Journal of Proteome Research ARTICLE Figure 3. An. gambiae midgut brush border microvilli GPI-anchored subproteome. (a) PepArML identiﬁed 65 proteins from a combined search using Mascot, OMSSA, and X!Tandem at <1% FDR. Distribution of the identiﬁed GPI-anchored proteins (N = 65) into categories was based on function (see text and Table S5, Supporting Information, for details). (b) Venn diagram illustrating the common proteins that were identiﬁed in the GPI, extracellular DRM, and total DRM proteomes. *, two proteins, AGAP004944 and AGAP012745, that were identiﬁed in the GPI-anchored subproteome were not identiﬁed in the DRM proteome (<1%FDR). which protects the midgut epithelial cells from the enzymatic pro- cesses involved with digestion. O-glycans have also been shown to be important mediators of ookinete attachment and invasion.11,57 The amino acid sequences of all predicted extracellular DRM- associated proteins were analyzed for the presence of predicted glycosylation sites. A total of 121 proteins were predicted to have at least one O-linked glycosylation site and 145 proteins were predicted to have at least one N-linked glycosylation site (Table S2, Supporting Information). Jacalin binds to galactose and GalNAc residues and has previously been shown to competi- tively inhibit ookinete attachment to the midgut.58 The GalNAc residues were also shown to be conserved between Aedes and Anopheles mosquitoes, and similarly involved in ookinete attach- ment.11,59 A follow-up study used jacalin column chromatography and LC MS/MS to identify the predominant heavily O-glyco- sylated midgut proteins that are recognized by this lectin on An. gambiae.11 Of the ﬁve proteins identiﬁed in that study, four of them, AGAP004809 (AgAPN1), AGAP001881 (AgAPN3), and two glycosyl hydrolases, AGAP003995 (alpha-glucosidase) and AGAP012401, a previously described maltase-like protein (AgM1),60 were detected in the DRM fractions indicating that O-glycosylated proteins are present in midgut DRMs. GPI-Anchored Proteins Are Enriched in the DRM Fractions Since the number of reagents available for conﬁrming anopheline homologues of common DRM-associated proteins are limited, an orthogonal approach was taken to validate our bioinformatics approach as well as to conﬁrm the technical validity of our DRM isolation approach. As GPI-anchored proteins are known to be enriched in DRMs,6 we examined the GPI proteome of An. gambiae midgut brush border microvilli to conﬁrm enrichment of GPI-anchored proteins in our DRM fractions. Sugar-fed midguts were used for three biological replicate BBMV preparations, which were subsequently treated with phosphatidylinositol-spe- ciﬁc phospholipase C (PI-PLC), an enzyme known to speciﬁcally release GPI-anchored proteins from the membrane. From three biological replicate preparations, a PepArML search against VectorBase identiﬁed 76 proteins that were ﬁnally clustered to 65 proteins (Figure 3a and Table S4, Supporting Information). Of the 65 proteins identiﬁed in the GPI-anchored protein subpro- teome, 96.9% are also present in the DRM proteome (Figure 3b). All GPI-anchored proteins contain both an N-terminal signal sequence and a C-terminal sequence, which is cleaved and eventually replaced with a GPI-anchor.61 Of the 65 proteins, 39 had a predicted signal sequence, which is a requirement for the addition of a GPI anchor. Among these 39 proteins, ﬁve proteins were predicted to be nonsecreted intracellular contaminants based on sequence homology. Thus, based on the presence of a signal peptide and the removal of 5 intracellular proteins, we predict that 34 proteins in the GPI proteome are extracellular. Proteins detected in the GPI-anchored protein fraction that were predicted to be extracellular included: peptidases (52.9%), glycosyl hydrolases (11.7%), proteins in the unknown function (14.7%), proteins containing an immunoglobulin-like domain (2.9%), and proteins categorized as “other” (17.6%). Alkaline phosphatases have been previously reported to be surface- associated and enriched in BBMVs from aedine and anopheline larvae but were, in general, regarded as inappropriate markers for the midgut surface.42,62 However, given that AGAP006400 and AGAP011302 were identiﬁed in the GPI-anchored midgut subproteome (Table S4, Supporting Information), these speciﬁc species of alkaline phosphatase may be appropriate as midgut surface marker. Also, it has been shown that PI-PLC treatment of BBMVs releases both the 125 kDa isoform of the GPI-anchored AgAPN1 as well as a truncated ∼60 kDa isoform.11 AgAPN1 was detected in our GPI-anchored subproteome conﬁrming this previous ﬁnding and also provides a known GPI-anchored marker to conﬁrm the isolation of GPI-anchored proteins in our subpro- teome. Taken together, these data demonstrate that our DRM fractions are enriched in GPI-anchored proteins, which are common markers of DRMs, thus providing additional supporting evidence that our midgut DRM fractions do indeed contain DRMs. Ookinete-Interacting Proteins Partition into the DRM Fractions The process of ookinete invasion of the midgut epithelium is likely to involve a hierarchy of multiple protein glycan and 5158 dx.doi.org/10.1021/pr2006268 |J. Proteome Res. 2011, 10, 5150–5162