Anopheles gambiae has a well-adapted system for host localization, feeding, and mating behavior, which are all governed by neuronal processes in the brain. However, there are no published reports characterizing the brain proteome to elucidate neuronal signaling mechanisms in the vector. To this end, a large-scale mapping of the brain proteome of An. gambiae was carried out using high resolution tandem mass spectrometry, revealing a repertoire of >1800 proteins, of which 15% could not be assigned any function. A large proportion of the identified proteins were predicted to be involved in diverse biological processes including metabolism, transport, protein synthesis, and olfaction. This study also led to the identification of 10 GPCR classes of proteins, which could govern sensory pathways in mosquitoes. Proteins involved in metabolic and neural processes, chromatin modeling, and synaptic vesicle transport associated with neuronal transmission were predominantly expressed in the brain. Proteogenomic analysis expanded our findings with the identification of 15 novel genes and 71 cases of gene refinements, a subset of which were validated by RT-PCR and sequencing. Overall, our study offers valuable insights into the brain physiology of the vector that could possibly open avenues for intervention strategies for malaria in the future.
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References
1.
AmenyaDA, ChouW, LiJ, et al. (2010). Proteomics reveals novel components of the Anopheles gambiae eggshell. J Insect Physiol, 56, 1414–1419.
2.
CareyAF, WangG, SuCY, ZwiebelLJ, and CarlsonJR. (2010). Odorant reception in the malaria mosquito Anopheles gambiae. Nature, 464, 66–71.
3.
CastellanaN, and BafnaV. (2010). Proteogenomics to discover the full coding content of genomes: A computational perspective. J Proteomics, 73, 2124–2135.
4.
ChaerkadyR, KelkarDS, MuthusamyB, et al. (2011). A proteogenomic analysis of Anopheles gambiae using high-resolution Fourier transform mass spectrometry. Genome Res, 21, 1872–1881.
5.
Chu-LaGraffQ, and DoeCQ. (1993). Neuroblast specification and formation regulated by wingless in the Drosophila CNS. Science, 261, 1594–1597.
6.
DaniFR, FranceseS, MastrobuoniG, et al. (2008). Exploring proteins in Anopheles gambiae male and female antennae through MALDI mass spectrometry profiling. PLoS One, 3, e2822.
7.
DinglasanRR, DevenportM, FlorensL, et al. (2009). The Anopheles gambiae adult midgut peritrophic matrix proteome. Insect Biochem Mol Biol, 39, 125–134.
8.
FarabaughPJ. (1996). Programmed translational frameshifting. Annu Rev Genet, 30, 507–528.
9.
FerminD, AllenBB, BlackwellTW, et al. (2006). Novel gene and gene model detection using a whole genome open reading frame analysis in proteomics. Genome Biol, 7, R35.
10.
HarshaHC, MolinaH, and PandeyA. (2008). Quantitative proteomics using stable isotope labeling with amino acids in cell culture. Nat Protoc, 3, 505–516.
11.
HeN, BotelhoJM, McNallRJ, et al. (2007). Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry. Insect Biochem Mol Biol, 37, 135–146.
12.
HernandezC, WaridelP, and QuadroniM. (2014). Database construction and peptide identification strategies for proteogenomic studies on sequenced genomes. Curr Top Med Chem, 14, 425–434.
13.
HernandezLG, LuB, da CruzGC, et al. (2012). Worker honeybee brain proteome. J Proteome Res, 11, 1485–1493.
14.
HillCA, FoxAN, PittsRJ, et al. (2002). G protein-coupled receptors in Anopheles gambiae. Science, 298, 176–178.
15.
HoltRA, SubramanianGM, HalpernA, et al. (2002). The genome sequence of the malaria mosquito Anopheles gambiae. Science, 298, 129–149.
16.
HuangY, HowlettE, SternM, and JacksonFR. (2009). Altered LARK expression perturbs development and physiology of the Drosophila PDF clock neurons. Mol Cell Neurosci, 41, 196–205.
