Primary structure of CD52.
The CD52 antigen was extracted from human spleens with organic solvents and purified by immunoaffinity and reverse-phase chromatography. The latter step resolved two CD52 species, called CD52-I and CD52-II. Both species were found to contain similar N-linked oligosaccharides and glycosylphosphatidylinositol (GPI) anchor glycans. The N-linked oligosaccharides were characterized by methylation linkage analysis and, following exhaustive neuraminidase and endo-beta-galactosidase digestion, by the reagent array analysis method. The results showed that the single CD52 N-glycosylation site is occupied by large sialylated, polylactosamine-containing, core-fucosylated tetraantennary oligosaccharides. The locations of the phosphoryl substituents on the GPI anchor glycan were determined using a new and sensitive method based upon partial acid hydrolysis of the GPI glycan. The difference between CD52-I and CD52-II was in the phosphatidylinositol (PI) moieties of the GPI anchors. The phosphatidylinositol-specific phospholipase C-sensitive CD52-I contained exclusively distearoyl-PI, while the PI-phospholipase C-resistant CD52-II contained predominantly a palmitoylated stearoyl-arachidonoyl-PI, as judged by electrospray ionization mass spectrometry. Tandem mass spectrometric studies indicated that the palmitoyl residue of the CD52-II anchor is attached to the 2-position of the myo-inositol ring. Both the CD52-I and CD52-II PI structures are unusual for GPI anchors and the possible significance of this is discussed. The alkali-lability of the CD52 epitope recognized by the Campath-1H monoclonal antibody was studied. The data suggest that the alkali-labile hydroxyester-linked fatty acids of the GPI anchor are necessary for antibody binding.
Treumann
A
A
Department of Biochemistry, University of Dundee, Scotland, United Kingdom.
Lifely
M R
MR
Schneider
P
P
Ferguson
M A
MA
D015703:Antigens, CD / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D000951:Antigens, Neoplasm; D000074301:CD52 Antigen; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D013154:Spleen / Q000276:immunology*
D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000074301:CD52 Antigen; C000613862:CD52 protein, human; D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols; D009844:Oligosaccharides; D010446:Peptide Fragments