碩士 / 國立彰化師範大學 / 生物學系 / 98 / Sco-CHH and Sco-CHH-L (CHH-like peptide), two structural variants of the crustacean hyperglycemic hormone family identified in the mud crab (Scylla olivacea), are identical up to the 40th residue, but different from each other in the remaining sequence. Analyses of transcript sequences indicated they are encoded by alternatively spliced transcripts of the chh gene. Recombinant protein rSco-CHH were successfully produced using an E. coil expression system, refolded, purified, and confirmed by Western blotting, mass spectrometric analyses, circular dichroism analyses and subsequently used to confer the function of Sco-CHH. To reveal the structural bases of functional divergence, site-directed mutagenesis was used to produced mutated rSco-CHH (mutated sites located at 2, 3, 4, 12, 13, 51, 54, 60, 69, 70, 72 amino acid of the mature Sco-CHH) and tested the biological activity by using the established bioassays. The hyperglycemic activity of I2G (The second amino acid of mature Sco-CHH isoleucine mutated to glycine) decreased by 54.9 ± 50.5 %, F3A (phenylalanine mutated to alanine) decreased by 38.2 ± 57.9 %, Q70A (glutamine mutated to alanine) decreased by 27.1 ± 47.7 %, and V72G (valine mutated to glycine) decreased by 34.1 ± 45.9 %. The substitutions of critical residues at the N-terminus and C-terminus inferred that the N and C-terminal hydrophobic activity of Sco-CHH was important to receptor binding. Furthermore, D12N (aspartic acid mutated to asparagine) lost the hyperglycemic activity. Q51A (glutamine mutated to alanine) and E54Q (glutamic acid mutated to glutamine) not only decreased hyperglycemic activity but also changed the secondary structure of Sco-CHH. The result indicated that these critical residues contributed to the stability of Sco-CHH. The combined results support the notion that Sco-CHH and Sco-CHH-L are functionally divergent, and might reveal the structural bases of functional divergence.
| Identifer | oai:union.ndltd.org:TW/098NCUE5112014 |
| Date | January 2009 |
| Creators | Chang Cheng-Yen, 張政彥 |
| Contributors | Lee Chi-Ying, 李奇英 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 135 |
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