Isolation and identification of insect intestinal mucin HaIIM86--the new target for Helicoverpa armigera biocontrol

Abstract

There are many more glycoproteins in Helicoverpa armigera peritrophic membrane than midgut separated by SDS-PAGE analysis after Periodic acid-Schiff (PAS) and coomassie staining. The peritrophic membrane (PM) of H. armigera larvae contains about forty associated proteins. A cDNA library was constructed from H. armigera midgut mRNA to study the new target for pest biocontrol. An antiserum against Spodoptera exigua integral/total PM proteins cross reacted with several H. armigera PM proteins and was used to isolate a complete cDNA encoding an insect intestinal mucin (HaIIM86). The deduced protein sequence of the cDNA contained one potentially glycosylated, mucin-like domain, five cysteine-rich chitin-binding domains (CBDs) and two D-G rich regions. Mucin domain was lined between the first and second CBDs; the two additional D-G rich regions were proposed to internal reside at the amino terminus of the protein flanked by three cysteine-rich CBDs. HaIIM86 contains two D-G-rich tandem repeat domains flanked by cysteine-rich sequences in peritrophic membrane proteins which is not present in all the currently known PM proteins. Howerer the functions of D-G rich domains require further investigation. HaIIM86 was shown as 200 kDa protein by SDS-PAGE analysis and appeared to be associated with the PM. HaIIM86 has chitin-binding activity and can be degraded into 90 and 70 kDa by HaGV Enhancin in vivo. The finding has shown that HaIIM86 is the target substrate for enhancin and the potential target for pest control.

Keywords: Helicoverpa armigera; Peritrophic matrix; biocontrol; cDNA expression library; insect intestinal mucin.

Fig 1

Protein composition by SDS-PAGE analysis for H. armigera PM & midgut. A&B: Gels stained by Periodic acid-Schiff (PAS) before and after coomassie staining; C: Silver staining; PM, peritrophic membrane; Mg, midgut; M, protein Marker, 220, 170, 116, 76, 49kDa

Fig 2

Nucleotide sequence of the cDNA for Helicoverpa armigera intestinal mucin and its deduced amino acid sequence. Signal peptide domains (grey background), threonine-rich domains (Muc1, wavy underlined), cysteine-rich regions (CBD1-5, underlined), and the translation stop codon (in box) are indicated. Additional aspartic acid-rich regions are marked with _ _ _ _ _

Fig 2

Nucleotide sequence of the cDNA for Helicoverpa armigera intestinal mucin and its deduced amino acid sequence. Signal peptide domains (grey background), threonine-rich domains (Muc1, wavy underlined), cysteine-rich regions (CBD1-5, underlined), and the translation stop codon (in box) are indicated. Additional aspartic acid-rich regions are marked with _ _ _ _ _

Fig 3

Prediction of O-glycosylation sites and structure in H. armigera intestinal mucin HaIIM86. Residues with an O-glycosylation potential exceeding the threshold level (red line) are expected to be glycosylated. SIP, signal peptide sequence; Muc, mucin-like domain; ChtBD2, chitin-binding domain; D-r, Glycine-Aspartic acid-rich domains. Asn-Xaa-Ser/Thr sequons in the sequence output below are highlighted in blue.

Fig 4

Alignment of insect intestinal mucin chitin-binding domains (CBD) from Mamestra configurata intestinal mucin, (Mc, genbank accession number AY057052), Plutella xylostella intestinal mucin (Px) (Genbank accession number AF545582), Trichoplusia ni intestinal mucin IIM14 (Tni) (Genbank accession number AF000605), and Helicoverpa armigera intestinal mucin (Ha) (Genbank accession number EU047712). A: The alignment of CBDs from known IIMs. B: Dendrogram shows the relationship among CBDs from known IIMs. The carboxyl-terminal CBD from each IIM formed a single group (in box).

Fig 5

Western Blot analysis of expression of HaIIM86 in insect cells. 1-4, supernatant from cell culture infected by recombinant baculovirus for 24, 72, 96, 48hs; CK+, cells infected by recombinant baculovirus; CK-, Sf9 cells.

Fig 6

Analysis of chitin-binding activity of recombinant HaIIM86. M, protein marker, 220, 116, 96, 66, 43kDa; HaIIM86 treated with 0.2% Calcoflour (1) and 1% Calcoflour (2).

Fig 7

HaIIM86 is the target for enhancin action. The degradation of HaIIM86 was analyzed by Western blot analysis using anti-HaIIM86 antiserum. Lane 1, HaIIM86; lane 2, HaIIM86 treated by enhancin

Fig 8

Detection of HaIIM86 in H. armigera tissues. 1, fat bodies; 2, pupa; 3, fecal pellets; 4-5, PM from early and middle of fifth instar larvae; 6, hemolymph; 7-8, midgut from early and middle of fifth instar larvae;9, DH5a(pMal- SeCBP66); M, Protein Marker

Fig 9

Structure analysis of known insect intestinal mucin proteins