archives of oral biology 59 (2014) 302–309
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Isolation, biochemical characterization and anti-bacterial activity of BPIFA2 protein Vladimir Prokopovic a, Milica Popovic a, Uros Andjelkovic b, Aleksandra Marsavelski a, Brankica Raskovic a, Marija Gavrovic-Jankulovic a, Natalija Polovic a,* a Faculty of Chemistry, Department of Biochemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia b Institute for Chemistry, Technology and Metallurgy, Department of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
article info
abstract
Article history:
Objective: Human BPIFA2 (parotid secretory protein) is a ubiquitous soluble salivary protein,
Accepted 15 December 2013
which belongs to the PLUNC family of proteins. Having sequence similarity to bactericidal/ permeability-increasing protein and lipopolysaccharide-binding protein, PLUNC proteins
Keywords:
are probably involved in local antibacterial response at mucosal sites, such as oral cavity.
Parotid secretory protein
The aim of the study was to isolate and characterize human BPIFA2.
BPIFA2
Design: In this paper, we report one-step affinity chromatography method for BPIFA2
Saliva
purification from whole human saliva. The isolated BPIFA2 was identified by trypsin mass
Bactericidal activity
fingerprinting and characterized by electrophoretic methods. Antibacterial activity of BPIFA2 against model microorganism Pseudomonas aeruginosa was shown in minimum inhibitory concentration and time kill study assays. Results: The protein showed microheterogeneity, both in molecular weight and pI value. BPIFA2 inhibited the growth of P. aeruginosa in microgram concentration range determined by minimum inhibitory concentration assay. In the time kill study, 32 mg/mL BPIFA2 showed clear bactericidal activity and did not cause any aggregation of bacteria. Conclusion: Affinity chromatography is well suited for isolation of functional BPIFA2 with a potent bactericidal activity against P. aeruginosa. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Saliva is a colourless fluid whose content includes mainly water, sodium, potassium, calcium, bicarbonate and chloride ions. However, around 2% of saliva are proteins, many of which have an unknown function.1 Proteins such as alkaline salivary proteins, immunoglobulin A, statherine, acidic salivary proteins, amylase, high and low molecular weight mucins, cistatines, histatines and lipases are just some of the 1200 proteins known to compose saliva.2
Human BPIFA2 (parotid secretory protein – PSP) is a soluble salivary protein expressed in salivary glands and gingival epithelial cells.3–5 The protein has a molecular weight of 25 kDa and was first identified as a product of parotid salivary glands in mice and rats.6 Up to now, orthologous BPIFA2 proteins were found in saliva of mice,7 rat,8 hamster,9 pig,10 cattle11 and dog12 and BPIFA2 related genes were identified in genomes of chimpanzee, orangutan, rhesus macaque, marmoset and giant panda.13 Presence of BPIFA2 in different (evolutionary remote) species suggests that this protein could potentially exert an important role for salivary function.
* Corresponding author. Tel.: +381 113336721; fax: +381 112184330. E-mail address: [email protected] (N. Polovic). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.12.005
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BPIFA2 belongs to the PLUNC protein family (PLUNC – palate, lung and nasal epithelium clone). Human BPIFA2 gene (c20orf70) is located in the PLUNC locus. The PLUNC family comprises of nine genes located in a single locus on chromosome 20 which are expressed in the embryonic palate, nasal epithelium and trachea.14 In the past decade in related literature duplicate names for PLUNC proteins were used, but the systematic nomenclature has been recently established.15 According to the present nomenclature, human parotid secretory protein (PSP or SPLUNC2) is designated as BPIFA2. PLUNC proteins have sequence similarity to the related proteins, lipopolysaccharide-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI), key members of the systemic innate immune response to Gram-negative bacteria.16 From the pattern of gene expression and the predicted structure, it is stipulated that PLUNC proteins may play a key role in the local antibacterial response in nose, mouth and upper respiratory pathways. Human BPIFA2 has recently been expressed in the rat cell line GH4C1 and was shown that it possesses antibacterial activity against Pseudomonas aeruginosa.17 Human BPIFA2 can bind bacterial lipopolysaccharide (LPS).18 PLUNC proteins are hydrophobic (leucine, isoleucine and valine residues represent 34% of total aminoacid content of PLUNC), soluble in 75% ethanol solutions.19 The general method of fractionation of PLUNC proteins from bronchoalveolar lavage (BAL) fluids comprise of several precipitation steps, yielding the mixture of PLUNC proteins.19 In the case of purification of BPIFA2 from human saliva, the obtained ethanol soluble protein fraction contained BPIFA2 but also a number of non-PLUNC salivary proteins.18 Immunoaffinity chromatography of human saliva had also been used in BPIFA2 purification with the antibodies raised against C-terminal BPIFA2 peptide. The purified protein could be detected in immunoblot but the obtained quantities were insufficient for precise identification and functional studies.17 In this report we established one step procedure for purification of salivary human BPIFA2. Since it has been recently shown that BPIFA2 can bind LPS,18 we tested whether BPIFA2 could be purified by LPS-affinity chromatography. Furthermore, LPS-free affinity chromatography support was synthesized and used in BPIFA2 purification from the whole human saliva. Purified BPIFA2 was characterized by means of electrophoretic behaviour and antibacterial activity.
2.
Materials and methods
2.1.
Chemicals
Bovine serum albumin (BSA), sequencing grade trypsine, LPS from Escherichia coli 0111:B4 and octadecylamine (ODA) were purchased from Sigma–Aldrich (Steinheim, Germany), Sepharose 6B was obtained from GE Healthcare (Uppsala, Sweden). All other chemicals were of analytical grade and were used without further purification.
2.2.
Saliva collection
Whole saliva samples were collected from 10 healthy volunteers, 5 males (mean age 26.0 1.7) and 5 females
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(mean age 27.8 3.5). All volunteers gave informed consent to participate in the research study. All volunteers were healthy and free of oral diseases. Eating and drinking was not permitted for 2 h prior to sampling. Saliva was collected using mechanical stimulation i.e. chewing action. The equal volumes of the salivary samples were mixed to obtained mixed human saliva. Mixed saliva was diluted with the equal volume of 100 mM Na–phosphate buffer pH 7.4 and filtered three times on Bu¨chner funnel through the Whatman No. 54 filter paper. The filtrates were immediately used in affinity chromatography.
2.3.
Protein concentration
Total protein concentration in the whole mixed human saliva and the concentration of the purified protein were determined using both Lowry and Bradford method with bovine serum albumin (BSA) as standard.20
2.4.
Chromatography
2.4.1.
Affinity matrix preparation
Preswollen Sepharose 6B beads were washed with water and 0.5 M Na–phosphate buffer pH 7.4. Sepharose beads were mixed with one volume of 25% glutaraldehyde and incubated overnight at 37 8C with moderate shaking. Water suspensions of E. coli LPS or ODA (both in concentration 0.67 mg per 1 mL of matrix) were added and left to incubate overnight at 37 8C with agitation. Synthesized matrices were washed with 0.5 M Na–phosphate buffer pH 7.4 and afterwards with 50 mM Na–phosphate buffer pH 7.4 containing 1 M NaCl on Bu¨chner funnel through the Whatman No. 54 filter paper. Two volumes of 100 mM ethanolamine were added to block residual reactive glutaraldehyde groups. The blocking reaction proceeds overnight at 37 8C with moderate shaking. A schematic representation of affinity beads preparation is presented below.
