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• W1968339504 abstract "The enamel protein amelogenin binds to GlcNAc (Ravindranath, R. M. H., Moradian-Oldak, R., and Fincham, A.G. (1999) J. Biol. Chem. 274, 2464–2471) and to the GlcNAc-mimicking peptide (GMp) (Ravindranath, R. M. H., Tam, W., Nguyen, P., and Fincham, A. G. (2000) J. Biol. Chem. 275, 39654–39661). The GMp motif in the N-terminal region of the cytokeratin 14 of ameloblasts binds to trityrosyl motif peptide (ATMP) of amelogenin (Ravindranath, R. M. H., Tam, W., Bringas, P., Santos, V., and Fincham, A. G. (2001) J. Biol. Chem. 276, 36586 – 36597). K14 (Type I) pairs with K5 (Type II) in basal epithelial cells; GlcNAc-acylated K5 is identified in ameloblasts. Dosimetric analysis showed the binding affinity of amelogenin to K5 and to GlcNAc-acylated-positive control, ovalbumin. The specific binding of [3H]ATMP with K5 or ovalbumin was confirmed by Scatchard analysis. [3H]ATMP failed to bind to K5 after removal of GlcNAc. Blocking K5 with ATMP abrogates the K5-amelogenin interaction. K5 failed to bind to ATMP when the third proline was substituted with threonine, as in some cases of human X-linked amelogenesis imperfecta or when tyrosyl residues were substituted with phenylalanine. Confocal laser scan microscopic observations on ameloblasts during postnatal (PN) growth of the teeth showed that the K5-amelogenin complex migrated from the cytoplasm to the periphery (on PN day 1) and accumulated at the apical region on day 3. Secretion of amelogenin commences from day 1. K5, similar to K14, may play a role of chaperone during secretion of amelogenin. Upon secretion of amelogenin, K5 pairs with K14. Pairing of K5 and K14 commences on day 3 and ends on day 9. The pairing of K5 and K14 marks the end of secretion of amelogenin. The enamel protein amelogenin binds to GlcNAc (Ravindranath, R. M. H., Moradian-Oldak, R., and Fincham, A.G. (1999) J. Biol. Chem. 274, 2464–2471) and to the GlcNAc-mimicking peptide (GMp) (Ravindranath, R. M. H., Tam, W., Nguyen, P., and Fincham, A. G. (2000) J. Biol. Chem. 275, 39654–39661). The GMp motif in the N-terminal region of the cytokeratin 14 of ameloblasts binds to trityrosyl motif peptide (ATMP) of amelogenin (Ravindranath, R. M. H., Tam, W., Bringas, P., Santos, V., and Fincham, A. G. (2001) J. Biol. Chem. 276, 36586 – 36597). K14 (Type I) pairs with K5 (Type II) in basal epithelial cells; GlcNAc-acylated K5 is identified in ameloblasts. Dosimetric analysis showed the binding affinity of amelogenin to K5 and to GlcNAc-acylated-positive control, ovalbumin. The specific binding of [3H]ATMP with K5 or ovalbumin was confirmed by Scatchard analysis. [3H]ATMP failed to bind to K5 after removal of GlcNAc. Blocking K5 with ATMP abrogates the K5-amelogenin interaction. K5 failed to bind to ATMP when the third proline was substituted with threonine, as in some cases of human X-linked amelogenesis imperfecta or when tyrosyl residues were substituted with phenylalanine. Confocal laser scan microscopic observations on ameloblasts during postnatal (PN) growth of the teeth showed that the K5-amelogenin complex migrated from the cytoplasm to the periphery (on PN day 1) and accumulated at the apical region on day 3. Secretion of amelogenin commences from day 1. K5, similar to K14, may play a role of chaperone during secretion of amelogenin. Upon secretion of amelogenin, K5 pairs with K14. Pairing of K5 and K14 commences on day 3 and ends on day 9. The pairing of K5 and K14 marks the end of secretion of amelogenin. Ameloblasts synthesize and secrete the proteins involved in the microstructure and biomineralization of the enamel. Amelogenin constitutes 90% of the total enamel proteins secreted by ameloblasts. It is a non-glycosylated single polypeptide of about 180 amino acids (1Termine J.D. Belcourt A.B. Miyamoto M.S. Conn K.M. J. Biol. Chem. 1980; 255: 9769-9772Abstract Full Text PDF PubMed Google Scholar, 2Fincham A.G. Belcourt A.B. Termine J.D. Butler W.T. Cothran W.C. Biochem. J. 1983; 211: 149-154Crossref PubMed Scopus (97) Google Scholar, 3Akita H. Fukae M. Shimoda S. Aoba T. Arch. Oral. Biol. 1992; 37: 953-962Crossref PubMed Scopus (42) Google Scholar). The N-terminal 45 amino acid residues are referred to as tyrosine-rich amelogenin polypeptide (TRAP). 