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J. Biol. Chem., Vol. 276, Issue 39, 36586-36597, September 28, 2001

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Amelogenin-Cytokeratin 14 Interaction in Ameloblasts during Enamel Formation^*

Rajeswari M. H. Ravindranath, Wai-Yin Tam, Pablo Bringas Jr., Valentino Santos, and Alan G. Fincham

From the Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, California 90033

Received for publication, May 22, 2001, and in revised form, June 18, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The enamel protein amelogenin binds 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 is found in the N-terminal region of CK14, a differentiation marker for ameloblasts. The binding affinity of CK14 and amelogenin was confirmed by dosimetric binding of CK14 to recombinant amelogenin (rM179), and to the tyrosine-rich amelogenin polypeptide. The specific binding site for CK14 was identified in the amelogenin trityrosyl motif peptide (ATMP) of tyrosine-rich amelogenin polypeptide and specific interaction between CK14 and [³H]ATMP was confirmed by Scatchard analysis. Blocking rM179 with GlcNAc, GMp, or CK14 with ATMP abrogates the CK14-amelogenin interaction. CK14 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. Morphometry of developing teeth distinguished three phases of enamel formation; growth initiation phase (days 0-1), prolific growth phase (days 1-7), and growth cessation phase (post-day 7). Confocal microscopy revealed co-assembly of CK14/amelogenin in the perinuclear region of ameloblasts on day 0, migration of the co-assembled CK14/amelogenin to the apical region of the ameloblasts from day 1, reaching a peak on days 3-5, and a collapse of the co-assembly. Autoradiography with [³H]ATMP and [³H]GMp corroborated the dissociation of the co-assembly at the ameloblast Tomes' process. It is proposed that CK14 play a chaperon role for nascent amelogenin polypeptide during amelogenesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Dental enamel is formed within a protein matrix secreted by ameloblast cells of ectodermal origin (1). Ameloblasts synthesize several matrix proteins (2) and also cytokeratins 5 and 14 (3-5). Ninety percent of the enamel matrix protein constitutes amelogenins (6-8). Amelogenins are tissue-specific, non-glycosylated proteins, rich in proline, glutamine, leucine, and histidine. Post-secretory processing of amelogenins involves a series of discrete steps including supramolecular self-assembly and progressive proteolytic reduction in molecular size, facilitating enamel biomineralization and maturation (9). Little is known about the presecretory and secretory stages of amelogenesis. Understanding of these events may shed light on the significance of the interaction of amelogenins with other ameloblast proteins, and the functional roles of the different domains of amelogenin polypeptide structures, including the possible significance of the highly conserved phosphorylation locus at serine 16, which remains enigmatic.

While seeking to define the functional role of different domains of amelogenins, we have observed that the conserved tri-tyrosyl motif (amelogenin tri-tyrosyl motif peptide (ATMP):¹ PYPSYGYEPMGGW) of the N-terminal region of amelogenins binds specifically to N-acetylglucosamine (GlcNAc) of glycoconjugates (10). Furthermore, we have demonstrated that the ATMP also recognizes peptide mimics of GlcNAc (11). Most importantly, one of the GlcNAc mimicking peptides (GMp: SFGSGFGGGY) binds avidly to ATMP. Modifications of the ATMP motif, including substitution of proline 3 by threonine as observed in a case of human X-linked amelogenesis imperfecta (12), resulted in the loss of binding to both GlcNAc and GMp (10, 11). Interestingly, the GMp sequence is localized in the highly conserved N-terminal domain of cytokeratins 14, 16, and 17. Since CK14 is a known marker for ameloblasts in a developing tooth prior to synthesis of amelogenins (3-5) and it contains the GMp that binds specifically to the ATMP sequence of amelogenins, we hypothesize that interactions between CK14 and amelogenins may play an important role in amelogenesis, enamel development, and disease.

In the present investigation, we demonstrate that CK14 binds specifically to amelogenins through the ATMP. Furthermore, we show that putative loss of function mutations of ATMP (e.g. substitution of a proline residue with threonine, as noted above) abrogates binding of amelogenins to CK14. Using confocal laser microscopy, we demonstrate co-assembly of amelogenin-CK14, its migration to the apical region of ameloblasts, and subsequent dissociation at Tomes' process. Our findings suggest that CK14 functions as a chaperon for nascent amelogenin polypeptides during amelogenesis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals-- Twenty-five normal, healthy, female Swiss Webster pregnant mice (Charles River Breeding) were used to obtain a sufficient number of litters for this study. All protocols involving mice were approved by the Institutional Animal Care and Use Committee (Los Angeles, CA). Mandibles were obtained from Swiss Webster mice at different developmental stages ranging from E18-day pregnant mice through newborn (NB) day "0," postnatal days (PN) 1, 3, 5, 7, and 9. Mandibular incisors were used for morphometric studies.

Amelogenin Proteins-- The recombinant mouse amelogenin rM179 (20.16 kDa) was prepared by expression in Escherichia coli, isolated, and purified by high performance liquid chromatography as previously described (13). The protein was further purified by analytical reversed phase HPLC and homogeneity was assessed by SDS-PAGE (10, 11). The amino acid sequence of this protein (ATMP in bold) is as follows: rM179, PLPPHPGSPGYINLSYEVLTPLKWYQSMIRQPYPSYGYEPMGGW- LHHQIIPVLSQQHPPSHTLQPHHHLPVVPAQQPVAPQQPMMPVP- GHHSMTPTQHHQPNIPPSAQQPFQQPFQPQAIPPQSHQPMQP- QSPLHPMQPLAPQPPLPPLFSMQPLSPILPELPLEAWPATDKTKR- EEVD¹⁷⁹_..

The polypeptides used in this investigation include: TRAP (5.20 kDa), a synthetic polypeptide representing the N-terminal conserved 45 amino acid residues of amelogenins; LRAP (6.82 kDa), a synthetic leucine-rich amelogenin polypeptide (a polypeptide sharing 33 amino acid residues of the N terminus and 26 amino acid residues of the C terminus of full-length amelogenins); ATMP (1.45 kDa) (see sequence above), a synthetic polypeptide, representing the 13 C-terminal residues of the TRAP amelogenin and the two altered ATMP peptides; T-ATMP (PYPSYGYETMGGW) and F-ATMP (PFPSFGFEPMGGW). P162 (23 kDa) and P148 (20 kDa) (Porcine amelogenins) were isolated from unerupted mandibular pig molars and purified as previously described (10).