2.4.2.
Affinity chromatography
One millilitre of ODA- or LPS-Sepharose matrix was washed with 20 column volumes (CV) of water and pre-equilibrated with 20 CV of 100 Na–phosphate buffer pH 7.4. Twenty CV of saliva filtrates (corresponding to 10 mL of mixed human saliva) were applied to column with peristaltic pump (flow rate 60 mL/h). The flow-through was applied twice on the column. The unbound salivary proteins were eluted with 30 CV of 100 mM Na–phosphate buffer pH 7.4. Bound protein fraction was eluted with 20 CV of 100 mM Na– phosphate buffer pH 7.4 containing 0.2% SDS. Collected fractions (5 CV) were analyzed by measuring A280 nm. Fractions containing bound proteins were pooled and stored at 20 8C.
2.5.
Biochemical methods
2.5.1. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) The presence of protein in fractions collected after affinity chromatography was analyzed by SDS-PAGE.
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SDS-PAGE was performed using a discontinuous buffer system according to the method of Laemmli21 using 12% polyacrylamide gel as the separating gel. Protein fractions were mixed with 5 times concentrated Laemmli non-reducing sample buffer for SDS-PAGE and incubated for 5 min at 95 8C. Twenty-five microlitres of each sample was applied per lane. The gels were run under constant current of 35 mA per gel in a Hoefer Dual Gel Caster Mighty small SE 245 system (Hoefer, Holliston, MA, USA). After electrophoresis, SDS-PAGE gel was stained with silver, except in the case of band excision for proteomic analysis where it was stained with Coomassie Brilliant Blue R250 (CBB). In the case of examining the electrophoretic behaviour in reducing and non-reducing conditions, ODA-purified protein sample was prepared both in reducing and non-reducing sample buffer, respectively. Molecular weights were determined using Unstained Protein MW Marker (Thermo Fisher Scientific, Rockford, IL, USA) and Prestained Precision Plus ProteinTM All Blue Standards (BioRad, Hercules, CA, USA).
2.5.2. 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) In the case of 2D-PAGE electrophoresis, the first dimension was carried out as native isoelectric focusing.20 Isoelectric focusing was run in 7% polyacrylamide gel containing 4% wide range ampholytes for 2D electrophoresis (Serva, Heidelberg, Germany). Twenty microlitres of the protein purified in ODA-Sepharose affinity chromatography was mixed with an equal volume of 2 times gel loading buffer (1% ampholytes and 0.1% bromphenol blue solution in Milli Q water). Thirty microlitres of the sample (0.6 mg of the protein) was applied per lane. The isoelectric focusing was run for 30 min at 150 V followed with additional 2.5 h at 200 V. After the focusing, the pH gradient was determined by cutting the empty gel stripe into 0.5 cm slices. Each slice was incubated in 10 mM KCl for 30 min and pH value of KCl solutions was measured. The stripe of the gel with the resolved protein sample was incubated in Laemmli nonreducing sample buffer for SDS-PAGE for 30 min and loaded on the top of the separating gel for SDS-PAGE analysis. SDSPAGE was run as described above and gel was silver stained afterwards.
2.5.3.
Trypsin mass fingerprinting (TMF)
Protein spots were excised from CBB stained gel to perform ingel trypsin digestion according to the manufacturer instructions. Obtained peptides were injected onto a reversed phase column (ACQUITY UPLC BEH 130, 1.7 mm, C18 2.1 mm 50 mm, Waters, Milford, MA, USA) installed on Acquity UPLC (ultra-performance liquid chromatography) system (Waters, Milford, MA, USA) and separated using acetonitrile gradient (0–50% for 40 min with 0.1% formic acid). After detection with photodiode array (UV) at 280 nm mass spectra were recorded on triple-quadrupole mass spectrometer (ACQUITY TQD, Waters, Milford, MA, USA) in positive ion mode with capillary voltage of 3 kV and drying gas flow rate of 8.3 L/min at 250 8C. The scan range was set from 400 to 2000m/z. The peptide masses were searched against the SwissProt protein sequence database using the MASCOT programme.
2.6.
Microbiology
2.6.1. Bacterial growth inhibitory assay and minimum inhibitory concentration (MIC) determination Strain of P. aeruginosa ATCC 27853 was kindly provided by Dr. Gordana Gojgic-Cvijovic, Department of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade. MIC was measured by broth microdilution according to the CLSI M7-A7 with slight modifications.22 Briefly, cells were grown on LB agar plates at 37 8C for 24 h before use. Inoculum was prepared by transferring single colony from the agar plate with a sterile tip to Luria–Bertani (LB) liquid medium and growing on 37 8C overnight. Inocula were diluted in LB liquid medium to approximately 105 colony forming units (cfu) and then grown in the presence of increasing BPIFA2 concentrations (0, 0.5, 1, 2, 4, 8, 16 and 32 mg/mL). The incubation was carried out in sterile 96 well microtiter plates and bacterial growth was monitored by measuring the absorbance (optical density, OD) at 620 nm using a LKB Micro plate reader 5060-006 (GDV, Roma, Italy). Results were expressed as percentage of bacterial growth compared to the control sample incubated in the appropriate buffer in the absence of BPIFA2 (100 mM Na–phosphate buffer pH 7.4 containing 0.2% SDS added to bacterial culture). The mean growth values of triplicates were obtained and then converted to the inhibition percentage of cell growth in relation to the control treatment by using the formula, MGI(%) = [(dc dt)/ dc] 100, where dc and dt represent A620 nm in control and treated wells, respectively. The first well in the series with no visible growth after the incubation period was taken as the MIC. The experiments were performed twice independently.
2.6.2.
Time-kill study
Time-kill assays were performed according to the guideline M26-A23 of the CLSI with the following change: polypropylene 1.5 mL microtubes were used for the time-kill assays with P. aeruginosa. Protein sample (40 mg/mL in 100 mM Na–phosphate buffer pH 7.4 containing 0.2% SDS) and control (100 mM Na– phosphate buffer pH 7.4 containing 0.2% SDS) were prepared by mixing 9 volumes of the sample with 1 volume of 10 times concentrated sterile LB media. Exponential-phase P. aeruginosa was diluted to 5 107 cfu/mL and exposed to BPIFA2 (final concentration 32 mg/mL after addition of LB media and bacterial culture) over a 24 h period in a total volume of 400 mL of appropriate media. Viable cell count was determined by plating serial dilution (onto LB agar). Bactericidal activity was defined as a 3 log decrease in cell counts.23 Time-kill assays were performed twice independently. Mean values of triplicate cfu/mL measurements from a single experiment (SD) are plotted.
2.6.3.
