On the multimeric nature of natural human interleukin-6.
Natural human interleukin-6 (IL-6) characterized under completely denaturing conditions consists of a set of differentially modified phosphoglycoproteins of molecular mass in the range from 23 to 30 kDa ("25-kDa" O-glycosylated species and "30-kDa" O- and N-glycosylated species). The 25-kDa O-glycosylated IL-6 (which contains only Ser- or Thr-GalNAc-Gal-NeuNAc and thus should not bind wheat germ or lentil lectins) bound to and was eluted from a wheat germ lectin affinity column by GlcNAc and from a lentil lectin affinity column by methyl-alpha-D-Man suggesting that the 25-kDa IL-6 species formed heteromeric complexes with the N-glycosylated 30-kDa IL-6. In non-denaturing gels (0.2% Nonidet P-40-polyacrylamide gel electrophoresis (PAGE)), even under reducing conditions (15 mM dithiothreitol or 1 M beta-mercaptoethanol and heating), fibroblast-derived IL-6 migrated as a predominant complex of mass approximately 85 kDa and additional minor 45-65-kDa complexes. Little IL-6 was detected in the size range 23-30 kDa. Elution of the major 85-kDa complex and re-electrophoresis through sodium dodecyl sulfate-PAGE revealed that it represented a heteromeric aggregate of the 25- and 30-kDa IL-6 species; the 45-65-kDa complexes were largely composed of the 25-kDa protein. The bulk of fibroblast-derived IL-6 eluted in the size range 45-85 kDa from a Sephadex G-200 gel filtration column further indicating that fibroblast-derived IL-6 was largely multimeric even in dilute solutions. Functionally, the high molecular mass IL-6 fractions from the G-200 column were less active in the B9 hybridoma growth factor assay than the lower molecular mass fractions but appeared to be equally active in the Hep3B hepatocyte-stimulating factor assay. Taken together, the data indicate that natural human IL-6 exists as a multimeric aggregate with varying biological activity.
May
L T
LT
Rockefeller University, New York, New York 10021.
Santhanam
U
U
Sehgal
P B
PB
D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry*; D037102:Lectins
D015850:Interleukin-6; D037102:Lectins