Molecular cloning and characterization of a hemolymph clottable protein from tiger shrimp (Penaeus monodon).
To investigate the coagulation system in crustacean decapoda, a homodimeric glycoprotein of 380 kDa was purified from the hemolymph of tiger shrimp (Penaeus monodon) by sequential DEAE anion exchange chromatography. The purified protein was coagulated by the shrimp hemocyte transglutaminase in the presence of Ca2+. The clottable protein contains 44% alpha helices and 26% beta sheets as determined by circular dichroism spectra. Its conformation is stable in buffer of pH 4-9. To solve its primary structure, partial sequences of the purified polypeptides from cyanogen bromide cleavage and endopeptidase digestion were also determined. A shrimp cDNA expression library was constructed. By combination with antibody screening, reverse transcriptase PCR using degenerate primers from determined amino acid sequences and cDNA library screening with digoxigenin-labeled DNA probes, the entire cDNA of 6124 bp was obtained. This cDNA encodes a protein of 1670 amino acids, including a 14-amino acid signal peptide. With four potential N-glycosylation sites, the clottable protein was found to contain 3.8% high-mannose glycan; and Man8GlcNAc and Man9GlcNAc were released upon endo-beta-N-acetylglucosaminidase hydrolysis. Upon conducting a protein sequence database survey, the shrimp clottable protein shows 36% identities to the crayfish clotting protein and lower similarities to members of insect vitellogenins, apolipoprotein B and mammalian von Willebrand factor. Notably, a region rich in Gln residues, a polyGln motif and five Ser-Lys-Thr-Ser repeats are present in the shrimp protein, suggesting this protein might be a transglutaminase substrate. Northern blot analysis revealed that the clottable protein is expressed in most of the shrimp tissues but not in the mature hemocytes.
Yeh
M S
MS
Institute of Biological Sciences, National Taiwan University, Taipei.
Huang
C J
CJ
Leu
J H
JH
Lee
Y C
YC
Tsai
I H
IH
D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D001777:Blood Coagulation; D015152:Blotting, Northern; D002241:Carbohydrates / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000378:metabolism; D015723:Gene Library; D006458:Hemolymph / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D033561:Penaeidae; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014018:Tissue Distribution
D002241:Carbohydrates; D018076:DNA, Complementary; D009844:Oligosaccharides; D011134:Polysaccharides