Properties of the cuticular proteins of Anopheles gambiae as revealed by serial extraction of adults

Abstract

How cuticular proteins (CPs) interact with chitin and with each other in the cuticle remains unresolved. We employed LC-MS/MS to identify CPs from 5–6 day-old adults of Anopheles gambiae released after serial extraction with PBS, EDTA, 2-8M urea, and SDS as well as those that remained unextracted. Results were compared to published data on time of transcript abundance, localization of proteins within structures and within the cuticle, as well as properties of individual proteins, length, pI, percent histidine, tyrosine, glutamine, and number of AAP[A/V/L] repeats. Thirteen proteins were solubilized completely, all were CPRs, most belonging to the RR-1 group. Eleven CPs were identified in both soluble fractions and the final pellet, including 5 from other CP families. Forty-three were only detected from the final pellet. These included CPRs and members of the CPAP1, CPF, CPFL, CPLCA, CPLCG, CPLCP, and TWDL families, as well as several low complexity CPs, not assigned to families and named CPLX. For a given protein, many histidines or tyrosines or glutamines appear to be potential participants in cross-linking since we could not identify any peptide bearing these residues that was consistently absent. We failed to recover peptides from the amino-terminus of any CP. Whether this implicates that location in sclerotization or some modification that prevents detection is not known. Soluble CPRs had lower isoelectric points than those that remained in the final pellet; most members of other CP families had isoelectric points of 8 or higher. Obviously, techniques beyond analysis of differential solubility will be needed to learn how CPs interact with each other and with chitin.

A comprehensive omics analysis and functional survey of cuticular proteins in the brown planthopper

“…It is possible that not all peptides could be detected because they were too long for detection or because cuticle components were cross-linked to each other ( 7 ). Different CPs might have different solubility ( 8 ), and sclerotization could also make some CPs unextractable ( 9 ).…”

Proteomics reveals localization of cuticular proteins in Anopheles gambiae

“…Searches were against two databases, An. gambiae peptides (AgamP4.3) from VectorBase and a locally constructed database of 304 putative and previously verified structural cuticular proteins used previously (Zhou et al, 2016, 2017). Decoy databases were created by reversing the protein sequences of corresponding target databases.…”

“…We also examined the total proteome of 5–6 day old adult An. gambiae in a study designed to probe the relative solubility of different CPs (Zhou et al, 2017). We have now examined two more adult structures, proboscises and palps, separating males and females, and have added a preparation enriched in larval tracheae as well as intact 4th instar larvae and justthe terminal segment of these larvae bearing spiracles, dorsal tufts, ventral brushes and tracheal gills.…”

Comparative Proteomics Studies of Insect Cuticle by Tandem Mass Spectrometry: Application of a Novel Proteomics Approach to the Pea Aphid Cuticular Proteins

“…To date, proteomics studies on cuticular structures have been essentially based on SDS extraction/precipitation, sometimes including a separation by 1D‐ or 2D‐SDS‐PAGE, and subsequent trypsin digestion of the obtained supernatants, gel bands, or SDS‐insoluble pellets prior to structural elucidation by bottom‐up proteomics of the CPs fractions. Such approaches have been used to identify CPs in different species and conditions . In recent studies, between 1100 and 1500 An.…”

“…In recent studies, between 1100 and 1500 An. gambiae adults were used to study the CPs of the complete body and between 80 and 330 insects to study the CPs composition of their appendages . Micas et al.…”