Salivary gland proteins of
Malaria has been prevalent for a long time in tropical developing regions causing great morbidity and mortality [
The salivary gland proteins are thus relevant for malaria research since the
Salivary gland tissues of
Unfortunately, to date, only limited studies exist to efficiently explore molecular interactions and role of salivary gland proteins of the mosquito and the sporozoites of the
Here we describe an in-gel proteomic approach using 1D and LC-MS/MS to characterize the proteome of the salivary gland extracts (SGEs) of
A total of 100 pairs of salivary glands of female
SGE samples were first fractionated on SDS-PAGE for separation. Briefly, 50–75
Proteins were reduced, alkylated with iodoacetamide, and digested with trypsin overnight at 37°C. Briefly, the excised gel slices were subjected to reduction and were dried in a vacuum centrifuge. DTT (10 mM) in ammonium bicarbonate (100 mM) was added to gel pieces and proteins were reduced for 1 hour at 56°C. After cooling to room temperature reduced proteins were alkylated with IAA (55 mM) in ammonium bicarbonate (100 mM) for 45 min at 25°C. After incubation in the dark with occasional vortexing the gel pieces were washed with 50–100
In-gel digested peptides were analyzed by nano LC-ESI-QTOF-MS/MS on a Bruker micrOTOF-Q II system. For LC-MS/MS analysis 15
Role of salivary glands and their proteins is important in the mosquito because parasites mature to form infectious sporozoites in salivary glands. Various active protein molecules must be annotated/expressed in salivary glands of mosquito which may help in food ingestion and digestion and facilitate blood feeding, immune defenses, and haemostasis [
Mass-spectrometry-based proteomics is now a powerful and reliable method that allows characterization of protein assemblies, and when this is combined with molecular, cellular, and bioinformatics techniques it provides a framework for translating complex molecules into simple molecules for in-depth analysis of expressed proteomes [
Availability of genome sequence for mosquito
In the present study, we employed a MS-based approach to categorize different putative functional proteins of salivary glands of an urban malaria vector
A catalogue of known proteins identified by using in-gel digestion strategy and LC/MS/MS using MASCOT algorithm.
S. number | Accession number/vector base accession number | Protein | Band number | Mol. weight | Peptides | Calculated pI | Sequence coverage | Domain/function |
---|---|---|---|---|---|---|---|---|
1 | gi: 37201975 | GE rich salivary protein | 16 | 15214 | 8 | 5.15 | 56% | No conserved domain |
2 | gi: 15718081 | D7 protein | 11 | 36396 | 14 | 8.79 | 34% | Protein |
3 | gi: 29501536 | SG1D salivary protein precursor | 7 | 46811 | 10 | 9.38 | 23% | No conserved domain |
4 | ASTM013042-PA | G1 family long form salivary protein 3 | 7 | 45829 | 9 | 6.85 | 22% | No conserved domain |
5 | gi: 27372941 | Putative salivary protein SG1C | 7 | 44292 | 8 | 6.73 | 16% | No conserved domain |
6 | gi: 27372911 | Salivary apyrase | 4 | 64248 | 8 | 6.77 | 12% | No conserved domain |
7 | ASTM006960-PA | Alpha amylase | 3 | 67923 | 2 | 5.27 | 4% | Alpha amylase |
8 | ASTM007102-PA | Salivary peroxidase | 3 | 67504 | 6 | 8.75 | 10% | Heme |
9 | gi: 27372939 | Putative salivary protein SG1A | 15 | 19725 | 2 | 4.94 | 11% | Nucleotide transport and metabolism |
10 | gi: 27372929 | Putative salivary protein SG1B | 6 | 48120 | 2 | — | 4% | No conserved domain |
11 | gi: 29501376 | Short D7-4 salivary protein precursor | 15 | 18412 | 1 | — | 7% | No conserved domain |
12 | gi: 27372895 | Salivary antigen-5 related protein | 14 | 28974 | 2 | 9.05 | 8% | CTD-interacting domain (polypeptide binding) |
13 | gi: 29501528 | TRIO salivary gland protein precursor | 7 | 44013 | 3 | 7.01 | 3% | SCP-like extracellular protein domain |
A catalogue of novel proteins identified by using in-gel digestion strategy and LC/MS/MS using MASCOT algorithm.
