Structural and functional comparisons and production of recombinant crustacean hyperglycemic hormone (CHH) and CHH-like peptides from the mud crab Scylla olivacea.

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 alpha-helices, and are similarly thermo-stable with a melting temperature of 74-75 degrees 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 alpha-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 alpha-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 alpha-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.

Molecular cloning and differential expression pattern of two structural variants of the crustacean hyperglycemic hormone family from the mud crab Scylla olivacea.

Two full-length cDNA sequences encoding a crustacean hyperglycemic hormone (CHH) precursor were cloned from tissues of the mud crab Scylla olivacea. Sco-CHH (S. olivacea CHH) was cloned from eyestalk ganglia, whereas Sco-CHH-L (S. olivacea CHH-like peptide) was cloned from extra-eyestalk tissues (pericardial organ and thoracic ganglia). Each conceptually translated precursor is expected to be processed into a signal peptide, a CHH precursor-related peptide (CPRP), and a mature CHH or CHH-like peptide. The two precursors are identical in amino acid sequence through the 40th residue of the mature peptide, but different from each other substantially in the C-terminus. Both CHH variants contain the six highly conserved cysteine residues characteristic of the CHH family peptides, and share higher sequence identities with other brachyuran CHH sequences than with those of other taxonomic groups. As determined by reverse transcription-polymerase chain reaction (RT-PCR), the transcripts of Sco-CHH and Sco-CHH-L were present in eyestalk ganglia and several extra-eyestalk tissues (the thoracic ganglia, pericardial organ, brain, circumesophageal connectives, and gut). Sco-CHH was the predominant form in eyestalk ganglia, while Sco-CHH-L was the predominant form in several extra-eyestalk tissues. Neither transcript was expressed in the muscle, hepatopancreas, ovary, testis, heart, or gill. Antisera were raised against synthetic peptides corresponding to a stretch of sequence-specific to the C-terminus of Sco-CHH or Sco-CHH-L. Western blot analyses of tissues expressing Sco-CHH and Sco-CHH-L detected a Sco-CHH immunoreactive protein in the sinus gland, and a Sco-CHH-L immunoreactive protein in the pericardial organ. Immunohistochemical analyses of the eyestalk ganglia localized both Sco-CHH and Sco-CHH-L immunoreactivity to the sinus gland, and only Sco-CHH immunoreactivity to the X-organ somata; analyses of the pericardial organs also localized both Sco-CHH and Sco-CHH-L immunoreactivity to the anterior and posterior bars, as well as to longitudinal trunks joining the two bars. The combined data provided supporting evidence that Sco-CHH and Sco-CHH-L are co-localized in the same tissue.