Abstract
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 presumably alternatively spliced gene products. In this study, Sco-CHH and Sco-CHH-L were isolated from the tissues using high performance liquid chromatography. Identity of the native peptides was confirmed using mass spectrometric (MS) analyses of purified materials and of trypsin-digested peptide fragments. Additionally, characterizations using circular dichroism (CD) spectrometry revealed that the 2 peptides have similar CD spectral profiles, showing they are composed mainly of α-helices, and are similarly thermo-stable with a melting temperature of 74-75°C. Results of bioassays indicated that Sco-CHH exerted hyperglycemic and molt-inhibiting activity, whereas Sco-CHH-L did not. Further, recombinant Sco-CHH-Gly (rSco-CHH-Gly, a glycine extended Sco-CHH) and Sco-CHH-L (rSco-CHH-L) were produced using an Escherichia coli expression system, refolded, and purified. rSco-CHH-Gly was further α-amidated at the C-terminal end to produce rSco-CHH. MS analyses of enzyme-digested peptide fragments of rSco-CHH-Gly and rSco-CHH-L showed that the two peptides share a common disulfide bond pattern: C7-C43, C23-C39, and C26-C52. Circular dichroism analyses and hyperglycemic assay revealed that rSco-CHH and rSco-CHH-L resemble their native counterparts, in terms of CD spectral profiles, melting curve profiles, and biological activity. rSco-CHH-Gly has a lower α-helical content (32%) than rSco-CHH (47%), a structural deviation that may be responsible for the significant decrease in the biological activity of rSco-CHH-Gly. Finally, modeled structure of Sco-CHH and Sco-CHH-L indicated that they are similarly folded, each with an N-terminal tail region and 4 α-helices. Putative surface residues located in corresponding positions of Sco-CHH and Sco-CHH-L but with side chains of different properties were identified. The combined results support the notion that Sco-CHH and Sco-CHH-L are functionally different, but resemble each other at higher-level structures. Functional diversity between the 2 peptides is probably due to critical residues located in the C-terminus. The availability of large amounts of recombinant proteins will permit additional functional and structural studies of these CHH family peptides.
| Original language | English |
|---|---|
| Pages (from-to) | 68-76 |
| Number of pages | 9 |
| Journal | General and Comparative Endocrinology |
| Volume | 167 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2010 May |
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All Science Journal Classification (ASJC) codes
- Animal Science and Zoology
- Endocrinology
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Structural and functional comparisons and production of recombinant crustacean hyperglycemic hormone (CHH) and CHH-like peptides from the mud crab Scylla olivacea. / Chang, Chih Chun; Tsai, Kuo Wei; Hsiao, Nai Wan; Chang, Cheng Yen; Lin, Chih Lung; Watson, R. Douglas; Lee, Chi Ying.
In: General and Comparative Endocrinology, Vol. 167, No. 1, 05.2010, p. 68-76.Research output: Contribution to journal › Article
TY - JOUR
T1 - Structural and functional comparisons and production of recombinant crustacean hyperglycemic hormone (CHH) and CHH-like peptides from the mud crab Scylla olivacea
AU - Chang, Chih Chun
AU - Tsai, Kuo Wei
AU - Hsiao, Nai Wan
AU - Chang, Cheng Yen
AU - Lin, Chih Lung
AU - Watson, R. Douglas
AU - Lee, Chi Ying
PY - 2010/5
Y1 - 2010/5
N2 - 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 presumably alternatively spliced gene products. In this study, Sco-CHH and Sco-CHH-L were isolated from the tissues using high performance liquid chromatography. Identity of the native peptides was confirmed using mass spectrometric (MS) analyses of purified materials and of trypsin-digested peptide fragments. Additionally, characterizations using circular dichroism (CD) spectrometry revealed that the 2 peptides have similar CD spectral profiles, showing they are composed mainly of α-helices, and are similarly thermo-stable with a melting temperature of 74-75°C. Results of bioassays indicated that Sco-CHH exerted hyperglycemic and molt-inhibiting activity, whereas Sco-CHH-L