Synthesis and Purification of Polypeptides-- All the polypeptides used (TRAP, LRAP, ATMP, T-ATMP, F-ATMP, and GMp (SFGSGFGGGY located at the highly conserved N-terminal region of CK14) 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 (14). 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 (B contained 60% (v/v) aqueous acetonitrile in 0.1% (v/v) trifluoroacetic acid) at a flow rate of 1.0 ml/min (10, 11).

Specificity of the Antibodies-- The murine monoclonal antibody for CK14 (clone LL002) is affinity purified and specifically recognizes the 14 C-terminal residues of CK14 (15). Anti-amelogenin antibody is developed in rabbits after immunizing with recombinant amelogenin rM179 (13). The specificity of the antibody was assessed using different fragments of amelogenin in ELISA (16-18). The polyclonal antibody (at 1/6000) binds better to rM166 (a construct that lacks the hydrophilic C-terminal 13-residue segment), than to rM179 at the same concentration (50 ng/well), suggesting that the C-terminal acidic residues are not essential for binding. The antibody did not recognize the N-terminal TRAP region in ELISA or in Western blots suggesting that it recognizes the hydrophobic central core of the amelogenin.

³H Labeling of ATMP and GMp-- The 13-residue ATMP (P[³H]YPSYGYEPMGGW) was prepared from tritium gas by Nycomed Amersham Plc. (Amersham Pharmacia Biotech) and after labeling, the material co-chromatographed with the ATMP synthesized by the University of Southern California Microchemical Core Laboratory. A mass spectrum is consistent with the proposed structure. The material was supplied as a water:ethanol (1:1, v/v) solution in a silanized borosilicate multidose vial with additional screw cap under nitrogen. GMp (SFGSGFGGG[³H]Y) was also labeled as noted previously (11). Polypeptides were purified by HPLC on a Vydac C18 300-Å (protein or peptide: 250 × 4.6 mm) column with a gradient of solution A (0.01 M aqueous trifluoroacetic acid) and solution B (0.01 M trifluoroacetic 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. The peptides were stored in the absence of light and air at 20 or 4 °C, respectively.

Binding of rM179 and TRAP to CK14 by Enzyme-linked Immunosorbent Assay (ELISA)-- ELISA was performed using CK14 (RDI Research Diagnostics, Inc.) or TRAP as antigen following the protocol previously described (16-18). Antigen coating was done by adding 100 µl of a solution containing varying amounts of CK14 (Research Diagnostics) in PBS (pH 7.2), TRAP in carbonate and bicarbonate buffer (pH 9.6) was added to wells (Falcon 3915, Fisher Scientific, Pitsburgh, PA) and incubated at room temperature overnight. Wells were blocked with PBS containing 1% HSA at 37 °C for 1 h. One-hundred microliters of a known amount of rM179 (5 pmol/100 µl)/CK14 (10 pmol/100 µl) was added to wells and incubated for 1 h at 37 °C. After washing the plates five times, primary antibody against the recombinant M179 protein (12) (at a dilution of 1:6000) or anti-CK14 affinity purified murine monoclonal (1:1000) (LL002 Neomarkers) (15) was added and incubated for 1 h at 37 °C and then incubated with the secondary antibody (goat anti-rabbit IgG; Jackson ImmunoResearch, West Grove, PA, rabbit anti-mouse IgG) for 1 h. After washing, substrate (o-phenylenediamine dihydrochloride; Life Technologies, Inc., Gaithersburg, MD) 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 H₂SO_4. The absorbence difference at 490-650 nm was measured after automix in a Bio-Tek microplate reader (Bio-Tek Instruments). The values were corrected for background (wells without antigen). BSA and LRAP were used as negative controls.

Assessment of Binding of CK14 with Amelogenins by Western Blot Analysis-- The proteins were resolved via SDS-PAGE using 12 or 15% resolving and 3.5% stacking gels (19) and electrotransfered to polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Immunolon-P Transfer Membrane) at 100 mA for 1 h using a semidry transblot apparatus (Hoefer Scientific Instruments, San Francisco, CA) (11, 20). Protein transfer was assessed by staining the PVDF strips with 0.1% Fast Green (Sigma) in 40% methanol and 10% acetic acid, and the strips were compared with Coomassie Blue-stained protein bands (11, 21). Replicas were treated with ligands (GlcNAc, GMp) after blocking the membrane with phosphate-buffered saline, 1% HSA for 1 h at 37 °C. The membranes were washed five times with phosphate-buffered saline containing 0.1% HSA (11, 22). After washing, the membranes were overlaid with CK14 alone or CK14 preincubated with ATMP for 1 h. The strips were washed (5 times) and immunostained with anti-CK14 monoclonal antibody for CK14 (clone LL002, 1/1000).

Dosimetric Binding of CK14 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 CK14 in Tris-buffered saline (pH 7.2) and the mixture was gently shaken every 20 min for 2 h at 37 °C. The proteins were precipitated with 1 ml of cold ethanol (200 proof; Gold Shield Chemical Co., Hayward, CA) at 4 °C for 20 min, centrifuged for 15 min at 12,000 × g, and the supernatant was removed. The unbound [³H]ATMP was removed completely by repeated vortex mixing and washing four times with ethanol. The final pellets were dissolved in 50 µl of 1 N NaOH, and bound radioactivity was measured 15 min after adding 4 ml of scintillation fluid (Amersham Pharmacia Biotech) in a -counter, as described elsewhere (10, 11).

Specific Binding of CK14 to [³H]ATMP as a Function of Increasing Concentration of ATMP-- The total binding of labeled ATMP to CK14 (350 pmol) was determined using increasing concentrations of [³H]ATMP (2-600 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. The specific binding was further analyzed by the Scatchard plot.

Loss of Function "Mutations" of ATMP Results in Loss of Binding to CK14 as Assessed by Western Blot Analysis-- Recombinant M179 on SDS-PAGE were electrotransfered to PVDF membranes at 100 mA for 1 h using a semidry transblot apparatus. Protein transfer was assessed as described in the legend to Fig. 3. After blocking the membrane with 1% HSA in PBS for 1 h at 37 °C and washing, the membranes were overlaid with CK14 alone or CK14 preincubated (for 1 h) with ATMP or CK14 preincubated with T-ATMP or F-ATMP. The strips were immunostained with murine monoclonal antibody for CK14 (clone LL002, 1/1000).

