Internal deletions in human interleukin-6: structure-function analysis

Abstract

By cDNA mutagenesis, we have constructed internal and C-terminal deletions (Δ 21–51, Δ 52–97, Δ 97–104, Δ 127–174, Δ 97–184 and Δ 134–184) in human interleukin-6 (hIL-6). All those deletion-carrying hIL-6 (ΔhIL-6) proteins were then produced in Xenopus laevis oocytes and examined by sodium dodecyi sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that, at least in frog oocytes, the first potential N-glycosylation site (Asn 45) is utilized exclusively. The IL-6 conformation of these deletion-carrying proteins has been studied by immunoprecipitation with two kinds of monoclonal antibodies (mAb's): mAb's that show preference towards denatured hIL-6, or conformation-specific mAb's. The binding pattern of these two series of mAb's indicated that the IL-6 conformation has been largely destroyed for four of our Δ-proteins. Proteins Δ 21–51 and Δ 127–174 have kept a part of the IL-6 tertiary structure since they are still recognized by some conformation-specific mAb's. All of these ΔhIL-6 proteins were inactive in the IL-6 hybridoma growth factor (HGF) assay and unable to inhibit the HGF activity of the recombinant human wild-type IL-6 (wt hIL-6). Moreover, the oocyte-synthesized ΔhIL-6 (Δ 21–51, Δ 127–174,Δ 97–184, Δ 134–184 did notbind to the IL-6 receptor. Finally, we have produced two proteins with aa 29–33 or 97–104 substituted by corresponding murine IL-6 (mIL-6) sequences. In contrast to mIL-6 that is able to act only on murine cells, these chimeric h/mIL-6 proteins are active toward both murine and human cells. However, drastic reduction in IL-6 activity observed for the 29–33-aa-substituted h/m protein toward both of these kinds of cells suggests that the 29–33-aa region may be in part implicated in modeling the receptor-binding site of IL-6. On the other hand, the 97–104-aa region seems to be involved in the overall structure of IL-6.