Cloning and Expression of Two Crustacean Hyperglycemic‐hormone mRNAs in the Eyestalk of the Crayfish Orconectes limosus

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Abstract
Crustacean hyperglycemic hormone (CHH) is a multifunctional neurohormone produced in the eyestalk of crustaceans and is primarily involved in the regulation of carbohydrate metabolism. In several crustacean species, CHH isoforms with identical amino acid sequences and molecular masses, but with different chromatographic elution patterns, are synthesized. To obtain sequence information on the CHH preprohormone in the crayfish Orconectes limosus we isolated two full-length cDNAs encoding two structurally different preproCHH species. The sequences of these precursors differ slightly in the signal peptide, the CHH-precursor-related peptide(CPRP)-coding sequences and in the non-coding regions, but are identical in the CHH peptide-coding sequence. Determination of the levels of preproCHH nRNAs and the amount of CHH peptide in the eyestalks of individual animals revealed that the ratio between the two preproCHH mRNAs varies for different individuals while the ratio between the two CHH peptide isoforms does not differ among animals. Our results suggest that the existence of two CHH isoforms in the crayfish O. limosus is due to a post-translational modification event. Northern-blot analysis showed only one band in eyestalk tissue with a size of approximately 2.4 kb, similar to the sizes of the cDNA sequences. Southern-blot analysis revealed the presence of at least two preproCHH genes in the crayfish suggesting a gene duplication event. Slight modifications in the duplicated genes could be responsible for the existence of the two preproCHH-encoding mRNAs.