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J Dent Res 84(2):149-153, 2005
© 2005 International and American Associations for Dental Research

RESEARCH REPORTS
Biological

Design and Validation of Anti-inflammatory Peptides from Human Parotid Secretory Protein

C. Geetha1,2, S.G. Venkatesh1,*, L. Bingle3, C.D. Bingle3, and S.-U. Gorr1,4

1 Department of Periodontics, Endodontics and Dental Hygiene, Room 209C, and
4 Department of Biochemistry and Molecular Biology, University of Louisville Health Sciences Center, School of Dentistry, Louisville, KY 40292, USA;
3 Academic Unit of Respiratory Medicine, Division of Genomic Medicine, The University of Sheffield Medical School, Royal Hallamshire Hospital, Sheffield S10 2JF, UK;

* corresponding author, venkatesh.srirangapatnam{at}louisville.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Parotid secretory protein (PSP) and palate-lung-nasal epithelium clone (PLUNC) are novel secretory proteins that are expressed in the oral cavity and upper airways. Both proteins are related to bactericidal/permeability increasing protein (BPI). Cationic peptides derived from BPI exhibit anti-inflammatory activity. To test if PSP (C20orf70 gene product) also contains anti-inflammatory peptides, we designed 3 cationic peptides based on the predicted structure of PSP and known active regions of BPI. Each peptide inhibited the lipopolysaccharide (LPS)-stimulated secretion of TNF{alpha} from RAW 264.7 macrophage cells. At 200 µg/mL, the peptide GK-7 exhibited inhibition similar to that achieved with 10 µg/mL of polymyxin B. PSP peptides directly inhibited the binding of LPS to LPS-binding protein. The cationic peptide Substance P had no inhibitory effect in these assays, confirming the specificity of the PSP peptides. These findings suggest that PSP peptides can serve as templates for the design of novel anti-inflammatory peptides.

KEY WORDS: cationic peptides • endotoxin • inflammation • lipopolysaccharide • PLUNC • saliva • C20orf70


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mucosal surfaces of the oral cavity and upper airways are major entry points for micro-organisms involved in both local and systemic diseases. In addition to infection, bacterial endotoxins (lipopolysaccharides, LPS) cause inflammation, fever, hypertension, and sepsis. Accordingly, a variety of anti-microbial and anti-inflammatory proteins and peptides has been identified in saliva and airway fluids (e.g., Weinberg et al., 1998; Amerongen and Veerman, 2002; Cole et al., 2002; Ganz, 2003). These proteins serve protective functions in the mouth and upper airways. Due to the complex microbial environment in these areas, and developing antimicrobial resistance, the identification of novel antimicrobial proteins and peptides is a continuing priority (Bradshaw, 2003). Thus, antimicrobial peptides alone, or in combination with antibiotics, hold promise in the treatment of bacterial infections and inflammation.

Parotid secretory protein (PSP) has been described in rodents, pigs, and cattle (Madsen and Hjorth, 1985; Mirels and Ball, 1992; Rajan et al., 1996; Gupta et al., 2000; Wheeler et al., 2002; Yin et al., 2004). PSP and palate-lung-nasal epithelium clone (PLUNC) are members of a recently identified family of human oral and airway epithelial proteins (LeClair, 2003). The PSP/PLUNC proteins exhibit a weak sequence similarity with lipopolysaccharide (LPS) binding proteins, including bactericidal permeability-increasing protein (BPI) and LPS-binding protein (LBP). Both BPI and the PSP/PLUNC genes are located on human chromosome 20q11 and may have arisen by gene duplication (Bingle and Craven, 2002). In fact, modeling of PSP and PLUNC on known x-ray structures revealed that these proteins are likely related to BPI proteins, despite the poor sequence conservation (Bingle and Craven, 2002; Wheeler et al., 2002; Geetha et al., 2003). Indeed, secreted recombinant PSP exhibits anti-bacterial activity against the Gram-negative bacterium P. aeruginosa (Geetha et al., 2003). Although PLUNC binds LPS (Ghafouri et al., 2003), a recent report showed that the protein failed to exhibit anti-inflammatory activity in an in vitro assay of endotoxin binding to LBP (Campos et al., 2004). A possible explanation for this discrepancy is that PSP/PLUNC proteins must be proteolytically processed to release anti-inflammatory peptides. Such anti-inflammatory peptides have been described in the BPI sequence (Little et al., 1994; Battafarano et al., 1995; Uknis et al., 1997; Dankesreiter et al., 2000). In this study, the PSP structure was modeled and synthetic peptides utilized to test the hypothesis that similar anti-inflammatory peptides are also found in the PSP sequence.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
RNA Isolation and RT-PCR Analysis
Total RNA was isolated from human nasal septal epithelium, trachea, and peripheral lung. Human tissue specimens were collected with appropriate consent and approval by the Local Ethical Committee at The University of Sheffield. Additional human RNA samples were purchased from Clontech (Palo Alto, CA, USA). RT-PCR analyses of hPLUNC and hPSP were performed with the following primers: hPLUNC (Forward, 5' ATG CCC TCA GCA ATG GCC TGC T 3'; Reverse, 5' AGG GTG ATG TCC AAG CCT CTG A 3') and hPSP (Forward, 5' ACG CGT GAC AAG ACA AAA GA 3'; Reverse, 5' CCA CAG TGG TCC TCC TCA TT 3') and 30 cycles of the following program (94°C for 1 hr, 60°C for 2 hrs, and 72°C for 3 hrs). Reaction products were resolved on 2.5% gels, and we confirmed the correct identity of each band by cloning and sequencing.