Slide-agglutination assay
Possible agglutination of P. aeruginosa in the presence of BPIFA2 was monitored as previously described.24 Briefly, bacterial agglutination is performed using exponential-phase culture of P. aeruginosa cells centrifuged and resuspended in saline to the concentration of 5 108 cfu/mL. The influence of BPIFA2 on bacterial agglutination was tested at MIC concentration of BPIFA2. Two microlitres of the cell suspension was mixed with 8 mL of appropriate concentration of
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BPIFA2 on the glass slide, left in humid atmosphere from 1 min to 2 h. After incubation, cells were fixed on the flame, visualized with 0.3% Methylene blue in 30% ethanol and observed by light microscopy under magnification of 200 times. The slides were photographed using a digital camera Olympus m-mini (2272 1704 pixels) and converted to black and white images in GIMP (GNU Image Manipulation Programme).
3.
Results
3.1.
Protein concentration in mixed human saliva
The protein concentrations in mixed human saliva were estimated by both Bradford and Lowry method using BSA as protein standard. The obtained concentrations were 0.7 0.2 mg/mL and 1.1 0.4 mg/mL respectively and were comparable to those published by Jenzano et al.25
3.2.
Purification of human BPIFA2
Matrices used for BPIFA2 purification from whole saliva were synthesized by coupling LPS and ODA, respectively, to Sepharose resulting in LPS-Sepharose and long hydrophobic chain affinity matrix, ODA-Sepharose (Scheme 1). Fig. 1 shows affinity chromatograms of BPIFA2 purification using both affinity matrices. Protein elution from LPS column was achieved immediately after including detergent SDS to the elution buffer. Identical chromatogram was achieved using ODA-Sepharose. The BPIFA2 yield in relation to total protein content of both LPS and ODA chromatography was around 5% (540 mg of purified protein was isolated from 10 mL of saliva). Fractions obtained by LPS and ODA-Sepharose affinity chromatography were analyzed by SDS-PAGE. Gels were silver-stained and presented in Fig. 2. The protein of around 25 kDa is present in bound fraction corresponding to the bound peak in affinity chromatographies. Also, a minor protein band of about 20 kDa was visible in both samples.
Fig. 1 – Fractionation of the whole human saliva on LPSSepharose (grey line) and ODA-Sepharose (black line) columns. Human saliva (10 mL), collected from healthy volunteers, was used as starting material for BPIFA2 purification. Unbound protein fraction was eluted with Buffer A (100 mM phosphate buffer pH 7.4). Bound protein elution at 30 CV was achieved in Buffer B (Buffer A containing 0.2% SDS). Fractions of 5 CV were collected at a flow rate of 60 mL/h and analyzed by absorbance measurements at 280 nm.
ODA-Sepharose purified protein was used in consequent analysis.
3.3.
Proteomic analysis of the purified BPIFA2
After SDS-PAGE and CBB staining of protein isolated by ODASepharose affinity chromatography, we sought to identify the isolated protein bands of 25 kDa and 20 kDa by proteomic analysis after trypsin digestion. According to Mascot, protein scores greater than 70 were considered as significant
Scheme 1 – Affinity matrix synthesis.
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Fig. 2 – SDS-PAGE analysis of LPS-Sepharose fractions (left) and ODA-Sepharose fractions (right). Bound protein fractions eluted in phosphate buffer containing 0.2% SDS are marked with asterisk. Lane M corresponds to molecular weight standards.
( p < 0.05). BPIFA2 was identified by 10 matching peptides, with sequence coverage of 31%, as the only significant hit (Table 1). Band of 20 kDa (Fig. 2) was also identified as BPIFA2 (identified peptides matched those presented in Table 1 and covered the sequence form amino acid positions G53 to K121) and probably correspond to a proteolytically processed protein.
3.4.
Biochemical characterization of the purified BPIFA2
The concentration of purified BPIFA2 was determined by both and Lowry method. The results were Bradford 0.000 0.007 mg/mL and 0.040 0.005 mg/mL, respectively. It appears that BPIFA2 cannot be detected in Bradford method. Nevertheless, the protein concentration could be estimated by Lowry method. In order to check if the reduction of disulfide bonds affects electrophoretic mobility, protein isolated by ODA-Sepharose affinity chromatography was prepared for SDS-PAGE both in reducing and non-reducing conditions. Results are shown in Fig. 3A. Protein does not exhibit differential electrophoretic mobility regardless of the reduction of disulfide bonds indicating the limited number or a complete absence of disulfide bridges.
The isolated protein molecular weight and pI value were determined in 2D-PAGE (Fig. 3B). The molecular weight of around 25 kDa is in good agreement with theoretical molecular mass for the soluble protein after the proteolysis of the signal peptide. However, it could be observed that the protein showed microheterogeneity in both SDS-PAGE and 2D-PAGE regarding both molecular weights and pI values. At least two different forms are present in the sample. The less abundant protein spot had a molecular weight below 25 kDa and a pI value of 5.3 (estimated by 2D PAGE). The more abundant spot showed molecular weight of about 25 kDa and acidic pI value of about 4.9 (estimated by 2D PAGE).
3.5.
Antibacterial activity of BPIFA2
The isolated BPIFA2 inhibited P. aeruginosa growth in low concentration range. MIC was identified at a mass concentration of 32 mg/mL. The EC50 value was identified in two separate experiments set with two different batches of ODA-Sepharose purified BPIFA2. The values did not differ significantly ( p > 0.05) and were found to be 3.7 0.2 mg/mL and 3.5 0.2 mg/mL, respectively. The dependence of % of bacterial growth from BPIFA2 concentration is shown in Fig. 4A.
Table 1 – Peptide mass fingerprint of the isolated BPIFA2. Values given in the brackets correspond to the protein without the signal peptide. Gel band
Protein names
Match-ing species
25 kDa
BPIFA2 BPIA2 SPLUNC2 PSP
Homo sapiens
Protein score P < 0.05 94
Peptide matched in peptide mass fingerprint 53
GILEKLK59 LKVDLGVLQK67 58 LKVDLGVLQKSSAWQLAK75 68 SSAWQLAKQK77 68 SSAWQLAKQKAQEAEK83 76 QKAQEAEK83 78 AQEAEK83 104 ISNSLILDVKAEPIDDGK121 206 STVSSLLQK214 222 IFIHSLDVNVIQQVVDNPQHK 242 58
Number of Theoretical Theoretical Sequence matched molecular pI value coverage peptides weight (Da) (%) 10
27011 (25054)
5.35 (5.19)
31 (35)
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Fig. 3 – (A) SDS-PAGE analysis of isolated BPIFA2 in non-reducing (NR) and reducing conditions (R) on 12% polyacrylamide gel. Lane M corresponds to molecular weight standards. (B) 2D-PAGE of BPIFA2. The first dimension was run as native isoelectric focusing, and second dimension was run as SDS-PAGE on 12% polyacrylamide gel.
Fig. 4 – Antibacterial activity of BPIFA2. (A) Inhibition of P. aeruginosa growth with BPIFA2 was monitored by measuring A620 nm in the presence of growing concentrations of BPIFA2. Results are presented as triplicates’ mean value W standard deviation. (B) Graph for time-kill assay of P. aeruginosa incubated with 32 mg/mL PSP. Bactericidal activity of BPIFA2 was determined by counting the number of colony forming units after incubation with BPIFA2 at MIC for 2, 6 and 24 h.
