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Insect neuropeptides: structure determination, chemical synthesis and biological activities


Our studies have focused on the following peptides which we have isolated from Locusta migratoria: (a) the C-peptide of locust insulin named 5 kDa and shown to induce a cationic current in locust neurones; and (b) two peptides of 35 and 36 amino acid residues, referred to as PMP-C and PMP-D2. PMP-C contains a fucose moiety on Thr9. The main goal of this program was to synthesise the peptides by solid phase synthesis (SPS) to enable us to determine their three dimensional (3-D) structure and biological activity as well as their functional architecture.
-It was determined that the shortest active fragment of 5kDa comprises residues 9 to 38.
-PMP-D2, fucosylated (Fuc-PMP-C) and non-fucosylated PMP-C have been synthesised by SPS.
-The 3-D structure of PMP-D2, PMP-C and Fuc-PMP-C has been determined using NMR spectroscopy. The three peptides exhibit a compact folding consisting of a triple stranded antiparallel beta-sheet stabilised by three disulphide bonds and regions not involved in beta sheets but exposed to the surface. This folding is also is seen in small canonical tight binding serine protease inhibitors and the N-type Ca2+ channel inhibitor omega-conotoxin GIVA.
-In Fuc-PMP-C the fucose moiety hinders a number of side chain hydroxyl groups and increases the stability of the PMP-C molecule by about 1 kcal mol{-1}.
-The activity of PMP-D2, Fuc-PMP-C and PMP-C on serine proteases and Ca2+ channels were studied. Both Fuc-PMP-C and PMP-C are potent inhibitors of alpha-chymotrypsin (Ki 1E-10 M) but weaker inhibitors of human leucocyte elastase (HLE) (Ki 1E-7 M). PMP-D2 is a weak inhibitor of alpha-chymotrypsin (Ki 10sup-6 M) and has no effect on HLE.
-Selective replacement of amino acid residues permitted us to locate the active site P1-P'1 of these peptides and develop PMP-D2 analogues with enhanced inhibitory capability.
-Whole-cell recording and 45Ca2+ influx studies showed that both PMP-D2 and PMP-C are inhibitors of high-voltage activated Ca2+ channels. In contrast, Fuc-PMP-C has no effect, suggesting that the fucose moiety targets the activity of PMP-C towards serine protease inhibition.
-Pharmacological studies indicated that PMP-D2 and PMP-C interact selectively on omega-conotoxin GVIA-sensitive N-type Ca2+ channels.
-Three peptides containing three S-S bonds have been successfully synthesised by SPS. We also fucosylated PMP-C on Thr9.
-Fucosylation increases the stability of PMP-C and abolishes its Ca2+ channel blocking properties with no effect on its serine protease inhibitory activity. This is the first time that it has been shown that glycosylation results in changes in the stability and biological activity of peptides.
-PMP-D2 and PMP-C are selective blockers of N-type Ca2+ channels. This is important as besides omega-conotoxin GIVA there is no other selective N-Type Ca2+ channel blocker available.
-PMP-C is unique in that it is a highly potent serine protease and a N-type Ca2+ channel blocker.


Institut National de la Santé et de la Recherche Médicale (INSERM)
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67084 Strasbourg

Participants (1)

Oxford Brookes University
United Kingdom
Gipsy Lane, Headington,
OX3 0BP Oxford