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

Buchko, Garry W.; Jayasinha Arachchige, Rajith; Tao, Jinhui; Tarasevich, Barbara J.; Shaw, Wendy J.

doi:10.1016/j.archoralbio.2018.06.001

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

Journal Article · Sat Sep 01 00:00:00 EDT 2018 · Archives of Oral Biology

DOI:https://doi.org/10.1016/j.archoralbio.2018.06.001· OSTI ID:1503496

; Jayasinha Arachchige, Rajith; Tao, Jinhui; Tarasevich, Barbara J.; Shaw, Wendy J.

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.

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE

DOE Contract Number:: AC05-76RL01830

OSTI ID:: 1503496

Report Number(s):: PNNL-SA-132678

Journal Information:: Archives of Oral Biology, Journal Name: Archives of Oral Biology Journal Issue: C Vol. 93; ISSN 0003-9969

Publisher:: Elsevier

Country of Publication:: United States

Language:: English

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