Identification and molecular characterization of a new member of the peritrophic membrane proteins from the meadow moth, loxostege sticticalis

Abstract

The peritrophic membrane (PM) plays an important role in protecting insects. The PM proteins are important to determinate the formation and function of the PM. A new PM protein, named Lsti99, was identified from the PM of Loxostege sticticalis larvae by cDNA library screening. The full cDNA of Lsti99 is 1392 bp in length, contains an open reading frame (ORF) of 1245 bp that encodes a preprotein of 415 amino acid residues with a 17-amino acid signal peptide. The sequence of Lsti99 showed no homology to other known PM proteins. The recombinant Lsti99 was successfully expressed in insect cells (Sf9) using recombinant baculoviruses and was used to isolate the antibodies to Lsti99 from the polyclonal antiserum. Lsti99 was expressed mainly in the PM, but weaker bands could be detected in the head and integument as well. The Lsti99 protein could be separated from the PM complex by chitinase in vitro, but M2R did not show effect in vitro confirming the chitin-binding activity of Lsti99. The biochemical and physiological functions of Lsti99 in L. sticticalis require further investigation.

Keywords: Chitin-binding activity; Loxostege sticticalis; Lsti99.; Peritrophic membrane protein; Recombinant protein.

Fig 1

Nucleotide sequence and deduced amino acid sequence of the Lsti99 cDNA in L. sticticalis. The suggested start codon ATG and stop codon TAG are indicated in boxes. A putative polyadenylation signal is shaded with grey. The predicted signal peptide is underlined. Arrows under the nucleotide sequences represent the position of the different synthetic primers used in recombinant expression. The N-glycosylation sites in Lsti99 were predicted using Expert Protein Analysis System, and shaded in black. The O-glycosylation sites in Lsti99 were predicted using the NetOglyc 3.1 server and showed with dotted line. The nucleotide sequence reported here is deposited in the GenBank database under the accession number FJ798745.

Fig 2

The predicted hydropathy profiles of the deduced amino acid sequences of the Lsti99. The hydropathy profiles was predicted using DNAman 6.3.0.99.

Fig 3

Western blotting analysis of expressed Lsti99 in Sf9 cells. M, represent the protein marker; lane 1, Western-blotting of the normal Sf9; lane 2, Western-blotting of the Sf9 being infected by recombinant virus.

Fig 4

Tissue distribution of the Lsti99 based on the western blot. 20 µg of protein samples extracted from several different laval tissues were loaded on each lane for immunoblot analysis.

Fig 5

Western blot analysis of the PM proteins treated with M2R and chitinase. A, the proteins from the pellet after treatment were used for analysis; B, the soluble solutions were used for analysis.

Fig S1

Alignment of the amino acid sequences of Lsti99 and Lsti201. The conserved amino acids were shaded. Lsti99, FJ798745; Lsti201 FJ798746.