ARTICLE |
Correspondence to: Antonio Nanci, Université de Montréal, Faculty of Dentistry/Stomatology, PO Box 6128, Station Centre-Ville, Montréal, QC, Canada H3C 3J7. E-mail: antonio.nanci@umontreal.ca
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Summary |
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Amelogenins represent the major component of the organic matrix of enamel, and consist of several intact and degraded forms. A precise knowledge of their respective distributions throughout the enamel layer could provide some insight into their functions. To date, no antibody exists that can selectively detect the secretory forms of amelogenin. In this study we used the chicken egg yolk system to generate an antibody to recombinant mouse amelogenin. Immunoblots of whole homogenates from rat incisor enamel organs and enamel showed that the resulting antibody (M179y) recognized proteins corresponding to the five known secretory forms of rat amelogenin. Immunogold cytochemistry demonstrated that reactivity was restricted to ameloblasts and enamel. Secretory forms of amelogenin persisted in significant amounts throughout the enamel layer. The density of labeling was highest over the surface portion of the enamel layer, but enamel growth sites in this region showed a localized paucity of gold particles. Immunoreactivity was lowest over the mid-portion of the layer and increased moderately near the dentinoenamel junction. These results indicate that intact forms of amelogenin probably have a more complex distribution in the enamel layer than was heretofore suspected.
(J Histochem Cytochem 49:285292, 2001)
Key Words: polyclonal antibody, amelogenin, secretory forms, immunoblotting, immunocytochemistry
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Introduction |
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During enamel formation, ameloblasts produce a number of matrix proteins that are believed to promote and regulate mineral ion deposition into unique and extremely long apatite crystals. Amelogenins represent the major secretory product of these epithelium-derived cells (reviewed in
The aim of this study was to determine whether this system could produce an antibody selective for secretory forms of amelogenin. Although such an antibody is beneficial for biochemical characterizations, it would be particularly useful for immunocytochemical mapping of the temporospatial distribution of the protein. This information is essential to understanding of its function and its implication in pathological alterations.
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Materials and Methods |
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All animal handling and experimental procedures were approved by the Comité de Déontologie de l'expérimentation sur les Animaux of the Université de Montréal.
Preparation of Chicken Egg Yolk Polyclonal Antibody
A mouse recombinant amelogenin [M179, lacking the N-terminal methionine and the Ser16-phosphate group found on the main native mouse amelogenin M180 isoform (
Sample Preparation for Immunoblotting
Male Wistar rats weighing 100150 g (Charles Rivers Canada; St-Constant, QC, Canada) were anesthetized with Metofane (methoxyfluorane; Janssen Pharmaceutica, North York, ON, Canada) and decapitated. The hemimandibles were dissected out and the enamel organ was partially exposed by cracking off some of the covering alveolar bone. They were immediately plunged into liquid nitrogen and maintained in it for a minimum of 5 hr before freeze-drying for at least 48 hr at -80C on a 12-liter cascade lyophilizer system (Labconco; Kansas City, MO). The enamel organ, with adhering labial connective tissue and enamel, was then transected on each incisor into a series of five sequential strips relative to the secretory (S) and maturation (M) stages of amelogenesis, using a molar reference line (-mercaptoethanol, and 0.005% bromophenol blue. The vials were immersed in a boiling water bath for 5 min, cooled, and stored at 4C.
Immunoblotting
Twenty µl of extraction fluid from each vial was applied to separate lanes of standard format (16 cm x 14 cm x 1 mm) 12% polyacrylamide slab gels. Broad-range molecular weight marker proteins (Bio-Rad; Mississauga, ON, Canada) were also loaded in one lane of each gel. Proteins were separated by electrophoresis at 20 mA per gel constant current using a discontinuous buffer system (
Tissue Processing for Immunohistochemistry
Male Wistar rats weighing 100 ± 10 g (Charles Rivers Canada) were anesthetized with chloral hydrate (0.4 mg/g bw) and sacrificed by intravascular perfusion with a fixative solution consisting of 1% glutaraldehyde in 0.1 M sodium phosphate (PB), pH 7.2. The hemimandibles were removed and immersed in the fixative overnight at 4C. They were then washed in 0.1 M PB, pH 7.2, and decalcified for 21 days in 4.13% EDTA at 4C (
Immunocytochemistry
Sections were floated for 15 min on a drop of 0.01 M PBS containing 1% ovalbumin (Oval; Sigma Chemical, St Louis, MO). They were transferred for 3 hr onto a drop of M179y diluted 1:100, washed with PBS, refloated on PBSOval, and then incubated for 1 hr with a rabbit anti-chicken IgG antibody (diluted 1:2000) (Cappel Research Products). Finally, they were washed again with PBS, refloated on PBSOval, and incubated with protein Agold complex for 30 min. After immunolabeling, the grids were extensively rinsed with PBS, followed by distilled water. Controls consisted of incubations with preimmune antibody followed by the secondary antibody and protein Agold, secondary antibody and protein Agold, or protein Agold alone. All incubations were carried out at room temperature. Grids were stained with 4% aqueous uranyl acetate and lead citrate for examination in a JEOL JEM-1200EX-II transmission electron microscope operated at 60 kV.