17.
HummonAB, RichmondTA, VerleyenP, et al. (2006). From the genome to the proteome: Uncovering peptides in the Apis brain. Science, 314, 647–649.
18.
HuybrechtsJ, BonhommeJ, MinoliS, et al. (2010). Neuropeptide and neurohormone precursors in the pea aphid, Acyrthosiphon pisum. Insect Mol Biol, 19, 87–95.
19.
KalumeDE, OkulateM, ZhongJ, et al. (2005a). A proteomic analysis of salivary glands of female Anopheles gambiae mosquito. Proteomics, 5, 3765–3777.
20.
KalumeDE, PeriS, ReddyR, et al. (2005b). Genome annotation of Anopheles gambiae using mass spectrometry-derived data. BMC Genomics, 6, 128.
21.
KelkarDS, KumarD, KumarP, et al. (2011). Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry. Mol Cell Proteomics 10, M111. 011627.
22.
KinsellaRJ, KahariA, HaiderS, et al. (2011). Ensembl BioMarts: A hub for data retrieval across taxonomic space. Database (Oxford), 2011, bar030.
23.
KrugK, NahnsenS, and MacekB. (2011). Mass spectrometry at the interface of proteomics and genomics. Mol Biosyst, 7, 284–291.
24.
KusterB, MortensenP, AndersenJS, and MannM. (2001). Mass spectrometry allows direct identification of proteins in large genomes. Proteomics, 1, 641–650.
25.
LawniczakMK, EmrichSJ, HollowayAK, et al. (2010). Widespread divergence between incipient Anopheles gambiae species revealed by whole genome sequences. Science, 330, 512–514.
26.
LiAQ, Popova-ButlerA, DeanDH, and DenlingerDL. (2007). Proteomics of the flesh fly brain reveals an abundance of upregulated heat shock proteins during pupal diapause. J Insect Physiol, 53, 385–391.
27.
LiJ, Hosseini MoghaddamSH, ChenX, ChenM, and ZhongB. (2010). Shotgun strategy-based proteome profiling analysis on the head of silkworm Bombyx mori. Amino Acids, 39, 751–761.
28.
LiJY, ChenX, FanW, et al. (2009). Proteomic and bioinformatic analysis on endocrine organs of domesticated silkworm, Bombyx mori L. for a comprehensive understanding of their roles and relations. J Proteome Res, 8, 2620–2632.
29.
LinMF, CarlsonJW, CrosbyMA, et al. (2007). Revisiting the protein-coding gene catalog of Drosophila melanogaster using 12 fly genomes. Genome Res, 17, 1823–1836.
30.
LiuC, PittsRJ, BohbotJD, JonesPL, WangG, and ZwiebelLJ. (2010). Distinct olfactory signaling mechanisms in the malaria vector mosquito Anopheles gambiae. PLoS Biol, 8, e1000467.
31.
LoureiroJ, and PeiferM. (1998). Roles of Armadillo, a Drosophila catenin, during central nervous system development. Curr Biol, 8, 622–632.
32.
MastrobuoniG, QiaoH, IovinellaI, et al. (2013). A proteomic investigation of soluble olfactory proteins in Anopheles gambiae. PLoS One, 8, e75162.
33.
McNeilGP, KaurM, PurrierS, and KangR. (2009). The Drosophila RNA-binding protein Lark is required for localization of Dmoesin to the oocyte cortex during oogenesis. Dev Genes Evol, 219, 11–19.
34.
OkulateMA, KalumeDE, ReddyR, et al. (2007). Identification and molecular characterization of a novel protein Saglin as a target of monoclonal antibodies affecting salivary gland infectivity of Plasmodium sporozoites. Insect Mol Biol, 16, 711–722.
35.
OlsenJV, de GodoyLM, LiG, et al. (2005). Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics, 4, 2010–2021.
36.
