博士 / 國立臺灣大學 / 生化科學研究所 / 88 / Abstract
The clottable protein was purified from the hemolymph of tiger shrimp (Penaeus monodon) by sequential DEAE-anion exchanger chromatography. The protein formed stable clots in the presence of Ca2+ and the transglutaminase contained in hemocyte lysate. The molecular mass of the clottable protein was determined to be 380 kDa by SDS-PAGE and MALDI-TOF mass spectrometry and the protein is a disulfide-linked homodimer. By PAS stain, the clottable protein was found to be a glycoprotein. The size and the amino acid composition and sequences of the clottable protein are similar to those of other crustaceans including lobster (Panulirus interruptus) and crayfish (Pacifastacus leniusculus). The clottable protein was found to contain 2.6% mannose and 1.2% N-acetylglucosamine. Endoglycosidase H digestion of the glycoprotein generated mainly Man8GlcNAc and Man9GlcNAc sugar chains, suggesting the presence of high-mannose type glycan structure. Its conformation is stable at temperature below 66 0C, as shown by UV-spectrophotometry. The protein contains 44% a-helices, 26% b sheets and 16% b turn as determined by circular dichroism spectra. Its conformation is stable in buffers of pH 4-9 and NaCl concentration up to 0.5 M.
The purified clottable protein was subjected to CNBr cleavage and Glu-C and Lys-C digestion. The peptides were separated by C18 column chromatography. We designed the degenrate primers from the peptide sequences and N-terminal sequence. PCR were performed with the synthetic primers and the total cDNA as template. We obtained a 500 bp clone encoding authentic amino acid sequences of the clottable protein. Moreover, the cDNA expression library was constructed and screened with the antiserum and the PCR product. We totally obtained 5 clones. These five clones were isolated and sequenced to complete the entire nucleotide sequence of the clottable protein. The complete cDNA coding for the entire protein is 6,124 base pairs in length and has an open reading frame encoding a protein of 1,670 amino acids, including a 14-amino acid signal peptide. Upon conducting a protein sequence database survey, the shrimp clottable protein shows 36% identities to the crayfish clotting protein and up to 20% identities to members of insect vitellogenins. In the C-terminal 200 residues of the clottable protein as well as of the insect vitellogenins there are 10 cysteines residues and a GICG motif in concensus, suggestive of genetic relationship between these genes. The clottable protein also contains a stretch with similarity to the D domain of mammalian von Willebrand factor, mucin 2 and silkworm hemocytin. A region rich in Gln residues (1079-1122), a polyGln motif (1635-1650) and five Ser-Lys-Thr-Ser repeats (197-231) are present in the shrimp protein, suggesting these regions might be involved in cross-linking of the clottable proteins in the presence of TGase.
The tissue distribution of clottable protein was detecteded by Western blot, immunohistochemistry and Northern blot analysis. The clottable protein is expressed in most of the shrimp tissue. The expression of the clottable protein is found with highest level in gill and heart of the shrimp, with a lower level in lymphoid organ, hepatopancreas and muscle but not in hemocytes.
| Identifer | oai:union.ndltd.org:TW/088NTU00103001 |
| Date | January 2000 |
| Creators | Yeh Maw-Sheng, 葉茂盛 |
| Contributors | Tsai Inn-Ho, 蔡蔭和 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 98 |
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