Cleavage site specificity of MMP-20 for secretory-stage ameloblastin.
Ameloblastin is processed by protease(s) during enamel formation. We tested the hypothesis that MMP-20 (enamelysin) catalyzes the cleavages that generate secretory-stage ameloblastin cleavage products. We isolated a 23-kDa ameloblastin cleavage product from developing enamel and determined its N-terminus sequence. Ameloblastin was stably expressed and secreted from HEK293-H cells, purified, and digested with MMP-20 or Klk4 (kallikrein 4). The digests were analyzed by SDS-PAGE and Western blotting, and cleavage products were characterized by N-terminal sequencing. Six fluorescent peptides were digested with MMP-20 and Klk4 and analyzed by RP-HPLC and by mass spectrometry. MMP-20 cleaved each peptide exactly at the sites corresponding to ameloblastin cleavages catalyzed in vivo. Klk4 cleaved ameloblastin and the fluorescent peptides at sites not observed in vivo, and cleaved at only a single correct site: before Leu(171). We conclude that MMP-20 is the enzyme that processes ameloblastin during the secretory stage of amelogenesis, and we present a hypothesis about the sequence of ameloblastin cleavages.
Chun
Y-H P
YH
Department of Biologic and Materials Sciences, University of Michigan, School of Dentistry, 1011 North University, Ann Arbor, MI 48109-1078, USA.
Yamakoshi
Y
Y
Yamakoshi
F
F
Fukae
M
M
Hu
J C-C
JC
Bartlett
J D
JD
Simmer
J P
JP
D000566:Amelogenesis / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003746:Dental Enamel Proteins / Q000378:metabolism*; D006801:Humans; D007610:Kallikreins / Q000378:metabolism; D013058:Mass Spectrometry; D053519:Matrix Metalloproteinase 20 / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D034421:Sus scrofa
D003746:Dental Enamel Proteins; D010455:Peptides; D011994:Recombinant Proteins; C490891:ameloblastin protein, Sus scrofa; D007610:Kallikreins; C120801:kallikrein 4; D053519:Matrix Metalloproteinase 20
J Dent Res. 2007 Feb;86(2):153-7
17251515
Arch Oral Biol. 2001 Nov;46(11):1005-14
11543707
Connect Tissue Res. 1998;39(1-3):101-9; discussion 141-9
11062992
J Dent Res. 2009 Sep;88(9):823-8
19767579
J Dent Res. 1999 Mar;78(3):743-50
10096449
J Cell Biol. 2004 Dec 6;167(5):973-83
15583034
J Bone Miner Res. 1996 Jul;11(7):883-91
8797107
Eur J Oral Sci. 2002 Oct;110(5):358-65
12664466
Calcif Tissue Int. 1994 Jan;54(1):69-75
8118757
J Biol Chem. 2009 Jul 10;284(28):19110-21
19578120
Biochem J. 2000 Nov 1;351 Pt 3:765-8
11042132
J Biol Chem. 2001 Aug 24;276(34):31871-5
11406633
Histochem Cell Biol. 1997 Jun;107(6):485-94
9243282
Gene. 1996 Dec 12;183(1-2):123-8
8996096
J Dent Res. 2007 Oct;86(10):962-7
17890672
Exp Cell Res. 2010 Apr 15;316(7):1202-12
20043904
J Histochem Cytochem. 1997 Oct;45(10):1329-40
9313795
Eur J Oral Sci. 2006 May;114 Suppl 1:139-46; discussion 164-5, 380-1
16674676
Eur J Oral Sci. 2008 Apr;116(2):124-34
18353005
J Dent Res. 1997 Feb;76(2):648-57
9062558
Crit Rev Oral Biol Med. 1998;9(2):128-61
9603233
J Dent Res. 2003 Dec;82(12):982-6
14630899
J Dent Res. 1998 Feb;77(2):377-86
9465170
Eur J Oral Sci. 2006 May;114 Suppl 1:266-71; discussion 285-6, 382
16674696
Histochemistry. 1991;96(2):129-38
1917569
J Biol Chem. 2002 Dec 20;277(51):49598-604
12393861
J Biol Chem. 2008 Apr 18;283(16):10858-71
18252720
Gene. 2000 Jun 13;251(1):1-8
10863090
J Biol Chem. 2009 Oct 2;284(40):27176-84
19648121
Adv Dent Res. 1987 Dec;1(2):261-6
3504175
Eur J Oral Sci. 2006 May;114 Suppl 1:45-51; discussion 93-5, 379-80
16674662
Eur J Oral Sci. 2006 May;114 Suppl 1:244-53; discussion 254-6, 381-2
16674693
Eur J Oral Sci. 2006 May;114 Suppl 1:212-8; discussion 254-6, 381-2
16674688