PalladinoMJ, BowerJE, KreberR, and GanetzkyB. (2003). Neural dysfunction and neurodegeneration in Drosophila Na+/K+ ATPase alpha subunit mutants. J Neurosci, 23, 1276–1286.
37.
PandeyA, and MannM. (2000). Proteomics to study genes and genomes. Nature, 405, 837–846.
38.
PaskewitzSM, and ShiL. (2005). The hemolymph proteome of Anopheles gambiae. Insect Biochem Mol Biol, 35, 815–824.
39.
PavlidesSC, PavlidesSA, and TammarielloSP. (2011). Proteomic and phosphoproteomic profiling during diapause entrance in the flesh fly, Sarcophaga crassipalpis. J Insect Physiol, 57, 635–644.
40.
PawarH, SahasrabuddheNA, RenuseS, et al. (2012). A proteogenomic approach to map the proteome of an unsequenced pathogen—Leishmania donovani. Proteomics, 12, 832–844.
41.
PrasadTS, HarshaHC, KeerthikumarS, et al. (2012). Proteogenomic analysis of Candida glabrata using high resolution mass spectrometry. J Proteome Res, 11, 247–260.
42.
PredelR, NeupertS, GarczynskiSF, et al. (2010). Neuropeptidomics of the mosquito Aedes aegypti. J Proteome Res, 9, 2006–2015.
43.
RenuseS, ChaerkadyR, and PandeyA. (2011). Proteogenomics. Proteomics, 11, 620–630.
44.
RoyM, Sivan-LoukianovaE, and EberlDF. (2013). Cell-type-specific roles of Na+/K+ ATPase subunits in Drosophila auditory mechanosensation. Proc Natl Acad Sci USA, 110, 181–186.
45.
RozenS, and SkaletskyH. (2000). Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol, 132, 365–386.
46.
RutzlerM, LuT, and ZwiebelLJ. (2006). Galpha encoding gene family of the malaria vector mosquito Anopheles gambiae: Expression analysis and immunolocalization of AGalphaq and AGalphao in female antennae. J Comp Neurol, 499, 533–545.
47.
SilvaJP, and UshkaryovYA. (2010). The latrophilins, “split-personality” receptors. Adv Exp Med Biol, 706, 59–75.
48.
SundramV, NgFS, RobertsMA, MillanC, EwerJ, and JacksonFR. (2012). Cellular requirements for LARK in the Drosophila circadian system. J Biol Rhythms, 27, 183–195.
49.
UnoY, FujiyukiT, MoriokaM, TakeuchiH, and KuboT. (2007). Identification of proteins whose expression is up- or down-regulated in the mushroom bodies in the honeybee brain using proteomics. FEBS Lett, 581, 97–101.
50.
VizcainoJA, CoteRG, CsordasA, et al. (2013). The PRoteomics IDEntifications (PRIDE) database and associated tools: Status in 2013. Nucleic Acids Res, 41, D1063–1069.
51.
WolschinF, MunchD, and AmdamGV. (2009). Structural and proteomic analyses reveal regional brain differences during honeybee aging. J Exp Biol, 212, 4027–4032.
52.
XiaD, SandersonSJ, JonesAR, et al. (2008). The proteome of Toxoplasma gondii: Integration with the genome provides novel insights into gene expression and annotation. Genome Biol, 9, R116.
53.
XiaY, WangG, BuscariolloD, PittsRJ, WengerH, and ZwiebelLJ. (2008). The molecular and cellular basis of olfactory-driven behavior in Anopheles gambiae larvae. Proc Natl Acad Sci USA, 105, 6433–6438.
54.
YatesJR3rd, EngJK, and McCormackAL. (1995). Mining genomes: Correlating tandem mass spectra of modified and unmodified peptides to sequences in nucleotide databases. Anal Chem, 67, 3202–3210.
55.
ZwiebelLJ, and TakkenW. (2004). Olfactory regulation of mosquito-host interactions. Insect Biochem Mol Biol, 34, 645–652.
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