The Enamel Protein Amelogenin Binds to the N-Acetyl-d-glucosamine-mimicking Peptide Motif of Cytokeratins

Abstract

Amelogenins bind to GlcNAc of the dentine-enamel matrix proteins (Ravindranath, R. M. H., Moradian-Oldak, J., Fincham, A. G. (1999) J. Biol. Chem. 274, 2464–2471). The hypothesis that amelogenins may interact with the peptides that mimic GlcNAc is tested. GlcNAc-mimicking peptide (SFGSGFGGGY) but not its variants with single amino acid substitution at serine, tyrosine, or phenylalanine residues inhibited hemagglutination of amelogenins and the terminal tyrosine-rich amelogenin polypeptide (TRAP). The binding affinity of SFGSGFGGGY to amelogenins was confirmed by dosimetric binding of amelogenins or TRAP with [3H]peptide, specific binding in varying concentrations of the peptide, Scatchard plot analysis, and competitive inhibition with the unlabeled peptide. The ability of the peptide or GlcNAc to stoichiometrically inhibit TRAP binding of [14C]GlcNAc or [3H]peptide indicated that both the peptide and GlcNAc compete for a single binding site. Using different fragments of amelogenins, we have identified the peptide-binding motif in amelogenin to be the same as the GlcNAc-binding "amelogenin trityrosyl motif peptide." The GlcNAc-mimicking peptide failed to bind to the amelogenin trityrosyl motif peptide when the tyrosyl residues were substituted with phenylalanine or when the third proline was replaced with threonine, as in some cases of human X-linked amelogenesis imperfecta. This study documents that molecular mimicry may play a role in stability and organization of amelogenin during amelogenesis. Amelogenins bind to GlcNAc of the dentine-enamel matrix proteins (Ravindranath, R. M. H., Moradian-Oldak, J., Fincham, A. G. (1999) J. Biol. Chem. 274, 2464–2471). The hypothesis that amelogenins may interact with the peptides that mimic GlcNAc is tested. GlcNAc-mimicking peptide (SFGSGFGGGY) but not its variants with single amino acid substitution at serine, tyrosine, or phenylalanine residues inhibited hemagglutination of amelogenins and the terminal tyrosine-rich amelogenin polypeptide (TRAP). The binding affinity of SFGSGFGGGY to amelogenins was confirmed by dosimetric binding of amelogenins or TRAP with [3H]peptide, specific binding in varying concentrations of the peptide, Scatchard plot analysis, and competitive inhibition with the unlabeled peptide. The ability of the peptide or GlcNAc to stoichiometrically inhibit TRAP binding of [14C]GlcNAc or [3H]peptide indicated that both the peptide and GlcNAc compete for a single binding site. Using different fragments of amelogenins, we have identified the peptide-binding motif in amelogenin to be the same as the GlcNAc-binding "amelogenin trityrosyl motif peptide." The GlcNAc-mimicking peptide failed to bind to the amelogenin trityrosyl motif peptide when the tyrosyl residues were substituted with phenylalanine or when the third proline was replaced with threonine, as in some cases of human X-linked amelogenesis imperfecta. This study documents that molecular mimicry may play a role in stability and organization of amelogenin during amelogenesis. tyrosine-rich amelogenin polypeptide leucine-rich amelogenin polypeptide wheat germ agglutinin GlcNAc mimicking peptide amelogenin trityrosyl motif peptide ATMP where proline is replaced by threonine ATMP where tyrosine is substituted by phenylalanine amelogenin carboxyl-terminal peptide hemagglutination hemagglutination inhibition human serum albumin high performance liquid chromatography Tris-buffered saline amelogenesis imperfecta GlcNAc-mimicking peptide cytokeratin-14 polyvinylidene difluoride bovine serum albumin Dental enamel is derived through the biomineralization