Morphological Analyses-- Mouse mandibular molar tissues for immunohistochemistry were fixed immediately in 10% neutral buffered formalin for 12 h at 4 °C. The fixed tissues were embedded in paraffin and 6-µm saggital sections were mounted on Histostik-coated slides (Accurate Chemical and Scientific Corp., Westbury, NY). Tissues for enamel morphometric studies were fixed in formalin for 24 h, decalcified with 10% EDTA. Day "0" mandibles were decalcified for 2 h (PN), day 1 for 4 h, day 3 for 30 h, day 5 for 66 h, day 7 for 108 h, and day 9 samples for 156 h, washed, and then processed for paraffin embedding.

Morphometry of Enamel during Tooth Development-- The serial cross-sections (6 µM) obtained from the whole length of the mouse mandibular incisor (PN 0, 1, 3, 5, 7, and 9) was taken as 100%. The sections were stained with Mallory's triple stain for 5 min and the width of enamel was measured at the 40 and 60% level from the base of the incisor using the software program Image-Pro Plus 4.0 (Media Cybernetics, L. P.) on 9 sections (three incisors from three mice) per day.

Immunochemical Localization of CK14 in Ameloblasts-- Saggital sections (6-µm) of mouse mandibular molar of day 0 (NB), day 1, 3, 5, 7, and 9 (PN) were deparaffinized and immunostained. Endogenous peroxidase activity was blocked with 3% H₂O_2. Tissue sections were treated with 1% HSA in PBS (pH 6.0) and then incubated with affinity purified mouse monoclonal antibody for CK14 (LL002, 1/500) at 37 °C for 1 h. Negative controls were performed by replacing the primary antibody with Tris-buffered saline and also IgG isotypes. Biotinylated anti-mouse secondary antibody was added to sections, incubated for 30 min at room temperature, and rinsed (3 times). The sections were treated with streptavidin-peroxidase conjugate for 30 min at room temperature and stained with hematoxylin:eosin. The images were photographed.

Co-localization of CK14 and Amelogenins with Confocal Laser Scanning Microscopy in Ameloblasts during Enamel Formation-- To examine the spatial distribution and co-localization of CK14 and amelogenin, saggital sections of mouse postnatal mandibular molars at different developmental stages were prepared as described earlier (23, 24). The sections were deparaffinized, rehydrated, and endogenous peroxidase activity was blocked with 3% H₂O_2. The sections were stained with primary antibody against CK14 and then incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (goat anti-mouse IgG, Jackson Immuno Research, West Grove, PA), 1:40 dilution, for 30 min at room temperature. The sections were sequentially stained with the primary antibody against recombinant mouse amelogenin for 1 h and then incubated with the secondary antibody coupled with tetramethylrhodamin isothiocyanate (TRITC) to goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, pA), 1:40 dilution for 30 min at room temperature. Replacing the primary antibody with Tris-buffered saline and also IgG isotypes performed as negative controls. Sections were then washed, mounted immediately with glycerol (95% glycerol with 5% phosphate buffer as mounting media), and examined with a Zeiss Laser Scan Microscope (LSM 510) (Carl Zeiss, Oberkochen, Germany) equipped with a 514 argon and a 543 helium-neon laser.

To determine coassembly of CK14 and amelogenin in vivo, co-localization was studied on sections stained sequentially with FITC-conjugated antibody for CK14 (green signal at 514) and TRITC-conjugated antibody for amelogenin (red signal at 543) (25-28). The 514- and 543-nm lines of an argon-helium-neon laser exited FITC and TRITC, respectively. Both lines pass through a dichroic mirror and a neutral density filter before passing through the cell. The emitted light reflected from the sample and fluorescence was collected by an oil immersion lens and imaged on to a photomultiplier tube after passing through a confocal aperture at an optical filter. The cells were scanned at 4 s/frame and averaged twice to reduce background noise. The laser power was adjusted to ensure any photodynamic effect on the cells was negligible.

Lectin Histochemistry-- Sections from mouse mandibular tooth organs (PN day 5) were treated with lectins before and after treatment with sialidase. The following lectins were used; Triticum vulgaris (wheat germ agglutinin) that binds to sialic acid and GlcNAc (29, 30) and Datura stramomium specific for GlcNAc (purchased from EY Laboratories) (31). Sections were treated with neuraminidase to rule out the staining due to sialic acids. Clostridium perfringens neuraminidase (type X) (purchased from Sigma) was used in all the major experiments. The enzyme treatments were done as described elsewhere (10, 22). Briefly, the sections were overlaid with 200 milliunits of enzyme in 200 µl of PBS/section and were incubated for 1 h at 37 °C. After washing, the sections were treated with lectin peroxidase-coupled wheat germ agglutinin or biotinylated D. stramomium (1 mg/ml, 1:10).

Autoradiography of [³H]ATMP to CK14 and ³H-Labeled GMp to Amelogenins-- Sections from mouse mandibular tooth organs (saggital sections, 6 µm) were used to assess the localization of free CK14 or free amelogenins in ameloblasts during enamel formation. Sections were treated with 50 milliunits of N-acetylglucosaminidase (Sigma) (22) to assess the location of free CK14 in ameloblasts. The sections were blocked with PBS (pH 6.0) with 1.0% HSA at 37 °C for 1 h and then incubated with [³H]ATMP or ³H-labeled GMp for 2 h at 37 °C to identify free CK14 or amelogenins. After washing the slides 3 times with PBS (0.1% HSA, pH 6.0), the slides were dried for 30 min at room temperature. Each slide was dipped separately in emulsion fluid diluted in water at 1:1 dilution (Kodak Autoradiography Emulsions, Type NTB2 for  emitters, International Biotech Inc. Eastman Kodak Co., New Haven, CT) in the dark for 1 min, dried, and stored at 4 °C. The autoradiographs were developed in Kodak DEKTOL Developer (Eastman Kodak Co., Rochester, NY) according to the manufacturers recommendations and counterstained with hematoxylin. Digital and phase contrast microscopy was used to identify the grains on sections.

