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MG2 and Lactoferrin Form a Heterotypic Complex in Salivary Secretions
R.V. Soares1,
C.C. Siqueira1,
L.S. Bruno1,
F.G. Oppenheim1,2,
G.D. Offner1,3 and
R.F. Troxler1,2,*
1 Departments of Periodontology and Oral Biology,
2 Biochemistry, and
3 Medicine, Boston University Medical Center, 80 East Concord Street, K-312, Boston, MA 02118;
Correspondence: *corresponding author, btrox{at}bu.edu
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ABSTRACT
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Protein-protein interactions are necessary for homeostasis to be maintained and for biological systems to be integrated. Heterotypic complexes occur in saliva, and a complex between MG2 and SIgA has been suggested to promote microbial clearance from the oral cavity. In this study, we used a peptide display library to investigate previously unrecognized heterotypic complexes involving MG2 and other proteins. The library was panned with MG2 12 times, and analyses of clones identified the sequence Ala-Leu-Leu-Cys-, which occurs in salivary lactoferrin. Blotting experiments confirmed that MG2 and lactoferrin form a heterotypic complex in viro and in vivo. Periodate treatment of MG2 did not affect the interaction. A synthetic lactoferrin peptide containing the motif Ala-Leu-Leu-Cys-blocked the interaction between MG2 and lactoferrin, confirming the specificity of the interaction identified by panning. This complex may enhance the properties of these salivary components in the oral environment.
Key Words: protein-protein interactions • saliva • salivary proteins
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INTRODUCTION
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A complete understanding of the biological function of proteins requires knowledge of protein-protein interactions. Putative interactions between a target protein and peptides encoded in plasmids in a peptide display library can be selected by a simple process called panning (Scott and Smith, 1990). A library can be made by cloning degenerate oligonucleotides encoding random peptide sequences into a non-essential portion of a gene that encodes a protein expressed on the surface of a phage or bacteria (Lu et al., 1995). The exposed fusion protein can then interact with an immobilized target protein. Analysis of clones selected by the panning process provides the peptide sequence that participated in the protein-protein interaction.
One of the first applications of random peptide display was to map epitopes of monoclonal antibodies (Philippe et al., 1993; Sibille and Strosberg, 1997; Murthy et al., 1998). A vast number of non-antigen-antibody protein-protein interactions was also identified by this technology (Blond-Elguindi et al., 1993; Koivunen et al., 1994; Pasqualini et al., 1995; Brown et al., 2000; Dintilhac and Bernues, 2002). In addition, display of genetically engineered proteins on cell surfaces has broad application in the field of biotechnology (Samuelson et al., 2002).
Saliva is necessary for the maintenance of oral health, and the unique properties of this fluid are derived in large part from the proteins that are present. In the oral cavity, salivary proteins participate in formation of the acquired enamel pellicle, occur in the biofilm covering oral surfaces, initiate digestion, and promote agglutination and clearance of bacteria (Scannapieco, 1994). Several reports have described heterotypic complexes between salivary proteins, and it has been suggested that such interactions may modulate the function of these molecules in vivo (Rundegren and Arnold, 1987; Biesbrock et al., 1991; Iontcheva et al., 1997).
A bacterial-binding motif exists in the N-terminal region of MG2 (Liu et al., 2000; Soares et al., 2002). This salivary mucin has also been reported to interact with a diverse number of oral microbes (Groenink et al., 1996; Liu et al., 1999, 2000, 2002) and to participate in a heterotypic complex with SIgA (Biesbrock et al., 1991). Together, these properties suggest that MG2 is likely to be an important component of the innate immune system.
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MATERIALS & METHODS
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Saliva Collection
Stimulated submandibular/sublingual secretion (SMSL) was collected from two healthy volunteers (ages 30-36) by means of a custom-fitted device in ice-chilled containers as described (Jensen et al., 1992; Liu et al., 1999). Gustatory stimulation was induced by lemon-flavored candies (Jolly Rancher, Hershey, PA, USA). This study was approved by the Institutional Review Board at Boston University Medical Center, informed consent was obtained from all subjects prior to their participation, and subjects’ rights were protected at all times.
Test Proteins
A monoclonal antibody directed against human Interleukin 8 (IL-8) was purchased from BIODESIGN (Saco, ME, USA). Salivary mucin MG2 was isolated from human SMSL secretion as described (Liu et al., 1999).
