PMID: 8670172

 

    Legend: Sugar

Title : Structural com position and functional characterization of soluble CD59 : heterogeneity of the oligosaccharide and glycophosphoinositol ( GPI ) anchor revealed by laser-desorption mass spectrometric analysis

Abstract :
  1. CD59 ( protectin ) is a glycophosphoinositol ( GPI )-anchored inhibitor of the membrane attack complex of complement found on blood cells, endothelia and epithelial cells
  2. In addition to the lipid-tailed CD59 , soluble lipid-free forms of CD59 are present in human body fluids
  3. We have investigated the detailed structural com position of the naturally occurring soluble urinary CD59 ( CD59u ) using peptide mapping, anion-exchange chromatography, sequential exoglycosidase digestion and matrix-assisted laser-desorption mass spectrometry (MALDI-MS)
  4. CD59u exhibited an average M(r) of 12444 in MALDI-MS
  5. Mass analysis of the isolated C-terminal peptide (T9) indicated that a GPI-anchor (at Asn-77 ) without an inositol-associated phospholipid was present in soluble CD59u
  6. By using residue-specific exoglycosidases, chemical modification and MALDI-MS structures of seven different GPI-anchor variants were determined
  7. Variant forms of the anchor had deletions and/or extensions of one or more monosaccharide units
  8. Sialic acid linked to an N-acetylhexosamine-galactose arm was found in two GPI-anchor variants
  9. The N-linked carbohydrate side chain of CD59u (at Asn-18 ) also displayed considerable heterogeneity
  10. The predominant oligosaccharide chains were fucosylated biantennary and triantennary complexes with variable sialylation
  11. Mono Q anion-exchange chromatography resolved urinary CD59 into nine different fractions that bound equally well to the terminal complement SC5b-8 complexes
  12. Despite binding to C5b-8, soluble CD59u inhibited complement lysis at an approx
  13. 200-fold lower efficiency than erythrocyte CD59
  14. These results document the structural heterogeneity of both the GPI anchor and N-linked oligosaccharide of CD59 and demonstrate that the phospholipid tail is needed for the full functional activity of CD59
  15. The site of cleavage between the diradylglycerol phosphate and inositol suggests that a mammalian phospholipase D could be involved in the solubilization of GPI-anchored proteins