BINDING INTERACTIONS OF HUMAN INTERLEUKIN-5 WITH ITS RECEPTOR-ALPHA SUBUNIT - LARGE-SCALE PRODUCTION, STRUCTURAL, AND FUNCTIONAL, STUDIES OF DROSOPHILA-EXPRESSED RECOMBINANT PROTEINS

被引:85
作者
JOHANSON, K
APPELBAUM, E
DOYLE, M
HENSLEY, P
ZHAO, B
ABDELMEGUID, SS
YOUNG, P
COOK, R
CARR, S
MATICO, R
CUSIMANO, D
DUL, E
ANGELICHIO, M
BROOKS, I
WINBORNE, E
MCDONNELL, P
MORTON, T
BENNETT, D
SOKOLOSKI, T
MCNULTY, D
ROSENBERG, M
CHAIKEN, I
机构
[1] SMITHKLINE BEECHAM PHARMACEUT, DEPT MOLEC IMMUNOL, KING OF PRUSSIA, PA 19406 USA
[2] SMITHKLINE BEECHAM PHARMACEUT, DEPT PROT BIOCHEM, KING OF PRUSSIA, PA 19406 USA
[3] SMITHKLINE BEECHAM PHARMACEUT, DEPT GENE EXPRESS SCI, KING OF PRUSSIA, PA 19406 USA
[4] SMITHKLINE BEECHAM PHARMACEUT, DEPT PHYS & STRUCT CHEM, KING OF PRUSSIA, PA 19406 USA
[5] SMITHKLINE BEECHAM PHARMACEUT, DEPT PHARMACOL TECHNOL, KING OF PRUSSIA, PA 19406 USA
[6] SMITHKLINE BEECHAM PHARMACEUT, DIV BIOPHARMACEUT, KING OF PRUSSIA, PA 19406 USA
[7] SMITHKLINE BEECHAM PHARMACEUT, DEPT MACROMOLEC SCI, KING OF PRUSSIA, PA 19406 USA
关键词
D O I
10.1074/jbc.270.16.9459
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Human interleukin 5 (hIL5) and soluble forms of its receptor alpha 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 hTL5 remained active, while similarly deglycosylated receptor alpha subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-Angstrom 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 alpha subunit (shIL5R alpha). Additionally, the relative abundance of ligand and receptor in the hIL5 . shIL5R alpha 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 nM, respectively, for the binding of hIL5 to shIL5R alpha and to a chimeric form of the receptor containing shIL5R alpha fused to the immunoglobulin Fc domain (shIL5R alpha-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 K-d values from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor alpha subunit, Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5R alpha in Drosophila membranes was found to be 6 nM, in accord with the affinities measured for the soluble receptor forms, Hence, most of the binding energy of the alpha 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 hIL5-receptor alpha beta complex and signal transduction.
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收藏
页码:9459 / 9471
页数:13
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