title: Identification of an ADAM17 cleavage region in human CD16 (FcγRIII) and the engineering of a non-cleavable version of the receptor in NK cells.
abstract: CD16a and CD16b are IgG Fc receptors expressed by human natural killer (NK) cells and neutrophils, respectively. Both CD16 isoforms undergo a rapid down-regulation in expression by ADAM17-mediated proteolytic cleavage upon cell activation by various stimuli. We examined soluble CD16 released from activated NK cells and neutrophils by mass spectrometric analysis, and identified three separate cleavage sites in close proximity at P1/P1' positions alanine195/valine196, valine196/serine197, and threonine198/isoleucine199, revealing a membrane proximal cleavage region in CD16. Substitution of the serine at position 197 in the middle of the cleavage region for a proline (S197P) effectively blocked CD16a and CD16b cleavage in cell-based assays. We also show that CD16a/S197P was resistant to cleavage when expressed in the human NK cell line NK92 and primary NK cells derived from genetically-engineered human induced pluripotent stem cells. CD16a is a potent activating receptor and despite blocking CD16a shedding, the S197P mutation did not disrupt IgG binding by the receptor or its activation of NK92 cells by antibody-treated tumor cells. Our findings provide further characterization of CD16 cleavage by ADAM17 and they demonstrate that a non-cleavable version of CD16a can be expressed in engineered NK cells.
authors: Department of Veterinary and Biomedical Sciences, University of Minnesota, Minneapolis and St. Paul, Minnesota, United States of America.
authors: Yawu
authors: Y
authors: Jing
authors: Department of Medicine, Stem Cell Institute, University of Minnesota, Minneapolis and St. Paul, Minnesota, United States of America.
authors: Zhenya
authors: Z
authors: Ni
authors: Department of Veterinary and Biomedical Sciences, University of Minnesota, Minneapolis and St. Paul, Minnesota, United States of America.
authors: Jianming
authors: J
authors: Wu
authors: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis and St. Paul, Minnesota, United States of America.
authors: LeeAnn
authors: L
authors: Higgins
authors: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis and St. Paul, Minnesota, United States of America.
authors: Todd W
authors: TW
authors: Markowski
authors: Department of Medicine, Stem Cell Institute, University of Minnesota, Minneapolis and St. Paul, Minnesota, United States of America.
authors: Dan S
authors: DS
authors: Kaufman
authors: Department of Veterinary and Biomedical Sciences, University of Minnesota, Minneapolis and St. Paul, Minnesota, United States of America.
authors: Bruce
authors: B
authors: Walcheck
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000072198:ADAM17 Protein; D019943:Amino Acid Substitution; D002460:Cell Line; D058851:GPI-Linked Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007694:Killer Cells, Natural / Q000166:cytology / Q000378:metabolism*; D013058:Mass Spectrometry; D059748:Proteolysis; D017452:Receptors, IgG / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*
chemical_list: C496124:FCGR3A protein, human; C428200:FCGR3B protein, human; D058851:GPI-Linked Proteins; D007074:Immunoglobulin G; D017452:Receptors, IgG; D012694:Serine; D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human