1The abbreviations used are: TRAP, tyrosine-rich amelogenin polypeptide; ATMP, ameloblasts binds to trityrosyl motif peptide; GlcNAc, N-acetylglucosamine; GMp, GlcNAc-mimicking peptide; AI, amelogenesis imperfecta; PN, postnatal; BME, buffered urea-β-mercaptoethanol; PVDF, polyvinylidene difluoride; PBS, phosphate-buffered saline; HSA, human serum albumin; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; GD2, disialoganglioside 2; GD3, disialoganglioside 3; GM2, monosialoganglioside 2; GD1b, disialoganglioside 1b.1The abbreviations used are: TRAP, tyrosine-rich amelogenin polypeptide; ATMP, ameloblasts binds to trityrosyl motif peptide; GlcNAc, N-acetylglucosamine; GMp, GlcNAc-mimicking peptide; AI, amelogenesis imperfecta; PN, postnatal; BME, buffered urea-β-mercaptoethanol; PVDF, polyvinylidene difluoride; PBS, phosphate-buffered saline; HSA, human serum albumin; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; GD2, disialoganglioside 2; GD3, disialoganglioside 3; GM2, monosialoganglioside 2; GD1b, disialoganglioside 1b. The C-terminal 13-amino acid sequence in the TRAP region called “amelogenin tyrosyl motif peptide” (ATMP: PYPSYGYEPMGGW) possesses unique ligand-binding properties, in that it binds specifically to N-acetylglucosamine (GlcNAc) (4Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2741Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and GlcNAc-mimicking peptides (GMps) (5Ravindranath R.M.H. Tam W. Nguyen P. Fincham A.G. J. Biol. Chem. 2000; 275: 39654-39661Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Mutations in the ATMP sequence are observed in the human X-linked amelogenesis imperfecta (AI) (6Collier P.M. Sauk J.J. Rosenbloom S.J. Yuan Z.A. Gibson C.W. Arch. Oral Biol. 1997; 42: 235-242Crossref PubMed Scopus (118) Google Scholar). The mutated forms of ATMP failed to bind with GlcNAc or the GMp (4Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2741Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 5Ravindranath R.M.H. Tam W. Nguyen P. Fincham A.G. J. Biol. Chem. 2000; 275: 39654-39661Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Loss-of-function “mutations” of ATMP correlated well with the loss of amelogenin-ligand interaction. A GMp motif is found in the N-terminal region of Type I cytokeratin 14 (K14), a differentiation marker for ameloblasts prior to amelogenin synthesis (7Tabata M.J. Matsumura T. Liu J.G. Wakisaka S. Kurisu K. Arch. Oral Biol. 1996; 41: 1019-1027Crossref PubMed Scopus (51) Google Scholar). The purified or recombinant amelogenin and TRAP bind to K14 in vitro dosimetrically (8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Scatchard analysis confirms the specific interaction between K14 and ATMP in vitro. GlcNAc and GMp blocked binding of rM179 or ATMP with K14. Mutated ATMP failed to bind to K14 when the third proline was substituted with threonine, as in some cases of human X-linked AI (6Collier P.M. Sauk J.J. Rosenbloom S.J. Yuan Z.A. Gibson C.W. Arch. Oral Biol. 1997; 42: 235-242Crossref PubMed Scopus (118) Google Scholar) or when tyrosyl residues were substituted with phenylalanine. Amelogenin co-assembled with K14 in the perinuclear region of ameloblasts on day 0. The K14-amelogenin complex migrated to the apical region of the ameloblasts on day 1 and accumulated there between days 3 and 5 and collapsed on day 9. Autoradiography with [3H]ATMP and [3H]GMp corroborated the dissociation of amelogenin and K14 at the Tomes' process of the ameloblast, suggesting