The results of time-kill studies are presented in Fig. 4B. Bactericidal activity of BPIFA2 was presented in terms of the log 10 cfu/mL change and is based on the conventional bactericidal activity standard, that is, 3 log 10 cfu/mL reduction in the viable colony number. The striking difference between cultures incubated in the presence and in the absence of BPIFA2 is noticeable after 6 h, when no cfu could have been detected in the BPIFA2 treated sample. Aggregation of P. aeruginosa cells in the presence of MIC concentration of BPIFA2 was monitored for up to 2 h. The bacterial aggregation could not be observed in the respected time range. The results presented in Fig. 5 are photographed after 1 h of incubation.
4.
Discussion
The affinity chromatography represents a simple, one-step method for isolation of a single protein from a complex mixture. The method is limited to biological pairs, except in the case of immunoaffinity methods. Despite successful
BPIFA2 purification, immunoaffinity chromatography was found to be inappropriate for large scale BPIFA2 production due to low protein yield.17 Also, because of unavailability of sufficient quantities of the whole protein, BPIFA2 peptides are usually used as immunogens for antibody production.4,17 It has been shown that antibodies raised against different BPIFA2 peptides differentially recognize BPIFA2 isoforms in saliva.4 Thus, in order to purify the functional protein, we sought to employ another strategy, affinity purification of BPIFA2 based on its ability to bind LPS hypothesizing that LPS binding is necessary for BPIFA2 function. For successful usage of affinity chromatography in purification of BPIFA2, it is necessary to identify and use the ligand that specifically binds BPIFA2, or to synthesize and use the close analogue of the ligand molecule. The structure and the biological role of human PLUNC proteins expressed in oral and upper airway mucosa were predicted and deduced mainly via indirect methodology. Thus, despite lacking the structural determinations, Bingle and colleagues16 suggested that PLUNC family members share the same structural features like distant relatives: bactericidal/permeability increasing protein and
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Fig. 5 – Agglutination assay of P. aeruginosa incubated for 1 h with 32 mg/mL BPIFA2. Control is incubated in buffer alone. Cells were visualized with Methylene blue staining.
lipopolysaccharide-binding protein. It has been recently shown that human BPIFA2 binds LPS via hydrophobic interactions.18 We exploited that finding by synthesizing affinity matrix based on LPS and ODA (Scheme 1) in order to affinity purify BPIFA2. Although LPS-Sepharose could be used for purification of BPIFA2 (Fig. 2A), we applied another strategy, keeping in mind LPS toxicity. ODA is a molecule that could mimic long hydrophobic chains of LPS (Scheme 1) and we showed that the parotid secretory protein (BPIFA2) can be successfully purified on the ODA-Sepharose column (Fig. 2B). The protein presence was authenticated by TMF (Table 1). Some of the unidentified peptides correspond to peptides with N-glycosylation sites.26 The purified protein exhibits different behaviour in two commonly used methods for protein concentration determination. Since saliva represents a complex mixture of proteins, which differ in their amino acid composition, it is already well known that determination of total protein concentration in mixed human saliva represents a formidable task. Acidic, basic, proline-rich proteins, or massively glycosylated proteins may react differently in both Biuret based methods (Lowry method) or dye-binding methods (Bradford method) for protein concentration determination.25 It appears that a significant portion of salivary proteins do not bind dye CBB, suggesting that Bradford method is not an appropriate one for the determination of total salivary protein concentration.25 Solubility of PLUNC proteins in ethanol-water mixtures could suggest that alcohol fixation and CBB-staining of SDSPAGE gels could not be the most sensitive methods for PLUNC identification. BPIFA2 did not show differential behaviour in reducing and non-reducing conditions, probably because the protein has just one disulfide bridge between Cys174 and Cys217, whose position is highly conserved in the whole PLUNC family.3
We detected at least two different spots corresponding to BPIFA2 in 2D-PAGE. Recombinant BPIFA2 produced in E. coli lacking post translational modifications exhibits a mass of 25 kDa17 and could represent the protein of 25 kDa and pI 4.9 that we detected in the isolated BPIFA2.4 Protein spot showing pI 5.3 and molecular weight below 25 kDa (Fig. 3B) could correspond to proteolytically processed BPIFA2. Despite many predictions and speculations, the exact function of BPIFA2 in saliva remains unknown. Robinson et al. showed in 1997 that murine BPIFA2 can bind E. coli.27 The studies with human BPIFA2 are limited just to recombinantly produced protein. In the case of mammalian expression of BPIFA2, cell culture medium of rat cell line GH4C1 transfected with BPIFA2 gene, limited the number of colonies of P. aeruginosa.17 However, the active concentration of recombinant BPIFA2 has been undetermined. BPIFA2 produced in inclusion bodies by expression in bacterial cells (E. coli) did not exhibit the bactericidal activity but preferably caused bacterial agglutination.13 However, the question of BPIFA2 purity, fold and concentration in the inclusion bodies solution remain unanswered. Heterologous production of human proteins in prokaryotic systems is often accompanied by misfolding, protein aggregation, and storage in the bacterial inclusion bodies,28 requiring subsequent solubilisation and troublesome in vitro refolding.29 The limitations of the host systems used in the recombinant production of BPIFA2 opens the door to the usage of native BPIFA2 in both structural and functional studies. Results presented in this paper proved that native human salivary BPIFA2 inhibited the growth of P. aeruginosa in microgram concentration range exerting bactericidal activity after 6 h of incubation without causing any visible aggregation of the bacterial cells thus designating this molecule an important tool in the future research of PLUNC protein family.
Funding This work was financially supported by the Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 172049.
Competing interests The authors have declared no conflict of interest.
Ethical approval The study did not require the approval of the ethics committee.
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BPI fold-containing superfamily. Biochem Soc Trans 2011;39(4):977–83. Bingle CD, Bingle L, Craven CJ. Distant cousins: genomic and sequence diversity within the BPI fold-containing (BPIF)/PLUNC protein family. Biochem Soc Trans 2011; 39(4):961–5. Geetha C, Venkatesh SG, Dunn BHF, Gorr SU. Expression and anti-bacterial activity of human parotid secretory protein (PSP). Biochem Soc Trans 2003;31(4):815–8. Abdolhosseini M, Sotsky JB, Shelar AP, Joyce PBM, Gorr SU. Human parotid secretory protein is a lipopolysaccharide-binding protein: identification of an anti-inflammatory peptide domain. Mol Cell Biochem 2012;359(1–2):1–8. Campos MA, Abreu AR, Nlend MC, Cobas MA, Conner GE, Whitney PL. Purification and characterization of PLUNC from human tracheobronchial secretions. Am J Respir Cell Mol Biol 2004;30(2):184–92. Bollag DM, Rozycki MD, Edelstein SJ. Protein methods. 2nd Ed. New York: Wiley-Liss; 1996. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680–5. Institute CLS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard M7A7. Wayne, PA, USA: CLSI; 2006. Standards NCCL. Methods for determining bactericidal activity of antimicrobial agents: approved guideline M26-A. Wayne, PA, USA: NCCLS; 1999. La´nyi B, Bergan T, Chapter IV Serological Characterization of Pseudomonas aeruginosa.Bergan T, Norris JR, editors. Methods in Microbiology, 10. Academic Press; 1978. p. 93–168.
