AEDES-AEGYPTI - CHARACTERIZATION OF A HEMOLYMPH POLYPEPTIDE EXPRESSEDDURING MELANOTIC ENCAPSULATION OF FILARIAL WORMS

We report on the initial characterization of an 84-kDa polypeptide tha t is differentially expressed in Aedes aegypti during melanotic encaps ulation immune reactions against filarial worms. [S-35]Methionine-labe led hemolymph from mosquitoes inoculated with saline, parasites that a re melanized, or parasites that are not melanized was analyzed by sodi um dodecyl sulfate polyacrylamide gel electrophoresis. Results show th at the level of the 84-kDa polypeptide increases considerably in those mosquitoes undergoing encapsulation reactions against parasites but r emains down-regulated in those mosquitoes exposed to parasites that ar e not melanized or are undergoing wound healing responses (saline-inoc ulated). Experiments involving glycosidase treatment of hemolymph samp les indicate that this polypeptide is not heavily glycosylated. Amino acid microsequencing was performed and two internal sequence fragments (15 continuous amino acids and 12 noncontinuous amino acids) were obt ained. Analysis of these sequences to known sequences in a protein dat abase did not yield any conclusive information as to the identity of t he 84-kDa polypeptide. Therefore, degenerate oligonucleotide primers w ere designed, based on the sequence of the 15-amino-acid fragment, and used with the polymerase chain reaction (PCR) to amplify from A, aegy pti genomic DNA the region between the primers. The PCR product was cl oned and sequenced to verify that the nucleic acid sequence matched th e known protein sequence. Screening of an A, aegypti cDNA library with this small PCR-generated clone resulted in the selection of an approx imately 540-bp clone. Northern analysis with this larger cDNA clone in dicates that it hybridizes to an approximately 2.0-kb message that is differentially expressed in mosquitoes undergoing melanotic encapsulat ion reactions against filarial worms. Furthermore, sequencing of this approximately 540-bp clone showed that it contained the 15-amino-acid sequence that had been used to design the degenerate PCR primers, indi cating that an appropriate clone was selected. However, sequence analy sis of this clone at the protein and nucleic acid level did not provid e any conclusive answers to the identity or function of the 84-kDa pol ypeptide. (C) 1994 Academic Press, Inc.