that K14 plays a chaperone role for nascent amelogenin polypeptide during secretion of amelogenin (8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). K14 (Type I) pairs with Type II K5 in basal epithelial cells. Such pairing of Type I and Type II cytokeratins are known to occur during epithelial cell differentiation (9Sun T.T. Eichner R. Schermer A. Cooper D. Nelson W.G. Weiss R.A. Levine A.J. VandeWoude G.F. Topp W.C. Watson J.D. The Transformed Phenotype 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1984: 169-176Google Scholar). K5 is also present in ameloblasts (10Kasper M. Karsten U. Stosiek P. Moll R. Differentiation. 1989; 40: 207-214Crossref PubMed Scopus (88) Google Scholar, 11Domingues M.G. Jaeger M.M.M. Araujo V.C. Araujo N.S. Eur. J. Oral. Sci. 2000; 108: 43-47Crossref PubMed Scopus (65) Google Scholar). The pairing of K5 with K14 requires N-terminal regions of both cytokeratins (12Wilson A.K. Coulombe P.A. Fuchs E. J. Cell Biol. 1992; 119: 401-414Crossref PubMed Scopus (113) Google Scholar). We hypothesize that, if the N-terminal region (GMp motif) of K14 is bound to the ATMP motif of amelogenin, it may not pair with K5 until amelogenin is disassociated from it. Furthermore, we hypothesize that K5, per se, might also bind to amelogenin, because it may carry GlcNAc at the N-terminal residues similar to other Type II keratins (K8) (13Omary M.B. Ku N.O. Liao J. Price D. Subcell. Biochem. 1998; 31: 105-140PubMed Google Scholar). If K5 were to bind to amelogenin, it might define the relative role of Type I and II cytokeratins in secretion of amelogenin. If pairing of K14 and K5 were to occur in ameloblasts, it would be important to define the event in the context of amelogenin secretion and enamel formation. We present evidence to show that K5 binds to amelogenin, leading to the secretion of amelogenin and pairing with K14. The in vitro and in situ observations, during different stages of enamel formation, define the interactions among amelogenin and cytokeratins. The pairing of K14 with K5 does occur after secretion of amelogenin and just prior to the disintegration of cytokeratins. These findings are novel and critical, and they define the relative role of cytokeratins in the secretion of amelogenin during enamel formation. The results are important for understanding the events taking place during secretion of amelogenin and enamel formation, and the development of corrective measures to prevent abnormal enamel development in genetic disorders like AI and different kinds of epidermolysis bullosa caused by mutations in K14 or K5 (14Coulombe P.A. Hutton M.E. Letai A. Hebert A. Paller A.S. Fuchs E. Cell. 1991; 66: 1301-1311Abstract Full Text PDF PubMed Scopus (524) Google Scholar, 15Bonifas J.M. Rothman A.L. Epstein Jr., E.H. Science. 1991; 254: 1202-1205Crossref PubMed Scopus (342) Google Scholar, 16Stephens K. Sybert V.P. Wijsman E.M. Ehrlich P. Spencer A. J. Invest. Dermatol. 1993; 101: 240-243Abstract Full Text PDF PubMed Google Scholar, 17Chan Y. Anton-Lamprecht I. Yu Q. Jackel A. Zabel B. Ernst J. Fuchs E. Genes Dev. 1994; 8: 2574-2587Crossref PubMed Scopus (158) Google Scholar, 18Rugg E.L. McLean W.I. lane E.B. Pitera R. McMillan J.R. Dopping-Hepenstal P.J. Navasaria H.A. Leagh I.M. Eady R.A.J. Genes Dev. 1994; 8: 2563-2573Crossref PubMed Scopus (149) Google Scholar). Mice—All observations were made on Swiss Webster mice at different developmental stages ranging from newborn day “0” through postnatal days (PN) 1, 3, 5, 7, and 9. A total of 25 normal, healthy, female pregnant Swiss Webster mice (Charles River Breeding) were used to obtain a sufficient number of litters as was carried out previously (8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The Institutional Animal Care and Use Committee (Los Angeles, CA) approved all protocols involving mice. Isolation and Purification of K5—We have isolated and purified K5 from HeLa cells (19Taddei A. Roche D. Sibarita J.