of an extracellular organic matrix secreted by the ameloblast cells of the inner enamel epithelium. Although ameloblasts synthesize several other proteins, including cytokeratin 14 prior to synthesis of amelogenins (1Tabata M.J. Matsumura T. Liu J.G. Wakisaka S. Kurisu K. Arch. Oral Biol. 1996; 41: 1019-1027Crossref PubMed Scopus (51) Google Scholar), the amelogenins constitute some 90% of the secretory stage enamel matrix proteins (2Eastoe J.E. Arch. Oral Biol. 1963; 8: 633-652Crossref PubMed Scopus (103) Google Scholar, 3Eastoe J.E. J. Dent. Res. 1979; 58: 753-764Crossref PubMed Google Scholar, 4Zeichner-David M. Diekwisch T. Fincham A.G. Lau E. MacDougall M. Moradian-Oldak J. Simmer J. Snead M. Slavkin H.C. Int. J. Dev. Biol. 1995; 39: 69-92PubMed Google Scholar). Previously, we have hypothesized that amelogenins may bind to sugar residues of enamel matrix glycoproteins facilitating the biomineralization process (5Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2471Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The hypothesis was supported by identification of a stoichiometric interaction specifically between amelogenins and the GlcNAc residues of glycoconjugates (5Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2471Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Further, we have identified the glycobinding locus of the amelogenin structure in a highly conserved motif (-PYPSYGY-) located at the carboxyl-terminal of the tyrosine-rich amelogenin polypeptide (TRAP).1Remarkably, this trityrosyl motif has a striking structural similarity to the GlcNAc-binding domain of several GlcNAc-specific lectins (5Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2471Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 6Wright C.S. J. Mol. Biol. 1984; 178: 91-104Crossref PubMed Scopus (205) Google Scholar, 7Yamamoto K. Konami Y. Osawa T. Irimura T. J. Biochem. ( Tokyo ). 1992; 111: 436-439Crossref PubMed Scopus (32) Google Scholar). Recent observations have indicated that the GlcNAc-binding motif of several lectins such as wheat germ agglutinin (WGA) and lectins from Datura stramonium, Lycopersicon esculentum,Solanum tuberosum, and Wisteria floribunda also recognize and bind to a specific peptide sequence that mimics GlcNAc found in cytokeratins (8Shikhman A.R. Greenspan N.S. Cunningham M.W. J. Immunol. 1994; 154: 5593-5606Google Scholar). The present study is based on the hypothesis that comparable interactions between the amelogenins and GlcNAc-mimicking peptides (GMp) may occur during amelogenesis with implications for the understanding of the control of normal enamel development and of the molecular lesions that underlie enamel pathologies such as the condition of amelogenesis imperfecta. In this investigation, we demonstrate that a conserved GMp motif of cytokeratins specifically binds to the amelogenin trityrosyl motif peptide (ATMP). Further, we have directly tested the likely biological relevance of GMp-amelogenin interactions showing that loss of function mutations of the ATMP sequence correlates with loss of interaction with GMp, specifically that the substitution of a proline residue of ATMP (with threonine) as recently observed in a case of human X-linked amelogenesis imperfecta (AI) (9Collier P.M. Sank J.J. Rosenbloom J. Yuan Z.A. Gibson C.W. Arch. Oral Biol. 1997; 42: 235-242Crossref PubMed Scopus (121) Google Scholar) strongly abrogates the GMp-amelogenin interaction. The following amelogenin polypeptides (Fig. 1) were used: (i) rM179 (20.16 kDa), a recombinant mouse amelogenin, which is identical to the native murine amelogenin, M180 (except for the lack of the amino-terminal methionine residue (10Lau E.C. Simmer J.P. Bringas P. Hsu D. Hu C.C. Zeichner-David M. Thiemann F. Snead M.L. Slavkin H.C. Fincham A.G. Biochem. Biophys. Res. Commun. 