title: Preferential and selective degradation and removal of amelogenin adsorbed on hydroxyapatites by MMP20 and KLK4 in vitro.
abstract: The hardest tooth enamel tissue develops from a soft layer of protein-rich matrix, predominated by amelogenin that is secreted by epithelial ameloblasts in the secretory stage of tooth enamel development. During enamel formation, a well-controlled progressive removal of matrix proteins by resident proteases, Matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), will provide space for the apatite crystals to grow. To better understand the role of amelogenin degradation in enamel biomineralization, the present study was conducted to investigate how the adsorption of amelogenin to hydroxyapatite (HAP) crystals affects its degradation by enamel proteinases, MMP20 and KLK4. Equal quantities of amelogenins confirmed by protein assays before digestions, either adsorbed to HAP or in solution, were incubated with MMP20 or KLK4. The digested samples collected at different time points were analyzed by spectrophotometry, SDS-PAGE, high performance liquid chromatography (HPLC), and LC-MALDI MS/MS. We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4). The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates. Using LC-MALDI MS/MS, more accessible cleavage sites and hydrolytic fragments from MMP20/KLK4 digestion were identified for the amelogenin adsorbed on HAP crystals as compared to the amelogenin in solution. These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 in vitro. Based on these findings, a new degradation model related to enamel crystal growth is proposed.
authors.affiliation: Department of Orofacial Sciences, School of Dentistry, University of California, San Francisco San Francisco, CA, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco San Francisco, CA, USA.
authors.forename: Haichuan
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco San Francisco, CA, USA.
authors.forename: H Ewa
authors.initials: HE
authors.lastname: Witkowska
authors.affiliation: Department of Oral Medicine, Guanghua School of Stomatology, Sun Yat-sen University Guangdong, China.
authors.forename: Yulei
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Departments of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences Hiroshima, Japan.
authors.forename: Kataro
authors.initials: K
authors.lastname: Tanimoto
authors.affiliation: Department of Orofacial Sciences, School of Dentistry, University of California, San Francisco San Francisco, CA, USA.
authors.forename: Wu
authors.initials: W
authors.lastname: Li
keywords: KLK4; MMP20; amelogenin; biomineralization; hydroxyapatites; protein interactions; proteolysis; tooth enamel
references.citation: Arch Oral Biol. 2011 Apr;56(4):331-6
references.pmid: 21081224
references.citation: J Dent Res. 2010 Apr;89(4):344-8
references.pmid: 20160068
references.citation: Bull Tokyo Med Dent Univ. 1992 Dec;39(4):71-80
references.pmid: 1333893
references.citation: J Dent Res. 2006 Sep;85(9):775-93
references.pmid: 16931858
references.citation: J Dent Res. 2008 Jan;87(1):39-44
references.pmid: 18096891
references.citation: J Dent Res. 1998 Aug;77(8):1580-8
references.pmid: 9719031
references.citation: J Am Chem Soc. 2011 Nov 2;133(43):17406-13
references.pmid: 21916473
references.citation: J Biosci Bioeng. 2006 May;101(5):434-9
references.pmid: 16781474
references.citation: J Struct Biol. 1999 Jun 30;126(3):270-99
references.pmid: 10441532
references.citation: J Dent Res. 2008 May;87(5):451-5
references.pmid: 18434575
references.citation: Curr Opin Struct Biol. 2004 Feb;14(1):110-5
references.pmid: 15102457
references.citation: Connect Tissue Res. 2002;43(2-3):441-9
references.pmid: 12489196
references.citation: J Biol Chem. 2001 Aug 24;276(34):31871-5
references.pmid: 11406633
references.citation: Gene. 1996 Dec 12;183(1-2):123-8
references.pmid: 8996096
references.citation: Connect Tissue Res. 2003;44 Suppl 1:72-8
references.pmid: 12952177
references.citation: Calcif Tissue Int. 1987 Aug;41(2):86-94
references.pmid: 3115550
references.citation: Anal Biochem. 2006 Sep 15;356(2):291-3
references.pmid: 16890905
references.citation: J Dent Res. 2002 Jan;81(1):69-73
references.pmid: 11820371
references.citation: J Dent Res. 2009 Sep;88(9):823-8
references.pmid: 19767579
references.citation: Arterioscler Thromb Vasc Biol. 2005 Dec;25(12):2679-84
references.pmid: 16210568
references.citation: Anat Rec. 1989 Jun;224(2):292-316
references.pmid: 2774208
references.citation: Ital J Anat Embryol. 1995 Oct-Dec;100(4):203-9
references.pmid: 8826798
references.citation: J Dent Res. 1979 Mar;58(Spec Issue B):950-75
references.pmid: 283137
references.citation: Calcif Tissue Int. 1980;31(3):247-51
references.pmid: 6781730
references.citation: Connect Tissue Res. 2003;44 Suppl 1:300-4
references.pmid: 12952213
references.citation: Connect Tissue Res. 1995;32(1-4):119-24
references.pmid: 7554907
references.citation: Cell Tissue Res. 1994 Jul;277(1):151-8
references.pmid: 8055533
references.citation: Curr Opin Biotechnol. 1996 Feb 1;7(1):72-7
references.pmid: 8791316
references.citation: Biopolymers. 2001 Jun;58(7):606-16
references.pmid: 11285557
references.citation: J Dent Res. 1999 Mar;78(3):743-50
references.pmid: 10096449
references.citation: Calcif Tissue Int. 1984 Sep;36(5):550-5
references.pmid: 6441627
references.citation: J Biomed Mater Res. 1999 Aug;46(2):245-52
references.pmid: 10380003
references.citation: Crit Rev Oral Biol Med. 1999;10(4):425-41
references.pmid: 10634581
references.citation: J Dent Res. 1984 Feb;63(2):98-105
references.pmid: 6582100
references.citation: J Dent Res. 2012 Oct;91(10):967-72
references.pmid: 22933608
references.citation: Eur J Oral Sci. 2002 Oct;110(5):358-65
references.pmid: 12664466
references.citation: J Histochem Cytochem. 1998 Aug;46(8):911-34
references.pmid: 9671442
references.citation: Cells Tissues Organs. 2009;189(1-4):203-6
references.pmid: 18701806
references.citation: Biosci Rep. 1981 Oct;1(10):771-8
references.pmid: 7306685
references.citation: Angew Chem Int Ed Engl. 2011 Aug 8;50(33):7541-5
references.pmid: 21710666
references.citation: Biopolymers. 2009 Feb;91(2):103-7
references.pmid: 19025992
references.citation: J Phys Chem B. 2009 Feb 19;113(7):1833-42
references.pmid: 19199690
references.citation: Arch Oral Biol. 2002 Apr;47(4):261-5
references.pmid: 11922869
references.citation: Eur J Oral Sci. 1999 Oct;107(5):352-9
references.pmid: 10515200
references.citation: Proc Natl Acad Sci U S A. 2010 Jan 5;107(1):11-5
references.pmid: 20018743
references.citation: Arch Oral Biol. 1974 May;19(5):381-6
references.pmid: 4527995
references.citation: Eur J Oral Sci. 1998 Jan;106 Suppl 1:282-91
references.pmid: 9541238