-B. Turner B.M. Almouzni G. J. Cell Biol. 1999; 147: 1153-1166Crossref PubMed Scopus (173) Google Scholar). We have obtained HeLa cells (CCL-2 obtained from ATCC) and have grown them in Eagle's minimum essential medium containing 10% fetal bovine serum at 37 °C. The cells were collected and frozen at -20 °C. The proteins were extracted as previously described (20Sun T.T. Eichner R. Nelson W.G. Vidrich A. Woodcock-Mitchell J. Curr. Probl. Dermatol. 1983; 11: 277-291Crossref PubMed Google Scholar, 21Vidrich A. Gilmartin M.E. Mitchell J. Freedberg I.M. Ann. N. Y. Acad. Sci. 1985; 455: 354-370Crossref PubMed Scopus (11) Google Scholar). The keratin fraction was obtained from cells that were homogenized in equal volumes of ice-cold Tris-Triton buffer (25 mm Tris-HCl, pH 7.4, 0.5% Triton X-100, 1 mm EDTA, 1 mm of phenylmethylsulfonyl fluoride). The extract was centrifuged at 10,000 × g for 2 min. The pellet was extracted three times with Tris-Triton buffer. The supernatant fractions contain most of the soluble cellular proteins, including many membrane proteins. The pellet was washed in Tris buffer and then dispersed in double the volume of Tris-HCl, pH 7.4, with 1 m KCl, incubated for 30 min at 37 °C, and once again centrifuged for 10,000 × g for 30 min. The final pellet was solubilized in about 1.5 volumes of buffered urea-β-mercaptoethanol (BME) solution (25 mm Tris-HCl, pH 7.4, 9 m urea, 1 mm EDTA, 100 mm BME, 1 mm phenylmethylsulfonyl fluoride). The extracted proteins were purified by the USC Microchemical Core Laboratory, using a reversed-phase high performance liquid chromatograph (C18 analytical column with a gradient of 0–100% B in 45 min; buffer B contained 70% (v/v) aqueous acetonitrile, 0.09% trifluoroacetic acid; buffer A contained 0.1% trifluoroacetic acid). Protein concentrations were determined by Lowry et al. (23Lowry O.H. Rosebrough N.H. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), and purity and homogeneity were assessed by affinity-purified monoclonal antibody for K5. The antibodies used included mouse anti-K5 (1:1000) (Chemicon International, Temecula, CA) and sheep monoclonal K5 antibody (1:100) (Binding Site, Birmingham, UK). The availability of a source of defined and purified K5 has now permitted us to study the binding properties of this molecule with amelogenins. Protein Preparations from Ameloblasts—We have isolated ameloblasts from mouse postnatal mandibular first molars at different developmental stages ranging from newborn day “0” through postnatal (PN) days 1, 3, 5, 7, and 9. Isolated ameloblasts were pooled, frozen, and thawed (four cycles) to extract the proteins as described with modifications previously used (20Sun T.T. Eichner R. Nelson W.G. Vidrich A. Woodcock-Mitchell J. Curr. Probl. Dermatol. 1983; 11: 277-291Crossref PubMed Google Scholar, 21Vidrich A. Gilmartin M.E. Mitchell J. Freedberg I.M. Ann. N. Y. Acad. Sci. 1985; 455: 354-370Crossref PubMed Scopus (11) Google Scholar). The cytokeratin fraction was obtained from cells that were homogenized with approximately three volumes of ice-cold Tris-HCl (Tris-HCl, pH 7.4, with 1 m urea, 100 μm EDTA, 10 mm BME). The homogenates were incubated for about 30 min at 37 °C and then centrifuged for 5 min at 8,000 × g in a Beckman Microfuge 12, and the supernatant was collected. The proteins from the supernatants were isolated using a Microcon concentrator with a cut-off size of 10,000 and centrifuged at 2,000 × g for 12 min. The protein fraction was collected and stored at -20 °C until further use. Equivalent amounts of isolated proteins (∼20 μg), as assessed by spectrophotometry (23Lowry O.H. Rosebrough N.H. