The CPCFC cuticular protein family: Anatomical and cuticular locations in Anopheles gambiae and distribution throughout Pancrustacea

Abstract

Arthropod cuticles have, in addition to chitin, many structural proteins belonging to diverse families. Information is sparse about how these different cuticular proteins contribute to the cuticle. Most cuticular proteins lack cysteine with the exception of two families (CPAP1 and CPAP3), recently described, and the one other that we now report on that has a motif of 16 amino acids first identified in a protein, Bc-NCP1, from the cuticle of nymphs of the cockroach, Blaberus craniifer (Jensen et al., 1997). This motif turns out to be present as two or three copies in one or two proteins in species from many orders of Hexapoda. We have named the family of cuticular proteins with this motif CPCFC, based on its unique feature of having two cysteines interrupted by five amino acids (C-X(5)-C). Analysis of the single member of the family in Anopheles gambiae (AgamCPCFC1) revealed that its mRNA is most abundant immediately following ecdysis in larvae, pupae and adults. The mRNA is localized primarily in epidermis that secretes hard cuticle, sclerites, setae, head capsules, appendages and spermatheca. EM immunolocalization revealed the presence of the protein, generally in endocuticle of legs and antennae. A phylogenetic analysis found proteins bearing this motif in 14 orders of Hexapoda, but not in some species for which there are complete genomic data. Proteins were much longer in Coleoptera and Diptera than in other orders. In contrast to the 1 and occasionally 2 copies in other species, a dragonfly, Ladona fulva, has at least 14 genes coding for family members. CPCFC proteins were present in four classes of Crustacea with 5 repeats in one species, and motifs that ended C-X(7)-C in Malacostraca. They were not detected, except as obvious contaminants, in any other arthropod subphyla or in any other phylum. The conservation of CPCFC proteins throughout the Pancrustacea and the small number of copies in individual species indicate that, when present, these proteins are serving important functions worthy of further study.

Distribution of cuticular proteins in different structures of adult Anopheles gambiae

“…Despite its limitations, He et al (2007) confirmed the authenticity of many of the CPs we had annotated and provided us with additional members of known CP families and several new families of CPs. Since then, three previously unrecognized families of CPs have been reported: CPAP1 and CPAP3 (Jasrapuria et al, 2010; Tetreau et al, 2015) and CPCFC (Vannini et al, 2015). Further annotation based on these results and a far more robust proteome (Vector Base AgamP4.2) resulted in a cuticulome of An.…”

“…These genes code for only 282 distinct CPs, because the protein products of several genes are identical. The CP genes have been classified into 13 distinct families and some orphans (Willis, 2010; Willis et al, 2012; Ioannidou et al, 2014; Tetreau et al, 2015; Vannini et al, 2015). This information is summarized in Table 1 and all CP sequences where we identified peptides in this study are in Supplementary File 1.…”

Immunolocalization of cuticular proteins in Johnston's organ and the corneal lens of Anopheles gambiae

“…Sixteen of the 19 primary antibodies tested in this study were used in previous studies by Vannini et al (2014b; 2015) and Vannini and Willis (2016). All the antibodies, except for the anti-3RC Cluster and anti-CPCFC1, were raised against synthetic peptides at Genscript Inc. (Piscataway, NJ) and affinity purified.…”

“…In attempts to learn why there are so many CPs, we have examined the precise location of a small number of these proteins within the cuticle with EM immunolocalization (Vannini et al, 2014b, 2015; Vannini and Willis, 2016). The structures we examined had long stretches of seemingly identical cuticle and the epitopes detected for individual CPs covered large areas of these structures.…”

“…We had also published results of EM immunolocalization using antibodies against individual members of the CPF, CPLCG, and CPCFC families (Vannini et al, 2014b, 2015). Thus we had a collection of 19 antibodies that we thought might give us fresh insights if used against the cuticle in the two specialized structures that are the focus of this report, JO and corneal lens.…”