Quantitative Analysis of Images using LSM 510-- The scatter diagram (co-localization) is created and analyzed for pixel distribution (27). All pixels having the same positions in both images are considered a pair. Of every pair of pixels (P1, P2) from the two images, the brightness level of pixel P1 is interpreted as the X coordinate, and that of pixel P2 as the Y coordinate of the scatter diagram. Non-co-localized P1 and P2 pixels and the background values were excluded to distinguish the pixels that are co-localized. The results were expressed as percentage of co-localization of amelogenins and CK14 at different developmental stages from day 0 to 9 from initiation, growth, and cessation (double-headed arrow) of enamel formation. The quantity of non-co-localized P1 and P2 pixels representing free amelogenin and free CK14, respectively, are also plotted for comparison.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recombinant amelogenin (rM179) is a 20.16-kDa polypeptide with 179 amino acids. It differs from native amelogenin only in the absence of the N-terminal methionine and that serine at position 16 is not phosphorylated. The ATMP sequence that interacts with CK14 is localized in the C-terminal region of TRAP. It binds to GlcNAc as well as the GMp localized in the N-terminal head region of CK14. The specific binding of ATMP-GMp was confirmed earlier (11).

rM179 and TRAP Interact with CK14-- Solid matrix immunoassay analysis of the interaction between CK14 and amelogenins showed dosimetric binding of recombinant M179 to CK14 (Fig. 1A). Low (<0.15) but no dose-dependent binding was observed with BSA. Fig. 1B indicates a similar dose-dependent binding of CK14 to TRAP. The titration does not reach saturation unlike in Fig. 1A. No binding was observed with LRAP.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   A, amelogenin (rM179) binding to varying concentrations of CK14 as measured by a solid matrix immunoassay. B, cytokeratin-14 (CK14) binding to varying concentrations of TRAP by a solid matrix immunoassay.

CK14- and Amelogenin Interaction on Western Blot-- The results (Fig. 2) show that CK14 binds to rM179 (lane 5) native porcine amelogenins (lanes 9 and 10) P148 and P162 and TRAP (lane 12). The binding of CK14 to amelogenin was abrogated when the Western blot of rM179 was pretreated (overlaid and washed) with GlcNAc or GMp (lanes 6 and 7 and Table I). Similarly when CK14 was preincubated with ATMP it failed to bind to rM179 (lane 8 and Table I), suggesting that the CK14-rM179 interaction involves ATMP binding with the GMp motif of CK14.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Western blot analysis of binding of CK14 with rM179 and TRAP in the presence and absence of inhibitors. Recombinant M179, TRAP, and BSA (negative control) on SDS-PAGE were electrotransfered to PVDF membranes at 100 mA for 1 h using a semidry transblot apparatus. Lanes 1-10, transblot from 12% gel. Lane 1, standards; Lane 2, CK14 plus anti-CK and 2nd antibody; Lane 3, CK14 plus 2nd antibody only; Lane 4, immunostaining rM179 without CK14 with anti-CK14 antibody and second antibody, no staining observed; Lane 5, immunostaining of CK14 bound to rM179; Lane 6, immunostaining of CK14 added to rM179 after overlay with GlcNAc, no staining observed; Lane 7, immunostaining of CK14 added to rM179 after overlay with GMp, no staining observed; Lane 8, immunostaining of CK14 after rM179 overlaid with CK14 preincubated with ATMP, no staining observed; Lane 9, immunostaining of CK14 on native (porcine) amelogenin P148; Lane 10, immunostaining of CK14 on native (porcine) P162; Lanes 11-13, transblot from 15% gel; Lane 11, standards; Lane 12, immunostaining of CK14 bound to TRAP; Lane 13, immunostaining of CK14 on BSA (negative control), no staining observed.

Specific Binding of [³H]ATMP to CK14-- To select the optimal concentration of CK14 for assessing the specific interaction between CK14 and ATMP, the dosimetry of [³H]ATMP binding to CK14 was determined (Fig. 3A). The purity and homogeneity of ATMP was assessed by reversed phase HPLC and a typical profile of the purified fraction is illustrated in the inset of Fig. 3A. Fig. 3B shows the specific binding of [³H]ATMP to CK14 as a function of increasing concentration of ATMP. The nonspecific binding was measured with unlabeled ATMP and subtracted from the total binding to obtain the specific ATMP-CK14 interaction. A Scatchard plot of the binding of [³H]ATMP to CK14 indicates that the peptide-binding site is homogenous with respect to the association constant. The significance of the slope (p < 0.001) and r² (0.96) are indicated in the Fig. 3B, inset.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   A, [3H]ATMP binding to varying concentrations of CK14. The mean values of duplicate analyses at each concentration were plotted. The position of 350 pmol of CK14, selected to study the specific binding of CK14 with ATMP is indicated on the graph as a vertical dotted line. BSA is used as a negative control. Inset provides the profile of synthetic ATMP, with the amino acid sequence "PYPSYGYEPMGGW" purified by C18-reversed phase HPLC. B, specific binding of [3H]ATMP to CK14 as a function of increasing concentration of ATMP-Scatchard plot analysis. The Scatchard analysis of this specific binding is shown as an inset. Each point represents the mean of duplicate determinations. r2 and p values of the slope are indicated in the graph.

ATMP but Not Mutations of ATMP Bind to CK14-- The binding of CK14 to rM179 was assessed in Western blot before and after preincubating CK14 with ATMP or loss of function mutations of ATMP. In one of the two mutant ATMP peptides (T-ATMP), the third proline is substituted with threonine, and in the other (F-ATMP) all three tyrosyl residues are replaced by phenylalanine (the T-ATMP mutation has been found in some cases of human X-linked amelogenesis imperfecta). The binding of CK14 to rM179 is abrogated after pretreatment with ATMP but not after pretreatment of CK14 with T-ATMP or F-ATMP suggesting that mutated ATMP is not capable of binding to CK14 as does ATMP (Fig. 4).

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Loss of function mutations of ATMP results in loss of binding to CK14 as assessed by Western blot analysis. Lanes 1-7, transblot from 12% gel. Lane 1, standards; Lane 2, rM179 stained with Fast Green; Lanes 3-7 were immunostained with affinity purified, murine monoclonal antibody for CK14 (clone LL002, 1/1000). Lane 3, CK14 after immunostaining. Lane 4, rM179 overlaid with CK14 alone stained. Lane 5, CK14 preincubated (for 1 h) with ATMP did not stain. Lane 6, CK14 preincubated with T-ATMP stained. Lane 7, CK14 preincubated with F-ATMP, stained.

