Garry W Buchko1, Rajith Jayasinha Arachchige2, Jinhui Tao3, Barbara J Tarasevich4, Wendy J Shaw5. 1. Pacific Northwest National Laboratory, Richland, WA 99352, USA; School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA. Electronic address: garry.buchko@pnnl.gov. 2. Pacific Northwest National Laboratory, Richland, WA 99352, USA. Electronic address: rajith.jayasinhaarachchige@pnnl.gov. 3. Pacific Northwest National Laboratory, Richland, WA 99352, USA. Electronic address: jinhui.tao@pnnl.gov. 4. Pacific Northwest National Laboratory, Richland, WA 99352, USA. Electronic address: barbara.tarasevich@pnnl.gov. 5. Pacific Northwest National Laboratory, Richland, WA 99352, USA. Electronic address: wendy.shaw@pnnl.gov.
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: Recombinant15N-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 h incubation of murine amelogenin with MMP20 were: S55-L163, P2-L147, P2-E162, P2-A167, and P2-R176. After 72 h 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.
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: Recombinant15N-labeled murineamelogenin 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 h incubation of murineamelogenin with MMP20 were: S55-L163, P2-L147, P2-E162, P2-A167, and P2-R176. After 72 h 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.
Authors: Jinhui Tao; Yongsoon Shin; Rajith Jayasinha; Garry W Buchko; Sarah D Burton; Alice C Dohnalkova; Zheming Wang; Wendy J Shaw; Barbara J Tarasevich Journal: Proc Natl Acad Sci U S A Date: 2019-06-25 Impact factor: 11.205