A PEPTIDE FROM THE MALE ACCESSORY-GLAND IN LEPTINOTARSA-DECEMLINEATA - PURIFICATION, CHARACTERIZATION AND MOLECULAR-CLONING

Our interest in the male accessory glands (MAGs) of Leptinotarsa decem lineata was raised recently by our finding that certain cells produce a secretory substance that is recognized by one of our monoclonal anti bodies (MAC-18), developed for the immunohistochemical demonstration o f peptidergic neurons in the brain, We undertook to isolate this subst ance, presumably a peptide, to find out more about its role in the pos t-mating physiology of the recipient of this peptide, the mated female , This paper describes the purification and chemical characterization of the immunoreactive peptide from 100 pairs of male accessory glands, The peptide was purified by two subsequent reversed-phase-HPLC runs, and fractions were analyzed on Western blots that were immunostained b y MAC-18, This indicated the presence of an 8 kDa peptide in the MAG. Partial analysis of the N-terminal amino acids by automated Edman degr adation revealed a sequence of 40 amino acid residues, To obtain the f ull amino acid sequence of this peptide, the technique of reverse tran scriptase PCR (3'RACE) was used, A PCR product of 350 bp was obtained, which encoded the 3'-end of the mRNA, After cloning and sequencing, t his product contained most of the genetic information of the MAG pepti de, The PCR product was also used as a probe for screening a cDNA libr ary constructed from mRNA extracted from MAGs. The nucleotide sequence coding for the signal peptide was elucidated by 5'RACE. The cDNA and 5'RACE clones were analyzed and sequenced, The sequence of the cDNA cl one contained an insert of 411 bp, which agreed well with the mRNA siz e measured by Northern blotting, Translation of the DNA sequences conf irmed the data from partial amino acid sequence analyses and also pred icted the remainder of the amino acid sequence, The entire peptide, de signated Led-MAGP, consists of 74 residues; its mass was calculated an d confirmed by mass spectrometry at 7971 Da, The peptide contains seve n imperfect hexa-repeats, and this hexa-repeat sequence shows remarkab le similarity to the hexa-repeat section of the chicken prion protein, The physiological function of the peptide has yet to be determined, b ut the hexa-repeat motif has recently been identified as the signal th at induces internalization of the prion protein by coated-pit mediated endocytosis, Possible implications for the control of reproductive ac tivities in L. decemlineata are discussed. (C) 1997 Elsevier Science L td.