Structural Analysis of PSP and PLUNC and Design of Anti-inflammatory Peptides
Protein sequence alignments were performed with the CLUSTALW program (Thompson et al., 1994). Similar results were obtained with BLASTP (Altschul et al., 1997).

The human PSP and PLUNC protein sequences were submitted to 3DPSSM, a software threading program that returns a list of structures and fit (http://www.sbg.bio.ic.ac.uk/~3dpssm/) (Kelley et al., 2000). The modeled structures were further analyzed with use of the Cn3D structure viewer from NCBI (http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml). Based on this analysis, cationic peptides were identified in a region that matched the location of peptides in BPI (Little et al., 1994; Dankesreiter et al., 2000) and LBP (Taylor et al., 1995) that inhibit LPS binding. The corresponding region in the PSP sequence forms one of three predicted hairpin structures in the molecule. The sequences of these predicted hairpins were searched for cationic peptides. The N- and C-terminal amino acids of these peptides were chosen to include multiple basic amino acid residues, while largely excluding acidic residues from the PSP sequence. A similar analysis was performed for the PLUNC sequence. Three peptides from the PSP sequence were selected for the experiments in this report. These are peptide KK-9 (-KLLNNVISK-), peptide GK-7 (-GQIINLK-), and peptide KN-11 (-KHSQIINKFVN-). The synthetic peptides were purchased from Peptides International (Louisville, KY, USA).

Circular Dichroism Spectroscopy
The peptides were separately dissolved in 0.1 M phosphate buffer, pH 7.0, at a concentration of 80 µM. Spectra were recorded in the wavelength range from 195 to 240 nm by means of a Jasco J710 spectropolarimeter. Trifluoro-ethanol (30%) was used to induce the formation of potential secondary structure.

Inhibition of TNF{alpha} Secretion
RAW 264.7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured to confluence in RAW culture medium (DMEM) (Dulbecco Modified Eagle’s Medium) containing 4 mM L-glutamine, 4.5 g/L glucose with 50,000 U/L penicillin, 50 mg/L streptomycin, and 10% fetal bovine serum (Hyclone, Logan, UT, USA).

RAW 264.7 cells were dislodged from the culture flask by being gently scraped and were then seeded at a density of 5 x 105 cells/mL in a 24-well plate in RAW culture medium. The plate was incubated overnight at 37°C. The cells were washed with DMEM for 30 min and then incubated for 6 hrs at 37°C in DMEM with 100 ng/mL LPS or LPS that had been pre-incubated with 200 µg/mL of the PSP peptides, substance P, or polymyxin B for 1 hr at 37°C. Secretion media were collected at the end of the incubation period and assayed for TNF{alpha} by means of a mouse TNF{alpha} ELISA kit (Biosource International, Camarillo, CA, USA).

Inhibition of LPS-LBP Binding
The endotoxin-inhibitory capacity of the synthetic peptides was measured with the use of the Endoblock test kit from Hycult biotechnology (Cell Sciences, Norwood, MA, USA). The assay has a minimum detection level of 5 ng/mL of polymyxin B, a potent inhibitor of endotoxin binding. The cationic peptide substance P was used as a further control. The assay was performed according to the manufacturer’s protocol. PSP peptides (100 µg/mL), substance P (100 µg/mL), or polymyxin B (0.01–10 µg/mL) was incubated with biotinylated LPS for 30 min at 37°C and then added to individual wells of a 96-well culture plate that was pre-coated with LBP antibody and 2.5 ng/well of LBP. The samples were incubated at room temperature for 1 hr. The wells were washed and then incubated with streptavidin-peroxidase conjugate for 1 hr at room temperature. The wells were then washed thoroughly and incubated with tetramethylbenzidine substrate in the dark for 25–30 min. The enzymatic color development reaction was stopped by the addition of 1.0 M citric acid, and the absorbance was quantitated at 450 nm in an ELISA plate-reader. A polymyxin B standard curve revealed maximum inhibition at 10 µg/mL.