1992; 188: 1253-1260Crossref PubMed Scopus (105) Google Scholar) and a phosphorylated serine at position 16) (11Fincham A.G. Hu Y. Lau E. Slavkin H.C. Snead M.L. Arch. Oral Biol. 1991; 36: 305-317Crossref PubMed Scopus (83) Google Scholar, 12Akita H. Fukae M. Shimoda S. Aoba T. Arch. Oral Biol. 1992; 37: 953-962Crossref PubMed Scopus (42) Google Scholar); (ii) rM166 (18.6 kDa), as rM179 but lacking the 13 C-terminal amino acid residues (13Simmer J.P. Lau E.C. Hu C.C. Aoba T. Lacey M. Nelson D. Zeichner-David M. Snead M.L. Slavkin H.C. Fincham A.G. Calcif. Tissue Int. 1994; 54: 312-319Crossref PubMed Scopus (166) Google Scholar); (iii) TRAP (5.20 kDa), a synthetic murine tyrosine-rich amelogenin polypeptide representing the N-terminal 45 amino acid residues of the M180 amelogenin; (iv) LRAP (6.82 kDa), synthetic leucine-rich amelogenin polypeptide, identical to the full-length (M180) amelogenin at its two termini but lacking the center portion of the protein (14Fincham A.G. Lau E.C. Simmer J. Zeichner-David M. Slavkin H.C. Price P. Chemistry and Biology of Mineralized Tissues. Elsevier, Amsterdam1992: 187-201Google Scholar); (v) amelogenin C-terminal peptide (ACP); (vi) P173 (25 kDa) and P148 (20 kDa) (porcine amelogenins were extracted and purified following the protocol of Fincham et al. (15Fincham A.G. Belcourt A.B. Termine J.D. Butler W.T. Cothran W.C. Biochem. J. 1983; 211: 149-154Crossref PubMed Scopus (98) Google Scholar) as described previously (5Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2471Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar)); and (vii) ATMP, PYPSYGYEPMGGW and two altered ATMP peptides in one of which the third proline is substituted with threonine (T-ATMP) (PYPSYGYETMGGW), and in another, all three tyrosine residues are replaced by phenylalanine (F-ATMP) (PFPSFGFEPMGGW) as described earlier (5Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2471Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) (see Fig. 1). A typical reverse phase HPLC profile of rM179 and electrophoretic homogeneity of rM179, rM166, and the synthetic peptides TRAP and LRAP used in this investigation were shown and described earlier (5Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2471Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The purified preparations (rM166) were devoid of or had negligible contamination with vector proteins. Synthetic GlcNAc-mimicking peptide (GM-peptide) with the cytokeratin amino acid sequence SFGSGFGGGY (GMp1) and its variants (GMp2 to -8) with single amino acid substitutions (Table I) were used in this investigation. These peptides are known to bind to anti-GlcNAc monoclonal antibody and also to GlcNAc-specific lectins such as WGA, U. europaeus 11 (UEA-II), and that of D. stramonium and S. tuberosum, expressing functional similarity to the carbohydrate (8Shikhman A.R. Greenspan N.S. Cunningham M.W. J. Immunol. 1994; 154: 5593-5606Google Scholar). The atomic mass units of these synthetic GM-peptides range from 935 to 888 (Table I). In order to identify the amino acid residues of the peptide sequence critical for binding to the tyrosyl motif of amelogenins, the pentamers (first and second half) of the GMp1 (SFGSGFGGGY), "SFGSG" and "FGGGY," were synthesized and used to inhibit the amelogenin or TRAP-mediated hemagglutination.Table IInhibition of hemagglutination of rM179 or TRAP by GlcNAc, its oligomers, GMps, and their fragmentsAmino acid sequence of peptidesGMpAtomic mass unitsrM179TRAPMinimal concentration requiredRelative inhibitory potencyMinimal concentration requiredRelative inhibitory potencySFGSGFGGGYGMp1935.15150 nm10500 