Quantitative Analysis of Immunocytochemical Labeling
Sections from secretory, early, and mid-maturation stage of amelogenesis (see = 0.05, using version 5.5A of Statistica for Windows (Statsoft; Tulsa, OK). Power tests of differences between means were done using version 1.01 I of GraphPad StatMate (GraphPad Software; San Diego, CA). The lowest sampling number overall (n = 66) was in the middle region of the enamel layer in early maturation (see Fig 8, EMAT, Mdl).
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Results |
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Immunoblotting
M179y revealed a band near 27 kD in the S1 enamel organ cell extracts and an additional weakly stained band near 29 kD in the M1 sample (Fig 1). No immunoreactive proteins were discernible in the M2 and M3 cell extracts. Enamel extracts, on the other hand, showed three bands at 23, 27, and 29 kD from S1 to M1 (Fig 1). Additional faint bands were present near 30 kD in S2 and M1 and near 31 kD only in S2 samples.
Immunolabeling
Many gold particles were observed over the saccules of the Golgi apparatus (Fig 2) and over secretory granules in Tomes' processes (Fig 3) of secretory stage ameloblasts. Enamel was intensely immunoreactive except at rod (Fig 3) and inter-rod (Fig 4) enamel growth sites, at which few particles were present. Early to mid-maturation stage ameloblasts still showed immunoreactivity over the Golgi apparatus and occasional secretory granules were found in these cells (Fig 5). The overall density of labeling over enamel gradually decreased towards late maturation. However, the general distribution was similar throughout (compare Fig 6A and Fig 6B). In the regions sampled, no immunoreactivity was seen in other cells of the enamel organ or in odontoblasts.
Statistical analyses of the three stages of amelogenesis confirmed that there was a general decline in the density of M179y labeling from secretory to early and mid-maturation stages (Fig 7, SEC to EMAT, p< 0.0000; EMAT to MMAT, p<0.0152). When the three regions in which the enamel layer was partitioned were considered, all stages showed a higher density of labeling over the surface portion of the enamel layer (Region 1) than over the middle part of the enamel layer (Region 2) (Fig 7, Near AM to Middle, p<0.0000; Fig 8, AM to Mdl for all stages, p<0.0000). An increase in the density of labeling near the DEJ (Region 3) was detected in secretory and mid-maturation stage samples (Fig 8; SEC, Mdl to DEJ, p<0.0000; MMAT, Mdl to DEJ is not significant). Early maturation stage samples, in contrast, showed fairly uniform density of labeling for most of the thickness of the enamel layer except near the surface, where the density of labeling was higher (Fig 8; Mmat, AM to Mdl or DEJ, p<0.0000).
In all cases, control incubations resulted in a major reduction of the labeling and in the presence of few randomly distributed gold particles throughout the tissue sections.
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Discussion |
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Using the chicken egg yolk system (
One past interpretation given to autoradiographic findings of a sharp increase of labeling over areas of enamel that initially show almost no radioactivity is that protein fragments cleaved from parent amelogenins diffuse into deeper regions of the enamel layer (discussed in
It has long been assumed that amelogenins are uniformly distributed throughout the enamel layer, a belief consistent with the notion that enamel proteins are arranged in a thixotropic gel that allows free mixing of all components (
The persistence of secretory forms of amelogenin throughout the enamel layer has some important functional implications. Because amelogenin fragments adsorb less efficiently to enamel crystals (
In conclusion, we have prepared a chicken egg yolk antibody that appears to recognize only secretory forms of amelogenins. This antibody has revealed a more complex distribution of parent amelogenins than had been previously suspected and represents a potentially useful tool for studying their functional relationship with enamel crystals.
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Acknowledgments |
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Supported by a grant from the Canadian Institute of Health Research (CIHR).
We thank Dr J.P. Simmer (University of Texas at San Antonio) for providing the recombinant mouse M179 amelogenin used for injection into chickens, Mireille Fyfe and Line Lespérance for their technical help with production of the antibody, and Sylvia Zalzal for advice on immunolabeling.
