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 :
- CD59 ( protectin ) is a glycophosphoinositol ( GPI )-anchored inhibitor of the membrane attack complex of complement found on blood cells, endothelia and epithelial cells
- In addition to the lipid-tailed CD59 , soluble lipid-free forms of CD59 are present in human body fluids
- 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)
- CD59u exhibited 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 ) without an inositol-associated phospholipid was present in soluble CD59u
- By using residue-specific exoglycosidases, chemical modification and MALDI-MS structures of seven different GPI-anchor variants were determined
- Variant forms of the anchor had deletions and/or extensions of one or more monosaccharide units
- Sialic acid linked to an N-acetylhexosamine-galactose arm was found in two GPI-anchor variants
- The N-linked carbohydrate side chain of CD59u (at Asn-18 ) also displayed considerable heterogeneity
- The predominant oligosaccharide chains were fucosylated biantennary and triantennary complexes with variable sialylation
- Mono Q anion-exchange chromatography resolved urinary CD59 into nine different fractions that bound equally well to the terminal complement SC5b-8 complexes
- Despite binding to C5b-8, soluble CD59u inhibited complement lysis at an approx
- 200-fold lower efficiency than erythrocyte CD59
- 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
- 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