Full length amelogenin binds to cell surface LAMP-1 on tooth root/periodontium associated cells.
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.
Zhang
Hai
H
Department of Restorative Dentistry, School of Dentistry, University of Washington, Seattle, WA 98195, USA. haizhang@u.washington.edu
Tompkins
Kevin
K
Garrigues
Jacques
J
Snead
Malcolm L
ML
Gibson
Carolyn W
CW
Somerman
Martha J
MJ
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
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
Biophys J. 2007 Nov 15;93(10):3664-74
17704165
Eur J Oral Sci. 2006 May;114 Suppl 1:239-43; discussion 254-6, 381-2
16674692
J Periodontol. 2003 Oct;74(10):1423-31
14653387
J Periodontol. 2004 Aug;75(8):1126-36
15455742
Cell Mol Life Sci. 2003 Jan;60(1):38-55
12613657
J Clin Periodontol. 1997 Sep;24(9 Pt 2):705-14
9310876
Biochem J. 1983 Apr 1;211(1):149-54
6870818
Arch Oral Biol. 2005 May;50(5):527-32
15777534
Curr Opin Struct Biol. 2009 Feb;19(1):31-8
19157855
Biochem Biophys Res Commun. 2008 Feb 29;367(1):1-6
18086559
J Bone Miner Metab. 2001;19(4):236-43
11448016
Crit Rev Oral Biol Med. 2002;13(4):323-34
12191959
Eur J Oral Sci. 2006 May;114 Suppl 1:276-9; discussion 285-6, 382
16674698
J Clin Periodontol. 1997 Sep;24(9 Pt 2):693-6
9310874
Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000 Aug;90(2):218-23
10936841
J Clin Periodontol. 1999 Dec;26(12):855-60
10599915
Curr Opin Cell Biol. 1996 Dec;8(6):844-50
8939666
J Clin Periodontol. 1999 Dec;26(12):833-40
10599912
Science. 2009 Apr 10;324(5924):198-203
19359576
Cell Tissue Res. 2007 Aug;329(2):283-94
17443352
Biochem Biophys Res Commun. 2001 Jan 19;280(2):460-5
11162539
Lancet. 2004 Jul 10-16;364(9429):149-55
15246727
J Biol Chem. 2003 Sep 12;278(37):35743-8
12851394
J Periodontol. 2001 May;72(5):679-87
11394405
J Biol Chem. 2001 Aug 24;276(34):31871-5
11406633
Dev Dyn. 2003 Dec;228(4):651-63
14648842
J Biol Chem. 1980 Oct 25;255(20):9760-8
7430099
Bone. 2006 Feb;38(2):172-80
16214432
J Periodontal Res. 2009 Jun;44(3):289-96
19462488
Curr Opin Struct Biol. 2008 Dec;18(6):756-64
18952168
Trends Cell Biol. 2003 Mar;13(3):137-45
12628346
J Periodontol. 2000 Dec;71(12):1829-39
11156039
J Clin Periodontol. 1997 Sep;24(9 Pt 2):669-77
9310871
J Mol Biol. 1999 Oct 22;293(2):321-31
10550212
J Periodontol. 2000 Jan;71(1):63-72
10695940
J Periodontol. 2000 Aug;71(8):1278-86
10972643
J Clin Periodontol. 1997 Sep;24(9 Pt 2):658-68
9310870
Biochem J. 2007 Dec 15;408(3):347-54
17708745
J Dent Res. 2006 Sep;85(9):814-8
16931863
J Periodontal Res. 1991 Jan;26(1):42-7
1825333
Biochem Biophys Res Commun. 2009 Sep 25;387(3):558-63
19615979
Curr Top Dev Biol. 2007;78:47-126
17338915
Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13625-30
11087820
J Biochem. 2008 Oct;144(4):531-7
18676354
Biosci Rep. 1981 Oct;1(10):771-8
7306685
J Cell Mol Med. 2009 Jun;13(6):1110-24
19228267
Proteins. 2009 Aug 15;76(3):560-9
19274734
J Biol Chem. 2000 Dec 29;275(52):41263-72
10998415
Matrix Biol. 1999 Apr;18(2):133-43
10372553
Biochemistry. 2009 Mar 17;48(10):2272-81
19236004
Orthod Craniofac Res. 2005 Nov;8(4):285-91
16238609
J Dent Res. 2006 Sep;85(9):775-93
16931858
Int J Periodontics Restorative Dent. 1999 Apr;19(2):157-63
10635181
Cell Mol Life Sci. 2007 Jan;64(2):244-56
17187173
Connect Tissue Res. 1995;32(1-4):131-6
7554909
Trends Biochem Sci. 2002 Oct;27(10):527-33
12368089
J Clin Periodontol. 2004 Mar;31(3):184-92
15016022
Curr Top Dev Biol. 2006;74:57-115
16860665
Crit Rev Oral Biol Med. 1997;8(1):4-39
9063623
Proc Natl Acad Sci U S A. 1988 Jun;85(11):3860-4
3287378
Bone. 2008 Jun;42(6):1072-9
18394981
J Periodontol. 2000 Jul;71(7):1110-6
10960017
Protein Sci. 2002 Apr;11(4):739-56
11910019
Mol Cell Biol. 2003 Feb;23(3):1075-84
12529411
J Bone Miner Res. 2010 May;25(5):1167-78
19888897