A Single Synonymous Variant (c.354G>A [p.P118P]) in ADAMTS13 Confers Enhanced Specific Activity
by
Ryan Hunt 1,†, Gaya Hettiarachchi 1,†, Upendra Katneni 1,†
, Nancy Hernandez 1, David Holcomb 1, Jacob Kames 1, Redab Alnifaidy 1, Brian Lin 1, Nobuko Hamasaki-Katagiri 1, Aaron Wesley 2,3, Tal Kafri 2, Christina Morris 4, Laura Bouché 5,6, Maria Panico 4,5, Tal Schiller 1, Juan Ibla 7, Haim Bar 8, Amra Ismail 9, Howard Morris 4,5, Anton Komar 9 and Chava Kimchi-Sarfaty 1,*
Ryan Hunt 1,†, Gaya Hettiarachchi 1,†, Upendra Katneni 1,†, Nancy Hernandez 1, David Holcomb 1, Jacob Kames 1, Redab Alnifaidy 1, Brian Lin 1, Nobuko Hamasaki-Katagiri 1, Aaron Wesley 2,3, Tal Kafri 2, Christina Morris 4, Laura Bouché 5,6, Maria Panico 4,5, Tal Schiller 1, Juan Ibla 7, Haim Bar 8, Amra Ismail 9, Howard Morris 4,5, Anton Komar 9 and Chava Kimchi-Sarfaty 1,*
1
Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA
2
Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
3
Present Address: Department of Emergency Medicine, Banner University Medical Center, The University of Arizona, Tucson, AZ 85724, USA
4
BioPharmaSpec Ltd., St. Saviour JE2 7LA, UK
5
Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
6
Present Address: Antikor Biopharma Ltd., Stevenage Bioscience Catalyst, Gunnels Wood Road, Stevenage SG1 2FX, UK
7
Departments of Cardiac Surgery and Anesthesiology, Perioperative and Pain Medicine, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA
8
Department of Statistics, University of Connecticut, Storrs, CT 06269, USA
9
Center for Gene Regulation in Health and Disease, Department of Biological, Geological & Environmental Sciences, Cleveland State University, Cleveland, OH 44115, USA
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2019, 20(22), 5734; https://doi.org/10.3390/ijms20225734
Received: 8 October 2019 / Revised: 9 November 2019 / Accepted: 13 November 2019 / Published: 15 November 2019
(This article belongs to the Collection Feature Papers in Molecular Biology)
Synonymous variants within coding regions may influence protein expression and function. We have previously reported increased protein expression levels ex vivo (~120% in comparison to wild-type) from a synonymous polymorphism variant, c.354G>A [p.P118P], of the ADAMTS13 gene, encoding a plasma protease responsible for von Willebrand Factor (VWF) degradation. In the current study, we investigated the potential mechanism(s) behind the increased protein expression levels from this variant and its effect on ADAMTS13 physico-chemical properties. Cell-free assays showed enhanced translation of the c.354G>A variant and the analysis of codon usage characteristics suggested that introduction of the frequently used codon/codon pair(s) may have been potentially responsible for this effect. Limited proteolysis, however, showed no substantial influence of altered translation on protein conformation. Analysis of post-translational modifications also showed no notable differences but identified three previously unreported glycosylation markers. Despite these similarities, p.P118P variant unexpectedly showed higher specific activity. Structural analysis using modeled interactions indicated that subtle conformational changes arising from altered translation kinetics could affect interactions between an exosite of ADAMTS13 and VWF resulting in altered specific activity. This report highlights how a single synonymous nucleotide variation can impact cellular expression and specific activity in the absence of measurable impact on protein structure.
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Keywords:
ADAMTS13; synonymous variant; translation; codon usage; specific activity; post-translational modifications; ribosome profiling
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MDPI and ACS Style
Hunt, R.; Hettiarachchi, G.; Katneni, U.; Hernandez, N.; Holcomb, D.; Kames, J.; Alnifaidy, R.; Lin, B.; Hamasaki-Katagiri, N.; Wesley, A.; Kafri, T.; Morris, C.; Bouché, L.; Panico, M.; Schiller, T.; Ibla, J.; Bar, H.; Ismail, A.; Morris, H.; Komar, A.; Kimchi-Sarfaty, C. A Single Synonymous Variant (c.354G>A [p.P118P]) in ADAMTS13 Confers Enhanced Specific Activity. Int. J. Mol. Sci. 2019, 20, 5734.
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