Identification of the crustacean hyperglycemic hormone (CHH) and CHH-like peptides in the crayfish Procambarus clarkii and localization of functionally important regions of the CHH

Four anti-peptide antibodies were raised each against a synthetic peptide corresponding in sequence to a short stretch of the crustacean hyperglycemic hormone (CHH) or CHH-like (CHH-L) peptides of the crayfish Procambarus clarkii. CHH and CHH-L are alternatively spliced products that share an identical sequence for the 1st 40 residues from the amino-terminus of the peptides. When used in Western blot analyses of tissue proteins, anti-CHH (1-10) recognized an immunoreactive protein band in both sinus gland (SGs) and thoracic ganglia (TGs), whereas anti-D-CHH (1-10) recognized an immunoreactive protein band only in SGs, but not in TGs; anti-CHH (59-72) recognized an immunoreactive protein band in SGs but not in TGs, and conversely, anti-CHH-L (58-72) recognized an immunoreactive protein band in TGs but not in SGs. Tissue homogenates were fractionated using high-performance liquid chromatography (HPLC). The immunoreactivity of the collected HPLC fractions was determined by an enzyme-linked immunosorbent assay and Western blotting, and the immunoreactive fractions were subjected to mass determination. A pair of stereoisomers, CHH and D-Phe³ CHH, both with a mass of 8386.4 and respectively immunoreactive to anti-CHH (1-10) and anti-DCHH (1-10), was identified in SGs; Western blot analyses showed that they were immunoreactive to anti-CHH (59-72), but not to anti-CHH-L (58-72). A CHH-L, with a mass of 8343.6 and immunoreactive to anti-CHH (1-10) but not to anti-D-CHH (1-10), was identified in TGs; Western blot analyses showed that it was immunoreactive to anti-CHH-L (58-72), but not to anti-CHH (59-72), and sequencing analysis of the peptide fragments generated by enzyme digestion of the immunoreactive protein revealed 3 sequences, which are contained within a CHH-L encoded by a previously identified transcript. Furthermore, anti-peptide antibodies were tested for the effects of blocking CHH-induced hyperglycemia. Results showed that anti-CHH (59-72) and anti-CHH (1-10) individually abolished CHH-induced hyperglycemia, whereas neither control treatments, pre-immune sera, nor anti-CHH-L (58-72) significantly affected CHH-induced hyperglycemia. In summary, these data reiterate the observations that CHH and CHH-L are preferentially expressed in different tissues; they also suggest that enzymes involved in L-to-D isomerization of CHH are expressed in tissue-specific manners. Finally, the data suggest the N- and C-terminal regions of CHH are important for its biological activity.