pm100SFGSGFGGGKGMp2900.1550 μm<10.5 μm0.1SFGSGKGGGYGMp3916.1530 μm<15 μm<0.01SFGSGFGGGDGMp4887.06150 nm100.5 μm0.1DFGSGFGGGYGMp5963.1660 μm<150 μm<0.01SDGSGFGGGYGMp6903.06500 μm<1500 μm<0.01SFGDGFGGGYGMp7963.16>500 μm<1500 μm<0.01SFGSGDGGGYGMp8993.1515 μm<150 μm<0.01FGGGY499.644 mm<1>5 μm<0.01SFGSG453.534 mm<1>0.2 μm0.25SFGSG and FGGGY935.154 mm<1>5 μm<0.01GlcNAc22115 nm10010 nm5Chitobiose445150 nm10NDChitotetraose890150 nm10NDGlcNH22161 mm00.01 mm0BSA60 kDA150 μm0ND0Purified peptides (12.5 μl), serially diluted in TBS, pH 6.3, were added to microtiter wells and mixed with rM179 (6 μg/12.5 μl) or TRAP (3 μg/12.5 μl) previously adjusted to give two-well agglutination. After 60 min of incubation of 25 °C, 25 μl of 1.5% suspension of fresh mouse erythrocytes (TBS, pH 7.2) were added to each microtiter well and mixed. The hemagglutination titer was determined after 2-h incubation at 25 °C. The relative inhibitory potency of each peptide is indicated. ND, not tested. Open table in a new tab Purified peptides (12.5 μl), serially diluted in TBS, pH 6.3, were added to microtiter wells and mixed with rM179 (6 μg/12.5 μl) or TRAP (3 μg/12.5 μl) previously adjusted to give two-well agglutination. After 60 min of incubation of 25 °C, 25 μl of 1.5% suspension of fresh mouse erythrocytes (TBS, pH 7.2) were added to each microtiter well and mixed. The hemagglutination titer was determined after 2-h incubation at 25 °C. The relative inhibitory potency of each peptide is indicated. ND, not tested. All of the polypeptides, (GMp variants, the two pentamers, TRAP, and LRAP) used in this investigation were synthesized by the USC microchemical Core Laboratory using an Applied Biosystems model 430A one-column peptide synthesizer with the modified Merrifield procedure (16Merrifield B. Science. 1996; 232: 341-347Crossref Scopus (794) Google Scholar). Peptides were purified by reversed-phase HPLC (C4–214TP54 column or C18–291HS54 column; Vydac/The Separations Group, Hesperia, CA) with a gradient of 35–50% B in 60 min (buffer B contained 60% (v/v) aqueous acetonitrile in 0.1% (v/v) trifluoroacetic acid, and buffer A contained 0.1% trifluoroacetic acid) at a flow rate of 1.0 or 0.5 ml/min for small peptides depending on their size (5Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2471Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The decapeptide SFGSGFGGGY (GMp1) was also labeled with 3H (Amersham Pharmacia Biotech) to identify the specific binding to amelogenins. SFGSGFGGG[3H]Y was prepared from tritium gas by Amersham Pharmacia Biotech. The product was purified by high performance liquid chromatography on a Vydac C18 300-Å (protein and peptide) (250 × 4.6 mm) column with a gradient of buffer A (0.01 mtrifluoroacetic acid, aqueous) and buffer B (0.01 mtrifluoroacetic acid in acetonitrile), 0–100% B over 30 min, at a flow rate of 1.0 ml/min. The peptide was supplied in an aqueous solution in a silanized borosilicate multidose vial with additional screw-cap under argon. The peptide was stored in the absence of light and air at 4 °C. GlcNAc, chitobiose, chitotetraose, and WGA (Sigma) were used as positive controls andd-(+)-glucosamine, LRAP, and BSA (Sigma) as negative controls. The HAI assays were performed in 8 × 12-microtiter plates, with U-bottomed wells, after assessing the HA activity of the recombinant amelogenin/TRAP molecules as described earlier (5Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2471Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Previously, we have screened a variety of mammalian erythrocytes and selected mouse erythrocytes for effective HA by amelogenins including both rM179 and TRAP. We have selected the concentration of rM179/TRAP that gives two-well agglutination. All GMps were diluted (1:10 in Tris-buffered saline (TBS), pH 6.3) in Eppendorf tubes and were warmed to 30 °C. To each well, 12.5 μl of peptide solutions were added. The final concentration of peptides/well was adjusted to and ranged from 1 pm to 1 mm at 10-fold dilution. 