Comparative studies on molecular characterizations and physiological functions of crustacean hyperglycemic hormone isoforms in the mud crab Scylla olivacea

• Main Authors: Kuo-Wei Tsai, 蔡國瑋
• Other Authors: Chi-Ying Lee
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/13309963145382498785

碩士 === 國立彰化師範大學 === 生物學系 === 93 === Crustacean hyperglycemic hormone (CHH) is a neuropeptide original identified from the X-organ (XO)-sinus gland (SG) complex in the crustacean eyestalk. In the present work, three full-length CHH cDNAs were cloned from the mud crab Scylla olivacea eyestalk ganglia (ES), thoracic ganglia (TG), and per- icardial organ (PO) by RT-PCR and rapid amplification of cDNA end (RACE). PO-CHH and TG-CHH have the same deduced amino acid sequence. TG/PO-CHH and sinus gland CHH (SG-CHH) share an identical N-terminal sequence (amino acids 1-40), but the remaining sequences (amino acids 41-73 or 41-75), differ considerably. The sequence evidence suggests that these two isoforms are derived a Chh gene transcribed in an alternative splicing manner. With the use of a two-step HPLC purification procedure and a CHH isoform- specific ELISA, the two CHH isoforms were isolated from sinus gland and pericardial organ, respectively. Mass spectrometric analysis indicates that the molecular masses for the native SG-CHH and TG/PO-CHH are 8379 Da and 8478 Da, respectively, which are consistent with the estimated values based on the deduced sequences. It also suggests that the two CHHs have a blocked N- terminus, and the C-terminus of SG-CHH is amidated, but TG/PO-CHH has a free C-terminus. The partial amino acid sequences of SG-CHH and TG/PO- CHH were determined by MALDI MS/MS analysis of trypsin-digested peptide fragments. The results show that the amino acid sequences of these peptide fragments were identical to the deduced amino acid sequences of the SG-CHH and TG/PO-CHH cDNAs. In addition, SG-CHH (10 pmole) exhibited a hyper- glycemic activity in vivo, whereas the same dosage of TG/PO-CHH did not elicit a hyperglycemic response. The physiological function of TG/PO-CHH remains to be clarified. We speculate that TG/PO-CHH may have regulatory functions differ from those of SG-CHH.