ROLE OF THE C-TERMINUS IN THE ACTIVITY, CONFORMATION, AND STABILITY OF INTERLEUKIN-6

被引:25
作者
WARD, LD
HAMMACHER, A
ZHANG, JG
WEINSTOCK, J
YASUKAWA, K
MORTON, CJ
NORTON, RS
SIMPSON, RJ
机构
[1] LUDWIG INST CANC RES,JOINT PROT STRUCT LAB,PO ROYAL MELBOURNE HOSP,PARKVILLE,VIC 3050,AUSTRALIA
[2] TOSOH CORP,BIOTECHNOL RES LAB,AYASE,KANAGAWA 252,JAPAN
[3] BIOMOLEC RES INST,NMR LAB,PARKVILLE,VIC 3052,AUSTRALIA
[4] ROYAL MELBOURNE HOSP,WALTER & ELIZA HALL INST MED RES,PARKVILLE,VIC 3050,AUSTRALIA
关键词
BIOSENSOR; CD; INTERLEUKIN-6; MUTANTS; NMR; RECEPTOR BINDING ACTIVITY;
D O I
10.1002/pro.5560020911
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
Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was <0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.
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页码:1472 / 1481
页数:10
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