Analysis of expression and chitin-binding activity of the wing disc cuticle protein BmWCP4 in the silkworm, Bombyx moil

The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin-binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic ＂R＆R＂ chitin-binding domain （CBD）. Western blotting showed that BmWCP4 was predominately expressed in the wing disc-containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full-length BmWCP4 protein, ＂R＆R＂ CBD peptide （CBD）, non-CBD peptide （BmWCP4-CBD^-）, four single site-directed mutated peptides （M1, M2, M3 and M4） and four-sites-mutated peptide （MF） were generated and purified, respectively, for in vitro chitin-binding assay. The results indicated that both the full-length protein and the ＂R＆R＂ CBD peptide could bind with chitin, whereas the BmWCP4-CBD- could not bind with chitin. The single residue mutants M1, M2, M3 and M4 reduced but did not completely abolish the chitin-binding activity, while four-sites-mutated protein MF completely lost the chitin-binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva-to-pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.

Insect group II chitinase OfChtll promotes chitin degradation during larva-pupa molting

The insect group ll chitinase(ChtIl,also known as Cht10)is a unique chitinasewith multiple catalytic and chitin-binding domains.It has been proven genetically to be anessential chitinase for molting.However,Chtll's role in chitin degradation during insectdevelopment remains poorly understood.Obtaining this knowledge is the key to fullyunderstanding the chitin degradation system in insects.Here,we investigated the roleof OfChtll during the molting of Ostrinia furnacalis,a model lepidopteran pest insect.OfChtll was expressed earlier than OfChtI(OfCht5)and OfChi-h,at both the gene andprotein levels during larva-pupa molting as evidenced by quantitative polymerase chainreaction and western blot analyses.A truncated OfChtII,OfChtII-B4C1,was recombinantlyexpressed in Pichia pastoris cells and purified to homogeneity.The recombinant OfChtll-B4C1 loosened compacted chitin particles and produced holes in the cuticle surface asevidenced by scanning electron microscopy.It synergized with OfChtl and OfChi-h whenhydrolyzing insoluble a-chitin.These findings suggested an important role for ChtIl duringinsect molting and also provided a strategy for the coordinatdd degradation of cuticularchitin during insect molting by Chtll,Chtl and Chi-h.