Peptide Display
A peptide display library was purchased from Invitrogen (Carlsbad, CA, USA). This library was comprised of E. coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active site loop of thioredoxin, which itself was inserted into a dispensable region of flagellin, the major constituent of flagellar filaments. When the fusion protein becomes an integral part of the flagellar filaments on the bacterial cell surface, the dodecapeptides become available to interact with target proteins. The peptide display library was panned 12 times with anti-IL-8 and MG2 according to manufacturer’s instructions, and resulting libraries were maintained as glycerol stocks. The panning procedure is illustrated in Fig. 1 .
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Figure 1. Panning procedure. Step 1. The target protein (20 µg of MG2) was plated for 1 hr. Step 2. The plate was blocked for 1 hr. Step 3. We added the pFlitrx library to the plate to allow for interaction of expressed dodecapeptides on E. coli flagellae with bound target protein for 1 hr. Step 4. E. coli (unbound) that did not interact with immobilized target protein were washed off. Step 5. Bound cells were eluted by mechanical agitation. Step 6. Eluted cells were grown overnight at 37°C. The panning procedure was repeated 11 times.
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DNA Sequence Analysis
Libraries were plated, and plasmid DNA from selected colonies was extracted by means of a Wizard Plus Miniprep DNA Purification System (Promega, Madison, WI, USA). Plasmid DNA was sequenced with the use of pFlitrx forward and reverse primers (Invitrogen, Carlsbad, CA, USA).
Gel Electrophoresis and Blotting
Stimulated SMSL (50 µL) and purified MG2 (3 µg) were examined by SDS-PAGE on 7.5% polyacrylamide gels under denaturing conditions or on pre-cast native 7.5% Tris-HCl gels (BIO-RAD, Hercules, CA, USA) under non-denaturing/non-reducing conditions. Proteins in gels were transferred electrophoretically to nitrocellulose membranes (Protran, Schleicher & Schuell, Keene, NH, USA). The blotting buffer was 0.19 M glycine, 0.025 M Tris-base, pH 8.3, and 20% methanol, and blotting was perfomed at 100 V for 1 hr at room tempertature. Blots were used for Western and Far-Western blotting experiments.
Western Blots
In control experiments suggested by the manufacturer, Western blots of cell extracts from libraries or individual colonies panned with an anti-IL-8 antibody (Lu et al., 1995) were probed with the same antibody. Blots were washed in 10 mM Tris-HCl, pH 7.5, containing 150 mM NaCl and 0.05% Tween 20 (TBST) for 5 min and blocked with 5% milk/TBST at room temperature for 1 hr. Blots were then washed with TBST 3 times for 10 min and incubated with the anti-IL-8 antibody diluted 1:1000 in 1% milk/TBST at room temperature for 1 hr. Blots were washed as above and incubated with goat anti-mouse IgG conjugated to alkaline phosphatase (AP; Promega) diluted 1:5000 in 1% milk/TBST at room temperature for 1 hr. The membrane was washed as above, and color development was obtained by the addition of BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and NBT (nitro blue tetrazolium) according to the manufacturer’s instructions (Promega). All colonies obtained after 12-time panning reacted with the IL-8 antibody, and sequence analysis showed that selected immunoreactive colonies all contained the IL-8 epitope (data not shown). Western blots of SMSL were probed with our rabbit anti-MG2 antibody directed against a synthetic peptide corresponding to residues 47-63 of secreted MG2 (Liu et al., 1999) diluted 1:1000 or with a rabbit anti-lactoferrin antibody (Jackson Immuno Research, West Grove, PA, USA) diluted 1:5000. The second antibody was goat anti-rabbit IgG conjugated to AP (Jackson Immuno Research) diluted 1:5000. Color development was performed as above.
Far-Western Blots
Blots of SMSL and purified MG2 were washed for 5 min with 10 mM Tris-HCl, pH 7.5, containing 100 mM NaCl and 0.1% Tween-20 (buffer A) and blocked with 5% BSA in buffer A for 1.5 hrs at room temperature. Blots were washed (3x, 10 min) with buffer A, and the membrane was incubated with 50 µg of lactoferrin (Sigma) dissolved in buffer A containing 1% BSA for 5 hrs at 4°C with gentle agitation. After the blots were washed with buffer A (6x, 10 min), immunodetection was performed with the anti-lactoferrin antibody diluted 1:5000 in buffer A containing 1% BSA for 1 hr at room temperature. Blots were washed with buffer A (3 times, 10 min), probed with goat anti-rabbit IgG conjugated to AP diluted 1:5000 in buffer A containing 1% BSA, and washed; color development was with BCIP and NBT.
Periodate Treatment of Glycans
Blots containing SMSL samples were rinsed with 50 mM sodium acetate buffer (pH 4.5) and incubated in the same buffer containing 20 mM periodate for 1 hr at 23°C in the dark (Woodward et al., 1985). Blots were then used to perform Far-Western blots as described above.
