Aedes aegypti: characterization of a hemolymph polypeptide expressed during melanotic encapsulation of filarial worms.
We report on the initial characterization of an 84-kDa polypeptide that is differentially expressed in Aedes aegypti during melanotic encapsulation immune reactions against filarial worms. [35S]Methionine-labeled hemolymph from mosquitoes inoculated with saline, parasites that are melanized, or parasites that are not melanized was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Results show that the level of the 84-kDa polypeptide increases considerably in those mosquitoes undergoing encapsulation reactions against parasites but remains down-regulated in those mosquitoes exposed to parasites that are not melanized or are undergoing wound healing responses (saline-inoculated). Experiments involving glycosidase treatment of hemolymph samples 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 obtained. Analysis of these sequences to known sequences in a protein database did not yield any conclusive information as to the identify of the 84-kDa polypeptide. Therefore, degenerate oligonucleotide primers were designed, based on the sequence of the 15-amino-acid fragment, and used with the polymerase chain reaction (PCR) to amplify from A. aegypti genomic DNA the region between the primers. The PCR product was cloned and sequenced to verify that the nucleic acid sequence matched the known protein sequence. Screening of an A. aegypti cDNA library with this small PCR-generated clone resulted in the selection of an approximately 540-bp clone. Northern analysis with this larger cDNA clone indicates that it hybridizes to an approximately 2.0-kb message that is differentially expressed in mosquitoes undergoing melanotic encapsulation 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, indicating that an appropriate clone was selected. However, sequence analysis of this clone at the protein and nucleic acid level did not provide any conclusive answers to the identity or function of the 84-kDa polypeptide.
Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison 53706.
B T
BT
Beerntsen
D W
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Severson
B M
BM
Christensen
D000330:Aedes / Q000235:genetics / Q000378:metabolism / Q000469:parasitology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D017179:Brugia pahangi / Q000502:physiology; D003001:Cloning, Molecular; D004183:Dirofilaria immitis / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005786:Gene Expression Regulation; D006026:Glycoside Hydrolases / Q000378:metabolism; D006458:Hemolymph / Q000737:chemistry*; D019476:Insect Proteins; D007303:Insect Vectors / Q000235:genetics / Q000378:metabolism / Q000469:parasitology*; D008297:Male; D008842:Microfilariae / Q000502:physiology; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D019476:Insect Proteins; D011506:Proteins; C090310:orf1 protein, Aedes aegypti; D006026:Glycoside Hydrolases