S. number | Accession number | Protein | Band number | Mol. weight | Peptides | Calculated pI | Sequence coverage | Domain/function |
---|---|---|---|---|---|---|---|---|
1 | gi: 94468834 | FOF1-type ATP synthase beta subunit (similar to |
5 | 53937 | 16 | 5.03 | 22% | Nucleotide-binding domain |
2 | gi: 170059752 | Histone H4 (similar to |
16 | 11374 | 2 | 11.36 | 21% | Nucleosome assembly |
3 | gi: 118784826 | AGAP005078-PA (similar to |
16 | 13032 | 2 | 11.16 | 13% | No conserved domain |
4 | gi: 347963754 | AGAP000403-PA (similar to |
15 | 19831 | 1 | 10.37 | 6% | Nucleotide binding |
5 | gi: 356578763 | Copper/zinc superoxide dismutase 3B (similar to |
16 | 15646 | 1 | 5.94 | 6% | Ion binding |
6 | gi: 118782571 | AGAP002575-PA (similar to |
14 | 27619 | 1 | 5.03 | 3% | Leucine rich repeats |
7 | gi: 129716442 | Rps7 (fragment) OS similar to |
16 | 15374 | 1 | 9.85 | 3% | Translation |
8 |
|
Molybdenum cofactor sulfurase 2 (similar to |
2 | 85615 | 2 | 6.76 | 3% | Pyridoxal phosphate- (PLP-) dependent enzymes |
9 | gi: 158285167 | AGAP007706-PA (kinesin-like protein) | 1 | 99191 | 3 | 9.2 | 2% | ATP activity |
10 | gi: 158299522 | Tetraspanin protein (similar to |
13 | 29075 | 1 | 8.96 | 2% | No conserved domain |
11 | gi: 58391886 | AGAP009833-PA (similar to |
13 | 30740 | 2 | 8.64 | 2% | Porin |
12 | gi: 170037149 | Apoptosis inhibitor (similar to |
4 | 61233 | 2 | 6.46 | 1% | No conserved domain |
Salivary gland protein profiling of
Different proteins are also assigned according to gel bands. In Table
Among all identified proteins by LC/MS/MS, further conserved domains were searched by using NCBI domain programs (
Among the known proteins, GE rich salivary gland protein was found with 56% sequence similarity with the highest score (609) and a total of 8 peptide matches. Further signal peptide for GE rich salivary gland protein was identified at amino acid positions 1 to 19 which depicts a secreted protein (Figure
Annotated sequences of salivary protein precursors from
A sort of salivary gland proteins termed as SG1 family [
13 novel hypothetical proteins were identified by MASCOT analysis that has features similar to proteins in other mosquito species like
All 16 digested samples were also analyzed by OMSSA algorithm after MS/MS analysis (
A catalogue of novel proteins identified by using in-gel digestion strategy and LC/MS/MS using OMSSA algorithm.
S. number | Accession | Features | MW | % Seq coverage |
|
Domain |
---|---|---|---|---|---|---|
1 | gi: 224037899 | Gambicin (similar to |
3373.62 | 38% | 0.02 | No conserved domain |
2 | gi: 126680249 | Unknown (similar to |
5546.78 | 34% | 0.02 | No conserved domain |
3 | gi: 53771806 | Glutaredoxin (similar to |
1932.89 | 34% | 0.005 | GRX domain |
4 | gi: 126680357 | Unknown (similar to |
3266.66 | 33% | 0.045 | No conserved domain |
5 | gi: 187440102 | CLIPB7 protein (similar to |
4640.28 | 31% | 0.01 | Clip domain |
6 | gi: 31281916 | Xanthine dehydrogenase (similar to |
1641.8 | 29% | 0.05 | Fe-S cluster binding domain |
7 | gi: 37576232 | Defender against programmed cell death (similar to |
3491.73 | 28% | 0.05 | Integral membrane protein |
8 | gi: 5834921 | ND4L gene product (mitochondrion) (similar to |
3456.69 | 28% | 0.01 | Oxidoreductases |
9 | gi: 54124659 | Peroxidase 12 (similar to |
3793.88 | 27% | 0.009 | Peroxidase domain |
10 | gi: 187440702 | GNBPB1 protein (similar to |
3776.06 | 26% | 0.02 | No conserved domain |
11 | gi: 87080401 | Putative TIL domain polypeptide (similar to |
3276.31 | 25% | 0.03 | Trypsin inhibitor-like cysteine rich domain |
12 | gi: 3139135 | Defensin (similar to |
2263 | 23% | 0.004 | Defensin superfamily |
13 | gi: 54124633 | Peroxidase 1 (similar to |
1978.95 | 23% | 0.02 | Animal heme peroxidases |
