Structure and regulation of the synthesis and secretion of an attacin like protein from larvae of Manduca sexta

Wei Dai, Purdue University

Abstract

An attacin-like protein (ALP) was identified in, and purified from, the hemolymph of M. sexta larvae injected with either formalin-killed bacteria or peptidoglycan fragments isolated from bacterial cell walls. The purity of the isolated protein was demonstrated by the criterion that a single amino-terminal residue and a single amino acid sequence were obtained when the protein was analyzed by automated Edman degradation. The reported purification procedure yielded a biologically active ALP. The protein was bacteriostatic in activity rather than bactericidal. A cDNA library was constructed using poly (A$\sp+$) RNA isolated from the induced fat body, and one cDNA clone encoding the ALP was isolated. The identity of the pALP-I clone was confirmed with the known N-terminal amino acid sequence obtained by protein sequencing. Specific features in the nucleotide sequence suggested a full length cDNA clone for the ALP had been isolated. The open reading frame of the cDNA specified a pre-ALP of 224 amino acid residues. Amino acid composition and N-terminal sequence comparisons suggested that the ALP was more similar to the acidic form of attacin from pupae of H. cecropia, although the deduced amino acid sequence showed a strong homology to both acidic and basic forms of attacins. ALP transcript abundance increased rapidly in fat body after induction (about 1 h), reaching a maximal level by about 8 h. The accumulation of ALP correlated with the rapid appearance and elevation of ALP transcripts. Transcriptional activation of the ALP gene following bacterial challenge of the insect has been strongly suggested. The induction of ALP was a specific response to treatment with peptidoglycan elicitor. Fat body of M. sexta was the major source of the serum ALP. The induced fat body was the most abundant source of ALP transcript, and in the presence of peptidoglycan elicitor, the induced tissue actively synthesized and secreted ALP into culture medium in vitro. While little or no ALP transcript was detected in six other induced tissues, the induced Malpighian tubules also contained a high level of ALP transcript. In vitro organ culture demonstrated that ALP mRNA in Malpighian tubules was translated and the translation product was secreted. (Abstract shortened with permission of author.)

Degree

Ph.D.

Advisors

Dunn, Purdue University.

Subject Area

Entomology|Genetics

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