Journal of Proteome Research Table 1. Known Ookinete-Interacting Proteins Identiﬁed in DRM Fractionsa accession no. annotation evidence for role of molecule in Plasmodium development AGAP004809 GPI-anchored Aminopeptidase N Anti-AgAPN1antibodies inhibited both P. falciparum (70 80%) 11 and P. berghei (70 80%) oocyst development AGAP003790 Annexin-like Anti-ANXB9 antibodies inhibited of P. berghei (30 38%) oocyst development 12 AGAP003721 Annexin-like Anti-ANXB10B antibodies inhibited P. berghei (36 40%) oocyst development 12 AGAP003722b Annexin-like Anti-ANXB10C antibodies inhibited 28.2 43.7% of P. berghei development in the midgut 12 AGAP006209 Carboxypeptidase B Antibodies against CpbAg1 inhibited both P. falciparum and P. berghei development 13 AGAP010133 Scavenger Receptor, Knock-down of SCRBQ2 results in a 62.5% inhibition of P. berghei 14 Croquemort Homologue oocyst formation a An. albimanus calreticulin (AaCrt) has been shown to localize to the apical surface of An. albimamus midguts and to interact with a recombinant form of the abundant P. vivax ookinete surface protein, Pvs25.15 The An. gambiae homologue of AaCrt, AGAP004212, was present in DRMs but was not included in Table 1, since to date, there is no direct evidence by either RNAi knock-down or the use of anti-AaCrt antibodies demonstrating the involvement of Anopheles midgut surface expressed calreticulin in Plasmodium invasion and establishment in the mosquito. b Only known ookinete- interacting protein not detected in the DRM proteome. ARTICLE reference Plasmodium ookinetes, a model is needed to explain mechan- istically how ookinetes coordinate multiple protein protein and protein glycan interactions with the apical surface of the midgut at a deﬁned point of cell entry. One idea is based on the hypothesis that host cell membrane microdomains mediate surface protein organization and that pathogens utilize these sites for adhesion complex formation and subsequent attachment and invasion.4 Lipid microdomains commonly referred to as “lipid rafts”, exhibit dynamic lateral movement on the cell surface and are enriched in proteins that facilitate various cellular functions including signal transduction, cell adhesion, and vesicle traﬃcking (reviewed in ref 5). Rafts compartmentalize these cellular processes by partitioning, both temporally and spatially, speciﬁc proteins into distinct phases of the plasma membrane. Biochemically, lipid rafts are character- ized by a high density of cholesterol and sphingolipids. The tight packing of sterols between the saturated sphingolipid acyl chains forms a lipid ordered phase within the plasma membrane.6 This intrinsic property allows rafts to be resistant to solubilization by nonionic detergents such as Triton X-100 at 4 C. Although a pure fraction of lipid rafts cannot be isolated, a detergent resistant membrane (DRM) fraction, which is enriched in lipid rafts and associated proteins, can be separated from other membrane proteins and lipids through detergent extraction followed by density gradient centrifugation.7,8 A variety of pathogens induce the fusion of multiple rafts to create large clusters of host receptors in a concentrated region of the membrane.9 This allows for the enhancement of multivalent protein protein9 and protein glycan10 interactions between the pathogen and the host cell that are necessary for attachment and invasion to occur. It is unknown if Plasmodium parasites engage Anopheles midgut lipid rafts in a similar fashion. However, given that exploitation of host lipid rafts by pathogens appears to be a common theme, we hypothesized that during the multistep process of midgut invasion, Plasmodium ookinetes promote the formation of an adhesion complex on the surface of the Anopheles midgut through the subversion of apical microvillar lipid rafts. The underlying premise is that midgut invasion requires the concentration of a diverse set of microvillar glycans and glyco- proteins that act cooperatively as adhesion and invasion ligands for the ookinete. To date, at least six midgut surface molecules11 15 have been shown to mediate parasite vector host interactions and consequently have been proposed as potent candidate TBV targets (Table 1). Therefore, a reasonable test of our hypothesis would be to determine if these targets are enriched in DRMs. If some or all of these targets are enriched in DRMs, then it is possible that several other uncharacterized molecules may prove to be important mediators for ookinete invasion and, as such, the handful of proteins in Table 1 represents only a fraction of the total number of potential TBV targets, and that other glycopro- teins that are resident or enriched in midgut DRMs, represent the untapped pool of vaccine candidates. We therefore used a pro- teomics approach to characterize the broad repertoire of lumenal glycoproteins that reside in an enriched sample of apical midgut surface DRMs. Using an extensive fractionation and informatics strategy, we were able to identify over 1400 proteins that will hopefully help us glean new insights into the mechanism(s) of Plasmodium ookinete invasion of the midgut and provide an extensive catalog of potentially new TBV targets and protein identities for the broader malaria and vector biology communities. ’ MATERIALS AND METHODS Preparation of An. gambiae Midguts for Analysis For each DRM preparation, approximately 1000 midguts were dissected and 1 mL of a solution of 1% Triton X-100 in TKM buﬀer (50 mM Tris-HCl, pH 8, 25 mM KCl, 5 mM MgCl2, 1 mM EDTA) was added to the sample. The sample was then homo- genized on ice with a Dounce homogenizer for 45 min. Following homogenization, the sample was incubated on ice for one hour. Then the midgut lysate was mixed with an equal volume of 80% sucrose and layered in the bottom of an ultra centrifuge tube. The sample was incubated on ice for one hour in the centrifuge tube. Next, 6 mL of 38% sucrose were layered slowly on top of the previous layer, followed by layering 4 mL of 5% sucrose. The sample was then subjected to ultracentrifugation for 18 h at 4 C at 100 000 g with no brake. After ultracentrifugation, 1 mL fractions were collected from the top of the sucrose gradient. The resulting fractions were precipitated in 15% w/v trichloroacetic acid overnight at 4 C and resuspended in 100 μL 1 Protea- seMax (Promega) in 55 mM NH4HCO3. Cholesterol Quantification Cholesterol detection and quantiﬁcation from An. gambiae midgut extracts was performed by using the Amplex Red cholesterol assay kit (Invitrogen) according to the manufacturer’s instruc- tions. Brieﬂy, using a cholesterol standard provided with the kit, 5151 dx.doi.org/10.1021/pr2006268 |J. Proteome Res. 2011, 10, 5150–5162