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 (P enaeus monodon) by sequential DEAE anion exchange chromatography. The purif ied protein was coagulated by the shrimp hemocyte transglutaminase in the p resence of Ca2+. The clottable protein contains 44% alpha helices and 26% b eta sheets as determined by circular dichroism spectra. Its conformation is stable in buffer of pH 4-9. To solve its primary structure, partial sequen ces of the purified polypeptides from cyanogen bromide cleavage and endopep tidase 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, incl uding a 14-amino acid signal peptide. With four potential N-glycosylation s ites, the clottable protein was found to contain 3.8% high-mannose glycan; and Man(8)GlcNAc and Man(9)GlcNAc were released upon endo-beta-N-acetylgluc osaminidase 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, apolipo protein B and mammalian von Willebrand factor. Notably, a region rich in Gi n residues, a polyGln motif and five Ser-Lys-Thr-Ser repeats are present in the shrimp protein, suggesting this protein might be a transglutaminase su bstrate. Northern blot analysis revealed that the clottable protein is expr essed in most of the shrimp tissues but not in the mature hemocytes.