14 | gi: 281186343 | Peptidoglycan recognition protein 3 short class (similar to |
4460.29 | 22% | 0.002 | Pattern recognition receptor |
15 | gi: 18139597 | Cytochrome P450 CYP4C28 (similar to |
3816.62 | 22% | 0.01 | cypX domain |
16 | gi: 187340440 | PGRPS1 protein (similar to |
1596.76 | 21% | 0.001 | Peptidoglycan recognition proteins (PGRPs) |
17 | gi: 7716428 | Thioredoxin 1 (similar to |
2425.01 | 21% | 0.02 | TRX domain |
18 | gi: 37677930 | agCP14332 (similar to |
2083.04 | 21% | 0.001 | No conserved domain |
19 | gi: 158452713 | Caspase short class, partial (similar to |
1869.74 | 21% | 0.038 | No conserved domain |
20 | gi: 48994192 | Putative odorant-binding protein OBPjj9 (similar to |
3964.71 | 20% | 0.02 | Olfactory receptor, OBP |
21 | gi: 6635469 | Immune-responsive trypsin-like serine protease-related protein ISPR10 (similar to |
2705.24 | 20% | 0.004 | No conserved domain |
22 | gi: 187441150 | SCRB2 protein (similar to |
2410.2 | 19% | 0.05 | Scavenger receptor |
23 | gi: 40019419 | Odorant-binding protein OBP5470 (similar to |
3655.7 | 18% | 0.01 | No conserved domain |
24 | gi: 28396160 | Putative antennal carrier protein AP-1 (similar to |
2793.37 | 18% | 0.03 | No conserved domain |
25 | gi: 310756184 | AGAP005196 (similar to |
2911.43 | 18% | 0.02 | Tryp_SPc domain |
26 | gi: 13509402 | Hypothetical protein (similar to |
2117 | 18% | 0.02 | No conserved domain |
27 | gi: 37703114 | Odorant receptor 1 (similar to |
4085.1 | 17% | 0.002 | Transmembrane receptor |
28 | gi: 187441612 | TEP2 protein (similar to |
3276.31 | 16% | 0.03 | Terpene cyclases domain |
29 | gi: 2564570 | NADH dehydrogenase subunit 5 (similar to |
4157.12 | 16% | 0.003 | Ubiquitin/PQ complex |
30 | gi: 187440738 | CLIPB13 protein (similar to |
2515.41 | 14% | 0.05 | Trypsin like serine protease |
31 | gi: 311985 | ANG12 precursor (similar to |
3171.52 | 14% | 0.06 | Insect allergen related repeat |
32 | gi: 187441890 | SCRBQ2 protein (similar to |
2356.32 | 14% | 0.001 | CD36 family |
33 | gi: 187444412 | FBN9 protein (similar to |
2425.3 | 14% | 0.01 | No conserved domain |
34 | gi: 1495237 | GSTD2 protein (similar to |
3391.77 | 14% | 0.005 |
|
35 | gi: 1369924 | Immune factor (similar to |
2675.3 | 13% | 0.07 | Rel homology domain |
36 | gi: 19071278 | Odorant-binding protein (similar to |
2129.03 | 13% | 0.06 | Olfactory receptor |
37 | gi: 117957967 | Beta carbonic anhydrase (similar to |
3784.98 | 13% | 0.008 | Carbonic anhydrase domain |
38 | gi: 33355867 | Odorant-binding protein AgamOBP52 (similar to |
2645.31 | 13% | 0.002 | No conserved domain |
39 | gi: 12007372 | Glutathione S-transferase E1 (similar to |
2884.43 | 12% | 0.001 | GSTC Delta epsilon |
40 | gi: 1245442 | Putative arylphorin precursor, partial (similar to |
2681.36 | 11% | 0.02 | Copper containing protein |
41 | gi: 240270034 | Serpin 7 inhibitory serine protease inhibitor (similar to |
3358.47 | 11% | 0.002 | Proteinase inhibitors |
42 | gi: 157042594 | suppressor of cytokine signaling 5 (similar to |
2507.2 | 11% | 0.03 | SH2 domains |
43 | gi: 169260669 | Vasa (similar to |
1458.78 | 11% | 0.03 | Helicase C terminal domain |
44 | gi: 71841593 | pk-1 receptor (similar to |
4041.93 | 11% | 0.001 | G protein coupled receptor |
45 | gi: 853701 | Serine proteinase (similar to |
2735.34 | 11% | 0.02 | Trypsin-like serine protease |
46 | gi: 1256440 | Hexamerin (similar to |
3847.48 | 5% | 0.03 | Hemocyanin Ig-like domain |
During LC/MS/MS analysis, one of the peptides eluted with an amino acid sequence NWATSGETVDECLEEMAGSACEQAYFFTRCVMTR was matched to putative odorant-binding protein OBPjj9 (
Another peptide with amino acid sequence LMTYFDYFDSDVSNVLPMQSTDKYFDYAVFAR was identified, that is, hexamerin, with signal peptide at position 1 to 18 (Figure
Peak spectrum analyzed by LC/MS/MS based on