Lactoferrin Synthetic Peptide
A peptide (KLADFALLCLDGKRK) corresponding to residues 587-601 of lactoferrin (Swiss-Protein database; accession number ANN 75578) was synthesized commercially (Invitrogen). Blots containing SMSL were blocked and washed (3x, 10 min) with buffer A, and the membrane was incubated with 50 µg of the synthetic peptide-dissolved buffer A containing 1% BSA for 3 hrs at 4°C with gentle agitation. After being washed with buffer A (6x, 10 min), the membrane was incubated with 50 µg of lactoferrin, and immunodetection with anti-lactoferrin antibodies was performed as described above.
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RESULTS
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Panning with MG2 Target Protein
When the pFlitrx library was constructed, a bias was introduced into the 36-base oligonucleotide encoding the random dodecapeptides (Lu et al., 1995). The sequence of the oligonucleotide used was 5'(XNN)12, where X is G:A:C:T in the ratio of 7:7:7:3, respectively, and N is G:A:C:T in the ratio of 1:1:1:1. Therefore, the number of codons having T at the first position is reduced, and the number of codons having G, A, or C at the first position is increased. Based on the foregoing, the predicted frequency of amino acids in dodecapeptides in the initial Flitrx library resulting from the T bias is given in Fig. 2A. The frequency of amino acids in dodecapeptides obtained after 12-day panning with MG2 is given in Fig. 2B. These results show that amino acid compositions of dodecapeptides obtained after panning with MG2 are notably different from those in the original library.
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Figure 2. Compositional analysis of dodecapeptides. (A) The solid bars represent the expected percentage of each amino acid in dodecapeptides in the initial random library. (B) The solid bars represent the observed percentage of each amino acid in dodecapeptides in the sub-library obtained after 12-day panning with MG2.
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Dodecapeptide sequences from 20 clones selected by MG2 are shown in the Table. Analyses of these sequences revealed a motif containing 3 or more consecutive amino acids with aliphatic side-chains or side-chains with aliphatic character (Table; clones 1-13; motifs in bold-face type). Inspection of the sequences of these motifs showed that the sequence X-Ala-Leu-Leu-Y- (Table; underlined) was the most abundant consensus sequence. A search of the Swiss-Protein database with the sequence X-Ala-Leu-Leu-Y, where X is His, Leu, or Cys and Y is Cys, Leu, or Arg revealed that the sequence Ala-Leu-Leu-Cys- found in clone-8 (Table) corresponds to residues 592-595 of lactoferrin.
MG2 and Lactoferrin Interactions
We conducted experiments to investigate complexing between MG2 and lactoferrin. When blots of SMSL were probed with anti-MG2 antibodies, a 180-kDa immunoreactive band was observed (Fig. 3A, lanes 1, 2), and when blots were probed with anti-lactoferrin antibodies, an 80-kDa immunoreactive band was observed (Fig. 3A, lanes 3, 4). When blots were pre-incubated with lactoferrin, washed, and subsequently probed with anti-lactoferrin antibodies (Far-Western blot), a strong 180-kDa immunoreactive band coincident with the electrophoretic mobility of MG2 was observed (Fig. 3A, lanes 5, 6). In addition, a smaller 80-kDa immunoreactive band corresponding to lactoferrin was observed. These results show that MG2 and lactoferrin can form a complex in vitro.
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Figure 3. Western and Far-Western blots showing interactions between MG2 and lactoferrin. SMSL (50 µL) from two subjects was subjected to SDS-PAGE on 7.5% polyacrylamide gels (A) or to electrophoresis on pre-cast native 7.5% Tris-HCl gels (B), and proteins were transferred to nitrocellulose membranes. (A) Western blots were probed with anti-MG2 antibodies (lanes 1, 2) and anti-lactoferrin antibodies (lanes 3, 4). Far-Western blots were incubated with lactoferrin and probed with anti-lactoferrin antibodies (lanes 5, 6). Far-Western blots oxidized with periodate prior to incubation with lactoferrin were probed with anti-lactoferrin antibodies (lanes 7, 8). Far-Western blots pre-incubated with the synthetic lactoferrin peptide were then incubated with lactoferrin and probed with anti-lactoferrin antibodies (lanes 9, 10). (B) Western blots from native gels were probed with anti-MG2 antibodies (lanes 1, 2) and anti-lactoferrin antibodies (lanes 3, 4). In panel A, the dashed line shows the interface between the stacking and separating gels. There is no stacking gel in the native gel system, and the top of the gel is indicated by an arrow. Abbreviations: Western blot, W.B.; Far-Western Blot, F.W.B.; Anti-lactoferrin antibody, Anti-LF.
