Processing of ameloblastin by MMP-20.
Ameloblastin (AMBN) cleavage products are the most abundant non-amelogenin proteins in the enamel matrix of developing teeth. AMBN N-terminal cleavage products accumulate in the sheath space between enamel rods, while AMBN C-terminal products localize within rods. We tested the hypothesis that MMP-20 is the protease that cleaves AMBN. Glycosylated recombinant porcine AMBN (rpAMBN) was expressed in human kidney 293F cells, and recombinant porcine enamelysin (rpMMP-20) was expressed in bacteria. The purified proteins were incubated together at an enzyme:substrate ratio of 1:100. N-terminal sequencing of AMBN digestion products determined that rpMMP-20 cleaved rpAMBN after Pro(2), Gln(130), Gln(139), Arg(170), and Ala(222). This shows that MMP-20 generates the 23-kDa AMBN starting at Tyr(223), as well as the 17-kDa (Val(1)-Arg(170)) and 15-kDa (Val(1)-Gln(130)) AMBN cleavage products that concentrate in the sheath space during the secretory stage. We conclude that MMP-20 processes ameloblastin in vitro and in vivo.
Iwata
T
T
Department of Biologic and Materials Sciences, Dental Research Lab, University of Michigan School of Dentistry, 1210 Eisenhower Place, Ann Arbor, MI 48108, USA.
Yamakoshi
Y
Y
Hu
J C-C
JC
Ishikawa
I
I
Bartlett
J D
JD
Krebsbach
P H
PH
Simmer
J P
JP
D000566:Amelogenesis / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003746:Dental Enamel Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007150:Immunohistochemistry; D053519:Matrix Metalloproteinase 20 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D034421:Sus scrofa
D003746:Dental Enamel Proteins; D011994:Recombinant Proteins; C490891:ameloblastin protein, Sus scrofa; D053519:Matrix Metalloproteinase 20