6 μg/12.5 μl of amelogenin or 3 μg/12.5 μl of TRAP (diluted in trifluoroacetic acid (0.04%) + TBS, pH 6.3), or TBS (pH 6.3) capable of two-well agglutination was added to each well and incubated for 1 h at 25 °C. After incubation, 25 μl of a 1.5% suspension of mouse erythrocytes (purchased from Crane Laboratories, Inc., Syracuse, NY) in TBS, pH 7.2, was added to all the wells. The plates were covered with parafilm, subjected to gentle low speed vortex for 10 s, and incubated at 25 °C, and scoring was done after 2 h. The HAI titers were reported as the reciprocal of the lowest concentration of the inhibitors giving complete HAI (button formation) after 2 h. A known amount of [14C]GlcNAc (2 × 104 cpm) in 100 μl of TBS (pH 7.2) was added to polypropylene microcentrifuge tubes containing 100 μl of increasing amounts of TRAP molecules in 0.04% trifluoroacetic acid. The mixture was incubated under constant agitation for 90 min at 25 °C and precipitated with 1 ml of cold ethanol (200 proof; Gold Shield Chemical Co., Hayward, CA) at 4 °C for 20 min. The tubes were then centrifuged for 15 min at 12,000 × g in a Beckman Microfuge 12, and the supernatant was removed. The unbound14C-labeled GlcNAc was removed completely by repeated vortex mixing and washing three times with cold ethanol. Washing three times with ethanol did not affect the bound peptides. The final pellets were dissolved in 1 n NaOH, and the radioactivity was measured 15 min after adding 4 ml of scintillation fluid (Bio-safe 11, Research Products International Corp., Mount Prospect, IL) in a β-counter (Beckman, LS-1801). WGA was used as a positive control and BSA as a negative control. For the competitive binding inhibition assay, 100 μl of unlabeled GMps or GlcNAc (TBS, pH 7.2) with increasing concentrations were prepared in duplicate in microcentrifuge tubes. To each concentration of the GMp or GlcNAc, 100 μl of [14C]GlcNAc (2 × 104 cpm in TBS, pH 7.2) was added and mixed. 100 μl of rM179 (7.5 nmol) or TRAP (20 nmol) in 0.04% trifluoroacetic acid were added to the mixture and incubated for 90 min at 25 °C on a rotator. After incubation, the proteins were precipitated with 1 ml of cold ethanol at 4 °C for 20 min and centrifuged for 15 min at 12,000 × g. The unbound radioactive GlcNAc was removed completely by repeated vortex mixing and washing, three times with ethanol. The final pellets were dissolved in 50 μl of 1 n NaOH, and the bound radioactivity was measured with 4 ml of scintillation fluid (Bio-safe 11) in a β-counter as mentioned earlier (5Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2471Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Glucosamine was used as a negative control. The values were expressed as percentage of bound [14C]GlcNAc to rM179 or TRAP. 100 μl of [3H]GMp1 (30 × 104 dpm in TBS, pH 7.2) was added to polypropylene microcentrifuge tubes containing increasing amounts of rM179 or TRAP molecules in 0.04% trifluoroacetic acid, and the mixture was gently shaken every 20 min for 2 h at 37 °C. After incubation, the samples were pelleted with 1 ml of cold ethanol at 4 °C for 20 min and centrifuged for 15 min at 12,000 ×g, and the supernatant was removed. The unbound radioactive peptides were removed completely by repeated vortex mixing and washing four times with ethanol, which did not affect the bound peptides. The final pellets were dissolved in 1 n NaOH, and the bound radioactivity was measured 15 min