did not. Further, recombinant Sco-CHH-Gly (rSco-CHH-Gly, a glycine extended Sco-CHH) and Sco-CHH-L (rSco-CHH-L) were produced using an Escherichia coli expression system, refolded, and purified. rSco-CHH-Gly was further α-amidated at the C-terminal end to produce rSco-CHH. MS analyses of enzyme-digested peptide fragments of rSco-CHH-Gly and rSco-CHH-L showed that the two peptides share a common disulfide bond pattern: C7-C43, C23-C39, and C26-C52. Circular dichroism analyses and hyperglycemic assay revealed that rSco-CHH and rSco-CHH-L resemble their native counterparts, in terms of CD spectral profiles, melting curve profiles, and biological activity. rSco-CHH-Gly has a lower α-helical content (32%) than rSco-CHH (47%), a structural deviation that may be responsible for the significant decrease in the biological activity of rSco-CHH-Gly. Finally, modeled structure of Sco-CHH and Sco-CHH-L indicated that they are similarly folded, each with an N-terminal tail region and 4 α-helices. Putative surface residues located in corresponding positions of Sco-CHH and Sco-CHH-L but with side chains of different properties were identified. The combined results support the notion that Sco-CHH and Sco-CHH-L are functionally different, but resemble each other at higher-level structures. Functional diversity between the 2 peptides is probably due to critical residues located in the C-terminus. The availability of large amounts of recombinant proteins will permit additional functional and structural studies of these CHH family peptides.
AB - 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 presumably alternatively spliced gene products. In this study, Sco-CHH and Sco-CHH-L were isolated from the tissues using high performance liquid chromatography. Identity of the native peptides was confirmed using mass spectrometric (MS) analyses of purified materials and of trypsin-digested peptide fragments. Additionally, characterizations using circular dichroism (CD) spectrometry revealed that the 2 peptides have similar CD spectral profiles, showing they are composed mainly of α-helices, and are similarly thermo-stable with a melting temperature of 74-75°C. Results of bioassays indicated that Sco-CHH exerted hyperglycemic and molt-inhibiting activity, whereas Sco-CHH-L did not. Further, recombinant Sco-CHH-Gly (rSco-CHH-Gly, a glycine extended Sco-CHH) and Sco-CHH-L (rSco-CHH-L) were produced using an Escherichia coli expression system, refolded, and purified. rSco-CHH-Gly was further α-amidated at the C-terminal end to produce rSco-CHH. MS analyses of enzyme-digested peptide fragments of rSco-CHH-Gly and rSco-CHH-L showed that the two peptides share a common disulfide bond pattern: C7-C43, C23-C39, and C26-C52. Circular dichroism analyses and hyperglycemic assay revealed that rSco-CHH and rSco-CHH-L resemble their native counterparts, in terms of CD spectral profiles, melting curve profiles, and biological activity. rSco-CHH-Gly has a lower α-helical content (32%) than rSco-CHH (47%), a structural deviation that may be responsible for the significant decrease in the biological activity of rSco-CHH-Gly. Finally, modeled structure of Sco-CHH and Sco-CHH-L indicated that they are similarly folded, each with an N-terminal tail region and 4 α-helices. Putative surface residues located in corresponding positions of Sco-CHH and Sco-CHH-L but with side chains of different properties were identified. The combined results support the notion that Sco-CHH and Sco-CHH-L are functionally different, but resemble each other at higher-level structures. Functional diversity between the 2 peptides is probably due to critical residues located in the C-terminus. The availability of large amounts of recombinant proteins will permit additional functional and structural studies of these CHH family peptides.
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UR - http://www.scopus.com/inward/citedby.url?scp=77952546099&partnerID=8YFLogxK
U2 - 10.1016/j.ygcen.2010.02.013
DO - 10.1016/j.ygcen.2010.02.013
M3 - Article
C2 - 20171218
AN - SCOPUS:77952546099
VL - 167
SP - 68
EP - 76
JO - General and Comparative Endocrinology
JF - General and Comparative Endocrinology
SN - 0016-6480
IS - 1
ER -