Morphometry and CK14 Distribution in Ameloblasts during Enamel Formation-- To determine CK14-amelogenin interaction in vivo during development, morphometry of enamel growth and the distribution and migration of CK14 was studied. Enamel growth was quantified by measuring the width of enamel in cross-sections of mouse incisors from day 0 to 9. The height of the tooth is distinguished into four zones from the base of the tooth as 20, 40, 60, and 80%. Fig. 5 shows that the width of enamel increases from day 1 to 5 at 60% level. It reaches a maximum on day 5 without much change thereafter on days 7 and 9. A similar pattern was observed at the 40% level. The enamel width steadily increased until day 7 and is constant thereafter. These morphometric observations indicated that tooth development is initiated on day 0 and prolific on day 3 and reaches a maximum between days 5 and 7 and ceases to increase after day 7. During the corresponding period, immunolocalization of CK14 in ameloblasts was carried out using a affinity purified monoclonal antibody specific for the C-terminal region of CK14 (15). Fig. 6 shows that CK14 is distributed throughout the cytoplasm on day 0. The immunostaining is increased in intensity at the apical region adjacent to the extracellular matrix on day 1. Staining is intense in the Tomes' process. CK14 immunostaining is maximum in the apical region on day 3. Such staining persisted until day 5 but is lost on day 7 and thereafter, when the enamel has reached its maximum thickness.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Morphometry of enamel during tooth development. The serial cross-sections (6 µm) obtained from the whole length of mouse mandibular incisor (PN 0, 1, 3, 5, 7, and 9) was taken as 100%. The sections were stained with Mallory's triple stain and the width of enamel was measured at the 40 and 60% level from the base of the incisor using the software program Image-Pro Plus 4.0 on nine sections (three incisors from three mice) per day. Mean (n = 9) and standard deviation (vertical bar) are indicated.

View larger version (140K): [in this window] [in a new window]   Fig. 6.   Distribution and migration of CK14 in ameloblasts during different stages of enamel formation. Saggital sections (6 µm) of mouse mandibular molar of day 0 (newborn), days 1, 3, 5, 7, and 9 (post-natal) were immunostained. A, negative control day 0 treated with IgG isotype shows no immunostaining; B, negative control on day 1 treated with IgG isotype shows no immunostaining; C, negative control, day 7 treated with IgG isotype shows no immunostaining. D, day 0, horizontal arrows point out the distribution of CK14 throughout the cytoplasm of ameloblasts (am). Vertical arrows show increased staining observed toward the periphery. E, day 1. Horizontal arrows show CK14 staining intensity at the apical region adjacent to the extracellular matrix. Long and vertical arrows indicate Tomes' processes. Note that enamel secretion has commenced. F, day 3. CK14 immunostaining is maximum at the apical region (horizontal arrows). Enamel secretion is evident. G, day 5 CK14 immunostaining is still intense at the apical region (vertical arrows). Enamel matrix is thickened. H, day 7 CK14 immunostaining declined (arrows). The enamel has reached maximum thickness. Horizontal bar refers to measurement. A, 20 µm; B, 20 µm; D, 20 µm; E, 30 µm; F, 30 µm.

Co-localization of CK14 and Amelogenin-- Three complete independent experiments were done using confocal microscopy. One for CK14-FITC, another for amelogenin-TRITC, and a third for the combination of the two dyes. Fig. 7 illustrates three fluorescent images of ameloblasts on day 0 with FITC (green) signal only (Fig. 7A) or TRITC (red) signal only (Fig. 7B) or with combination of signals (Fig. 7C). Coassembly of CK14 and amelogenin is indicated by the yellow signal (Fig. 7C). The yellow granules (pixel) are distributed all around the nucleus on day 0. Arrows indicate circular distribution or aggregation of the granules in the cytoplasm. Green signal (CK14 +/amelogenin ) is seen at the distal end of the ameloblasts (Fig. 7C). Fig. 8 demonstrates co-localization of CK14 and amelogenin in ameloblasts during different stages of enamel growth. Accumulation of co-localized CK14-amelogenin granules is observed in the apical region of ameloblasts on day 1 (Fig. 8C). Presence of yellow granules in the cytoplasm and increasing density of the granules toward the apical region suggests migration of co-assembled CK14 and amelogenin from the perinuclear region to the apical end of the cell. The sections showed a distinct green signal in the cytoplasm as well as in the distal region of ameloblasts indicative of free CK14 on day 1. Fig. 8D shows co-localization of CK14 and amelogenin on day 3. The accumulation of co-localized CK14-amelogenin in the apical region of ameloblasts is very prominent (yellow arrow). However, green signals were also observed at the apical end (green arrows). Further magnification (inset in Fig. 8D) revealed a mixture of signals in the apical region of the ameloblasts (Fig. 8E). The signals were predominantly yellow (yellow arrow). Green signals (dark green or green arrows) are observed toward the interior of the apical zone of ameloblast. Red signals (red arrows) are abundant toward the exterior of the apical zone. While the yellow signal showed the co-localization of CK14 and amelogenin, the red and green signals signify the presence of (non-co-localized) free CK14 and amelogenin in the apical zone. Free amelogenin (red signal) moves toward exterior while free CK14 (green signal) remains or moves toward interior of the ameloblasts. Fig. 8, F, G, and H, represent co-localization and secretion of amelogenin on days 5, 7, and 9, respectively. On day 9 (Fig. 8H) amelogenin forms a distinct layer at the formative zone of enamel. The yellow granules in the cytoplasm (yellow arrow) are very few. Red and green granules are also found in the disintegrating ameloblasts at days 7 and 9 (Fig. 8, G and H).

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Co-localization of CK14 and amelogenin in ameloblasts on day 0 as revealed by Lazer scan double-label confocal fluorescence microscopy. Saggital sections (6 µm) were sequentially stained and examined under confocal laser scan microscopy. A, green (FITC) signal (514) refers to CK14 immunostaining. Note the distribution of the granules throughout the cytoplasm of the ameloblasts. Arrows indicate the distribution of the cytoplasmic granules around the nucleus. B, red (TRITC) signal (543) refers to amelogenin immunostaining. Note the distribution of amelogenins in the cytoplasm. C, co-localization of CK14 and amelogenin near the nucleus (n) of ameloblasts is shown by a hybrid yellow signal (CK14+/amelogenin+). Horizontal bar refers to measurement. A, 30 µM; B, 30 µM; C, 30 µM.

