Biological activity of Manduca sexta paralytic and plasmatocyte spreading peptide and primary structure of its hemolymph precursor

A family of hemolymph peptides was previously identified in several lepidop teran insects, which exhibited multiple biological activities including rap id paralysis, blockage of growth and development, or stimulation of plasmat ocyte spreading and aggregation. We synthesized Manduca sexta paralytic pep tide 1 (PP1) and found that after it was injected into larvae, bleeding fro m wounds was dramatically reduced. PP1 also stimulated spreading and aggreg ation behavior of M. sexta plasmatocytes in vitro. Stimulation of plasmatoc yte aggregation and adherence to the body wall may explain a decrease obser ved in the number of circulating plasmatocytes after injection of PP1. Such aggregates might rapidly form plugs in wounds to prevent bleeding. We clon ed a cDNA for a Manduca paralytic peptide precursor, using polymerase chain reactions and cDNA Library screening. The active 23-residue PP2 peptide en coded by this clone is at the carboxyl-terminal end of a precursor protein predicted to be 107 amino acid residues long after cleavage of a secretion signal peptide. Active PP2 was produced by processing of recombinant proPP2 by bovine factor X-a. A single proPP2 mRNA was present in fat body but not in hemocytes. The level of this mRNA was not affected by injection of bact eria into larvae. We produced recombinant proPP2 in Escherichia coli and us ed this protein to produce an antiserum. The antiserum detected proPP2 in p lasma and was used to observe rapid proteolytic processing of proPP2 after hemolymph collection. (C) 1999 Elsevier Science Ltd. All rights reserved.