http://dx.doi.org/10.1016/S0580-9517(08)70661-5. ISSN 0580-9517, ISBN 9780125215107. 25. Jenzano JW, Hogan SL, Noyes CM, Featherstone GL, Lundblad RL. Comparison of five techniques for the determination of protein content in mixed human saliva. Anal Biochem 1986;159(2):370–6. 26. Ramachandran P, Boontheung P, Xie YM, Sondej M, Wong DT, Loo JA. Identification of N-linked glycoproteins in human saliva by glycoprotein capture and mass spectrometry. J Proteome Res 2006;5(6):1493–503. 27. Robinson CP, Bounous DI, Alford CE, Nguyen KH, Nanni JM, Peck AB, et al. PSP expression in murine lacrimal glands and function as a bacteria binding protein in exocrine secretions. Am J Physiol 1997;272(4 Pt 1):G863–71. 28. Villaverde A, Carrio MM. Protein aggregation in recombinant bacteria: biological role of inclusion bodies. Biotechnol Lett 2003;25(17):1385–95. 29. Singh SM, Panda AK. Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 2005;99(4):303–10.
Available online at www.sciencedirect.com
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Isolation, biochemical characterization and anti-bacterial activity of BPIFA2 protein Vladimir Prokopovic a, Milica Popovic a, Uros Andjelkovic b, Aleksandra Marsavelski a, Brankica Raskovic a, Marija Gavrovic-Jankulovic a, Natalija Polovic a,* a Faculty of Chemistry, Department of Biochemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia b Institute for Chemistry, Technology and Metallurgy, Department of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
article info
abstract
Article history:
Objective: Human BPIFA2 (parotid secretory protein) is a ubiquitous soluble salivary protein,
Accepted 15 December 2013
which belongs to the PLUNC family of proteins. Having sequence similarity to bactericidal/ permeability-increasing protein and lipopolysaccharide-binding protein, PLUNC proteins
Keywords:
are probably involved in local antibacterial response at mucosal sites, such as oral cavity.
Parotid secretory protein
The aim of the study was to isolate and characterize human BPIFA2.
BPIFA2
Design: In this paper, we report one-step affinity chromatography method for BPIFA2
Saliva
purification from whole human saliva. The isolated BPIFA2 was identified by trypsin mass
Bactericidal activity
fingerprinting and characterized by electrophoretic methods. Antibacterial activity of BPIFA2 against model microorganism Pseudomonas aeruginosa was shown in minimum inhibitory concentration and time kill study assays. Results: The protein showed microheterogeneity, both in molecular weight and pI value. BPIFA2 inhibited the growth of P. aeruginosa in microgram concentration range determined by minimum inhibitory concentration assay. In the time kill study, 32 mg/mL BPIFA2 showed clear bactericidal activity and did not cause any aggregation of bacteria. Conclusion: Affinity chromatography is well suited for isolation of functional BPIFA2 with a potent bactericidal activity against P. aeruginosa. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Saliva is a colourless fluid whose content includes mainly water, sodium, potassium, calcium, bicarbonate and chloride ions. However, around 2% of saliva are proteins, many of which have an unknown function.1 Proteins such as alkaline salivary proteins, immunoglobulin A, statherine, acidic salivary proteins, amylase, high and low molecular weight mucins, cistatines, histatines and lipases are just some of the 1200 proteins known to compose saliva.2
Human BPIFA2 (parotid secretory protein – PSP) is a soluble salivary protein expressed in salivary glands and gingival epithelial cells.3–5 The protein has a molecular weight of 25 kDa and was first identified as a product of parotid salivary glands in mice and rats.6 Up to now, orthologous BPIFA2 proteins were found in saliva of mice,7 rat,8 hamster,9 pig,10 cattle11 and dog12 and BPIFA2 related genes were identified in genomes of chimpanzee, orangutan, rhesus macaque, marmoset and giant panda.13 Presence of BPIFA2 in different (evolutionary remote) species suggests that this protein could potentially exert an important role for salivary function.
* Corresponding author. Tel.: +381 113336721; fax: +381 112184330. E-mail address: [email protected] (N. Polovic). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.12.005
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BPIFA2 belongs to the PLUNC protein family (PLUNC – palate, lung and nasal epithelium clone). Human BPIFA2 gene (c20orf70) is located in the PLUNC locus. The PLUNC family comprises of nine genes located in a single locus on chromosome 20 which are expressed in the embryonic palate, nasal epithelium and trachea.14 In the past decade in related literature duplicate names for PLUNC proteins were used, but the systematic nomenclature has been recently established.15 According to the present nomenclature, human parotid secretory protein (PSP or SPLUNC2) is designated as BPIFA2. PLUNC proteins have sequence similarity to the related proteins, lipopolysaccharide-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI), key members of the systemic innate immune response to Gram-negative bacteria.16 From the pattern of gene expression and the predicted structure, it is stipulated that PLUNC proteins may play a key role in the local antibacterial response in nose, mouth and upper respiratory pathways. Human BPIFA2 has recently been expressed in the rat cell line GH4C1 and was shown that it possesses antibacterial activity against Pseudomonas aeruginosa.17 Human BPIFA2 can bind bacterial lipopolysaccharide (LPS).18 PLUNC proteins are hydrophobic (leucine, isoleucine and valine residues represent 34% of total aminoacid content of PLUNC), soluble in 75% ethanol solutions.19 The general method of fractionation of PLUNC proteins from bronchoalveolar lavage (BAL) fluids comprise of several precipitation steps, yielding the mixture of PLUNC proteins.19 In the case of purification of BPIFA2 from human saliva, the obtained ethanol soluble protein fraction contained BPIFA2 but also a number of non-PLUNC salivary proteins.18 Immunoaffinity chromatography of human saliva had also been used in BPIFA2 purification with the antibodies raised against C-terminal BPIFA2 peptide. The purified protein could be detected in immunoblot but the obtained quantities were insufficient for precise identification and functional studies.17 In this report we established one step procedure for purification of salivary human BPIFA2. Since it has been recently shown that BPIFA2 can bind LPS,18 we tested whether BPIFA2 could be purified by LPS-affinity chromatography. Furthermore, LPS-free affinity chromatography support was synthesized and used in BPIFA2 purification from the whole human saliva. Purified BPIFA2 was characterized by means of electrophoretic behaviour and antibacterial activity.
2.
Materials and methods
2.1.
Chemicals
Bovine serum albumin (BSA), sequencing grade trypsine, LPS from Escherichia coli 0111:B4 and octadecylamine (ODA) were purchased from Sigma–Aldrich (Steinheim, Germany), Sepharose 6B was obtained from GE Healthcare (Uppsala, Sweden). All other chemicals were of analytical grade and were used without further purification.
2.2.