A total of 36 known proteins and 123 novel proteins were identified from both MASCOT and OMSSA algorithm. Putative functional annotation according to both biological approach and cellular approach was prepared among the identified proteins. These were identified by GO analysis (
Subcellular location of each identified protein was assigned. We found most of the proteins localized in plasma membrane (31), extracellular (13), cytoplasm (11), mitochondria (10), nucleus (8), intracellular (5), cytoskeleton (8), and so forth. We are unable to find location of a large number of proteins that were assigned under unknown category (65) (novel or known) (Figure
Depiction of identified salivary proteins of
On the basis of biological approach, the majority of proteins were scrutinized marked for their role in signal transduction, metabolism, cytoskeleton protein, transcription and translational regulation, energy pathways, regulation of blood coagulation cascade and intracellular trafficking and transport, stress response, and so forth (Figure
Various proteins that play an important role in immune responses were identified such as defensins, fibrinogen binding proteins (FBN9), majority of serine proteases, CLIPB (CLIPB 14, CLIPB 15, CLIPB 7, and CLIPB 13), serine protease 14, immune factor (rel homology domain), and lysozyme c6. Such proteins may also be responsible for reduction in microbial load in ingested blood. Among them defensin protein in
Long lists of enzymes were also identified that function as vasodilators, that is, peroxidases [
Among different tables some proteins with sequence coverage below 5% were identified which were actually not native proteins; in fact they are degraded product of the putative proteins.
We also presented the STRING network of some known/novel protein-protein interactions as an evidence view by using String 9.0 (Search Tool for the Retrieval of Interacting Genes/Proteins) database of physical and functional interactions (
STRING network of protein-protein interactions identified by OMSSA algorithm. (a) Protein Q9NGZ1_ANOGA is shown as TRX1 (thioredoxin 1) in the network showing functional interactions with other proteins. (b) Protein Q7QCC4_ANOGA is shown as OBPjj9 (odorant-binding protein). (c) Protein Q7QHS7_ANOGA is shown as pofut1 (O-fucosyltransferase 1 protein). Different line colors represent the types of evidence for the association. Green color depicts neighborhood; red color: gene fusion; pink color: experiments; light green color: text mining; blue color: cooccurrence; dark blue color: coexpression; purple color: homology. Circle nodes indicated different proteins.
Mass spectrometric based proteomics techniques coupled with high throughput bioinformatic analysis are a powerful platform to understand comprehensive biology and interaction of functional proteins. Salivary gland proteins of the
Such proteins may be used for development of novel antimalarial control strategies for improving innate protection against malaria and help to elucidate the various aspects of salivary gland-malaria parasite interactions.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Sonam Vijay carried out the experiments, participated in the data analysis, and drafted the paper. Manmeet Rawat participated in interpretation of data. Arun Sharma helped in design of the project, provided facilities and scientific environment for experimental work, and drafted the paper. All authors read and approved the final paper.
The authors would like to acknowledge their technical staff, Mr. Bhanu Arya (Technical Officer) and Mrs. Poonam Gupta (Technical Assistant), for their excellent technical help in proteomics experiments. This work has been financially supported by the National Institute of Malaria Research (Intramural), New Delhi, Government of India.