Studies on the structure and function of IL-6 and its receptor

Abstract

Interleukins area group of signalling molecules involved in the communication between cells. They are produced and secreted by many different cell types, particularly by those of the immune system. Interleukin-6 (IL-6) 1 elicits a wide spectrum of biological functions. In general, IL-6 represents a growth and differentiation factor acting on B-cells, T-cells, lymphoma cells, hybridoma/plasmacytoma cells, hematopoietic stem cells, and hepatocytes (for review see [1]). IL-6 consists of 184 amino acids. It is synthesized as a larger polypeptide precursor with a signal peptide of 28 amino acids. Studies on the structures of Nand O-linked carbohydrate chains and their location within the polypeptide backbone of human IL-6 synthesized and ;ecreted by NIH/3T3 cells stably transfected with a human IL-6-cDNA have shown that only one (Asn 45) of the two potential N-glycosylation sites carries an oligosaccharide side chain. Thr 138 was identified as Oglycosylation site. At the cDNA level we constructed deletion mutants of human IL-6, lacking one, two, three or four amino acids from the C-terminus. In the mouse B9 cell proliferation assay as well as in the 7-fibrinogen induction assay with the human hepatoma cell line HepG2 the removal of the last amino acid (Met 184) resulted in the loss of 80% of biological activity. When three amino acids were removed from the C-terminus, IL-6 activity was completely abbrogated. Studies with various point mutations at the C-terminus of IL-6 indicated that an c~helical structure and a positive charge (Arg 182) are essential for biological activity [2-4]. In order to define domains of interleukin-6 responsible for species specific receptor recognition, we have constructed a whole series of chimeric human/mouse interleukin-6 proteins. This approach is based on the fact that human IL-6 is active on human and murine cells whereas murine IL-6 is only active on murine cells. The proteins have been expressed in E. coli, purified to homogeneity and tested for bioactivity on human and murine cells. It turned out that a region in the NH2-terminal part of IL-6 (loop between helix A and B or helix B) as well as the carboxyterminal helix D (nomenclature taken from the threedimensional structure prediction for IL-6 of Bazan [5]) is impor-