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), were added to the gel (4–12% gradient SDS-PAGE). After confirming the homogeneity and purity, the gels were used for Western blot analysis. Expression and Purification of Recombinant Proteins—Preparation and purification of recombinant mouse amelogenin rM179 were carried out as previously described (4Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2741Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 22Simmer 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 (163) Google Scholar). Preparation and Purification of Synthetic Peptides—All the polypeptides (ATMP, T-ATMP, and F-ATMP) used in this study were synthesized by the University of Southern California Microchemical Core Laboratory using an Applied Biosystems model 430A single-column peptide synthesizer with the modified Merrifield procedure (24Merrifield B. Science. 1996; 232: 341-347Crossref Scopus (779) Google Scholar). Peptides were purified as mentioned previously (4Ravindranath R.M.H. Moradian-Oldak J. Fincham A.G. J. Biol. Chem. 1999; 274: 2464-2741Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). 3H Labeling of ATMP—The 13-residue ATMP “P[3H]YPSYGYEPMGGW” was prepared and purified by Amersham Biosciences as mentioned previously (8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Western Blot Analysis—K5 purified from HeLa cells and proteins isolated from ameloblast extracts during different developmental days (newborn to PN day 9) were resolved by gradient SDS-PAGE (4–12%) and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P Transfer Membrane, Millipore Corp.) at 100 mA for 35 min using a semi-dry transblot apparatus (Bio-Rad Scientific Instruments) (5Ravindranath R.M.H. Tam W. Nguyen P. Fincham A.G. J. Biol. Chem. 2000; 275: 39654-39661Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Protein transfer was assessed by staining the strips with 0.1% Fast Green (Sigma) in 40% methanol and 10% acetic acid (5Ravindranath R.M.H. Tam W. Nguyen P. Fincham A.G. J. Biol. Chem. 2000; 275: 39654-39661Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The membranes were washed (five times) and blocked with PBS (pH 7.2) containing -1% HSA, and were overlaid with appropriate antibodies. The antibodies used included mouse anti-K5 (1:1000, Chemicon International) and sheep monoclonal K5 antibody (1:100). The Western blots also were treated with monoclonal antibodies common for both N-linked and O-linked (IgM antibody, 1:100, Calbiochem) GlcNAc and with monoclonal antibody specific for O-linked GlcNAc (IgG1, Alexis Biochemicals, 1:100). Enzyme-linked secondary antibody from Roche Diagnostics (BM Chemiluminescence Western-blotting kit, 1:2000) were used. The presence of GlcNAc in proteins was also confirmed by staining the Western blots with Datura stramonium lectin (8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Binding of Amelogenin to Varying Concentrations of K5 by Enzyme-linked Immunosorbent Assay—enzyme-linked immunosorbent assay was performed using the purified K5 as an antigen following the protocol described previously (8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Antigen coating was done by adding 100 μl of a solution containing varying amounts of proteins in PBS (pH 7.2) to microtiter plates (Falcon 3915, Fisher Scientific) and incubating the plates at 4 °C overnight. Wells were blocked with PBS containing 1% HSA for 90 min at 37 °C. One-hundred microliters of a known amount of amelogenin protein (rM179, 5 pmol/100 μl) was added to wells, and the mixture was incubated for 1 h at 37 °C. After washing the plates five times, the primary antibody against the rM179 protein (8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) (at a dilution of 1:6000) (5Ravindranath R.M.H. Tam W. Nguyen P. Fincham A.G. J. Biol. Chem. 2000; 275: 39654-39661Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) was added, and the mixture was incubated for 1 h at 37 °C and then incubated with a secondary antibody (1:5000, goat anti-rabbit IgG, Jackson ImmunoResearch, West Grove, PA) for 1 