PURIFICATION AND CHARACTERIZATION OF THE SOLUBLE INTERLEUKIN-6 RECEPTOR FROM HUMAN PLASMA AND IDENTIFICATION OF AN ISOFORM GENERATED THROUGH ALTERNATIVE SPLICING

The soluble human interleukin-6 receptor (shIL6R) was purified from hu man plasma. In a single immunoaffinity purification step a 140000-fold enrichment with a yield of 95 % was achieved. A subsequent IL-6 affin ity chromatography resulted in a homogeneous receptor preparation but only in a yield of less than 5%. The biological activity of the solubl e receptor was clearly demonstrated by its ability to induce the synth esis of the acute-phase protein alpha(1)-antichymotrypsin in HepG2 cel ls stably transfected with IL-6. Upon gel filtration, the native shIL6 R showed an apparent molecular mass of 93 kDa. Analysis by SDS/PAGE re vealed an apparent molecular mass of 65 kDa for the soluble receptor. Deglycosylation with peptide N-glycosidase F led to a shift in molecul ar mass from 65 kDa to 45 kDa. It has previously been shown that the s hIL6R can be generated by shedding the membrane-bound form or by expre ssion of an alternatively spliced mRNA. Here we show that the shIL6R i solated from human plasma is recognized by an affinity-purified peptid e antibody raised against an amino acid sequence unique for the altern atively spliced isoform. Thus, the shIL6R isoform generated through al ternative splicing which has been previously detected in supernatants of cultured cell lines is also an in vivo product circulating in human plasma.