title: Full length amelogenin binds to cell surface LAMP-1 on tooth root/periodontium associated cells.
abstract: OBJECTIVE
  Lysosome-associated membrane protein-1 (LAMP-1) has been suggested to be a cell surface receptor for a specific amelogenin isoform, leucine-rich amelogenin peptide or LRAP. However, it is unclear if LAMP-1 is an amelogenin receptor for dental mesenchymal cells. The goal of this study was to determine if LAMP-1 serves as a cell surface binding site for full length amelogenin on tooth root/periodontium associated mesenchymal cells.
  
  
  METHODS
  Murine dental follicle cells and cementoblasts (OCCM-30) were cultured for 2 days followed by addition of full length recombinant mouse amelogenin, rp(H)M180. Dose-response (0-100 microg/ml) and time course (0-120 min) assays were performed to determine the optimal conditions for live cell surface binding using immunofluorescent microscopy. A competitive binding assay was performed to determine binding specificity by adding Emdogain (1 mg/ml) to the media. An antibody against LAMP-1 was used to detect the location of LAMP-1 on the cell surface and the pattern was compared to cell surface bound amelogenin. Both amelogenin and cell surface LAMP-1 were immuno-co-localized to compare the amount and distribution pattern.
  
  
  RESULTS
  Maximum surface binding was achieved with 50 microg/ml of rp(H)M180 for 120 min. This binding was specific as demonstrated by competitive inhibition (79% lower) with the addition of Emdogain. The binding pattern for rp(H)M180 was similar to the distribution of surface LAMP-1 on dental follicle cells and cementoblasts. The high co-localization coefficient (0.92) for rp(H)M180 and LAMP-1 supports rp(H)M180 binding to cell surface LAMP-1.
  
  
  CONCLUSIONS
  The data from this study suggest that LAMP-1 can serve as a cell surface binding site for amelogenin on dental follicle cells and cementoblasts.
authors.affiliation: Department of Restorative Dentistry, School of Dentistry, University of Washington, Seattle, WA 98195, USA. haizhang@u.washington.edu
authors.forename: Hai
authors.initials: H
authors.lastname: Zhang
authors.forename: Kevin
authors.initials: K
authors.lastname: Tompkins
authors.forename: Jacques
authors.initials: J
authors.lastname: Garrigues
authors.forename: Malcolm L
authors.initials: ML
authors.lastname: Snead
authors.forename: Carolyn W
authors.initials: CW
authors.lastname: Gibson
authors.forename: Martha J
authors.initials: MJ
authors.lastname: Somerman
mesh_terms: D053523:Amelogenin / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002478:Cells, Cultured; D003739:Dental Cementum / Q000166:cytology*; D003746:Dental Enamel Proteins / Q000378:metabolism; D003795:Dental Sac / Q000166:cytology*; D051907:Lysosomal Membrane Proteins / Q000378:metabolism*; D008648:Mesoderm / Q000166:cytology; D051379:Mice; D010513:Periodontal Ligament / Q000166:cytology / Q000254:growth & development; D011485:Protein Binding; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000378:metabolism; D012038:Regeneration / Q000502:physiology; D014092:Tooth Root / Q000166:cytology / Q000254:growth & development; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D053523:Amelogenin; D003746:Dental Enamel Proteins; D051907:Lysosomal Membrane Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C052255:Lamp1 protein, mouse; D052118:Lysosomal-Associated Membrane Protein 1; C109069:enamel matrix proteins
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