The three carboxy‐terminal amino acids of human interleukin‐6 are essential for its biological activity

Krüttgen, Alex; Rose-John, Stefan; Dufhues, G.; Bender, S.; Lütticken, Claudia; Freyer, P.; Heinrich, Peter C.

doi:10.1016/0014-5793(90)81059-w

Cited by 73 publications

(26 citation statements)

References 14 publications

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“…These results suggest that interactions involving the C-terminal amino acids stabilize the tertiary structure of mIL-6 and that the Ala-183, Leu-184, and Met-187 residues in pMC5H are more efficient in this respect than their counterparts in mIL-6 (Ser-183, Thr-184, Thr-187). Both Met-187 and Leu-I84 have been suggested to be of particular importance for receptor binding in hIL-6 (Kriittgen et al, 1990b;Yasueda et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

“…The C-terminal amino acids of hIL-6 have been shown to be essential for bioactivity. The removal of three C-terminal residues causes a drastic reduction of bioactivity (Kriittgen et al, 1990b). Without structural information, it is difficult to ascertain from these studies whether the observed changes in biological activity resulted from direct effects at the IL-6/IL-6R binding interface or, alternatively, from a gross perturbation of the conformation of the molecule.…”

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confidence: 99%

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Role of the C‐terminus in the activity, conformation, and stability of interleukin‐6

et al. 1993

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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 pMCS 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 pMCSH, 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 pMCSH 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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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Role of the C‐terminus in the activity, conformation, and stability of interleukin‐6

et al. 1993

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“…IL-6 has been expressed in rabbit reticulocyte lysate (Geiger et al, 1988), Escherichia coli (Brakenhoff et al, 1987;Tonouchi et al, 1988;Kriittgen et al, 1990a;Yasueda et al, 1990;Arcone et al, 1991a;Grennet et al, 1991;Zhang et al, 1992;Hammacher et al, 1994), Saccharomyces cerevisiae (Guisez et al, 1991), Pichia pastoris (this laboratory, unpublished data), Aspergillus nidulans , Xenopus laevis (Fontaine et al, 1991), baculovirus insect cells and Chinese hamster ovary (CHO) cells (Eisenthal et al, 1993;Orita et al, 1994).…”

Section: Post-translational Modifications Of Recombinant Il-6mentioning

confidence: 99%

“…Circular dichroism spectroscopy was used to show that recombinant IL-6 has a high a-helix content. The secondary structural compositions of human and mouse IL-6 expressed in E. coli are predicted to be 67% a-helix, 15% /?-sheet, 18% /?-turn and random coil (Kriittgen et al, 1990a), and 52% a-helix, 10% p-sheet, 19% p-turn and 19% random coil (Zhang et al, 1992), respectively. Estimations of the thermostability of IL-6 at physiological pH are hampered by the non-two-state equilibrium unfolding under these conditions.…”

Section: Physicochemical Characterization Of Il-6mentioning

confidence: 99%

Interleukin‐6: Structure‐function relationships

et al. 1997

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Interleukin-6 (IL-6) is a multifunctional cytokine that plays a central role in host defense due to its wide range of immune and hematopoietic activities and its potent ability to induce the acute phase response. Overexpression of IL-6 has been implicated in the pathology of a number of diseases including multiple myeloma, rheumatoid arthritis, Castleman's disease, psoriasis, and post-menopausal osteoporosis. Hence, selective antagonists of IL-6 action may offer therapeutic benefits. IL-6 is a member of the family of cytokines that includes interleukin-1 1, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, and ciliary neurotrophic factor. Like the other members of this family, IL-6 induces growth or differentiation via a receptor-system that involves a specific receptor and the use of a shared signaling subunit, gp130. Identification of the regions of IL-6 that are involved in the interactions with the IL-6 receptor and gp130 is an important first step in the rational manipulation of the effects of this cytokine for therapeutic benefit. In this review, we focus on the sites on IL-6 which interact with its low-affhity specific receptor, the IL-6 receptor, and the high-affinity converter gp130. A tentative model for the IL-6 hexameric receptor ligand complex is presented and discussed with respect to the mechanism of action of the other members of the IL-6 family of cytokines.

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“…Epitope mapping studies of hIL-6 have shown that neutralizing monoclonal antibodies against hIL-6 were directed toward the N-and C-terminal regions of IL-6, suggesting that these regions are in close proximity (Brakenhoff et al, 1990) and that residues 153-162 are important for biological activity on human cells (Ida et al, 1989). More direct evidence implicating the C-terminal region in receptor binding was obtained by site-directed mutagenesis studies (Nishimura et al, 1991Yasueda et al, 1992;Li et al, 1993;Savino et al, 1993), from deletion mutants (Kriittgen et al, 1990;Fontaine et al, 1993), and from murinelhuman IL-6 chimeras (Fiorillo et al, 1992a;Van Dam et al, 1993). By contrast, the N-terminal 28 amino acid residues of hIL-6 can be deleted without loss of biological activity (Brakenhoff et al, 1989).…”

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confidence: 99%

Structure‐Function analysis of human IL‐6: Identification of two distinct regions that are important for receptor binding

et al. 1994

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Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 a-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability , Protein Sci 2:1472-1481). To further probe the structurefunction relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of a-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.

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The three carboxy‐terminal amino acids of human interleukin‐6 are essential for its biological activity

Cited by 73 publications

References 14 publications

Role of the C‐terminus in the activity, conformation, and stability of interleukin‐6

Role of the C‐terminus in the activity, conformation, and stability of interleukin‐6

Interleukin‐6: Structure‐function relationships

Structure‐Function analysis of human IL‐6: Identification of two distinct regions that are important for receptor binding

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