Cloning and characterisation of a serine proteinase from the haemocytes of mud crab Scylla serrata.
A serine proteinase (SP) cDNA was cloned from the haemocytes of mud crab Scylla serrata using oligonucleotide primers and RT-PCR. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1,131 bp encoding a protein of 376 amino acids. The calculated molecular mass of the SP mature protein is 39.54 kDa with an estimated pI of 5.37. The C-terminal half of S. serrata SP is composed of a trypsin-like domain, with a sequence similar to that of other invertebrate and vertebrate SP domain. The typical catalytic triad of SP required for functional activity (His150, Asp217 and Ser331) was conserved in the polypeptide sequence. Sequence comparison showed that SP deduced amino acid has an overall similarity of 55%, 51% and 50% to SP deduced amino acid from spiny lobster Panulirus argus, horseshoe crab Tachypleus tridentatus and crayfish Pacifastaus leniusculus, respectively. The SP was strongly expressed in haemocytes, but was weakly expressed in heart, eyestalk and antennules. The SP transcript decreased significantly for the S. serrata following 3 days exposure to pH 9.5. However, the SP transcript increased significantly 24 h post-zymosan injection.
Vaseeharan
Baskaralingam
B
Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, No: 2 Pei-Ning Road, Keelung 202, Taiwan, ROC.
Lin
Yong-Chin
YC
Ko
Chi-Fong
CF
Chen
Jiann-Chu
JC
D000199:Actins / Q000032:analysis; D000595:Amino Acid Sequence; D000825:Animal Structures / Q000201:enzymology / Q000502:physiology; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D003386:Brachyura / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D006434:Hemocytes / Q000201:enzymology* / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment / Q000662:veterinary; D017385:Sequence Homology; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D013997:Time Factors; D015054:Zymosan / Q000008:administration & dosage / Q000494:pharmacology*
D000199:Actins; D017931:DNA Primers; D018076:DNA, Complementary; D015054:Zymosan; D012697:Serine Endopeptidases