after adding 4 ml of scintillation fluid (Amersham Pharmacia Biotech) in a β-counter. BSA and WGA were used as negative and positive controls, respectively. Purified recombinant murine amelogenins (rM179 and rM166) were resolved via SDS-polyacrylamide gel electrophoresis using 15% resolving and 3.5% stacking gels (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp.; Immobilon-P Transfer Membrane) at 100 mA for 1 h using a semidry transblot apparatus (Hoefer Scientific Instruments, San Francisco) (18Towbin H. Staehelin T. Garden J. Proc. Natl. Acad. Sci . U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). Protein transfer was assessed by staining the PVDF strips with 0.1% Fast Green (Sigma) in 40% acetic acid and 10% methanol, and the strips were compared with Coomassie Blue-stained protein bands (19Ravindranath R.M.H. Graves M.C. Virology. 1992; 188: 143-151Crossref PubMed Scopus (8) Google Scholar). Replicas were treated with [3H]GMp1 (7 × 107 dpm/ml) resuspended in phosphate-buffered saline (pH 6.0) for 18 h at 25 °C, after blocking the membrane with phosphate-buffered for 1 h at 37 °C. The membranes were times with phosphate-buffered 0.1% R.M.H. Graves M.C. J. PubMed Google Scholar). After washing and the membranes were to (Amersham Pharmacia Biotech) for 8 at 25 °C, and the were The binding of [3H]GMp1 to TRAP was determined in using increasing concentrations of GMp1 to 20 of TRAP The binding of [3H]GMp1 was determined in duplicate in the of of unlabeled and was from the binding to the specific A Scatchard plot of this specific binding was 100 μl of unlabeled GMp1 or GlcNAc in TBS (pH 7.2) with increasing concentrations were prepared in in microcentrifuge tubes. To each concentration of unlabeled peptide or GlcNAc, 100 μl of [3H]GMp1 (30 × 104 dpm in TBS, pH 7.2) was added and mixed. TRAP (25 μl in 0.04% trifluoroacetic acid) was added to the mixture and gently shaken every 20 min for 2 h at 37 °C. After incubation, the samples were precipitated with 1 ml of cold ethanol and centrifuged for 15 min at 12,000 × g. The unbound [3H]GMp1 was removed completely by repeated vortex mixing and washing four times with ethanol, and the bound radioactivity was measured with 4 ml of scintillation fluid (Amersham Pharmacia Biotech) in a β-counter as mentioned earlier (5Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2471Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The values were expressed as percentage of bound [3H]GMp1 to TRAP. The binding of [3H]GMp1 to different amelogenins was measured by matrix assay, using strips in a microtiter Scientific was done by adding a solution of 100 μl of polypeptides rM166, TRAP, LRAP, ATMP, amelogenins, 20 and 25 kDa) in buffer (pH to microtiter wells in and at 25 °C polypeptides are in the range of pH J. Moradian-Oldak J. Zeichner-David M. Fincham A.G. J. Dent. Res. PubMed Scopus Google Scholar). WGA and BSA were used as positive and negative controls, respectively. were with 0.1% in TBS, pH for 2 h at 37 °C. 100 μl of [3H]GMp1 (30 × 104 in TBS (pH 7.2) were added to wells and incubated for 2 h at 37 °C with gentle every 20 min. After incubation, the unbound peptide was removed and three times with TBS (pH The wells were transferred to scintillation The bound radioactive peptide was the 4 ml of scintillation fluid Research Products International by and for 15 min and for bound radioactivity in a β-counter. The was also used to inhibition of [14C]GlcNAc binding to TRAP by were In and and and of of were using values were to the of GMp with the amino acid sequence SFGSGFGGGY (GMp1) and its variants to -8) with single amino acid substitution in in were purified by reverse phase and a typical profile of GMp1 is in Fig. The peptide