Purification and characterization of an isoform of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii

Abstract

Isolation of the crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii was performed using 5,000 ground eye-stalks extracted in methanol-acetic acid-water (90:1:9). After the extract was partially purified using C18 cartridges, it was further purified by eight steps of RP-HPLC using four kinds of columns: C18, C8, cyano and phenyl, and three solvent systems: acetonitrile (ACN)/trifluoroacetic acid, ACN/heptafluoroacetic acid and ACN/triethylammonium acetate. The bioassay of CHH during purification was done by injection of eluate fractions into eyestalk-ablated prawns and determination of the ability of the fractions to elevate glucose in the haemolymph. A complete amino acid sequence analysis was performed on one isoform of CHH (Mar-CHH-1), consisting of 71 residues. The sequence of Mar-CHH-1 shows considerable similarity (45-68%) to CHHs reported in other crustaceans. It is expected that there might be more than one isoform of CHH in M. rosenbergii.

Cloning of Two Crustacean Hyperglycemic Hormone Isoforms in Freshwater Giant Prawn ( Macrobrachium rosenbergii ): Evidence of Alternative Splicing

“…The deduced amino acid sequences of the possible chh cDNA candidates fit the partial amino acid sequence previously reported (Lin et al, 1998;Sithigorngul et al, 1999) and the other known characters of CHH family members. Partial sequences of chh cDNA were obtained, and chh-specific primers for further experiments were then designed.…”

“…Five possible translational initiation sites in sense direction were detected by the translational initiation site analysis. Reading frames of the 3-scoring predictions, 135, 129, and 125 residues, respectively, matched the reported CHH sequence from M. rosenbergii (Lin et al, 1998;Sithigorngul et al, 1999). The 3 possible open reading frames (ORFs) were further examined for the appearance of signal peptide on both SPScan and SignalP 2.0.…”

An antibody to recombinant crustacean hyperglycaemic hormone of Nephrops norvegicus cross-reacts with neuroendocrine organs of several taxa of malacostracan Crustacea

“…RT-PCR reaction was performed in 50 µl at 45°C for 1 h with 250 pmol each of degenerated oligonucleotides HACHH1 Homarus americanus cHH-A and -B (de Kleijn et al 1995), Procambarus bouvieri cHH (Huberman et al 1993), Orconectes limosus cHH (de Kleijn et al 1994), Procambarus clarkii cHH (Yasuda et al 1994), Procambarus bouvieri moult-inhibiting hormone (MIH; Aguilar et al 1996), Jasus lalandii cHH (Marco et al 1998), Macrobrachium rosenbergii cHH (Sithigorngul et al 1999b), and Carcinus maenas cHH (Weidemann et al 1989). The alignment was constructed on the basis of Fasta3 results (Pearson and Lipman 1988).…”

Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp (Litopenaeus vannamei) against bacterial infection

“…The sequence of CHH hormone from Carcinus maenus was first reported by Kegel and coworkers [9]. Subsequently, CHH peptides sequences have been described and characterized in lobster [10], crabs [11], crayfishes [12], prawns [13,14] and shrimp [8,15]. CHH functions mainly in the regulation of glucose levels, as well as in the metabolism of carbohydrates and lipids [16].…”