STRUCTURAL COMPOSITION AND FUNCTIONAL-CHARACTERIZATION OF SOLUBLE CD59 - HETEROGENEITY OF THE OLIGOSACCHARIDE AND GLYCOPHOSPHOINOSITOL (GPI) ANCHOR REVEALED BY LASER-DESORPTION MASS-SPECTROMETRIC ANALYSIS

CD59 (protectin) is a glycophosphoinositol (GPI)-anchored inhibitor of the membrane attack complex of complement found on blood cells, endot helia and epithelial cells. In addition to the lipid-tailed CD59, solu ble lipid-free forms of CD59 are present in human body fluids. We have investigated the detailed structural composition of the naturally occ urring soluble urinary CD59 (CD59(U)) using peptide mapping, anion-exc hange chromatography, sequential exoglycosidase digestion and matrix-a ssisted laser-desorption mass spectrometry (MALDI-MS). CD59(U) exhibit ed an average M(r) of 12444 in MALDI-MS. Mass analysis of the isolated C-terminal peptide (T9) indicated that a GPI-anchor (at Asn-77) witho ut an inositol-associated phospholipid was present in soluble CD59(U). By using residue-specific exoglycosidases,chemical modification and M ALDI-MS structures of seven different GPI-anchor variants were determi ned. Variant forms of the anchor had deletions and/or extensions of on e or more monosaccharide units. Sialic acid linked to an N-acetylhexos amine-galactose arm was found in two GPI-anchor variants. The N-linked carbohydrate side chain of CD59(U) (at Asn-18) also displayed conside rable heterogeneity, The predominant oligosaccharide chains were fucos ylated biantennary and triantennary complexes with variable sialylatio n. Mono Q anion-exchange chromatography resolved urinary CD59 into nin e different fractions that bound equally well to the terminal compleme nt SC5b-8 complexes. Despite binding to C5b-8, soluble CD59(U) inhibit ed complement lysis at an approx. 200-fold lower efficiency than eryth rocyte CD59. These results document the structural heterogeneity of bo th the GPI anchor and N-linked oligosaccharide of CD59 and demonstrate that the phospholipid tail is needed for the full functional activity of CD59. The site of cleavage between the diradylglycerol phosphate a nd inositol suggests that a mammalian phospholipase D could be involve d in the solubilization of GPI-anchored proteins.