Data Analysis
Data were analyzed by one-way ANOVA with Dunnett’s post-test for comparison of the effects of PSP peptides with the effect of substance P. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PLUNC and PSP [also named SPLUNC1 and SPLUNC2, respectively (Bingle and Craven, 2002)] are members of a novel family of oral and airway proteins. The expression of these genes was analyzed by RT-PCR of 22 human tissues. Both genes were expressed in salivary glands and tracheal epithelium. However, only PLUNC was expressed in nasal epithelium and weakly in the thymus, while PSP was additionally expressed in the uterus, thyroid, and testes (Fig. 1Go). On the basis of these and other studies, PSP appears to be the most widely distributed member of this gene family in humans. The overlapping expression patterns and the finding that the protein sequences of PLUNC and PSP are 52% similar and 22% identical, including two conserved Cys residues, suggested that they may exhibit similar, but not identical, biological activities.



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  		Figure 1. PLUNC and PSP expression in human tissues. Expression of PLUNC and PSP was investigated by the use of RT-PCR with exon-spanning primer pairs as described in MATERIALS & METHODS. The negative control was a reverse-transcription reaction performed in the absence of RT enzyme.

 
PSP and the N-terminal domain of bactericidal permeability increasing protein (BPI1) are only 15% identical and 34% similar (Fig. 2AGo). Nevertheless, the BPI crystal structure (1ewf.pdb) is the only significant fit (E-value of 0.0149 and 95% certainty) when the PSP (Fig. 2BGo) or PLUNC sequences (Bingle and Craven, 2002) are threaded through the PDB structural database by use of the 3D-PSSM program (Kelley et al., 2000). This finding suggests that PSP and PLUNC may both act as anti-inflammatory proteins.



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  		Figure 2. Molecular modeling of human PSP. (A) Sequence alignment of hPSP, hPLUNC, and the N-terminal domain of BPI (BPI1). The PSP and PLUNC peptides and a peptide in the LPS-binding domain of BPI (Dankesreiter et al., 2000) are underlined. The putative signal peptides of PSP (20 residues) and BPI are not shown. (B) The PSP sequence was modeled with the 3D-PSSM package. The locations of the 3 PSP peptides are highlighted in black.

 
Comparison of the LPS-binding region of BPI and the predicted structure of PSP identified 3 cationic peptides (GK-7, KK-9, and KN-11) (see METHODS for details) located in 3 hairpins which enclose the opening of the PSP ‘barrel’ (Figs. 2AGo, 2BGo, end view). A similar analysis of the predicted PLUNC structure revealed 2 cationic peptides (LNNIIDIK and GASLLRLAVKL; Fig. 2AGo), which were located in hairpins corresponding to the PSP peptides KK-9 and GK-7, respectively (Bingle and Craven, 2002). For these studies, the 3 PSP peptides (GK-7, KK-9, and KN-11) were designed and synthesized. Circular dichroism spectroscopy of the peptides revealed random coil confirmation (not shown).

TNF{alpha} is a key inflammatory cytokine secreted from macrophages in response to stimulation by LPS. Thus, high levels of TNF{alpha} secretion resulted when the macrophage cell line RAW 264.1 was incubated with LPS (Fig. 3Go). This secretion was significantly inhibited when LPS was pre-incubated with polymyxin B (Fig. 3Go), a known inhibitor of LPS action (e.g., Stokes et al., 1989). To test if PSP peptides inhibit endotoxin-stimulated secretion of TNF{alpha}, we pre-incubated LPS with each peptide and used it to stimulate RAW 264.1 macrophage cells (Fig. 3Go). The peptides inhibited LPS-induced TNF{alpha} secretion by 30–80%, while the cationic peptide substance P had no effect on LPS-induced TNF{alpha} secretion. Thus, the PSP peptides exhibit anti-inflammatory activity.



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  		Figure 3. Inhibition of TNF{alpha} secretion from RAW 264.7 cells. RAW 264.7 cells were incubated with lipopolysaccharide (LPS) and substance P, the PSP-derived peptides KN-11, KK-9, or GK-7, or with polymyxin B. The secretion media were assayed for TNF{alpha}. Values obtained were normalized to TNF{alpha} secretion in the presence of LPS alone (100%). Values presented are the mean ± SEM of 3 independent experiments. *Different from secretion in the presence of LPS and substance P. P < 0.05.