Saliva collection
Whole saliva samples were collected from 10 healthy volunteers, 5 males (mean age 26.0 1.7) and 5 females
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(mean age 27.8 3.5). All volunteers gave informed consent to participate in the research study. All volunteers were healthy and free of oral diseases. Eating and drinking was not permitted for 2 h prior to sampling. Saliva was collected using mechanical stimulation i.e. chewing action. The equal volumes of the salivary samples were mixed to obtained mixed human saliva. Mixed saliva was diluted with the equal volume of 100 mM Na–phosphate buffer pH 7.4 and filtered three times on Bu¨chner funnel through the Whatman No. 54 filter paper. The filtrates were immediately used in affinity chromatography.
2.3.
Protein concentration
Total protein concentration in the whole mixed human saliva and the concentration of the purified protein were determined using both Lowry and Bradford method with bovine serum albumin (BSA) as standard.20
2.4.
Chromatography
2.4.1.
Affinity matrix preparation
Preswollen Sepharose 6B beads were washed with water and 0.5 M Na–phosphate buffer pH 7.4. Sepharose beads were mixed with one volume of 25% glutaraldehyde and incubated overnight at 37 8C with moderate shaking. Water suspensions of E. coli LPS or ODA (both in concentration 0.67 mg per 1 mL of matrix) were added and left to incubate overnight at 37 8C with agitation. Synthesized matrices were washed with 0.5 M Na–phosphate buffer pH 7.4 and afterwards with 50 mM Na–phosphate buffer pH 7.4 containing 1 M NaCl on Bu¨chner funnel through the Whatman No. 54 filter paper. Two volumes of 100 mM ethanolamine were added to block residual reactive glutaraldehyde groups. The blocking reaction proceeds overnight at 37 8C with moderate shaking. A schematic representation of affinity beads preparation is presented below.
2.4.2.
Affinity chromatography
One millilitre of ODA- or LPS-Sepharose matrix was washed with 20 column volumes (CV) of water and pre-equilibrated with 20 CV of 100 Na–phosphate buffer pH 7.4. Twenty CV of saliva filtrates (corresponding to 10 mL of mixed human saliva) were applied to column with peristaltic pump (flow rate 60 mL/h). The flow-through was applied twice on the column. The unbound salivary proteins were eluted with 30 CV of 100 mM Na–phosphate buffer pH 7.4. Bound protein fraction was eluted with 20 CV of 100 mM Na– phosphate buffer pH 7.4 containing 0.2% SDS. Collected fractions (5 CV) were analyzed by measuring A280 nm. Fractions containing bound proteins were pooled and stored at 20 8C.
2.5.
Biochemical methods
2.5.1. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) The presence of protein in fractions collected after affinity chromatography was analyzed by SDS-PAGE.
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SDS-PAGE was performed using a discontinuous buffer system according to the method of Laemmli21 using 12% polyacrylamide gel as the separating gel. Protein fractions were mixed with 5 times concentrated Laemmli non-reducing sample buffer for SDS-PAGE and incubated for 5 min at 95 8C. Twenty-five microlitres of each sample was applied per lane. The gels were run under constant current of 35 mA per gel in a Hoefer Dual Gel Caster Mighty small SE 245 system (Hoefer, Holliston, MA, USA). After electrophoresis, SDS-PAGE gel was stained with silver, except in the case of band excision for proteomic analysis where it was stained with Coomassie Brilliant Blue R250 (CBB). In the case of examining the electrophoretic behaviour in reducing and non-reducing conditions, ODA-purified protein sample was prepared both in reducing and non-reducing sample buffer, respectively. Molecular weights were determined using Unstained Protein MW Marker (Thermo Fisher Scientific, Rockford, IL, USA) and Prestained Precision Plus ProteinTM All Blue Standards (BioRad, Hercules, CA, USA).
2.5.2. 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) In the case of 2D-PAGE electrophoresis, the first dimension was carried out as native isoelectric focusing.20 Isoelectric focusing was run in 7% polyacrylamide gel containing 4% wide range ampholytes for 2D electrophoresis (Serva, Heidelberg, Germany). Twenty microlitres of the protein purified in ODA-Sepharose affinity chromatography was mixed with an equal volume of 2 times gel loading buffer (1% ampholytes and 0.1% bromphenol blue solution in Milli Q water). Thirty microlitres of the sample (0.6 mg of the protein) was applied per lane. The isoelectric focusing was run for 30 min at 150 V followed with additional 2.5 h at 200 V. After the focusing, the pH gradient was determined by cutting the empty gel stripe into 0.5 cm slices. Each slice was incubated in 10 mM KCl for 30 min and pH value of KCl solutions was measured. The stripe of the gel with the resolved protein sample was incubated in Laemmli nonreducing sample buffer for SDS-PAGE for 30 min and loaded on the top of the separating gel for SDS-PAGE analysis. SDSPAGE was run as described above and gel was silver stained afterwards.
2.5.3.
Trypsin mass fingerprinting (TMF)
Protein spots were excised from CBB stained gel to perform ingel trypsin digestion according to the manufacturer instructions. Obtained peptides were injected onto a reversed phase column (ACQUITY UPLC BEH 130, 1.7 mm, C18 2.1 mm 50 mm, Waters, Milford, MA, USA) installed on Acquity UPLC (ultra-performance liquid chromatography) system (Waters, Milford, MA, USA) and separated using acetonitrile gradient (0–50% for 40 min with 0.1% formic acid). After detection with photodiode array (UV) at 280 nm mass spectra were recorded on triple-quadrupole mass spectrometer (ACQUITY TQD, Waters, Milford, MA, USA) in positive ion mode with capillary voltage of 3 kV and drying gas flow rate of 8.3 L/min at 250 8C. The scan range was set from 400 to 2000m/z. The peptide masses were searched against the SwissProt protein sequence database using the MASCOT programme.
2.6.
Microbiology
2.6.1. Bacterial growth inhibitory assay and minimum inhibitory concentration (MIC) determination Strain of P. aeruginosa ATCC 27853 was kindly provided by Dr. Gordana Gojgic-Cvijovic, Department of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade. MIC was measured by broth microdilution according to the CLSI M7-A7 with slight modifications.22 Briefly, cells were grown on LB agar plates at 37 8C for 24 h before use. Inoculum was prepared by transferring single colony from the agar plate with a sterile tip to Luria–Bertani (LB) liquid medium and growing on 37 8C overnight. Inocula were diluted in LB liquid medium to approximately 105 colony forming units (cfu) and then grown in the presence of increasing BPIFA2 concentrations (0, 0.5, 1, 2, 4, 8, 16 and 32 mg/mL). The incubation was carried out in sterile 96 well microtiter plates and bacterial growth was monitored by measuring the absorbance (optical density, OD) at 620 nm using a LKB Micro plate reader 5060-006 (GDV, Roma, Italy). Results were expressed as percentage of bacterial growth compared to the control sample incubated in the appropriate buffer in the absence of BPIFA2 (100 mM Na–phosphate buffer pH 7.4 containing 0.2% SDS added to bacterial culture). The mean growth values of triplicates were obtained and then converted to the inhibition percentage of cell growth in relation to the control treatment by using the formula, MGI(%) = [(dc dt)/ dc] 100, where dc and dt represent A620 nm in control and treated wells, respectively. The first well in the series with no visible growth after the incubation period was taken as the MIC. The experiments were performed twice independently.
