Neuropeptides sequence determination

Jacquin-Joly E., Burnet M., Frangois M., Ammar D., Meillour P. and Descoins C. (1998) cDNA cloning and sequence determination of the pheromone biosynthesis activating neuropeptide of Mamestra brassicae a new member of the PBAN family. Insect Biochem. Mol. Biol. 28, 251-258. 

RfteeN years have elapsed since Alvin N. Starratt and Brian E. Brown released their pioneering publication announcing the initial determination of a primary structure for an insect neuropeptide—the penta-peptide proctolin. In the early 1980s, only a few American, Japanese, and European universities, as well as the Zoecon Corporation in the United States, were engaged in insect neuropeptide research. Neuropeptide discoveries in vertebrates and molluscs—and the advent of exquisitely sensitive techniques for isolation, sequence determination, and synthesis of neuropeptides—rapidly accelerated discoveries in both the mammalian and insect arenas. More than SO insect neuropeptides have been sequenced and reported in the literature, and that number is increasing rapidly. Concomitant with these developments have been several symposia, conferences, and workshops that address various aspects of insect neuroscience. 

Advancing methods in mass spectrometry (MS) have made it easier to determine the amino acid sequence in these peptides, and on ever smaller amounts of the compound. Direct tissue and single neuron analyses by matrix-assisted laser desorption/ionization-MS are particularly successful (Chapter 9.10). By these methods about 440 neuropeptides have been identified, and some 450 by electrospray ionization (ESI) techniques. Further techniques hold promise for more peptides.10 The neuropeptides have great importance for the insect physiologist,11 but hold less interest for the structural chemist. Their three-dimensional folding is largely undetermined. 

The related achetakinins double the rate of fluid secretion by isolated Acheta Malpighian tubules (Coast, G. M., et al. J. Insect Phvsiol.. in press.)- In addition, some leucokinins stimulate fluid secretion and/or depolarize transepithelial membrane potentials in the malpighian tubules of the yellow fever mosquito, Aedes aegvpti. in a fashion similar to several uncharacterized peptides isolated from mosquito head extracts (23). Thus, the neuropeptide family of leucokinins, achetakinins, and homologs may function in the control of water and ion balance, as well as myotropic activity, in a number of insects. To determine the relationships of the C-terminal sequence of the leucokinins to myotropic activity, analogs were synthesized and tested on the isolated cockroach hindgut. 

PBAN has been reported to control sex pheromone biosynthesis in many other moth species, and the peptide itself has been found in many Lepidopteran species as well as in other insect orders. Since 1989 the primary sequence of PBAN has been determined in numerous other moth species (Bombyx mori [89], Lymantria dispar [90], Helicoverpa assuita [91], Agrotis ipsilon [92], Mamestra brassicae [93] and Spodoptera littoralis [94] either by sequencing of the purified neuropeptide or from cloned cDNA or gene sequence [95-97]. 

In order to be efficient, the determination of the sequences of neuropeptides [221] requires a high sensitivity as a result of the small quantities of samples analyzed (on the pmol level). 

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