Received for publication September 14, 2000; accepted October 5, 2000.
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Literature Cited |
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Bartlett JD, Simmer JP (1999) Proteinases in developing dental enamel. Rev Oral Biol Med 10:425-441
Bendayan M (1995) Colloidal gold post-embedding immunocytochemistry. Prog Histochem Cytochem 29:1-163[Medline]
Chen W-Y, Bell AW, Simmer JP, Smith CE (2000) Mass spectrometry of native rat amelogenins: Primary transcripts, secretory isoforms, and C-terminal degradation. J Dent Res 79:840-849[Abstract]
Chen W-Y, Nanci A, Smith CE (1995) Immunoblotting studies on artifactual contamination of enamel homogenates by albumin and other proteins. Calcif Tissue Int 57:145-151[Medline]
Eastoe JE (1979) Enamel protein chemistry. Past, present and future. J Dent Res 58:753-763[Medline]
Fincham AG, MoradianOldak J, Simmer JP (1999) The structural biology of the developing dental enamel matrix. J Struct Biol 126:270-299[Medline]
Gassmann M, Thömmes P, Weiser T, Hübscher U (1990) Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J 4:2528-2532
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685[Medline]
Lau EC, Simmer JP, Bringas P, Jr, Hsu DDJ, Hu C-C, ZeichnerDavid M, Thiemann F, Snead ML, Slavkin HC, Fincham AG (1992) Alternative splicing of the mouse amelogenin primary RNA transcript contributes to amelogenin heterogeneity. Biochem Biophys Res Commun 188:1253-1260[Medline]
Lösch U, Schranner I, Wanke R, Jürgens L (1986) The chicken egg, an antibody source. J Vet Med B 33:609-619
Nanci A, Ahluwalia JP, Pompura JR, Smith CE (1989) Biosynthesis and secretion of enamel proteins in the rat incisor. Anat Rec 224:277-291[Medline]
Nanci A, Hashimoto J, Zalzal S, Smith CE (1996) Transient accumulation of proteins at interrod and rod enamel growth sites. Adv Dent Res 10:135-149[Medline]
Nanci A, Smith CE (2000) Matrix-mediated mineralization in enamel and the collagen-based hard tissues. In Goldberg M, Boskey A, Robinson C, eds. Chemistry and Biology of Mineralized Tissues. Rosemont, IL, American Academy of Orthopaedic Surgeons, 217-224
Nanci A, Zalzal S, Lavoie P, Kunikata M, Chen W-Y, Krebsbach PH, Yamada Y, Hammarström L, Simmer JP, Fincham AG, Snead ML, Smith CE (1998) Comparative immunochemical analyses of the developmental expression and distribution of ameloblastin and amelogenin in rat incisors. J Histochem Cytochem 46:911-934
Ryu OH, Hu CC, Simmer JP (1998) Biochemical characterization of recombinant mouse amelogenins: protein quantitation, proton absorption, and relative affinity for enamel crystals. Connect Tissue Res 39:207-214[Medline]
Schmidt P, Erhard MH, Schams D, Hafner A, Folger S, Lösch U (1993) Chicken egg antibodies for immunohistochemical labeling of growth hormone and prolactin in bovine pituitary gland. J Histochem Cytochem 41:1441-1446
Simmer JP, Lau EC, Hu C-C, Aoba T, Lacey M, Nelson D, ZeichnerDavid M, Snead ML, Slavkin HC, Fincham AG (1994) Isolation and characterization of a mouse amelogenin expressed in Escherichia coli.. Calcif Tissue Int 54:312-319[Medline]
Simmer JP, Snead ML (1995) Molecular biology of the amelogenin gene. In Robinson C, Kirkham J, Shore R, eds. Dental Enamel, Formation to Destruction. Boca Raton, FL, CRC Press, 59-83
Smith CE, Nanci A (1989) A method for sampling the stages of amelogenesis on mandibular rat incisors using the molars as a reference for dissection. Anat Rec 225:257-266[Medline]
Smith CE, Nanci A (1996) The protein dynamics of amelogenesis. Anat Rec 245:219-234[Medline]
Smith CE, Pompura JR, Borenstein S, Fazel A, Nanci A (1989) Degradation and loss of matrix proteins from developing enamel. Anat Rec 224:292-316[Medline]
Warshawsky H, Moore G (1967) A technique for the fixation and decalcification of rat incisors for electron microscopy. J Histochem Cytochem 15:542-549[Medline]
Warshawsky H, Smith CE (1974) Morphological classification of rat incisor ameloblasts. Anat Rec 179:423-446[Medline]