BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 248, 578–583 (1998) ARTICLE NO. RC989025 Elucidation of the Amino Acid Sequence of a Crustacean Hyperglycaemic Hormone from the Spiny Lobster, Jasus lalandii Heather G. Marco,* Wolf Brandt,† and Gerd Ga ¨ de* ,1 *Department of Zoology and †Department of Biochemistry, University of Cape Town, Rondebosch 7701, South Africa Received June 15, 1998 hormone, MIH), glucose metabolism (crustacean hy- We have isolated a peptide from extracts of sinus perglycaemic hormone, cHH) and movement of pig- glands of Jasus lalandii, a South African spiny lobster, ments (pigment-concentrating and pigment-dispers- by high-performance liquid chromatography (HPLC) ing hormones). The structurally related cHH, MIH and identified it as crustacean hyperglycaemic hor- and VIH peptides constitute a neuropeptide family mone (cHH) by (i) a conspecific bioassay measuring that share certain characteristics: (a) 6 cysteine resi- glucose elevation in the haemolymph and (ii) an immu- dues at conserved positions, (b) assignment of disul- noassay using an antiserum raised against cHH of the fide bridges (C 7 -C 43 ,C 23 -C 39 ,C 26 -C 52 ), (c) chain length American lobster. The J. lalandii peptide has a free N- of 72-78 amino acid residues, and (d) a number of terminus as evidenced by sequencing the first 30 areas with conserved residues (1). amino acid residues of the intact peptide. Further pri- The hyperglycaemic hormone of crustaceans has mary structural data were obtained from sequencing been sequenced from 4 decapod infraorders and ap- HPLC-purified tryptic and Asp-N proteolytic digests pears to be present in multiple forms (see 2). The pri- and by cyanogen bromide cleavage of the native, unre- mary structure of cHHs from different species and even duced peptide. In this way, less than 400 sinus glands different infraorders share very close sequence homol- were used to provide the full sequence. Mass spectro- metric analysis in conjunction with inferences based ogy, 60-80% between crab, crayfish and lobster. Despite on interspecies sequence homology of cHH molecules the fact that cHH is the most abundant neuropeptide unequivocally assigned the complete primary struc- present in the SG, most studies have used between 600 ture of cHH in a member of the crustacean infraorder and 6000 SGs for primary sequence elucidation when Palinura for the first time. Our results show 51-76% 200-60 ng purified cHH per SG was available (3-6). It homology with cHHs known from other decapod in- is well-known now that most of the peptide material is fraorders, the major difference being a free N-termi- lost after reduction of the disulfide bonds when the nus and several amino acid substitutions interspersed hydrophobic sites of the molecule are exposed and the in the non-conserved regions of the molecule. The J. peptide is retained on the HPLC column packing mate- lalandii cHH sequence presented here differs from a rial (7, 8). The objective of the present study was, to partial cHH sequence previously reported from alleg- determine for the first time the complete primary struc- edly the same species.  1998 Academic Press ture of the major cHH isoform (cHH-I) from a palinurid lobster and to use as little sinus gland material as pos- sible. To achieve this we have used the knowledge of sequence homology, combined with mass spectrometry In decapod crustaceans, the major neuroendocrine and traditional sequence analysis to elucidate the pri- centre is situated in the eyestalks, consisting of the mary structure using only native, i.e. non-reduced X-organ (XO)-sinus gland (SG) system which is analo- cHH-I, purified from SGs of the South African spiny gous to the vertebrate hypothalomo-neurophyseal sys- lobster, Jasus lalandii in which the average yield of tem. The neuropeptide hormones synthesised and se- cHH-I is approximately 100 ng/SG ({12 pmol/SG). creted by the XO-SG complex regulate many physio- logical processes such as reproduction (vitellogenesis- inhibiting hormone, VIH), moulting (moult-inhibiting MATERIALS AND METHODS Tissue collection, extraction and purification of peptides. Eye- 1 To whom correspondence should be addressed. Fax: /2721 6503301. E-mail: ggade@botzoo.uct.ac.za. stalks were excised from live adult specimens of Jasus lalandii at 0006-291X/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved. 578