Generation of a high-affinity Fcgamma receptor by Ig-domain swapping between human CD64A and CD16A.
A recombinant soluble version of the human high-affinity receptor for IgG, rh-FcgammaRIA or CD64A, was expressed in mammalian cells and purified from their conditioned media. As assessed by circular dichroism, size exclusion chromatography and dynamic light scattering, incubation of rh-FcgammaRIA at 37 degrees C resulted in time-dependent formation of soluble aggregates caused by protein unfolding and loss of native structure. Aggregate formation was irreversible, temperature-dependent and was independent of rh-FcgammaRIA concentration. Aggregated rh-FcgammaRIA lost its ability to inhibit immune complex precipitation and failed to bind to IgG-Sepharose. Addition of human IgG1 to rh-FcgammaRIA prior to incubation at 37 degrees C blocked the formation of rh-FcgammaRIA aggregates. Production of soluble monomeric rh-FcgammaRIA was limited by aggregate formation during cell culture. Substitution of the membrane distal D1 Ig domain of FcgammaRIA with the D1 Ig domain of FcgammaRIIIA or CD16A resulted in a chimeric receptor, FcgammaR3A1A, with enhanced temperature stability. Relative to native rh-FcgammaRIA, FcgammaR3A1A exhibited less aggregation in Chinese hamster ovary cell-conditioned media or when purified receptor was incubated for up to 24 h at 37 degrees C. Both receptors bound to immobilized human IgG1 with high affinity and were equipotent at blockade of immune complex-mediated cytokine production from cultured mast cells. Equivalent dose-dependent reductions in edema and neutrophil infiltration in the cutaneous Arthus reaction in mice were noted for rh-FcgammaRIA and FcgammaR3A1A. These data demonstrate that the D1 Ig domains of FcgammaRIA and FcgammaRIIIA are functionally interchangeable and further suggest that the chimeric receptor FcgammaR3A1A is an effective inhibitor of type III hypersensitivity in mice.
Department of Immunology, ZymoGenetics, Inc., Seattle, WA 98102, USA. jeff.ellsworth@hotmail.com
Jeff L
JL
Ellsworth
Nels
N
Hamacher
Brandon
B
Harder
Mark
M
Maurer
Thomas R
TR
Bukowski
Megan
M
Lantry
Claire
C
Noriega
Mark W
MW
Rixon
Brian
B
Fox
Ken
K
Lewis
Brent
B
Meengs
Emily
E
Rollins
Karlien
K
Greeff
Jeff
J
Meyer
Carl
C
Birks
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D017434:Protein Structure, Tertiary; D017452:Receptors, IgG / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism
C489017:FCGR1A protein, human; C496124:FCGR3A protein, human; D017452:Receptors, IgG