View larger version (128K): [in this window] [in a new window]   Fig. 8.   Co-localization, migration and dissociation of CK14 and amelogenin in ameloblasts during enamel development. Saggital sections of mouse postnatal mandibular molars at days 1, 3, 5, 7, and 9 were deparaffinized, rehydrated, and endogenous peroxidase activity was blocked with 3% H2O2. The sections were stained and examined as described in the legend to Fig. 9. Green (FITC) signal (514) refers to CK14 immunostaining (CK14+/amelogenin). Red (TRITC) signal (543) refers to amelogenin immunostaining (CK14/amelogenin+) co-localization of CK14 and amelogenin is shown by a hybrid yellow signal (CK14+/amelogenin+). A-C, day 1. Note the distribution and co-localization of CK14 and amelogenin toward the secretary end of the ameloblast (am). There is a clear and distinct accumulation of CK14+/amelogenin+ pixels at the apical end. Note also the presence of CK14+/amelogenin as well as CK14+/amelogenin+ pixels in the cytoplasm. CK14/amelogenin+ pixels are restricted to the site of enamel secretion. D, day 3, accumulation of CK14+/amelogenin+ pixels at the apical end. Note the presence of CK14+/amelogenin pixels (green arrow) in the apical region. CK14/amelogenin+ pixels appear as a layer at the site of enamel (e) secretion. E, day 3, white rectangular insert seen in D is magnified to show co-localization as well as dissociation of CK14 and amelogenin at the zone of enamel secretion. Yellow arrow refers to pixel indicative of co-localization (CK14+/amelogenin+). Green arrows indicate to pixels with green signal (CK14+/amelogenin). Red arrows indicate pixels with red signal (CK14/amelogenin+). Note the presence of red signal toward at the site of enamel formation. F, G, and H, distribution of green, yellow, and red signals on days 5, 7, and 9, respectively. Note the gradual disruption and disintegration of ameloblasts on these days. H, red signal is seen as a distinct layer at the formative zone of enamel on day 9. Arrows in F, G, and H indicate the presence of free CK14. Am, ameloblast; d, dentine; e, enamel; n, nucleus. Horizontal bar refers to measurement. A, 20 µm; B, 20 µm; D, 20 µm; E, 30 µm; F, 30 µm.

Collapse of Co-assembled CK14 and Amelogenin during Amelogenesis-- Fig. 8E (magnification of inset in 8D) provides evidence to suggest collapse of coassembled CK14 and amelogenin after accumulation at the apical region of ameloblasts by day 3. To verify whether such dissociation occurs at the apical zone, free CK14 and amelogenin were localized with [³H]ATMP and [³H]GMp. The rational for this investigation is that the coassembly of CK14 and amelogenin involves binding of ATMP (PYPSYGYEPMGGW) with the GMp of CK14. Dissociation would expose these peptide sequences in CK14 and amelogenin, which can be monitored using [³H]ATMP and [³H]GMp. [³H]ATMP may bind to both GlcNAc and GMp of CK14. The presence of GlcNAc is identified with D. stromonium lectin and wheat germ agglutinin (data not shown). The staining indicated diffused distribution of GlcNAc containing glycoconjugates in the cytoplasm of ameoloblasts toward the distal zone. To eliminate the interaction of GlcNAc-glycoconjugates with [³H]ATMP, autoradiography was performed after removing the GlcNAc residues with N-acetylglucosaminidase (Fig. 9). The figure shows the localization of free CK14 with [³H]ATMP at days 0, 1, and 3. [³H]ATMP was observed throughout the cytoplasm at all days. On day 0, the free CK14 is abundant in the cytoplasm (Fig. 9A). Accumulation of free CK14 in the apical end is observed on day 1 (Fig. 9, B and C). Concomitant with an increase in enamel growth on day 3, free CK14 accumulated in the apical region of ameloblasts, as evidenced by the increased accumulation of [³H]ATMP. Fig. 10, C and D, indicate accumulation of [³H]ATMP within the Tomes' process on day 5. In contrast, [³H]GMp failed to show such accumulation in the apical region of the cytoplasm either on day 5 or 3 (Fig. 10, A and E). However, magnification of the apical region of the ameloblasts on day 5 revealed accumulation of [³H]GMp within the Tomes' process and at the exterior of the ameloblasts (Fig. 10B). These observations suggest that the coassembled CK14-amelogenin may dissociate in the Tomes' process. The free amelogenin moves out of ameloblasts, whereas free CK14 may remain within the ameloblasts.

View larger version (132K): [in this window] [in a new window]   Fig. 9.   Binding of [3H]ATMP to free CK14 in N-acetylglucosaminidase-treated ameloblasts. Sections from mouse mandibular tooth organ (6 µm, saggital sections) were treated with 50 milliunits of GlcNAcase to assess the localization CK14 in ameloblasts during the enamel formation. A, day 0. Arrows indicate binding of ATMP to CK14 in the cytoplasm of ameloblasts. Note the uniform distribution of [3H]ATMP in the cytoplasm. B, day 1 early. Arrows indicate distribution of [3H]ATMP (site of CK14) at the apical end of the ameloblasts, the site of secretion of enamel. C, day 1, late. Arrows indicate accumulation of [3H]ATMP (site of CK14) at the apical end of the ameloblasts. D, day 3. Arrows indicate intensity in the accumulation of [3H]ATMP at the apical end of the ameloblasts. Am, ameloblasts; e, enamel; d, dentine; pd, predentine; si, stratum intermedium.

View larger version (118K): [in this window] [in a new window]   Fig. 10.   Binding of [3H]GMp to free amelogenins or [3H]ATMP to free CK14 at the Tomes' process in ameloblasts. A, B, and E, binding [3H]GMp to free amelogenins. On day 5 (A) and on day 3 (E) no accumulation of [3H]GMp is observed. Inset in A is magnified in B. Arrows indicate [3H]GMp, suggestive of free amelogenin, in Tomes' process of ameloblasts. C and D, binding [3H]ATMP on day 5 in GlcNAcase-treated sections. Inset is magnified in D and the arrows indicate [3H]ATMP, suggestive of free-CK14, in Tomes' process of ameloblasts.

Quantitation of Co-localized and Free Amelogenin and CK14-- To evaluate co-assembly and dissociation of CK14 and amelogenin the percentage of green, yellow, and red signals (pixels) were measured during the growth of enamel at different days of development of teeth (Fig. 11).

View larger version (15K): [in this window] [in a new window]   Fig. 11.   Quantitation of CK14-amelogenin paired pixels during different stages of development using confocal laser scan microscopy. The quantitation is based on the analyses of the scatter diagram (co-localization) obtained with confocal laser scan microscopy. All pixels having the same positions in both images are considered a pair. Of every pair of pixels (P1, P2) from the two images, the brightness level of pixel P1 is interpreted as X coordinate, and that of pixel P2 as Y coordinate of the scatter diagram. Non-co-localized P1 and P2 pixels and the background values were excluded to distinguish the pixels that are co-localized. The results were expressed as percentage of co-localization of amelogenins and CK14 at different developmental stages from day 0 to 9 from initiation, growth, and cessation (double-head arrows) of enamel formation. The quantity of non-co-localized P1 and P2 pixels representing free amelogenin and free CK14, respectively, are also plotted for comparison.

