeptidases