Does the N‐Terminal Pyroglutamate Residue have Any Physiological Significance for Crab Hyperglycemic Neuropeptides?

Abstract

A characteristic feature of all crustacean hyperglycemic hormones (CHH) is that they are always present in the sinus gland as multiple forms or isoforms. The amino acid sequence of the minor form of CHH from the green shore crab, Curcinus maenas, was determined by automated microsequencing and MS, and was almost identical to that of the major form, except that the N-terminal residue was glutamine rather than pyroglutamate. Limited analysis (electrospray MS and amino acid composition) of the two corresponding forms of CHH from the edible crab, Cancer pugurus, suggested a similar phenomenon in this species. For C. muenus, both forms were indistinguishable in terms of their ability to cause sustained hyperglycemia in vivo and repression of ecdysteroid synthesis in vitro. Similarly, the two forms were immunologically identical in RIA, and exhibited similar binding characteristics in competitive-receptorbinding assays. CD studies showed only minor differences in presumed secondary structure. In vitro release experiments with isolated sinus glands demonstrated that both forms are probably released in a stoichiometric manner and that both peptides are present in the haemolymph at the same ratio as that in the sinus gland. Preliminary results suggest that the in vivo clearance/degradation rates of both peptides are similar. The unblocked (Gln) terminus is of particular significance, since the presence of this amino acid indicates that this peptide is derived from a precursor that does not possess the same structure of those of established preproCHH, or that N-terminal processing is slow, which results in the presence of unblocked CHH in sinus glands. The similar biological activity of the unblocked CHH to that of the blocked CHH suggests that the N-terminal pyroglutamate residue has no obvious biological significance (with respect to the known functions of CHH), an observation which is in contrast to the widely accepted paradigms concerning the stability and biological activity of N-terminally blocked and unblocked peptides.Keywords: crustacean hyperglycemic hormone ; isoform; pyroglutamate ; crab.In decapod crustaceans, many structurally related neurohormones, which are synthesised by neurosecretory cells in the Xorgan of the eyestalk and released from a neurohaemal tissue, the sinus gland, have been identified and fully characterised in terms of amino acid sequence by means of sequence analysis and cDNA-cloning strategies [I -61. These have been functionally identified as hyperglycemic hormones (CHH), moult-inhibiting hormones, vitellogenesis/gonad-inhibiting hormones and mandibular-organ-inhibiting hormones, and evidence suggests that several of these hormones may be multifunctional [7]. With particular regard to the hormones primarily identified as CHH, (based upon their ability to profoundly elevate haemolymph glucose levels in vivo), it has long been apparent thatmultiple forms (2-4 peaks are separable by HPLC) of CHH peptides are found in the sinus gland of all decapods examined [8-lo]. In astacurans (crayfish an...

Moult cycle‐related changes in biological activity of moult‐inhibiting hormone (MIH) and crustacean hyperglycaemic hormone (CHH) in the crab, Carcinus maenas

“…Peptides were quantified by peak area with reference to standard MIH and CHH. For CHH, both CHH-I (N-terminally unblocked) and CHH-II (N-terminally blocked) peak areas were combined as they are indistinguishable with respect to biological activity [15].…”

Different transcription regulation routes are exerted by L- and D-amino acid enantiomers of peptide hormones

“…It is a 72 amino acid neuropeptide, characterized by six cysteines forming three intradisulfide bridges, p-glutamyl at its N-terminus and valyl amide at its C-terminus (Mosco et al, 2008). The Astacidea CHH is present in the circulation as a mix of two chiral isomers differing by the presence of D-or L-phenylalanine at the third N-terminus position in contrast to the exclusively L-CHH peptide of crabs (Chung and Webster, 1996). The two enantiomers were simultaneously discovered in Homarus americanus (Soyez et al, 1994) and Procambarus clarkii (Yasuda et al, 1994).…”

The specific binding sites of eyestalk- and pericardial organ-crustacean hyperglycaemic hormones (CHHs) in multiple tissues of the blue crab,Callinectes sapidus

“…CHH in many crustacean species is associated with multiple functions and tissue-specific structural isoforms (Yasuda et al, 1994;Aguilar et al, 1995;Chung and Webster, 1996;Chang et al, 1999;Ollivaux and Soyez, 2000;Chung and Webster, 2004). Eyestalk-CHH (ES-CHH), present in a moult stage-independent manner, is associated with hyperglycaemia, osmoregulation and the inhibition of ecdysteroid and methyl farnesoate synthesis (Webster and Keller, 1986;Liu et al, 1997;Khayat et al, 1998;Chung et al, 1999;Webster et al, 2000;Spanings-Pierrot et al, 2000;Tsutsui et al, 2005).…”

“…I-rPO or 125 I-ES-CHH was eluted on a pre-conditioned PD-10 column (Bio-Rad) as described previously (Chung and Webster, 1996) and kept in a 1:1 ratio of glycerol at 4°C. The specific activity of both CHHs was approximately 500-700 Ci mmol l -1 .…”