Structures of the major carbohydrates of natural human interleukin‐2

Abstract

Purified human interleukin-2 secreted by peripheral blood lymphocytes from healthy donors was found to exist in several forms. These forms were (partially) resolved by reversed-phase high-performance liquid chromatography and sodium dodecy1 sulfate/polyacrylamide gel electrophoresis. Two major plypeptide species (interleukin-2 N₁ and N₂, 16.5 kDa) were shown to be glycosylated on the basis of [³H]galactose/[³H]glucosamine incorporation and determination of amino sugars after acid hydrolysis. A third component (interleukin-2 M, 14.5 kDa) represents a nonglycosylated form. The amino acid composition and the NH₂-terminal sequence of both forms are consistent with the data deduced from the cDNA coding for interleukin-2 after removal of a leader peptide of 20 amino acids. Carbohydrates are O-linked to the IL-2 protein via threonine-3 of the polypeptide chain. The ologosaccharides were released by reuctive β-elimination and were purified by gel filtration and high performance liquid chomatography. Applying methylation analysis, exoglycosidase digestion and fast atom bombardment mass spectrometry the following major carbohydrate structures were identified: N₁, NeuAc(α 2-3) Gal(β 1-3) GalNAc-ol; and N₂, NeuAc(α 2-3) Gal(β 1-3) [NeuAc(α 2-6)] GalNAc-ol.