Cloning and characterization of hemolymph clottable proteins of kuruma prawn (Marsupenaeus japonicus) and white shrimp (Litopenaeus vannamei).
The hemolymph clottable protein (CP) of Marsupenaeus japonica (designated as Mj-CP) was purified by a DEAE anion-exchanger and a Sepharose CL-6B gel filtration column. In the presence of Ca(2+), it formed stable clots in vitro upon the addition of the hemocytes lysate containing transglutaminase. Results of gel filtration chromatography and SDS-PAGE indicated that Mj-CP mainly existed as disulfide-linked homodimers of 390 kDa. Specific primers were designed; PCR as well as RACE help to clone and sequence Mj-CP cDNA of 5660 bp. The predicted CP-precursor contains a signal peptide followed by a subunit of 1671 amino acids (isoelectric point 5.6), including two RGD motifs and three potential N-glycosylation sites. Mj-CP is structurally 80% and 38% identical to the CPs of tiger shrimp and crayfish, respectively. Likewise, CP cDNA of white shrimp (Litopenaeus vannamei) was also cloned and sequenced; the predicted CP has 1666 amino acid residues and an isoelectric point of 5.2. Both CPs bear potential transglutaminase cross-linking sites, i.e. seven Ser-Lys-Thr repeats near the N-terminus, a Ser- and Gln-rich region in the middle, and polyGln (n=8-11) near the C-terminus. Phylogenetic analyses of crustacean CPs and vitellogenins revealed divergent evolution of the two protein families. By RT-PCR, the sub-cuticular epidermis was identified as one of the major tissues that express CP in M. japonica.
Cheng
Winton
W
Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, Taiwan.
Tsai
Inn-Ho
IH
Huang
Chang-Jen
CJ
Chiang
Pei-Chi
PC
Cheng
Chia-Hsiung
CH
Yeh
Maw-Sheng
MS
D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D001779:Blood Coagulation Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling; D006458:Hemolymph / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001779:Blood Coagulation Factors; D018076:DNA, Complementary; D002122:Calcium Chloride