2.6.2.
Time-kill study
Time-kill assays were performed according to the guideline M26-A23 of the CLSI with the following change: polypropylene 1.5 mL microtubes were used for the time-kill assays with P. aeruginosa. Protein sample (40 mg/mL in 100 mM Na–phosphate buffer pH 7.4 containing 0.2% SDS) and control (100 mM Na– phosphate buffer pH 7.4 containing 0.2% SDS) were prepared by mixing 9 volumes of the sample with 1 volume of 10 times concentrated sterile LB media. Exponential-phase P. aeruginosa was diluted to 5 107 cfu/mL and exposed to BPIFA2 (final concentration 32 mg/mL after addition of LB media and bacterial culture) over a 24 h period in a total volume of 400 mL of appropriate media. Viable cell count was determined by plating serial dilution (onto LB agar). Bactericidal activity was defined as a 3 log decrease in cell counts.23 Time-kill assays were performed twice independently. Mean values of triplicate cfu/mL measurements from a single experiment (SD) are plotted.
2.6.3.
Slide-agglutination assay
Possible agglutination of P. aeruginosa in the presence of BPIFA2 was monitored as previously described.24 Briefly, bacterial agglutination is performed using exponential-phase culture of P. aeruginosa cells centrifuged and resuspended in saline to the concentration of 5 108 cfu/mL. The influence of BPIFA2 on bacterial agglutination was tested at MIC concentration of BPIFA2. Two microlitres of the cell suspension was mixed with 8 mL of appropriate concentration of
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BPIFA2 on the glass slide, left in humid atmosphere from 1 min to 2 h. After incubation, cells were fixed on the flame, visualized with 0.3% Methylene blue in 30% ethanol and observed by light microscopy under magnification of 200 times. The slides were photographed using a digital camera Olympus m-mini (2272 1704 pixels) and converted to black and white images in GIMP (GNU Image Manipulation Programme).
3.
Results
3.1.
Protein concentration in mixed human saliva
The protein concentrations in mixed human saliva were estimated by both Bradford and Lowry method using BSA as protein standard. The obtained concentrations were 0.7 0.2 mg/mL and 1.1 0.4 mg/mL respectively and were comparable to those published by Jenzano et al.25
3.2.
Purification of human BPIFA2
Matrices used for BPIFA2 purification from whole saliva were synthesized by coupling LPS and ODA, respectively, to Sepharose resulting in LPS-Sepharose and long hydrophobic chain affinity matrix, ODA-Sepharose (Scheme 1). Fig. 1 shows affinity chromatograms of BPIFA2 purification using both affinity matrices. Protein elution from LPS column was achieved immediately after including detergent SDS to the elution buffer. Identical chromatogram was achieved using ODA-Sepharose. The BPIFA2 yield in relation to total protein content of both LPS and ODA chromatography was around 5% (540 mg of purified protein was isolated from 10 mL of saliva). Fractions obtained by LPS and ODA-Sepharose affinity chromatography were analyzed by SDS-PAGE. Gels were silver-stained and presented in Fig. 2. The protein of around 25 kDa is present in bound fraction corresponding to the bound peak in affinity chromatographies. Also, a minor protein band of about 20 kDa was visible in both samples.
Fig. 1 – Fractionation of the whole human saliva on LPSSepharose (grey line) and ODA-Sepharose (black line) columns. Human saliva (10 mL), collected from healthy volunteers, was used as starting material for BPIFA2 purification. Unbound protein fraction was eluted with Buffer A (100 mM phosphate buffer pH 7.4). Bound protein elution at 30 CV was achieved in Buffer B (Buffer A containing 0.2% SDS). Fractions of 5 CV were collected at a flow rate of 60 mL/h and analyzed by absorbance measurements at 280 nm.
ODA-Sepharose purified protein was used in consequent analysis.
3.3.
Proteomic analysis of the purified BPIFA2
After SDS-PAGE and CBB staining of protein isolated by ODASepharose affinity chromatography, we sought to identify the isolated protein bands of 25 kDa and 20 kDa by proteomic analysis after trypsin digestion. According to Mascot, protein scores greater than 70 were considered as significant
Scheme 1 – Affinity matrix synthesis.
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Fig. 2 – SDS-PAGE analysis of LPS-Sepharose fractions (left) and ODA-Sepharose fractions (right). Bound protein fractions eluted in phosphate buffer containing 0.2% SDS are marked with asterisk. Lane M corresponds to molecular weight standards.
( p < 0.05). BPIFA2 was identified by 10 matching peptides, with sequence coverage of 31%, as the only significant hit (Table 1). Band of 20 kDa (Fig. 2) was also identified as BPIFA2 (identified peptides matched those presented in Table 1 and covered the sequence form amino acid positions G53 to K121) and probably correspond to a proteolytically processed protein.
3.4.
Biochemical characterization of the purified BPIFA2
The concentration of purified BPIFA2 was determined by both and Lowry method. The results were Bradford 0.000 0.007 mg/mL and 0.040 0.005 mg/mL, respectively. It appears that BPIFA2 cannot be detected in Bradford method. Nevertheless, the protein concentration could be estimated by Lowry method. In order to check if the reduction of disulfide bonds affects electrophoretic mobility, protein isolated by ODA-Sepharose affinity chromatography was prepared for SDS-PAGE both in reducing and non-reducing conditions. Results are shown in Fig. 3A. Protein does not exhibit differential electrophoretic mobility regardless of the reduction of disulfide bonds indicating the limited number or a complete absence of disulfide bridges.
The isolated protein molecular weight and pI value were determined in 2D-PAGE (Fig. 3B). The molecular weight of around 25 kDa is in good agreement with theoretical molecular mass for the soluble protein after the proteolysis of the signal peptide. However, it could be observed that the protein showed microheterogeneity in both SDS-PAGE and 2D-PAGE regarding both molecular weights and pI values. At least two different forms are present in the sample. The less abundant protein spot had a molecular weight below 25 kDa and a pI value of 5.3 (estimated by 2D PAGE). The more abundant spot showed molecular weight of about 25 kDa and acidic pI value of about 4.9 (estimated by 2D PAGE).
3.5.
Antibacterial activity of BPIFA2
The isolated BPIFA2 inhibited P. aeruginosa growth in low concentration range. MIC was identified at a mass concentration of 32 mg/mL. The EC50 value was identified in two separate experiments set with two different batches of ODA-Sepharose purified BPIFA2. The values did not differ significantly ( p > 0.05) and were found to be 3.7 0.2 mg/mL and 3.5 0.2 mg/mL, respectively. The dependence of % of bacterial growth from BPIFA2 concentration is shown in Fig. 4A.
