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Title: Identification of major matrix metalloproteinase-20 proteolytic processing products of murine amelogenin and tyrosine-rich amelogenin peptide using a nuclear magnetic resonance spectroscopy based method

Abstract

Objective: The aim of this study was to identify major matrix metalloproteinase-20 (MMP20) proteolytic processing products of amelogenin over time and determine if the tyrosine-rich amelogenin peptide (TRAP) was a substrate of MMP20. Design: Recombinant 15N-labeled murine amelogenin and 13C,15N-labeled TRAP were incubated with MMP20 under conditions where amelogenin self-assembles into nanospheres. Digestion products were fractionated by reverse-phase high-performance liquid chromatography at various time points. Product identification took advantage of the intrinsic disorder property of amelogenin that results in little change to its fingerprint 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance spectrum in 2% acetic acid upon removing parts of the protein, allowing cleavage site identification by observing which amide cross peaks disappear. Results: The primary product in five out of the six major reverse-phase high-performance liquid chromatography bands generated after a 24 hour incubation of murine amelogenin with MMP20 were: S55-L163, P2-L147, P2-E162, P2-A167, and P2-R176. After 72 hours these products were replaced with five major reverse-phase high-performance liquid chromatography bands containing: L46-A170, P2-S152, P2-F151, P2-W45, and short N-terminal peptides. TRAP was completely digested by MMP20 into multiple small peptides with the initial primary site of cleavage between S16 and Y17. Conclusions: Identification of the major MMP20 proteolytic productsmore » of amelogenin confirm a dynamic process, with sites towards the C-terminus more rapidly attacked than sites near the N-terminus. This observation is consistent with nanosphere models where the C-terminus is exposed and the N-terminus more protected. One previously reported end-product of the MMP20 proteolytic processing of amelogenin, TRAP, is shown to be an in vitro substrate for MMP20.« less

Authors:
ORCiD logo; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1503496
Report Number(s):
PNNL-SA-132678
Journal ID: ISSN 0003-9969
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Archives of Oral Biology
Additional Journal Information:
Journal Volume: 93; Journal Issue: C; Journal ID: ISSN 0003-9969
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
amelogenesis imperfecta, protease, amelogenin, enamel, biomineralization

Citation Formats

Buchko, Garry W., Jayasinha Arachchige, Rajith, Tao, Jinhui, Tarasevich, Barbara J., and Shaw, Wendy J. Identification of major matrix metalloproteinase-20 proteolytic processing products of murine amelogenin and tyrosine-rich amelogenin peptide using a nuclear magnetic resonance spectroscopy based method. United States: N. p., 2018. Web. doi:10.1016/j.archoralbio.2018.06.001.
Buchko, Garry W., Jayasinha Arachchige, Rajith, Tao, Jinhui, Tarasevich, Barbara J., & Shaw, Wendy J. Identification of major matrix metalloproteinase-20 proteolytic processing products of murine amelogenin and tyrosine-rich amelogenin peptide using a nuclear magnetic resonance spectroscopy based method. United States. doi:10.1016/j.archoralbio.2018.06.001.
Buchko, Garry W., Jayasinha Arachchige, Rajith, Tao, Jinhui, Tarasevich, Barbara J., and Shaw, Wendy J. Sat . "Identification of major matrix metalloproteinase-20 proteolytic processing products of murine amelogenin and tyrosine-rich amelogenin peptide using a nuclear magnetic resonance spectroscopy based method". United States. doi:10.1016/j.archoralbio.2018.06.001.
@article{osti_1503496,
title = {Identification of major matrix metalloproteinase-20 proteolytic processing products of murine amelogenin and tyrosine-rich amelogenin peptide using a nuclear magnetic resonance spectroscopy based method},
author = {Buchko, Garry W. and Jayasinha Arachchige, Rajith and Tao, Jinhui and Tarasevich, Barbara J. and Shaw, Wendy J.},
abstractNote = {Objective: The aim of this study was to identify major matrix metalloproteinase-20 (MMP20) proteolytic processing products of amelogenin over time and determine if the tyrosine-rich amelogenin peptide (TRAP) was a substrate of MMP20. Design: Recombinant 15N-labeled murine amelogenin and 13C,15N-labeled TRAP were incubated with MMP20 under conditions where amelogenin self-assembles into nanospheres. Digestion products were fractionated by reverse-phase high-performance liquid chromatography at various time points. Product identification took advantage of the intrinsic disorder property of amelogenin that results in little change to its fingerprint 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance spectrum in 2% acetic acid upon removing parts of the protein, allowing cleavage site identification by observing which amide cross peaks disappear. Results: The primary product in five out of the six major reverse-phase high-performance liquid chromatography bands generated after a 24 hour incubation of murine amelogenin with MMP20 were: S55-L163, P2-L147, P2-E162, P2-A167, and P2-R176. After 72 hours these products were replaced with five major reverse-phase high-performance liquid chromatography bands containing: L46-A170, P2-S152, P2-F151, P2-W45, and short N-terminal peptides. TRAP was completely digested by MMP20 into multiple small peptides with the initial primary site of cleavage between S16 and Y17. Conclusions: Identification of the major MMP20 proteolytic products of amelogenin confirm a dynamic process, with sites towards the C-terminus more rapidly attacked than sites near the N-terminus. This observation is consistent with nanosphere models where the C-terminus is exposed and the N-terminus more protected. One previously reported end-product of the MMP20 proteolytic processing of amelogenin, TRAP, is shown to be an in vitro substrate for MMP20.},
doi = {10.1016/j.archoralbio.2018.06.001},
journal = {Archives of Oral Biology},
issn = {0003-9969},
number = C,
volume = 93,
place = {United States},
year = {2018},
month = {9}
}