lobes of the locust corpora cardiucu comprise a homogen- eous population of .... or canavanine to load the neuroendocrine cells with the ana- logs. ... Amino acid analogs canavanine and thialysine were tested for their ability to block ...
Eur. J. Biochem. 217, 905-911 (1993) 0 FEBS 1993
Structural requirements for processing of pro-adipokinetic hormone I Richard C. RAYNE and Michael O’SHEA Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Brighton, England (Received July 20/August 23, 1993) - EJB 93 1077/2
We found that a seven-residue sequence in pro-adipokinetic hormone I (proAKH I) which precedes the endopeptidase cleavage site is predicted to form an 52 loop. Molecular modelling experiments indicated that a stable 52 loop may form at this site, and suggested that loop stability may depend on the C-terminal loop residue, Lysl2. The importance of this residue in proAKH I processing was confirmed by the observation that replacement of Lysl2 by thialysine, a Lys analog with an altered side chain, prevented processing in vivo. In addition we showed by molecular modelling that this side-chain alteration may prevent formation of an 52 loop. Together, these approaches lead us to propose that an 52 loop may serve as a recognition motif in proAKH I processing.
Fundamental questions concerning how neuropeptides neuroendocrine center associated with the insect brain. A reare cleaved from their precursor polypeptides remain unans- lated peptide called AKH II (Glp-Leu-Asn-Phe-Ser-Thr-Glywered. Notably, while it is well known that prohormones are Ti-pNH,; Siegert et al., 1985) is also synthesized by the same subjected to specific proteolysis (Docherty and Steiner, neurosecretory cells, being processed from a different pro1982; Loh et al., 1984; Darby and Smyth, 1990), little is hormone (Fischer-Lougheed et al., 1993). The glandular known about the molecular signals that determine which pep- lobes of the locust corpora cardiucu comprise a homogentide bonds will be cleaved by processing endoproteases. eous population of AKH-producing cells and as such repreOften prohormones are cut at the peptide bond immediately sent a favourable system for direct studies of prohormone C-terminal to pairs of basic amino acid residues, Lys-Arg processing in an intact neuroendocrine gland (O’Shea and being the most common. Not all such sites are used, indicat- Rayne, 1992). Moreover, proAKH I is only 41 amino acids ing that the basic residue pair alone does not represent the in length and possesses only a single dibasic processing site processing signal (Gluschankof and Cohen, 1987 ; Fisher and (Schulz-Aellen et al., 1989; Hekimi et al., 1989; O’Shea et Scheller, 1988; Darby and Smyth, 1990). This, together with al., 1990). The prohormone sequence begins with a single the lack of evidence for consensus primary sequence around copy of AKH I, followed by glycine, a basic processing site paired basic residues, suggests that secondary structure deter- pair (Lys-Arg) then a 28-residue C-terminal sequence conmines which sites will be cleaved (Geisow and Smyth, 1980; taining an unused dibasic site (Arg-Lys; see Fig. 1). The seRholam et al., 1986). For example, it has been shown that quence of proAKH 11 is also provided because it is relevant some cleavage sites are associated with exposed secondary to some control experiments described here. Prior to processstructures in prohormones such as p turns (Rholam et al., ing the AKH prohormones form dimers in the endoplasmic 1986, 1990; Paolillo et al., 1992) and L? loops (Bek and reticulum which are the direct precursors of AKH I and AKH Berry, 1990). Such secondary structures are believed to be I1 (Hekimi et al., 1989, 1991). Here we show that alteration of the side chain of Lys, but required for recognition of appropriate cleavage sites by pronot Arg, at the processing site in proAKH I prevents cleavage cessing endopeptidases (Brakch et al., 1993). We have investigated the processing of the precursor to of the prohormone by processing endopeptidases in vivo. a neuropeptide called adipokinetic hormone I (AKH I) in Structure prediction and molecular modelling indicated that the locust, Schistocercu greguriu. AKH I is a 10-amino-acid Lys, but not Arg, may participate in the formation of an 52 loop in the vicinity of the processing site. Moreover, it neuropeptide, Glp-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-ThrNH, (Glp, 5-oxoproline; Stone et al., 1976), produced by a showed that structural alteration of the Lys side chain could group of approximately 10000 neurosecretory cells which prevent formation of an 52 loop in proAKH I. These results form the glandular lobes of the corpora cardiaca, the major lead us to propose that an 52 loop directs the processing endopeptidase to the appropriate cleavage site in proAKH I. Correspondence to M . O’Shea, Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Brighton, England, BN1 9QG Fax: +44 273 678535. Abbreviations. AKH, adipokinetic hormone; E-64, N-[N-(L-~truns-carboxyoxiram-2-carbonyl)-~-leucyl]-agmatine; proOT/Np, pro-oxytocinheurophysin.
Experimental Procedures Animals Adult locusts (S. gregaria) were supplied by Blades Biological or were obtained from our own animal culture facility.
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ProAKH I
AKH I
R-Loop