The level of free cytokeratin within ameloblast as evidenced by the green signal appeared constant throughout and remained <20%. Free amelogenin (red signal) in ameloblast increased on day 3. This finding suggests that the dissociation of the CK14-amelogenin assembly may commence on day 3. The decline and the steady-state level of free amelogenin (red signals) between days 3 and 7 could be due to continuous and prolific secretion of amelogenin. The quantitative variation of co-assembled amelogenin-CK14 within ameloblasts showed a pattern concomitant with enamel growth. The yellow granules although abundant on day 0 tend to decline on day 3 concomitant with increase in free amelogenin (red signals). The decrease in the percent of pixels on day 3 could be due to the accumulation of the granules. Increase in co-localized granules on days 5 and 7 are consistent with enamel growth during this period. It also suggests active synthesis and transport of nascent amelogenin polypeptides to the sites of secretion.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Properties of the N-terminal "Head" Region of CK14-- Expression of cytokeratin is specific for each epithelial cell type and its state of differentiation (32). CK14, a member of the family of acidic type I cytokeratins, consists of a conserved rod domain with four -helical regions separated by short non-helical linker sequences and flanked by non-helical globular sequences commonly referred to as head (N-terminal) and tail (C-terminal) domains (33). Self-assembly of CK14 leads to formation of intermediate filaments of 10 nm diameter. The self-assembly is facilitated by the -helical region (33). Removal of the head domain does not affect self-assembly (34) suggesting that the head region may have a role different from that of CK14 self-assembly, which is necessary for formation of the cytoskeleton or intermediate filaments. The presence of the GMp sequence in the head region of CK14 favors a role in binding to GlcNAc-binding lectins. Indeed, the ATMP motif of the N-terminal domain of amelogenins, that specifically binds to GlcNAc residues (10), also recognizes the GMp sequence (11). The binding specificity of the ATMP sequence (PYPSYGYEPMGGW) to the GMp motif (SFGSGFGGGY) was confirmed by dosimetric binding of amelogenin or TRAP with [³H]GMp, specific binding in varying concentrations of labeled GMp, Scatchard plot analysis, and competitive inhibition with unlabeled GMp (11). These findings lend support to the binding capability of the ATMP motif of amelogenins with the GMp sequence in the N-terminal head region of CK14.

Specificity of Amelogenin-CK14 Interaction and the Biological Significance-- The dosimetric interaction between CK14 and amelogenin (Fig. 1A), CK14 and TRAP (Fig. 1B), and CK14 and the ATMP motif (Fig. 2) further favor the sequence-sequence interaction between the two proteins. Inability of amelogenin to bind to CK14, if blocked by GlcNAc or GMp sequence and inhibition of CK14 binding to amelogenin by ATMP (Table I), confirms the sequence-sequence interaction between the ATMP motif of amelogenins and the GMp motif of CK14. The possible biological significance of amelogenin-CK14 interaction is revealed by the loss of function mutations of ATMP (T-ATMP and F-ATMP) failing to interact with CK14. One of these mutations involves substitution of the third proline with threonine as has been reported in the inherited enamel defect of human amelogenesis imperfecta (12). Similarly, substitution of tri-tyrosyl residues with phenylalanine affected the binding of ATMP with CK14, suggesting that mutational alterations of the ATMP sequence that cause enamel defects could be due to loss of binding of the mutant amelogenin to CK14 and a consequent inability of the amelogenin to reach the site of secretion. Thus, CK14 may play an important role in amelogenesis by interacting with the ATMP motif of amelogenin. The immunofluorescence studies made in this investigation suggest a possible role of CK14 in amelogenesis.

CK14 and Amelogenin as Ameloblast-differentiation Markers-- While amelogenin is the postnatal differentiation marker of ameloblasts, the presence of CK14 in ameloblasts 48-96 h earlier than the expression of amelogenin indicates that CK14 is a stage-specific differentiation marker for ameloblasts (3). Morphometric measurements of developing enamel define the stage-specific secretion of the enamel matrix by ameloblasts. Three phases of enamel formation can be distinguished at different levels of a growing murine incisor tooth. Enamel secretion commences (stage I) on day 0 and remains slow until day 1. Phase II commences after day 1 when the enamel growth is prolific by day 3 and reaches a maximum between days 5 or 7 (Fig. 5). In phase III enamel growth ceases and ameloblasts lose their distinctive morphology. Correlated with sequential development of enamel formation, a change in the pattern of distribution of CK14 is observed in ameloblasts. While CK14 immunostaining is highly prevalent in the perinuclear region, the migration of CK14 to the apical region of the cell from day 1 is evident in both light and confocal microscopy. Intense fluorescent green signal, at dual wavelengths in confocal microscopy, at the distal region of ameloblasts provides evidence of self-assembly of CK14. Amelogenin is also found around the perinuclear region of ameloblasts on days 1 to 3, but amelogenin is always co-assembled with CK14.

Co-assembly of Amelogenin and CK14 during Enamel Formation-- Co-localization of CK14-amelogenin was evident by the yellow signal or pixels in confocal microscopy (Fig. 7). Absence of red signals at dual wavelengths on day 0 in the cytoplasm suggests either the absence of free amelogenin or masking of amelogenin by CK14. The perinuclear distribution of yellow granules in the cytoplasm of ameloblasts on day 0 indicates a co-localization of CK14 and amelogenin. Progressive accumulation of the same granules at the apical region of ameloblasts from days 1 to 3 suggests co-migration of the CK14-amelogenin complex from the perinuclear region to the apical region. Co-localization of CK14-amelogenin is prominently seen at the apical region until day 5.

Co-assembly of CK14-Amelogenin Is Transient-- During the prolific phase (phase II) of enamel matrix secretion between days 3 and 5 (Fig. 8E), three distinct signals are observed at the apical zone. They are yellow signals indicative of co-assembly of CK14-amelogenin, emigrating or red signals indicative of free amelogenin, and retention or green signals indicative of free CK14. The augmentation of the red signal outside the periphery of ameloblasts on day 7 as a distinct layer of red signal at the interphase between the apical region of ameloblasts and enamel matrix indicates newly secreted extracellular amelogenin that is yet to undergo postsecretory modifications. All these observations suggest that amelogenin may be translocated from the site of synthesis in the perinuclear region to the apical region of the cell in association with CK14, and at the apical region the dissociation of the co-assembly complex of CK14-amelogenin may occur, prior to amelogenin secretion.