 
One pathway of LPS action is initiated by the binding of LPS to LPS-binding protein, followed by binding to CD14 and activation of TLR4. To test if the PSP peptides block the binding of LPS to LPS-binding protein, we pre-incubated the peptides with LPS and then incubated them with LBP. Each peptide inhibited LPS binding to LBP, suggesting that these peptides act early in the inflammatory cascade (TableGo). As a control, the cationic peptide Substance P was tested. This peptide did not inhibit the binding of LPS to LBP (TableGo).


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  		Table. Inhibition of LPS-LBP Bindinga by PSP Peptides
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Due to their specificity and a relative lack of bacterial resistance, antimicrobial peptides are attractive alternatives to other antibiotics (Bradshaw, 2003). Multiple studies have shown that cationic peptides derived from the sequences of BPI and LBP exhibit potent anti-inflammatory and anti-microbial effects, and these peptides are under active study as novel therapeutics. The family of proteins to which BPI, LBP, PLUNC, and PSP belong has recently been predicted to share significant structural similarities, leading to the suggestion that they may serve related, but not identical, host defense functions in multiple epithelia (Bingle and Craven, 2002; Geetha et al., 2003; LeClair, 2003; Bingle and Gorr, 2004). Such a hypothesis is also supported by the sites of expression of members of this family which are found in epithelial cells at locations where multiple pathogens are found. Based on these structural similarities, novel cationic peptides have been identified in the sequences of human PSP and PLUNC. The PSP peptides were synthesized, and each inhibited the action of LPS in two distinct in vitro assays.

Depending on the type of assay, differences in the activities of the 3 PSP peptides were observed. Peptide GK7, which showed the least activity in the LBP-LPS binding assay, was, in contrast, the most effective peptide when used in the TNF{alpha} release assay. It is important to note that the assays used in the present study measure two different aspects of peptide behavior toward LPS. The LPS-LBP binding assay directly determines the effect of each peptide on the binding of LPS to LBP. In the macrophage assay, in contrast, LPS may interact with LBP, CD14, or possible other macrophage-associated proteins (Ulmer et al., 2002). Thus, the tested peptides may have inhibitory functions in these additional binding reactions, resulting in different inhibitory effects in the two assays. Consistent with this interpretation, the LBP-derived peptide H-14 inhibits binding of lipid A to Limulus anti-LPS factor, but does not inhibit LPS-stimulated secretion of TNF{alpha} from a macrophage cell line (Dankesreiter et al., 2000).

The PSP peptides were less potent than polymyxin B. However, further refinement of the peptide sequences may increase the anti-inflammatory activity, as recently reported for peptides derived from other anti-inflammatory proteins (Battafarano et al., 1995; Uknis et al., 1997; Dankesreiter et al., 2000). As an example, the LBP peptide H14, which served as one starting point for the search for PSP/PLUNC peptides, is a cyclic peptide. Such a structure may be more consistent with the location of these peptides in a turn of the native protein chain than the linear peptides used here. Thus, we consider the peptides tested in this report as templates for future modifications.

Saliva contains several antimicrobial proteins and peptide, including defensins, histatins, lactoferrin, peroxidase, and lysozyme (Tenovuo, 1998). It is not clear if intact PSP exerts biological activity in saliva, but purified, intact PLUNC does not block LPS binding to LBP (Campos et al., 2004). Moreover, analysis of preliminary data suggests that PSP is cleaved by matrix metalloproteinase-9 (Geetha, unpublished observations). This enzyme is secreted by macrophages in response to LPS stimulation, and by salivary acinar cells in response to pro-inflammatory cytokines (Wu et al., 1997; Azuma et al., 2002), suggesting that PSP/PLUNC may be proteolytically processed during the inflammatory response. These putative PSP/PLUNC-derived peptides could serve to control inflammation in the oral cavity (Bingle and Gorr, 2004). However, the testing of this model must await the purification of sufficient amounts of native PSP to allow for the isolation and biological testing of the cleavage products.


   ACKNOWLEDGMENTS
 
We thank Dr. Robert D. Gray, University of Louisville, for assistance with circular dichroism spectrometry. This work was supported by PHS grant R01 DE 12205 from NIDCR, by a grant from the University of Louisville School of Medicine, and by Research Incentive Funds from the University of Louisville. Lynne Bingle is an Allen & Hanburys/British Lung Foundation Research Scientist.


   FOOTNOTES
 
2 present address, Avestha Gengraine Technologies Pvt. Ltd., Discoverer 9th Floor, Unit 3, International Tech Park, Bangalore 560 066, India; and Back

Received December 5, 2003; Last revision November 29, 2004; Accepted December 2, 2004


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