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Human interleukin 5 (hIL5) and soluble forms of its receptor
Volume 270,
Number 16,
Issue of April 21, pp. 9459-9471, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit
LARGE SCALE PRODUCTION, STRUCTURAL, AND FUNCTIONAL STUDIES OF
DROSOPHILA-EXPRESSED RECOMBINANT PROTEINS
subunit were expressed in Drosophila cells and purified
to homogeneity, allowing a detailed structural and functional analysis.
B cell proliferation confirmed that the hIL5 was biologically active.
Deglycosylated hIL5 remained active, while similarly deglycosylated
receptor subunit lost activity. The crystal structure of the
deglycosylated hIL5 was determined to 2.6-Å resolution and found
to be similar to that of the protein produced in Escherichia
coli. Human IL5 was shown by analytical ultracentrifugation to
form a 1:1 complex with the soluble domain of the hIL5 receptor
subunit (shIL5R). Additionally, the relative abundance of ligand
and receptor in the hIL5shIL5R
complex was determined to be
1:1 by both titration calorimetry and SDS-polyacrylamide gel
electrophoresis analysis of dissolved cocrystals of the complex.
Titration microcalorimetry yielded equilibrium dissociation constants
of 3.1 and 2.0 n
M, respectively, for the binding of hIL5 to
shIL5R and to a chimeric form of the receptor containing
shIL5R fused to the immunoglobulin Fc domain (shIL5R-Fc).
Analysis of the binding thermodynamics of IL5 and its soluble receptor
indicates that conformational changes are coupled to the binding
reaction. Kinetic analysis using surface plasmon resonance yielded data
consistent with the Kvalues from
calorimetry and also with the possibility of conformational
isomerization in the interaction of hIL5 with the receptor
subunit. Using a radioligand binding assay, the affinity of hIL5 with
full-length hIL5R in Drosophila membranes was found to be
6 n
M, in accord with the affinities measured for the soluble
receptor forms. Hence, most of the binding energy of the receptor
is supplied by the soluble domain. Taken with other aspects of hIL5
structure and biological activity, the data obtained allow a prediction
for how 1:1 stoichiometry and conformational change can lead to the
formation of hIL5receptor
complex and signal
transduction.
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