Evidence for Dissociation of Co-assembled CK14-Amelogenin-- While confocal microscopy indicated co-distribution and co-migration of CK14-amelogenin, autoradiographic studies with [³H]ATMP provided information on the dissociation of the amelogenin-CK14 complex. Free CK14 is restricted within the ameloblast as evidenced by the distribution of [³H]ATMP. Accumulation of free CK14 at the apical region of ameloblasts commenced early on day 1 and increased on day 3 and remained constant until day 5. On the other hand, no prominent accumulation of radiolabeled GMp could be seen in the apical region of ameloblasts. However, upon magnification, a specific accumulation of [³H]GMp is observed in the apical cytoplasmic projections (Tomes' process) of the ameloblasts. Further examination revealed that [³H]ATMP is also seen in the same region, suggesting that these apical cellular projections of the ameloblasts are a specialized zone of the cytoplasm facilitating the dissociation of CK14-amelogenin complex.

Transient Co-expression between Cytokeratins and Other Proteins-- Similar to co-expression of CK14 and amelogenin, transient coexpression of cytokeratins and vimentin was observed during ontogenic development (4), in the cornea (4), fetal tongue epithelia (35), and in an adenomatoid odontogenic tumor (36). Interestingly such co-expression of cytokeratin and vimentin in epithelial cells is considered a typical feature of a proliferative situation and the secretory functions of vimentin (4). Robinson and co-workers (37) envisaged a similar interaction between tuft proteins and keratins in ameloblasts during enamel formation. Although none of these investigations documented sequence-sequence interaction between cytokeratin and vimentin or tuft proteins, it appears such coexpressions are in many cases not random but are intrinsic and cell type related. Several proteins have been identified to be associated with cytokeratins (38). A list of such cytokeratin-associated proteins is presented in Table II. Most of these proteins are associated with the C-terminal end of cytokeratins but no specific sequence-sequence interaction was established.

Significance of CK14-Amelogenin Interaction-- The observations that amelogenin binds specifically to GlcNAc (10) led to a study of its interaction with the GMp (11). The finding that the sequence of GMp has 100% homology with CK14 but not with any other known proteins or ameloblasts (present study) and that CK14 is a differentiation marker of ameloblasts before synthesis of amelogenin (3) led to the serendipitous findings reported in this investigation. The biological significance of the CK14-amelogenin association may not only be significant, in promoting translocation of amelogenin to the site of secretion but may also serve as a repressor of co-assembly and migration of CK14-amelogenin and amelogenesis, as in the cases of X-linked inherited diseases like amelogenesis imperfecta. This finding contrasts to our earlier assumption that mutations at the ATMP site may affect post-secretory interactions of amelogenin. Similarly, mutations at the GMp site of CK14 may also suppress the secretion of amelogenin. In support of this concept Rugg et al. (39) have reported the presence of discolored and notched front teeth in a child with a functional "knockout" of CK14, displaying clinical symptoms of epidermolysis bullosa. However, amelogenins mutated at sites other than the ATMP and non-GMp mutations of CK14 may not affect the co-assembly and migration of CK14-amelogenin.

In conclusion, our investigation appears unique in documenting a specific sequence-sequence interaction between CK14 and amelogenins. Based on this interaction between amelogenins and CK14 and their co-assembly, distribution, migration, and dissociation of the co-assembled proteins, we envisage that the functional role of CK14 may be similar to heat shock proteins (40) in binding to nascent peptides and carrying them to the cell surface. Our observations reveal that the ATMP motif at the N-terminal region of amelogenins function as a signal peptide for GMp-ligand of CK14 and CK14 may perform a chaperon role during amelogenesis. A schematic for the putative role of CK14 during amelogenesis is presented in Fig. 12. An additional point of note is that it has been shown that cytokeratin-associated proteins may become dissociated following phosphorylation of the associated protein (38). In this context it is of interest that serine 16 is phosphorylated in the native amelogenins extracted from the enamel, although evidence for the presence of a non-phosphorylated entity has also been reported (41). It remains unclear whether amelogenin phosphorylation contributes to the dissociation of the amelognein-CK14 complex prior to amelogenin secretion. A study of the enzymes involved in serine phosphorylation in the apical region of ameloblasts, particularly in the Tomes' process is under investigation.

View larger version (25K): [in this window] [in a new window]   Fig. 12.   Scheme of the putative functional role for CK14 in amelogenesis. CK14 binds to the ATMP motif of the N-terminal region of the nascent amelogenin polypeptide in the perinuclear region. ATMP motif serves as a signal peptide. The GMp motif at the N-terminal region of CK14 acts as ligand to bind to the signal peptide and form a co-assembly. Co-assembled CK14-amelogenin migrates to the Tomes' process, where collapse of co-assembly of CK14-amelogenin occurs resulting in release of amelogenin. Phosphorylation (P) at the serine residue of amelogenin suggests that it may facilitate the dissociation of amelogenin from CK14.

	ACKNOWLEDGEMENTS

We thank Dr. Charles F. Shuler, Chairman, Center for Craniofacial Molecular Biology, University of Southern California, for encouragement and support, and Ernesto Barron at the EM core facility at Doheny EYE institute for assistance in confocal laser scanning microscopy.

	FOOTNOTES

^* This work was supported by National Institutes for Health NIDR Grant DE-03660.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, 2250 Alcazar St., Los Angeles, CA 90033. Tel.: 323-442-3171; Fax: 323-442-2981; E-mail: rravindr@hsc.usc.edu.

Published, JBC Papers in Press, June 25, 2001, DOI 10.1074/jbc.M104656200

	ABBREVIATIONS

The abbreviations used are: ATMP, amelogenin tri-tyrosyl motif peptide; CK, cytokeratin; TRAP, tyrosine-rich amelogenin polypeptide; LRAP, leucine-rich amelogenin polypeptide; T-ATMP, where proline is substituted by threonine; F-ATMP, where all three tyrosyl residues are replaced by phenylalanine; HPLC, high performance liquid chromatography; NB, newborn; PN, post-natal; GlcNAc, N-acetyl-D-glucosamine; GMp, GlcNAc mimicking peptide; PVDF, polyvinylidene difluoride; HSA, human serum albumin; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamin isothiocyanate; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES