Cloning, sequencing, temporal expression and tissue-specificity of two serine proteases from the midgut of the blood-feeding fly Stomoxys calcitrans.
Using highly degenerate, serine-protease-specific PCR primers on a midgut-specific cDNA library it was estimated that a minimum of 24 independent serine proteases were expressed in the midgut of Stomoxys calcitrans. The relative abundance of these 24 independent serine proteases has been estimated by restriction analysis of PCR products, showing that 69% fall into six almost equally abundant groups. Two highly abundant serine protease cDNAs (Ssp1 and Ssp2) were isolated and sequenced. They encode preproenzymes of 272 amino acids (Mr 28521) and 255 amino acids (Mr 27097) with putative signal peptides of 17 amino acids and 16 amino acids, putative activation peptides of 15 amino acids and 10 amino acids and mature enzymes of 239 amino acids (Mr 25322; pI 4.89) and 228 amino acids (Mr 24182; pI 7.59), respectively. Both deduced amino acid sequences contain the Asp/His/Ser catalytic triad and the highly conserved sequences surrounding it. Ssp2 also has the aspartate and two glycine residues in the specificity pocket, marking this as a typical trypsin. The positioning of the residues in the specificity pocket of Sspl is unusual; aspartate and glycine residues are present, which is typical of trypsin, but both are separated from surrounding conserved residues by additional amino acids; the second glycine found in the specificity pocket of trypsin is replaced by a serine, which is typical of chymotrypsin. Although a serine protease, the precise substrate specificity of Sspl remains to be determined. Northern analysis shows that both serine proteases are expressed constitutively with only a 20% change in the levels of expression of Ssp1 and Ssp2 through the digestive cycle, and that expression occurs predominantly in the opaque region of the midgut, the region responsible for secretion of digestive enzymes.
Lehane
S M
SM
School of Biological Sciences, University of Wales, Bangor, UK. m.j.lehane@bangor.ac.uk
Assinder
S J
SJ
Lehane
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004064:Digestive System / Q000201:enzymology; D004435:Eating / Q000235:genetics / Q000502:physiology; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D017344:Genes, Insect; D008969:Molecular Sequence Data; D009117:Muscidae / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014018:Tissue Distribution
D017931:DNA Primers; D018076:DNA, Complementary; D012697:Serine Endopeptidases