Characterization of cDNAs encoding serine proteinases from the soybean cyst nematode Heterodera glycines.
Three cDNAs encoding serine proteinases (HGSPI-III) were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with three separate serine proteinase gene fragments amplified from cDNA of H. glycines using consensus oligonucleotide primers. Each predicted protein contains a secretion signal sequence, a propeptide and a mature protein of 226-296 amino acids. One of the predicted enzymes, HGSP-II has 41% identity to a chymotrypsin-like enzyme from the mollusc, Haliotis rufescens, and analysis of key residues involved in substrate binding also suggests a chymotrypsin-like specificity. HGSP-I and HGSP-III show greatest homology to kallikreins but sequence analysis does not allow prediction of their substrate preferences. Southern blot analysis suggests that HGSP-II and HGSP-III are encoded by single-copy genes in contrast to HGSP-I which may have two or more homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in Escherichia coli. Both HGSP-I and HGSP-II were shown, after refolding, to cleave the synthetic peptide N-CBZ-Phe-Arg-7-amido-4-methylcoumarin, and this activity could be inhibited by the cowpea trypsin inhibitor, CpTI. HGSP-III showed no activity against the synthetic substrates tested. The information gained from these studies indicates that serine proteinases are an important group of enzymes in H. glycines and further characterization will aid the development of a proteinase inhibitor-based approach for transgenic plant resistance to plant parasitic nematodes.
Lilley
C J
CJ
Centre for Plant Biochemistry and Biotechnology, University of Leeds, UK.
Urwin
P E
PE
Atkinson
H J
HJ
McPherson
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D018509:DNA, Helminth / Q000235:genetics*; D005260:Female; D018628:Gene Dosage; D017238:Genes, Helminth / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009348:Nematoda / Q000201:enzymology / Q000235:genetics*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013025:Glycine max / Q000469:parasitology*
D018076:DNA, Complementary; D018509:DNA, Helminth; D011994:Recombinant Proteins; C109321:HGSP-I protein, Heterodera glycines; C109322:SP-II protein, Heterodera glycines; C109323:SP-III protein, Heterodera glycines; D012697:Serine Endopeptidases