binding of purified rM179/TRAP molecules was by peptide inhibition of HAI was used as a positive control. the of GMps that inhibited HA of amelogenin and of TRAP. the peptides GMp1 and inhibited the HA of of the other GMps inhibited the HA of amelogenins at The substitution of the serine or phenylalanine residues of GMp1 the GMp1 and inhibited the HA at which is the same as with the and of The binding of GlcNAc to rM179 is 10-fold that of that GlcNAc is the of rM179 In GMp1 is the inhibitory peptide of The binding of GMp1 to the TRAP is that of GlcNAc, that GMp1 is the of TRAP. of GMp1 at a concentration as low as of is to such The inhibitory potency of GMp1 is that of or The substitution of serine in and or phenylalanine residues in or or the terminal tyrosine in and The binding affinity for GlcNAc GMp1 to with of the amelogenins to TRAP. To identify and the amino acid sequence for binding to the tyrosyl TRAP motif of amelogenins, the pentamers were used to inhibit the or TRAP-mediated The did not affect or TRAP-mediated representing the amino-terminal of the GMp1 inhibited the TRAP-mediated HA compared with the full-length GMp1 (see that of the functional amino acid sequence of GMp1 by TRAP. The loss of binding affinity of the peptide when GMp1 is two that the binding of amelogenin or TRAP amino acid residues of In order to the concentration of rM179 or TRAP for GMp inhibition of [14C]GlcNAc the of [14C]GlcNAc interaction with rM179/TRAP was The binding of [14C]GlcNAc with the concentration of rM179 (5Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2471Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and TRAP (Fig. on the we selected of rM179 and 20 of TRAP for inhibition of binding of [14C]GlcNAc by The of GMps inhibition of the interaction of [14C]GlcNAc to rM179 and TRAP are in Fig. A and respectively. GMp1 and inhibited the binding of [14C]GlcNAc to of the other GMps inhibited the interaction of rM179 with inhibition of GMp1 and at and inhibition by cold GlcNAc a concentration (250 (Fig. 4 The relative inhibitory of GMp1 and are cold GlcNAc, a different from that for HAI in which GlcNAc a 10-fold in inhibitory both GMp1 and were of rM179 in both GMp1 strongly and inhibited the binding of [14C]GlcNAc to TRAP. The relative inhibitory potency of GMp1 is cold GlcNAc (Fig. 4 a in with HAI observations in which GMp1 is GlcNAc in TRAP-mediated of the other peptides inhibited the interaction of TRAP with [14C]GlcNAc in the matrix GMp1 is the peptide the activity of rM179 and the TRAP The binding of [3H]GMp1 as the concentration of rM179/TRAP in a (Fig. A as by a with 2 for rM179 and for WGA used as the positive control also a (Fig. table LRAP, a negative and binding to [3H]GMp1 and as a control. the binding of [3H]GMp1 to amelogenins. the binding of [3H]GMp1 to TRAP in to the potency of inhibition GlcNAc in HA and GlcNAc binding of TRAP, we selected TRAP to the specific binding of GMp1 (Fig. Fig. 6 the specific binding of [3H]GMp1 to TRAP as a function of increasing concentration of The binding was measured with unlabeled GMp1 and to specific GMp1 A Scatchard plot of the binding of [3H]GMp1 to TRAP that the peptide-binding is with to the GMp1 inhibited the binding of [3H]GMp1 to TRAP The inhibition for GMp1 and GlcNAc and 2 for GMp1 and GlcNAc (Fig. of the or the inhibition that GMp1 is cold GlcNAc in binding to TRAP, a in with GMp1 inhibition of HA (Table I) and binding of [14C]GlcNAc to TRAP (Fig. 4 The specific binding of GMp1 to TRAP is confirmed in a matrix in which [3H]GMp1 bound to rM179 and rM166 the but failed to bind to that the is not for amino of the 45 amino acid residues of the N-terminal TRAP are also by LRAP (see Fig. 1). In LRAP amino acid residues of