PRIMARY STRUCTURE OF CD52

The CD52 antigen was extracted from human spleens with organic solvent s and purified by immunoaffinity and reverse-phase chromatography, The latter step resolved two CD52 species, called CD52-I and CD52-II. Bot h species were found to contain similar N-linked oligosaccharides and glycosylphosphatidylinositol (GPI) anchor glycans, The N-linked oligos accharides were characterized by methylation linkage analysis and, fol lowing exhaustive neuraminidase and endo-beta-galactosidase digestion, by the reagent array analysis method TM. The results showed that the single CD52 N-glycosylation site is occupied by large sialylated, poly lactosamine-containing, core-fucosylated tetraantennary oligosaccharid es. The locations of the phosphoryl substituents on the GPI anchor gly can were determined using a new and sensitive method based upon partia l acid hydrolysis of the GPI glycan. The difference between CD52-I and CD52-II was in the phosphatidylinositol (PI) moieties of the GPI anch ors. The phosphatidylinositol-specific phospholipase C-sensitive CD52- I contained exclusively distearoyl-PI, while the PI-phospholipase C-re sistant CD52-II contained predominantly a palmitoylated stearoyl-arach idonoyl-PI, as judged by electrospray ionization mass spectrometry. Ta ndem mass spectrometric studies indicated that the palmitoyl residue o f the CD52-II anchor is attached to the S-position of the myo inositol ring, Both the CD52-I and CD52-II PI structures are unusual for GPI a nchors and the possible significance of this is discussed. The alkali- lability of the CD52 epitope recognized by the Campath-1H monoclonal a ntibody was studied. The data suggest that the alkali-labile hydroxyes ter-linked fatty acids of the GPI anchor are necessary for antibody bi nding.