Altered O-glycan synthesis in lymphocytes from patients with Wiskott-Aldrich syndrome

Abstract

The only molecular defect reported for the X-linked immunodeficiency Wiskott-Aldrich syndrome (WAS) is the abnormal electrophoretic behavior of the major T lymphocyte sialoglycoprotein CD43. Since the 70 to 80 O-linked carbohydrate chains of CD43 are known to influence markedly its electrophoretic mobility, we analyzed the structure and the biosynthesis of O-glycans of CD43 in lymphocytes from patients with WAS. Immunofluorescence analysis with the carbohydrate dependent anti-CD43 antibody T305 revealed that in 10 out of the 12 WAS patients tested increased numbers of T lymphocytes carry on CD43 an epitope which on normal lymphocytes is expressed only after activation. Other activation antigens were absent from WAS lymphocytes. Western blots of WAS cell lysates displayed a high molecular mass form of CD43 which reacted with the T305 antibody and which could be found on in vivo activated lymphocytes but was absent from normal unstimulated lymphocytes. To examine the O-glycan structures, carbohydrate labeled CD43 was immunoprecipitated and the released oligosaccharides identified. WAS lymphocyte CD43 was found to carry predominantly the branched structure NeuNAcα2→3Galβ1→3 (NeuNAcα2→3Galβ1→4GlcNAcβ1→6) GalNAcOH whereas normal lymphocytes carry the structure NeuNAcα2→3Galβ1→3 (NeuNAcα2→6) GalNAcOH. Only after activation NeuNAcα2→3Galβ1→3 (NeuNAcα2→3Galβ1→4GlcNAcβ1→6) GalNAcOH becomes the principal oligosaccharide on CD43 from normal lymphocytes. Analyzing the six glycosyltransferases involved in the biosynthesis of these O-glycan structures it was found that in WAS lymphocytes high levels of β1→6 N-acetyl-glucosaminyl transferase are responsible for the expression of NeuNAcα2→3Galβ1→3 (NeuNAcα2→3Gal/β1→4GlcNAcβ1→6) GalNAcOH on CD43. The gene responsible for WAS has not yet been identified but the results presented in this study suggest that the primary defect in WAS may affect a gene which is involved in the regulation of O-glycosylation.