Characterization and sequence elucidation of a novel peptide with molt- inhibiting activity from the South African spiny lobster, Jasus lalandii Heather G. Marco a , Stanka Stoeva b , Wolfgang Voelter b , Gerd Ga ¨de a, * a Department of Zoology, University of Cape Town, Rondebosch 7701, South Africa b Department of Physical Biochemistry, Institute of Physiological Chemistry, University of Tu ¨bingen, D-72076 Tu ¨bingen, Germany Received 1 March 2000; accepted 19 May 2000 Abstract We have isolated a peptide from extracts of sinus glands from a South African spiny lobster species, Jasus lalandii, by high-performance liquid chromatography (HPLC) and identified it as a putative molt-inhibiting hormone (MIH) by (i) an in vitro assay with J. lalandii Y-organs to measure the inhibition of ecdysteroid synthesis and (ii) an immunoassay using antiserum raised against MIH of the edible crab. The MIH of J. lalandii has 74 amino acid residues, a molecular mass of 9006 Da, a free N-terminus and an amidated C-terminus. The full primary sequence has been obtained from sequencing various digest fragments (tryptic, endoproteinase Asp-N, cyanogen bromide) of the unreduced (native) peptide: RFTFDCPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCLQATMREHETPRFDI WRSIILKA-NH 2 . Structural comparisons with other peptides show that the J. lalandii MIH belongs to the peptide family which includes the crustacean hyperglycemic hormone, molt-inhibiting hormone and vitellogenesis-inhibiting hormone (cHH/MIH/VIH). This novel peptide has 36 – 43% sequence identity to putative MIHs from other decapod crustaceans and 32–34% identity to the two cHH peptides previously identified in this spiny lobster species. This is the first report of a peptide with MIH activity in the Palinuridae infraorder. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Molt-inhibiting hormone; Neuropeptide; Crustacean; Jasus lalandii; Spiny lobster; Sinus gland; Y-organ; Ecdysteroid synthesis 1. Introduction Molting, i.e. shedding of the exoskeleton, is associated with growth in crustaceans and is negatively regulated by a neuropeptide hormone released from the sinus gland (SG) in the eyestalk [4]. This molt-inhibiting hormone (MIH) is synthesized by a cluster of neurons in the eyestalk, collec- tively called the X-organ (XO), and is effective in suppress- ing the synthesis/secretion of molting hormones (ecdys- teroids) from the molting glands, i.e. Y-organs (YOs), as demonstrated by bioassays in vitro [16,20]. In 1990, the amino acid sequence of a putative MIH was first published from the American lobster Homarus americanus (Hoam- MIH, using the terminology in [15]) [5]. This peptide also displayed significant hyperglycemic activity and it is now speculated, based on sequence identity, that this Hoam-MIH may indeed be one of the crustacean hyperglycemic hor- mones (cHH) of this lobster species [4] which had been identified as CHH A [22]. Since this first report on the full structure of a putative MIH, several others have been pub- lished from 5 brachyuran crab species, 2 astacuran crayfish species and 3 penaeid prawn species [1,3,10,23,26]. In stud- ies where these putative MIHs were functionally character- ized, the MIHs did not exhibit cHH activity, whereas the corresponding cHHs from the different crustacean species could elicit some MIH activity. This shows, contrary to earlier speculations [6] that, like Brachyura, other crusta- cean infraorders may also have distinct MIH and cHH neuropeptides. We provide further support for this in the present study by reporting on the primary structure of a putative MIH from a member of the Palinuridae infraorder, Jasus lalandii. On the basis of sequence homology of the mature pep- tide, the molt-inhibiting hormone belongs to the so-called cHH/MIH/VIH (vitellogenesis-inhibiting hormone) peptide family [24]. This family can be subdivided into 2 groups at the preprohormone level and the peptide level. (1) cHH peptides are characterized by the disulfide linkages C 7 -C 43, C 23 -C 39, C 26 -C 52 and the preprohormone is composed of a signal peptide, a precursor-related peptide and the sequence * Corresponding author. Tel.: +27-21-650-3615; fax: +27-21-650- 3301. E-mail address: GGADE@BOTZOO.UCT.AC.ZA (G. Ga ¨de). Peptides 21 (2000) 1313–1321 0196-9781/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0196-9781(00)00273-4