Isolation,identification,andsynthesisofadisulfatedsulfakininfromthe central nervous system of an arthropod, the white shrimp Litopenaeus vannamei P. Torfs, a G. Baggerman, a T. Meeusen, a J. Nieto, b R.J. Nachman, c J. Calderon, b A. De Loof, a and L. Schoofs a, * a Laboratory of Developmental Physiology and Molecular Biology, K.U. Leuven, Naamsestraat 59, Leuven B-3000, Belgium b Centro Nacional de Acuicultura e Investigaciones Marinas, Guayaquil, Ecuador c Southern Plains Agriculture Research Center, U.S. Department of Agriculture, College Station, TX 77845, USA Received 13 October 2002 Abstract Two myotropic peptides displaying tyrosyl sulfation have been isolated from an extract of central nervous systems (brain, suboesophageal ganglion, thoracic ganglia, and ventral nerve cord) of the white shrimp Litopenaeus vannamei. Both peptides were identified by mass spectrometry and belong to the sulfakinin family of neuropeptides, which are characterized by the C-terminal hexapeptide Y(SO 3 H)GHMRF-NH 2 preceded by two acidic amino acid residues. Pev-SK 1 (AGGSGGVGGEY(SO 3 H)DDY (SO 3 H)GH(L/I) RF-NH 2 ) has two sulfated tyrosyl residues and a unique (L/I) for M substitution in the C-terminal sequence. Pev-SK 2 (pQFDEY(SO 3 H)GHMRF-NH 2 ) fully complies with the typical sulfakinin core sequence and is blocked by a pyro- glutamyl residue. Synthetic analogs (sulfated and unsulfated) were synthesized and the tyrosyl sulfations were confirmed by myotropic activity studies and co-elution with the native fractions. Pev-SK 1 is the first disulfated neuropeptide elucidated in the phylum of the arthropoda, with the only other reported disulfated neuropeptide, called cionin, found in a protochordate. The similarities in amino acid sequence and posttranslational modifications of the crustacean sulfakinins and protochordate cionin provide further evidence for the hypothesis stating that gastrin/CCK, cionin, and sulfakinins originate from a common ancestral gastrin/CCK-like peptide. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Mass spectrometry; Cholecystokinin; Gastrin/CCK; FaRP; Crustacea; Litopenaeus vannamei; Neuropeptide In recent years, a number of invertebrate FMRFa- mide-like peptides containing a sulfated tyrosine residue have been identified. The first sulfated invertebrate peptides were isolated and sequenced from heads of the cockroach Leucophaea maderae [1,2]. These peptides display strong myoactivity on the Leucophaea hindgut and were therefore designated as sulfakinins. Subse- quently, sulfakinin-like peptides were isolated from other insects including the cockroach Periplaneta americana [3], the locust Locusta migratoria [4], the fruitfly Drosophila melanogaster [5,6], the fleshfly Neo- bellieria bullata [7], and the blowflies Calliphora vomi- toria and Lucilia cuprina [8]. All insect sulfakinins found so far display the C-terminal DY(SO 3 )GHMRF-NH 2 consensus sequence (Table 1) and all are potent stimu- lators of spontaneous hindgut contractions in the Leu- cophaea hindgut assay. The insect sulfakinins have structural and physio- logical similarities to the vertebrate peptides gastrin and cholecystokinin (CCK). The consensus C-terminus of the sulfakinins displays sequence homology with gastrin and cholecystokinin. Gastrin and CCK are known to stimulate contraction of smooth muscles as well as en- zyme secretion. Sulfakinins have similar properties: they are potent stimulators of the spontaneous contraction of the cockroach hindgut and are reported to stimulate Biochemical and Biophysical Research Communications 299 (2002) 312–320 www.academicpress.com BBRC * Corresponding author. Fax: +32-16-323902. E-mail address: liliane.schoofs@bio.kuleuven.ac.be (L. Schoofs). 0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0006-291X(02)02624-4