Journal of Proteome Research Table 2. Midgut Detergent Resistant Membrane Proteins Containing an Immunoglobulin-like Domain protein conserved immunoglobulin-like domain signal P AGAP000550a Ig E-set Yes 1 AGAP000720a Ig C2-set; Ig I-set; Ig V-set; Ig V-set, subgroup; Ig subtype 2 Yes 1 AGAP001633 Ig I-set; Ig V-set; Ig subtype 2 No 0 AGAP001662 Ig I-set; Ig V-set; Ig subtype 2 No 0 AGAP001892 Ig E-set No 0 AGAP001894 Ig E-set No 0 AGAP002336 Ig I-set; Ig V-set subgroup; Ig subtype 2 No 1 AGAP002802 Ig I-set; Ig V-set subgroup; Ig subtype 2 No 1 AGAP002848a Ig E-set Yes 0 AGAP003656 Ig MHC, conserved site; Ig I-set; Ig V-set; Ig subtype 2 No 0 AGAP004335 Ig E-set Inc. seq. 0 AGAP005471 Ig I-set; Ig V-set Ig V-set subgroup; Ig subtype 2 No 0 AGAP005549 Ig MHC, conserved site No 0 AGAP007562 Ig I-set; Ig V-set; Ig subtype 2 No 0 AGAP007563 Ig C1-set; Ig I-set; Ig V-set; Ig subtype 2 No 0 AGAP008408 Ig I-set; Ig subtype 2 No 1 AGAP008813 Ig I-set; Ig subtype Inc. seq. 1 AGAP009515 Ig E-set No 0 AGAP010428 Ig E-set No 0 AGAP010821 Ig C2-set Inc. seq. 0 AGAP010823a Ig C2-set Yes; Inc. seq. 1 AGAP010877 Ig E-set Inc. seq. 0 AGAP010964 Ig Cl-set Inc. seq. 0 AGAP011578a Ig-like Yes; Inc. seq. 0 AGAP011700 Ig I-set; Ig V-set; Ig subtype 2 No 0 AGAP011859 Glycoside hydrolase, family 2, Ig-like beta-sandwich No 0 AGAP012168 Ig E-set No 0 AGAP012343 Ig C2-set; Ig Cl-set; Ig I-set; Ig V-set; Ig subtype 2 Inc. seq. 1 AGAP013106a Ig E-set Yes 0 a Denotes proteins which are predicted to be extracellular. Transmembrane Domain (TMD). Ig, immunoglobulin; Inc. seq., incomplete sequence; MHC, major histocompatibility complex. ARTICLE TMD or surface-associated proteins. Members of the immunoglobulin superfamily play roles in cell cell adhesion and recognition, function as cell-surface receptors, and in higher eukaryotes, are involved in the adaptive immune system.54 Approximately 138 genes in the An. gambiae genome are predicted to contain a minimum of one immunoglobulin domain.55 Of the Ig-like proteins identiﬁed in the DRM proteome, 45% contained I-set (intermediate, IPR013098) domains, which are found but not limited to several canonical cell adhesion and junction adhesion molecules in eukaryotes.54 The precise function of the majority of these Anopheles proteins is unknown. However, genetic silencing by RNA interference demonstrated a role for a subset of these genes in controlling bacterial and parasitic infection. In particular, knock down of IRD4 (AGAP003656) and IRD6 (AGAP005471, resulted in an approximate 2-fold increase of P. falciparum oocysts compared to controls, suggesting that these genes play a role in restricting Plasmodium infection.55 Both AGAP003656-PA and AGAP005471-PA were detected in the DRM fractions, and further examination of these immunoglo- bulin domain-containing proteins identiﬁed in DRMs may reveal a role for DRMs in mosquito immunity. Moreover, the identi- ﬁcation of an immunoglobulin domain containing proteins as well as the protein products of previously characterized immune- responsive genes in the midgut DRM proteome suggests that the observed up-regulation in transcript levels for a subset of these “immune genes” 18 24 h post infectious blood feeding29,30 may in fact represent an intrinsic midgut response to replenish the tissues apical epithelial surface architecture following blood feeding, and not necessarily a result of a direct response to the malaria parasite. For example, an interrogation of the available angaGEDU- CI: An. gambiae gene expression database (http://www.angaged. bio.uci.edu)56 using accession numbers from our DRM pro- teome, representing proteins that contain either a leucine-rich domain (AGAP005744; AGAP006643), an immunoglobulin domain (AGAP000550; AGAP002848; AGAP012168) or adhesion/lec- tin-like domains (AGAP000341; AGAP006280; AGAP005404) underscore the possibility of expression discordance in transcript and protein presence in the midgut of sugar-fed and blood fed An. gambiae (data not shown). Therefore, we contend that caution must be taken since an observed upregulation of transcript during the ﬁrst 3 24 h following blood feeding may not necessarily correspond to a commensurate expression of protein. Without empirical validation of protein presence in sugar-fed midguts, one cannot say that a protein is only present in blood fed midguts. Glycosylation of DRM-Associated Proteins The apical membrane of the midgut is heavily glycosylated and coated with a glycocalyx, an electron-dense layer rich in glycans, 5157 dx.doi.org/10.1021/pr2006268 |J. Proteome Res. 2011, 10, 5150–5162