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We treated blots containing SMSL with periodate prior to Far-Western blotting to assess whether sugars in MG2 were involved in the interaction between this mucin and lactoferrin. No significant difference was observed in the interaction between these salivary proteins after periodate treatment (Fig. 3A ; lanes 7, 8), suggesting that glycans do not participate in complex formation.
We synthesized a peptide (KLADFALLCLDGKRK) corresponding to residues 587-601 of lactoferrin and used it to verify the specificity of the interaction between lactoferrin and MG2. This synthetic peptide contained the motif Ala-Leu-Leu-Cys-, identified in panning experiments described above (Table, clone 8). When membranes were pre-incubated with this peptide prior to being exposed to lactoferrin, the interaction between MG2 and lactoferrin on Far-Western blots was nearly abolished (Fig. 3A; lanes 9, 10). These results confirm that the motif identified by panning is involved in complexing between MG2 and lactoferrin.
We conducted a parallel series of experiments to determine whether purified MG2 could form a complex with purified lactoferrin and to determine the effects of periodate oxidation and pre-incubation with synthetic peptide on complex formation. The results obtained in these experiments were identical to those described above with MG2 in SMSL (data not shown).
So that we could determine whether MG2 and lactoferrin form a complex in vivo, SMSL was electrophoresed on native gels under non-denaturing conditions, and proteins were transferred to nitrocellulose membranes. When the blot was probed with anti-MG2 antibodies, an immunoreactive band was observed near the top of the gel (Fig. 3B; lanes 1, 2). When an identical blot was probed with anti-lactoferrin antibodies, two immunoreactive bands were observed (Fig. 3B; lanes 3, 4). The band near the top of the gel had the same electrophoretic mobility as MG2 and likely represents lactoferrin molecules complexed with MG2. The lower band had the same electrophoretic mobility as standard lactoferrin in this gel system (data not shown). These results suggest that MG2 and lactoferrin form a heterotypic complex in SMSL.
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DISCUSSION
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Interactions between salivary proteins are an important property of these macromolecules and modulate several processes, including establishment of the enamel pellicle on tooth surfaces (Rykke et al., 1990; Yao et al., 2001), maintenance of the biofilm coating epithelial surfaces in the oral cavity (Collins and Dawes, 1987), and colonization of oral surfaces by microbes (Marcotte and Lavoie, 1998). Previously, we have reported that MG1 (the MUC5B gene product), a high-molecular-weight salivary mucin, selectively forms heterotypic complexes with several salivary proteins, including amylase, proline-rich proteins, statherin, and histatins (Iontcheva et al., 1997). Yeast two-hybrid mapping studies have identified statherin- and histatin-binding domains on non-glycosylated regions of MG1 (Iontcheva et al., 2000). MG2, the other major salivary mucin, has also been reported to form a heterotypic complex with SIgA (Biesbrock et al., 1991). In the present investigation, we used random peptide display to search for putative complexes between MG2 and other proteins occurring in the oral environment.
Analyses of clones interacting with MG2 showed that 4 of 20 dodecapeptides contained a motif suggesting lactoferrin as a potential candidate to complex with MG2. Several experiments confirmed the formation of a heterotypic complex between MG2 and lactoferrin. This is interesting because lactoferrin binds ferric ions and possesses antibacterial, antimycotic, antiviral, antineoplastic, and anti-inflammatory properties (Weinberg, 2001). MG2 interacts with several oral microbes, exhibits candidacidal and bactericidal activity (Liu et al., 2002), is present in the biofilm that covers mucosal surfaces (Collins and Dawes, 1987), has affinity for hydroxyapatite (Tabak et al., 1985), and has been identified in pellicle formed on enamel and cementum (Fisher et al., 1987). Migration of a heterotypic complex between MG2 and lactoferrin to enamel and cementum in gingivitis and periodontal disease could, in principle, result in localization of an antibacterial and an anti-inflamatory protein to sites challenged by oral microbes. The high content of carbohydrate in MG2 could possibly enhance the resistance of lactoferrin to proteolytic attack when complexed to this salivary mucin. Complexing could also modulate removal of these two proteins from the oral cavity, thereby extending the time for them to exert their important biological functions in the oral environment.
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ACKNOWLEDGMENTS
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This study was supported by NIH grants DE 11691, DE 14080, DE 07652, and DK 44619.
Received for publication October 20, 2002.
Revision received March 4, 2003.
Accepted for publication March 5, 2003.
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Journal of Dental Research, Vol. 82, No. 6,
471-475 (2003)
DOI: 10.1177/154405910308200613
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ABSTRACT
INTRODUCTION