Identification and molecular characterization of a novel protein Saglin as a target of monoclonal antibodies affecting salivary gland infectivity of Plasmodium sporozoites

Abstract

Molecular mechanisms underlying the interaction between malarial sporozoites and putative receptor(s) on the salivary glands of Anopheles gambiae remain largely unknown. In previous studies, a salivary gland protein of ~100 kDa was identified as a putative target based on recognition of the protein by a monoclonal antibody (mAb) 2A3 that caused a >/= 70% reduction in the average number of sporozoites per infected salivary gland when fed to mosquitoes. Using affinity purification we purified the target of this mAb from extracts of female A. gambiae salivary glands and it was found to be a novel protein by tandem mass spectrometric analysis. Biochemical and molecular characterization of the 100 kDa protein showed that this molecule, designated Saglin, exists as a disulphide-bonded homodimer of 50 kDa subunits. The ability to form homodimers was retained even in the recombinant Saglin expressed in mammalian cells (HEK293). The amino acid sequence of Saglin contains a signal peptide suggesting that Saglin is a secreted protein. If Saglin is indeed involved in the process of invasion of A. gambiae salivary glands by sporozoites of Plasmodium, it could provide a novel target for future investigations aimed at interruption of malaria transmission.

Salivary Gland Proteome Analysis Reveals Modulation of Anopheline Unique Proteins in Insensitive Acetylcholinesterase Resistant Anopheles gambiae Mosquitoes

“…The three saglin isoforms identified on the 2-DE gels are thus likely unglycosylated and partially to totally glycosylated forms of the ∼50 kDa subunits of the protein. A similar hypothesis of an heterogeneous mixture of unglycosylated and partially glycosylated forms of saglin was reported from 2D Western blot analysis and treatment with peptide N-glycosidase F (PNGase F) of the recombinant protein [27]. No transmembrane domain or GPI-anchoring signal indicative of cell surface protein localization could be identified.…”

“…Among these proteins, saglin presented the highest differences in protein expression with an average value of 5.7 fold for the three isoforms. Saglin was first identified as the target of monoclonal antibodies that reduce salivary gland infectivity of Plasmodium sporozoites, suggesting that it may represent one of the molecules involved during the invasion of salivary glands by sporozoites [27]. This 100 kDa-salivary protein was described as a disulphide bond-linked homodimer of ∼50 kDa subunits, and then reported as the receptor for sporozoites in salivary gland, binding to the thrombospondin-related anonymous protein (TRAP) expressed in sporozoites and conserved in all Plasmodium species [28].…”

Members of the Salivary Gland Surface Protein (SGS) Family Are Major Immunogenic Components of Mosquito Saliva

“…The electrophoretic pattern of the latter protein suggests that it is a disulfide-linked dimer, and interestingly, this pattern of migration is identical to that seen for Saglin, an Anopheles salivary protein that was shown to be involved in Plasmodium invasion of the salivary glands, along with some evidence that it is a component of mosquito saliva (35,36). The detection of putative Saglin is strong after only 3 weeks of exposure to bites, suggesting that Saglin may also be a sensitive target for an exposure surveillance strategy.…”

A proteogenomic analysis of Anopheles gambiae using high-resolution Fourier transform mass spectrometry

“…Several studies have demonstrated the use of mass spectrometrybased proteomic approaches to validate or correct gene annotations in Homo sapiens (Molina et al 2005;Suzuki and Sugano 2006;Sevinsky et al 2008;Menon et al 2009), Caenorhabditis elegans (Merrihew et al 2008), Drosophila melanogaster (Brunner et al 2007;Tress et al 2008), An. gambiae (Pandey and Mann 2000;Kalume et al 2005a,b;Okulate et al 2007), Toxoplasma gondii (Xia et al 2008), and Arabidopsis thaliana (Kuster et al 2001;Baerenfaller et al 2008).…”