Abstract
Our recent study on the functional analysis of the Knickkopf protein from T. castaneum (TcKnk), indicated a novel role for this protein in protection of chitin from degradation by chitinases. Knk is also required for the laminar organization of chitin in the procuticle. During a bioinformatics search using this protein sequence as the query, we discovered the existence of a small family of three Knk-like genes (including the prototypical TcKnk) in the T. castaneum genome as well as in all insects with completed genome assemblies. The two additional Knk-like genes have been named TcKnk2 and TcKnk3. Further complexity arises as a result of alternative splicing and alternative polyadenylation of transcripts of TcKnk3, leading to the production of three transcripts (and by inference, three proteins) from this gene. These transcripts are named TcKnk3-Full Length (TcKnk3-FL), TcKnk3-5′ and TcKnk3-3′. All three Knk-family genes appear to have essential and non-redundant functions. RNAi for TcKnk led to developmental arrest at every molt, while down-regulation of either TcKnk2 or one of the three TcKnk3 transcripts (TcKnk3-3′) resulted in specific molting arrest only at the pharate adult stage. All three Knk genes appear to influence the total chitin content at the pharate adult stage, but to variable extents. While TcKnk contributes mostly to the stability and laminar organization of chitin in the elytral and body wall procuticles, proteins encoded by TcKnk2 and TcKnk3-3′ transcripts appear to be required for the integrity of the body wall denticles and tracheal taenidia, but not the elytral and body wall procuticles. Thus, the three members of the Knk-family of proteins perform different essential functions in cuticle formation at different developmental stages and in different parts of the insect anatomy.
Highlights
Chitin, a homopolymer of b-1,4 linked N-acetyl glucosamine units, is an essential component of the extracellular matrix of insect cuticle [1]
The two newly identified members of this family, T. castaneum Knk-2 (TcKnk2) and T. castaneum Knk-3 (TcKnk3), have distinctly different but related functions. They are essential for adult morphogenesis including specialized ‘‘Velcro-like’’ cuticular denticles found in the lateral body wall, as well as the proper development of the tracheal lining
The TcKnk3 gene gives rise to multiple transcripts as a result of alternative polyadenylation and/or splicing, but only one of these transcripts is essential for adult development
Summary
A homopolymer of b-1,4 linked N-acetyl glucosamine units, is an essential component of the extracellular matrix of insect cuticle [1]. The major component of the procuticle, is synthesized by the integral membrane protein, chitin synthase-A (Chs-A) and is deposited outside of the cell in the form of bundles of fibers [2,3]. Several such bundles of nanofibers and proteins are arranged in the form of a chitin/protein sheet, or lamina [4,5]. Several groups of cuticle-associated proteins have been implicated in organizing the cuticle into a complex multi-layered structure with distinctly different properties in different parts of the insect’s body plan [6,7]. The role, if any, of specific cuticular and epidermal cell plasma membrane proteins in organizing chitin laminae into such helicoidal or orthogonal bundles has not been investigated in detail
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