How do enamelysin and kallikrein 4 process the 32-kDa enamelin?

The activities of two proteases – enamelysin (MMP‐20) and kallikrein 4 (KLK4) – are necessary for dental enamel to achieve its high degree of mineralization. We hypothesize that the selected enamel protein cleavage products which accumulate in the secretory‐stage enamel matrix do so because they are...

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Bibliographic Details
Published in:European journal of oral sciences 2006-05, Vol.114 (s1), p.45-51
Main Authors: Yamakoshi, Yasuo, Hu, Jan C.-C., Fukae, Makoto, Yamakoshi, Fumiko, Simmer, James P.
Format: Article
Language:English
Subjects:
amelogenesis
Amelogenesis - physiology
Amelogenin
Animals
Blotting, Western
Chromatography, High Pressure Liquid - methods
Dental Enamel - enzymology
Dental Enamel - secretion
Dental Enamel Proteins - analysis
Dental Enamel Proteins - metabolism
Dentistry
Electrophoresis, Polyacrylamide Gel
enamel
enamelin
Kallikreins - metabolism
KLK4
Mass Spectrometry
Matrix Metalloproteinase 20
Matrix Metalloproteinases - metabolism
MMP-20
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - metabolism
Protein Isoforms - analysis
Sequence Analysis, Protein
Swine
Time Factors
Tooth Calcification - physiology
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Summary:The activities of two proteases – enamelysin (MMP‐20) and kallikrein 4 (KLK4) – are necessary for dental enamel to achieve its high degree of mineralization. We hypothesize that the selected enamel protein cleavage products which accumulate in the secretory‐stage enamel matrix do so because they are resistant to further cleavage by MMP‐20. Later, they are degraded by KLK4. The 32‐kDa enamelin is the only domain of the parent protein that accumulates in the deeper enamel. Our objective was to identify the cleavage sites of 32‐kDa enamelin that are generated by proteolysis with MMP‐20 and KLK4. Enamelysin, KLK4, the major amelogenin isoform (P173), and the 32‐kDa enamelin were isolated from developing porcine enamel. P173 and the 32‐kDa enamelin were incubated with MMP‐20 or KLK4 for up to 48 h. Then, the 32‐kDa enamelin digestion products were fractionated by reverse‐phase high‐performance liquid chromatography (RP‐HPLC) and characterized by Edman sequencing, amino acid analysis, and mass spectrometry. Enamelysin cleaved the 32‐kDa enamelin only after it was deglycosylated. Kallikrein 4 digestion of the 32‐kDa enamelin generated nine major cleavage products, six of which were successfully characterized. After 12 h of digestion with KLK4, all of the 32‐kDa enamelin had been cleaved, but some cleavage products persisted after 48 h of digestion.
ISSN:0909-8836
1600-0722
DOI:10.1111/j.1600-0722.2006.00281.x