Functional Assessment of Residues in the Amino- and Carboxyl-Termini of Crustacean Hyperglycemic Hormone (CHH) in the Mud Crab Scylla olivacea Using Point-Mutated Peptides.
To assess functional importance of the residues in the amino- and carboxyl-termini of crustacean hyperglycemic hormone in the mud crab Scylla olivacea (Sco-CHH), both wild-type and point-mutated CHH peptides were produced with an amidated C-terminal end. Spectral analyses of circular dichroism, chromatographic retention time, and mass spectrometric analysis of the recombinant peptides indicate that they were close in conformation to native CHH and were produced with the intended substitutions. The recombinant peptides were subsequently used for an in vivo hyperglycemic assay. Two mutants (R13A and I69A rSco-CHH) completely lacked hyperglycemic activity, with temporal profiles similar to that of vehicle control. Temporal profiles of hyperglycemic responses elicited by 4 mutants (I2A, F3A, D12A, and D60A Sco-CHH) were different from that elicited by wild-type Sco-CHH; I2A was unique in that it exhibited significantly higher hyperglycemic activity, whereas the remaining 3 mutants showed lower activity. Four mutants (D4A, Q51A, E54A, and V72A rSco-CHH) elicited hyperglycemic responses with temporal profiles similar to those evoked by wild-type Sco-CHH. In contrast, the glycine-extended version of V72A rSco-CHH (V72A rSco-CHH-Gly) completely lost hyperglycemic activity. By comparing our study with previous ones of ion-transport peptide (ITP) and molt-inhibiting hormone (MIH) using deleted or point-mutated mutants, detail discussion is made regarding functionally important residues that are shared by both CHH and ITP (members of Group I of the CHH family), and those that discriminate CHH from ITP, and Group-I from Group-II peptides. Conclusions summarized in the present study provide insights into understanding of how functional diversification occurred within a peptide family of multifunctional members.
Department of Biology, National Changhua University of Education, Changhua, Taiwan.
Chun-Jing
CJ
Liu
Department of Biology, National Changhua University of Education, Changhua, Taiwan.
Shiau-Shan
SS
Huang
Sorbonne Universités, UPMC Université Paris 06, UMR 7144 CNRS, Equipe ABICE, Station Biologique de Roscoff, Roscoff, France; CNRS, UMR 7144, Adaptation et Diversité en Milieu Marin, Station Biologique de Roscoff, Roscoff, France.
Jean-Yves
JY
Toullec
Department of Biology, National Changhua University of Education, Changhua, Taiwan.
Cheng-Yen
CY
Chang
Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.
Yun-Ru
YR
Chen
Department of Biology, National Museum of Natural Science, Taichung, Taiwan; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.
Wen-San
WS
Huang
Department of Biology, National Changhua University of Education, Changhua, Taiwan.
Chi-Ying
CY
Lee
D000818:Animals; D060829:Arthropod Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003386:Brachyura / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D007447:Invertebrate Hormones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D011994:Recombinant Proteins
D060829:Arthropod Proteins; D007447:Invertebrate Hormones; D009419:Nerve Tissue Proteins; D010455:Peptides; D011994:Recombinant Proteins; C015947:hyperglycemic hormone, crustacean