Structure/function analysis of interleukin 4: interspecies comparison and site-directed mutagenesis

Abstract

Interleukin 4 (IL4) has a central role in allergic disease as it is involved in control of secretion of IgE. It is a pleiotropic cytokine which stimulates proliferation of T and B lymphocytes and induces expression of the low affinity IgE receptor (CD23) on the surface of B lymphocytes. The secondary structure of IL4 was recently solved using both nmr and Xray approaches [I]: IL4 was shown to have four major a helices and one short section of double-stranded p pleated sheet. IL4 is thus similar in overall topology to several other haemopoietic cytokines (eg GM-CSF). Initial mutational analysis of IL4 has revealed a possible role for the D helix in signal transduction [2, 31, but the ILWreceptor interaction remains to be fully defined. Murine and human IL4, which show 43% amino acid identity, do not cross-react. Interestingly, the area of greatest sequence divergence (including a 'relative' deletion of 7 amino acids) occurs in the B-C loop region, making this a prime target of interest particularly in terms of species specificity. Recently, bovine 1L4 cDNA was cloned [41. This shows 65% amino acid identity with human IL4, but its biological activity had not been defined. Notably, there was considerable difference in the B-C loop region (including a 'relative' deletion of 17 amino acids). We felt that the intermediate sequence divergence could rapidly yield useful structure/function information if bovine IL4 were tested in biological systems. Bovine and human IL4 were expressed in a HeLa transient system [ 5 ] . Harvest media from this expression system were tested in both a T cell proliferation assay[6) and in a receptor binding assay[7]. In the former, mononuclear cells were separated from peripheral blood by centrifugation through FicollPaque. The cells were cultured for 3 days with 10kg/ml PHA. The PHA-activated T blast celis were then washed and cultured for a further 3 days with serial dilutions of human or bovine IL4.