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Author Notes:

Correspondence and requests for materials should be addressed to X.M. (email: xi.mo@shsmu.edu.cn) or R.L. (email: renhao.li@emory.edu).

Y.T. and X.Z. contributed equally to this work.

Y.T. and X.Z. designed the study, performed the experiments, analyzed the data and wrote the paper; X.L. performed the experiments and analyzed the data; J.Z. provided key reagents; R.L. and X.M. initiated and designed the study, analyzed the data and wrote the paper.

We thank Chengliang Wang and the staff at beamline BL17U1 of the Shanghai Synchrotron Radiation Facility (SSRF) for X-ray data collection, and Emory Children’s Pediatric Research Center Flow Cytometry Core for technical support.

The authors declare no competing financial interests.

Subjects:

Research Funding:

This work was supported by Outstanding Youth of Shanghai Municipal Commission of Health and Family Planning (XYQ2013069, to XM), China Natural Science Fundation (81100346, to XM) and NIH grants (HL082808 and HL123984, to RL).

Keywords:

  • Science & Technology
  • Multidisciplinary Sciences
  • Science & Technology - Other Topics
  • INDUCED PLATELET-AGGREGATION
  • VONWILLEBRAND-FACTOR BINDING
  • IN-VITRO
  • IX-V
  • HEMOSTATIC FUNCTION
  • NORMAL PLASMA
  • STORAGE
  • GLYCOCALICIN
  • COMPLEX
  • ACTIVATION
  • Blood proteins
  • X-ray crystallography
  • Hematology

Structural basis for the specific inhibition of glycoprotein Ib alpha shedding by an inhibitory antibody

Tools:

Journal Title:

Scientific Reports

Volume:

Volume 6

Publisher:

, Pages 24789-24789

Type of Work:

Article | Final Publisher PDF

Abstract:

Ectodomain shedding of glycoprotein (GP) Ibα is thought to mediate the clearance of activated, aged or damaged platelets. A monoclonal antibody, 5G6, has been developed recently to specifically bind to the GPIbα shedding cleavage site and to inhibit its shedding. However, the molecular mechanism underlying antigen recognition and inhibitory specificity is not clear. To elucidate the structural basis for 5G6 binding to GPIbα, we determined the crystal structure of 5G6 Fab fragment in complex with its epitope peptide KL10 (GPIbα residues 461-470, KLRGVLQGHL), to 2.4-Å resolution. Key residues in both 5G6 and KL10 were mutated to validate their effects in antibody binding by using isothermal titration calorimetry. The 5G6 Fab-KL10 peptide complex structure confirmed the direct association of 5G6 with its target GPIbα residues and elucidated the molecular basis underlying its binding specificity and high affinity. The similar binding properties of 5G6 Fab fragment to GPIbα on human platelets as those to KL10 suggests that such an interaction may not be affected by the plasma membrane or nearby GPIbβ. This structural information may facilitate further antibody optimization and humanization.

Copyright information:

© 2016, Macmillan Publishers Limited.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

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