title: A novel slow-tight binding serine protease inhibitor from eastern oyster (Crassostrea virginica) plasma inhibits perkinsin, the major extracellular protease of the oyster protozoan parasite Perkinsus marinus.
abstract: A serine protease inhibitor was purified from plasma of the eastern oyster, Crassostrea virginica. The inhibitor is a 7609.6 Da protein consisting of 71 amino acids with 12 cysteine residues that are postulated to form 6 intra-chain disulfide bridges. Sequencing of the cloned cDNA identified an open reading frame encoding a polypeptide of 90 amino acids, with the 19 N-terminal amino acids forming a signal peptide. No sequence similarity with known proteins was found in sequence databases. The protein inhibited the serine proteases subtilisin A, trypsin and perkinsin, the major extracellular protease of the oyster protozoan parasite, Perkinsus marinus, in a slow binding manner. The mechanism of inhibition involves a rapid binding of inhibitor to the enzyme to form a weak enzyme-inhibitor complex followed by a slow isomerization to form a very tight binding enzyme-inhibitor complex. The overall dissociation constants K(i) with subtilisin A, perkinsin and trypsin were 0.29 nM, 13.7 nM and 17.7 nM, respectively. No inhibition of representatives of the other protease classes was detected. This is the first protein inhibitor of proteases identified from a bivalve mollusk and it represents a new protease inhibitor family. Its tight binding to subtilisin and perkinsin suggests it plays a role in the oyster host defense against P. marinus.
authors.affiliation: Cooperative Aquatic Animal Health Research Program, Department of Veterinary Science, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA.
authors.forename: Qing-Gang
authors.initials: QG
authors.lastname: Xue
authors.forename: Grover L
authors.initials: GL
authors.lastname: Waldrop
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Schey
authors.forename: Naoki
authors.initials: N
authors.lastname: Itoh
authors.forename: Masahiro
authors.initials: M
authors.lastname: Ogawa
authors.forename: Richard K
authors.initials: RK
authors.lastname: Cooper
authors.forename: Jack N
authors.initials: JN
authors.lastname: Losso
authors.forename: Jerome F
authors.initials: JF
authors.lastname: La Peyre
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D049896:Crassostrea / Q000166:cytology / Q000378:metabolism* / Q000469:parasitology; D018076:DNA, Complementary / Q000737:chemistry; D056890:Eukaryota / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000097:blood / Q000302:isolation & purification / Q000494:pharmacology*; D020860:Subtilisin / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; D020860:Subtilisin