Processing of ameloblastin by MMP-20

Area of Science:

• Biochemistry
• Developmental Biology
• Dental Research

Background:

• Ameloblastin (AMBN) is a key non-amelogenin protein in developing tooth enamel.
• AMBN cleavage products are abundant and their localization suggests specific processing.
• The protease responsible for AMBN cleavage remains to be definitively identified.

Purpose of the Study:

• To test the hypothesis that matrix metalloproteinase-20 (MMP-20) is the protease responsible for cleaving AMBN.
• To identify the specific cleavage sites on AMBN by MMP-20.
• To understand the generation of AMBN fragments during tooth enamel development.

Main Methods:

• Expression and purification of glycosylated recombinant porcine AMBN (rpAMBN).
• Expression and purification of recombinant porcine enamelysin (rpMMP-20).
• In vitro incubation of rpAMBN and rpMMP-20 at a 1:100 enzyme:substrate ratio.
• N-terminal sequencing of AMBN digestion products.

Main Results:

• Recombinant MMP-20 (rpMMP-20) was confirmed to cleave recombinant AMBN (rpAMBN).
• Specific cleavage sites were identified at Pro(2), Gln(130), Gln(139), Arg(170), and Ala(222) of AMBN.
• MMP-20 generates a 23-kDa AMBN fragment (starting at Tyr(223)) and other fragments (17-kDa, 15-kDa) crucial for enamel structure.

Conclusions:

• MMP-20 is the protease that processes ameloblastin.
• The in vitro findings are relevant to in vivo processes during tooth enamel formation.
• MMP-20 activity explains the localization of AMBN fragments in developing enamel.