Table 1 – Peptide mass fingerprint of the isolated BPIFA2. Values given in the brackets correspond to the protein without the signal peptide. Gel band
Protein names
Match-ing species
25 kDa
BPIFA2 BPIA2 SPLUNC2 PSP
Homo sapiens
Protein score P < 0.05 94
Peptide matched in peptide mass fingerprint 53
GILEKLK59 LKVDLGVLQK67 58 LKVDLGVLQKSSAWQLAK75 68 SSAWQLAKQK77 68 SSAWQLAKQKAQEAEK83 76 QKAQEAEK83 78 AQEAEK83 104 ISNSLILDVKAEPIDDGK121 206 STVSSLLQK214 222 IFIHSLDVNVIQQVVDNPQHK 242 58
Number of Theoretical Theoretical Sequence matched molecular pI value coverage peptides weight (Da) (%) 10
27011 (25054)
5.35 (5.19)
31 (35)
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Fig. 3 – (A) SDS-PAGE analysis of isolated BPIFA2 in non-reducing (NR) and reducing conditions (R) on 12% polyacrylamide gel. Lane M corresponds to molecular weight standards. (B) 2D-PAGE of BPIFA2. The first dimension was run as native isoelectric focusing, and second dimension was run as SDS-PAGE on 12% polyacrylamide gel.
Fig. 4 – Antibacterial activity of BPIFA2. (A) Inhibition of P. aeruginosa growth with BPIFA2 was monitored by measuring A620 nm in the presence of growing concentrations of BPIFA2. Results are presented as triplicates’ mean value W standard deviation. (B) Graph for time-kill assay of P. aeruginosa incubated with 32 mg/mL PSP. Bactericidal activity of BPIFA2 was determined by counting the number of colony forming units after incubation with BPIFA2 at MIC for 2, 6 and 24 h.
The results of time-kill studies are presented in Fig. 4B. Bactericidal activity of BPIFA2 was presented in terms of the log 10 cfu/mL change and is based on the conventional bactericidal activity standard, that is, 3 log 10 cfu/mL reduction in the viable colony number. The striking difference between cultures incubated in the presence and in the absence of BPIFA2 is noticeable after 6 h, when no cfu could have been detected in the BPIFA2 treated sample. Aggregation of P. aeruginosa cells in the presence of MIC concentration of BPIFA2 was monitored for up to 2 h. The bacterial aggregation could not be observed in the respected time range. The results presented in Fig. 5 are photographed after 1 h of incubation.
4.
Discussion
The affinity chromatography represents a simple, one-step method for isolation of a single protein from a complex mixture. The method is limited to biological pairs, except in the case of immunoaffinity methods. Despite successful
BPIFA2 purification, immunoaffinity chromatography was found to be inappropriate for large scale BPIFA2 production due to low protein yield.17 Also, because of unavailability of sufficient quantities of the whole protein, BPIFA2 peptides are usually used as immunogens for antibody production.4,17 It has been shown that antibodies raised against different BPIFA2 peptides differentially recognize BPIFA2 isoforms in saliva.4 Thus, in order to purify the functional protein, we sought to employ another strategy, affinity purification of BPIFA2 based on its ability to bind LPS hypothesizing that LPS binding is necessary for BPIFA2 function. For successful usage of affinity chromatography in purification of BPIFA2, it is necessary to identify and use the ligand that specifically binds BPIFA2, or to synthesize and use the close analogue of the ligand molecule. The structure and the biological role of human PLUNC proteins expressed in oral and upper airway mucosa were predicted and deduced mainly via indirect methodology. Thus, despite lacking the structural determinations, Bingle and colleagues16 suggested that PLUNC family members share the same structural features like distant relatives: bactericidal/permeability increasing protein and
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Fig. 5 – Agglutination assay of P. aeruginosa incubated for 1 h with 32 mg/mL BPIFA2. Control is incubated in buffer alone. Cells were visualized with Methylene blue staining.
lipopolysaccharide-binding protein. It has been recently shown that human BPIFA2 binds LPS via hydrophobic interactions.18 We exploited that finding by synthesizing affinity matrix based on LPS and ODA (Scheme 1) in order to affinity purify BPIFA2. Although LPS-Sepharose could be used for purification of BPIFA2 (Fig. 2A), we applied another strategy, keeping in mind LPS toxicity. ODA is a molecule that could mimic long hydrophobic chains of LPS (Scheme 1) and we showed that the parotid secretory protein (BPIFA2) can be successfully purified on the ODA-Sepharose column (Fig. 2B). The protein presence was authenticated by TMF (Table 1). Some of the unidentified peptides correspond to peptides with N-glycosylation sites.26 The purified protein exhibits different behaviour in two commonly used methods for protein concentration determination. Since saliva represents a complex mixture of proteins, which differ in their amino acid composition, it is already well known that determination of total protein concentration in mixed human saliva represents a formidable task. Acidic, basic, proline-rich proteins, or massively glycosylated proteins may react differently in both Biuret based methods (Lowry method) or dye-binding methods (Bradford method) for protein concentration determination.25 It appears that a significant portion of salivary proteins do not bind dye CBB, suggesting that Bradford method is not an appropriate one for the determination of total salivary protein concentration.25 Solubility of PLUNC proteins in ethanol-water mixtures could suggest that alcohol fixation and CBB-staining of SDSPAGE gels could not be the most sensitive methods for PLUNC identification. BPIFA2 did not show differential behaviour in reducing and non-reducing conditions, probably because the protein has just one disulfide bridge between Cys174 and Cys217, whose position is highly conserved in the whole PLUNC family.3
We detected at least two different spots corresponding to BPIFA2 in 2D-PAGE. Recombinant BPIFA2 produced in E. coli lacking post translational modifications exhibits a mass of 25 kDa17 and could represent the protein of 25 kDa and pI 4.9 that we detected in the isolated BPIFA2.4 Protein spot showing pI 5.3 and molecular weight below 25 kDa (Fig. 3B) could correspond to proteolytically processed BPIFA2. Despite many predictions and speculations, the exact function of BPIFA2 in saliva remains unknown. Robinson et al. showed in 1997 that murine BPIFA2 can bind E. coli.27 The studies with human BPIFA2 are limited just to recombinantly produced protein. In the case of mammalian expression of BPIFA2, cell culture medium of rat cell line GH4C1 transfected with BPIFA2 gene, limited the number of colonies of P. aeruginosa.17 However, the active concentration of recombinant BPIFA2 has been undetermined. BPIFA2 produced in inclusion bodies by expression in bacterial cells (E. coli) did not exhibit the bactericidal activity but preferably caused bacterial agglutination.13 However, the question of BPIFA2 purity, fold and concentration in the inclusion bodies solution remain unanswered. Heterologous production of human proteins in prokaryotic systems is often accompanied by misfolding, protein aggregation, and storage in the bacterial inclusion bodies,28 requiring subsequent solubilisation and troublesome in vitro refolding.29 The limitations of the host systems used in the recombinant production of BPIFA2 opens the door to the usage of native BPIFA2 in both structural and functional studies. Results presented in this paper proved that native human salivary BPIFA2 inhibited the growth of P. aeruginosa in microgram concentration range exerting bactericidal activity after 6 h of incubation without causing any visible aggregation of the bacterial cells thus designating this molecule an important tool in the future research of PLUNC protein family.
Funding This work was financially supported by the Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 172049.
Competing interests The authors have declared no conflict of interest.
Ethical approval The study did not require the approval of the ethics committee.
references
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