h. After washing, a substrate (O-phenylenediamine dihydrochloride, Invitrogen) in citrate-phosphate buffer and hydrogen peroxidase was added to the plates for color development. The enzymatic oxidation was arrested after 30 min in the dark, with 6 n H2SO4. The absorbency difference at 490 – 650 nm was measured after automix in a microplate reader (Molecular Devices, Sunnyvale, CA). The values were corrected for background (wells without antigen). Ovalbumin was used as a positive control because it contains two residues of terminal GlcNAc (25Silva M.L.C. Stubbs H.J. Tamura T. Rice K.G. Arch. Biochem. Biophys. 1995; 318: 465-475Crossref PubMed Scopus (68) Google Scholar) and BSA as a negative control. Binding of [3H]ATMP to K5 by Western Blot Analysis and Autoradiography—Purified K5 was resolved via SDS-PAGE and transferred to PVDF membranes at 100 mA for 35 min using a semi-dry transblot apparatus (Bio-Rad Scientific Instruments) (5Ravindranath R.M.H. Tam W. Nguyen P. Fincham A.G. J. Biol. Chem. 2000; 275: 39654-39661Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Protein transfer was assessed by staining the PVDF strips with 0.1% Fast Green as mentioned previously (5Ravindranath R.M.H. Tam W. Nguyen P. Fincham A.G. J. Biol. Chem. 2000; 275: 39654-39661Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Replicas were treated with [3H]ATMP (8 × 105 dpm/ml) and resuspended in PBS (pH 7.2) for 18 h at 25 °C, after blocking the membrane with PBS containing, 1% HSA for 1 h at 37 °C. The membranes were washed five times with PBS containing 0.1% HSA (5Ravindranath R.M.H. Tam W. Nguyen P. Fincham A.G. J. Biol. Chem. 2000; 275: 39654-39661Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 26Moll R. Werner W.F. Schiller D.L. Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4474) Google Scholar). After washing and drying, the membranes were exposed to hyperfilm-3H (Amersham Biosciences) for 5 days at 4 °C, and the films were developed manually. To assess whether [3H]ATMP binds GlcNAc residues of K5, we treated blots of K5 on PVDF membranes, after blocking with PBS, 1% HSA for 1 h at 37 °C, N-acetyl glucosaminidase recombinant from Escherichia coli (A6805, Sigma). 100 μl (5 units) of the enzyme suspension (in PBS, pH 7.00) was overlaid on two strips. Control strips were overlaid with blocking buffer. A thin strip of Parafilm was placed over the solution to retain the enzyme at one location. The overlaid strips were incubated at room temperature (1 h) and at 37 °C for 90 min. After washing with the PBS, 0.1% HSA, 0.005% Tween 20, the strips were incubated in sealer bags with [3H]ATMP (8 × 105 dpm/ml) resuspended in PBS (pH 7.2) for 18 h at 4 °C. The membranes were washed five times with PBS, 0.1% HSA. After washing and drying, the membranes were exposed to hyperfilm-3H (Amersham Biosciences) for 5 days at 25 °C, and the films were developed manually. Ovalbumin was used as positive control. Dosimetric Binding of K5 to [3H]ATMP—100 μl of [3H]ATMP (30 × 104 dpm in Tris-buffered saline, pH 7.2), was added to 1.5-ml polypropylene microcentrifuge tubes containing increasing amounts of K5 in TBS (pH 7.2), and the mixture was gently shaken every 20 min for 2 h at 37 °C as described previously (5Ravindranath R.M.H. Tam W. Nguyen P. Fincham A.G. J. Biol. Chem. 2000; 275: 39654-39661Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Ovalbumin was used as positive control and BSA as a negative control. Specific Binding of K5 to [3H]ATMP as a Function of Increasing Concentration of ATMP—The total binding of labeled ATMP to K5 (333 pmol) was determined using increasing concentrations of [3H]ATMP (2–200 pmol). The nonspecific binding of labeled ATMP was determined in the presence of 40 nmol of unlabeled ATMP at 37 °C for 2 h and was subtracted from the total binding to obtain the specific binding. Ovalbumin was used as positive control. The total binding