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.