Reconstitution of adipokinetic hormone biosynthesis in vitro indicates steps in prohormone processing

Abstract

We have used a complete, synthetic precursor to adipokinetic hormone I (AKH I) and oligopeptides derived from this precursor as substrates for prohormone-processing enzymes extracted from AKH-synthesizing neurosecretory cells to reconstitute the post-translational steps in AKH biosynthesis in vitro. The results demonstrate the existence of endoproteolytic activity which cleaves the precursor only at the appropriate processing site (at the C-terminal side of Argl3). Further proteolytic processing of C-terminally extended AKH I (AKH-Gly-Lys-Arg) by a carboxypeptidase H-like activity removes the basic residues producing AKH-Gly-Lys, followed by AKH-Gly. Finally, a peptidylglycine-a-amidating-monooxygenase activity produces the amidated bioactive product from the glycine-extended peptide in a two-step process, the first of which requires ascorbate and Cu". Our results show that all steps in AKH precursor processing can be reconstituted and studied in vitro, providing a system to characterize the processing enzymes and to investigate the development of enzyme inhibitors for use as potential insecticides.Neuropeptides are produced by specific, limited proteolysis of larger precursor polypeptides, or prohormones (Docherty and Steiner, 1982;Sossin et al., 1989;Darby and Smyth, 1990). Prohormones are cleaved by endoproteases, often at sites adjacent to pairs of basic amino acid residues (e.g. lysine, arginine). This initial step has been attributed in some cases to members of a recently identified superfamily of subtilisin-like endopeptidases referred to as prohormone convertases (Steiner et al., 1992). Endoproteolytic cleavage is followed by removal of basic residues from C-terminally extended processing intermediates by carboxypeptidase H (also referred to as carboxypeptidase E; Fricker, 1988;Skidgel, 1988). For neuropeptides which require C-terminal amidation, glycine is normally the residue which, via the action of the peptidylglycine-a-amidating-monooxygenase (PAM) enzymes, contributes its amino group to form an amidated C-terminus (Eipper et al., 1992).

The neuropeptide complement of the marine annelid Platynereis dumerilii

“…We annotated all pNP sequences with various sequence features, including the presence of a SP, cysteine-rich stretches (potentially also involved in processing [72]), prohormone-convertase cleavage-sites, modified (amidated or pyroglutaminated) or non-modified active peptides and the presence of a MS-hit. For all Platynereis pNPs containing repetitive peptides we also generated sequence logos (Figure  1 and Additional file 8).…”

Individual carboxypeptidase D domains have both redundant and unique functions in Drosophila development and behavior

“…AKH-Gly-Lys-Arg is first processed from the AKH propeptide by the Drosophila prohormone convertase-2 amontillado (J.M. Rhea, C. Wegener, M. Bender, unpublished), then C-terminally trimmed by a carboxypeptidase and amidated at the Gly-site by amidating enzymes [37]. Previous studies have detected both AKH and AKH-Gly-Lys by direct MALDI-TOF mass spectrometric profiling in the larval and adult corpora cardiaca (CC), neurohemal organs that also accommodate the AKH-producing cells [38,39].…”

“…After assessing viability of the various constructs, we examined a variety of other phenotypes that were suspected of involving CPD. We also examined the ability of CPD overexpression to process adipokinetic hormone (AKH), which was previously shown to be processed by carboxypeptidase activity [37], and found to exist in partially-processed forms that contain C-terminal basic residues [38,39]. Taken together, our results support the predictions that CPD is involved in the processing of AKH and other peptides involved in viability, wing shape, cold-sensitivity, ethanol-sensitivity, and long term memory.…”

Modeling dietary influences on offspring metabolic programming in Drosophila melanogaster

“…The ring gland houses the neurosecretory cells of the corpora cardiaca, which, like pancreatic α-cells, secrete the glucagon-like protein adipokinetic hormone (AKH) (Kim and Rulifson, 2004). Like glucagon, AKH is derived from the processing of a preprohormone and in its mature form binds to the AKH G-protein-coupled transmembrane receptor located on the plasma membrane of fat body cells (Rayne and O’Shea, 1994; Noyes et al , 1995; Staubli et al , 2002). AKH receptor binding promotes glycogenolysis, the synthesis and subsequent release of trehalose, the major circulating sugar in the fly, and lipolysis (Staubli et al , 2002; Van der Horst, 2003; Rhea et al , 2010).…”