of [3H]ATMP to ovalbumin (700 pmol) was determined using increasing concentration of the labeled ATMP (0.5–250 pmol). The Scatchard plot further analyzed the specific binding as described previously (8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Slope (y), Bmax, r, or r2, and the levels of significance of the slopes are presented. Loss-of-function Mutations of ATMP Result in Loss of Binding to K5 as Assessed by Western Blot Analysis—rM179 on SDS-PAGE were electrotransferred to PVDF membranes at 100 mA for 35 min using a semi-dry transblot apparatus. Protein transfer was assessed as described previously. After blocking the membrane with 1% HSA in PBS for 1 h at 37 °C and washing, the membranes were overlaid with K5 alone or K5 preincubated (for 1 h) with ATMP or K5 preincubated with T-ATMP or F-ATMP. The strips were immunostained with affinity-purified murine monoclonal antibody for K5 (1:1000). Morphometry of Enamel and Ameloblast during Development—The serial sections (6 μm) were obtained from paraffin-embedded mouse mandibular incisors on different postnatal days and stained with Mallory. The width of enamel and length of ameloblasts were measured at the 20%, 40%, 60%, and 80% levels from the base of the incisor using the software program Image-Pro Plus 4.0 (Media Cybernetics). At each level, the mean width and length were obtained after measuring nine sections (three incisors from three mice). Immunochemical Localization of K5 in Ameloblasts—For immunochemical analyses, sagittal sections of mandibular molar tissues (6 μm) were mounted on Histostik-coated slides (Accurate Chemical and Scientific Corp., Westbury, NY). Details of immunolocalization methodology are the same as described previously (8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Tissue sections were stained with primary sheep monoclonal antibody for K5 (IgG specific for the first 16 sequences at N-terminal (Binding Site) diluted in phosphate-buffered saline (PBS, pH 7.2) (1:50) and incubated at 37 °C for 1 h. After washing with PBS containing 0.1% HSA and 0.1% Tween 20, the sections were incubated with peroxidase-conjugated secondary antibody (donkey anti-sheep IgG) diluted in PBS pH 7.2 (1:1000). IgG isotype 1 was used as a negative control. The sections were counter-stained with hematoxylin, and images were examined under digital microscopy. Colocalization of Amelogenins, K5 and K14, with Laser Scanning Confocal Fluorescence Microscopy in Ameloblasts during Enamel Formation—To examine the spatial distribution and colocalization of K14, K5, and amelogenin, sagittal sections of mouse postnatal mandibular molars at different developmental stages were prepared as described previously (8Ravindranath R.M.H. Tam W. Bringas Jr., P. Santos V. Fincham A.G. J. Biol. Chem. 2001; 276: 36586-36597Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The sections were deparaffinized and rehydrated, and endogenous peroxidase activity was blocked with 3% H2O2. The sections were stained with primary antibody against K14 (affinity-purified murine monoclonal antibody specifically recognizing the 14 amino acid sequences of C-terminal sequences of K14). The sections were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (goat anti-mouse IgG, Jackson ImmunoResearch), 1:40 dilution, for 30 min at room temperature. The sections were sequentially stained with the primary antibody against recombinant mouse amelogenin (1:500) for 1 h and then incubated with the secondary antibody coupled with tetramethylrhodamine isothiocyanate (TRITC) to goat anti-rabbit IgG (Jackson ImmunoResearch), 1:40 dilution for 30 min at room temperature. The sections also were stained sequentially with the primary affinity purified human anti-sheep K5 antibody at 1:100 dilution (Binding Site) for 1 h at 37 °C. Rabbit anti sheep IgG coupled wit" @default.
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