Identification and molecular characterization of a chitin-binding protein from the beet webworm, Loxostege sticticalis L

Abstract

As the first crucial barrier in the midgut of insects, the peritrophic membrane (PM) plays an important role in preventing external invasion. PM proteins, as the major components of the PM, determine the structure and function of this membrane. A new PM protein, named LstiCBP, from the PM of Loxostege sticticalis larvae was identified using cDNA library screening. The full cDNA of LstiCBP is 2606 bp in length and contains a 2403 bp ORF that encodes an 808-amino acid preprotein with a 15-amino acid as signal peptide. The deduced protein sequence of the cDNA contains 8 cysteine-rich chitin-binding domains (CBDs). Recombinant LstiCBP was successfully expressed in BL21 cells using recombinant plasmid DNA and showed high chitin-binding activity. LstiCBP expression was detected in the midgut at both the transcriptional and translational levels; however, the biochemical and physiological functions of LstiCBP in L. sticticalis require further investigation.

Figure 1

Nucleotide sequence of the cDNA for L. sticalitis CBP and its deduced amino acid sequence. Signal peptide domains (grey background), cysteine (red background)-rich regions (CBD1-8, underlined), the initiation and translation stop codon (in box) are indicated. The potential polyadenylation signal sequence is double lined. Eight chitin binding domains are underlined from N- to C-terminus of the protein.

Figure 2

Alignment of the amino acid similarity of LstiCBP from known insects. Trichoplusia ni CBP1: AAR06265.1; Trichoplusia ni CBP2: AAR06266.1; Spodoptera exigua CBP: ABW98673.1; Spodoptera litura CBP: ADV03161.1.

Figure 3

Alignment of chitin-binding domains (CBDs) from Loxostege sticticalis LstiCBP protein. The conserved amino acids are shaded. The consensus sequence is shown at the bottom.

Figure 4

SDS-PAGE and Western blot analysis of expressed recombine LstiCBP. Mw. Molecular weight marker; 1. 1.0 mmol/L IPTG induced E. coli pET/LstiCBP; 2. 2.0 mmol/L IPTG induced E. coli pET/LstiCBP; 3. IPTG non-induced E.coli pET/LstiCBP; 4. Western blot.

Figure 5

Analysis of chitin-binding activity of recombinant LstiCBP. LstiCBP treated with 1% Calcoflour, 2% Calcoflour, PBS, 6 M Urea, 0.5 M NaCl, 20 mM acetic acid, 2% SDS, Sodium carbonate buffer.

Figure 6

qPCR analysis of LstiCBP expressed in different tissues of L. sticticalis larvae. cDNAs were amplified with specific primers from midgut, hemolymph, ecdysis, fecal pellets, fat body and integument.

Figure 7

Identification of LstiCBP from L. sticticalis larval midgut proteins by Western blot analysis with antibodies specific to LstiCBP. Proteins were from the midgut, PM, hemolymph, ecdysis, fecal pellets, fat body and integument.