Structure of a soluble, glycosylated form of the human complement regulatory protein CD59

Abstract

The main features of the protein structure are two antiparallel beta-sheets (a central one with three strands and another with two), a short helix that packs against the three-stranded beta-sheet, and a carboxy-terminal region that, although lacking regular secondary structure, is well defined and packs against the three-stranded beta-sheet, on the opposite face to the helix. We have used the structure, in combination with existing biochemical data, to identify residues that may be involved in C8 binding.

The Glycosylation of the Complement Regulatory Protein, Human Erythrocyte CD59

“…After incubation of native, undenatured CD59 with PNGase F, the protein migrated as a narrow band with an apparent molecular mass of approximately 15 kDa. This indicated that the glycoform population had been digested by the enzyme and that the Asn 18 -sugar amide linkage is fully accessible to PNGase F. These data are consistent with NMR analyses of glycosylated forms of CD59, which provided little evidence for protein-sugar interactions beyond the first N-acetylglucosamine residue of the N-linked glycan (17,18). Moreover, incorporation of model oligosaccharides in structure calculations (18) suggested that the N-glycan projects away from the protein in the plane of the disc-like extracellular domain.…”

“…The full signif- icance of glycosylation for the structure and function of CD59 can only be explored properly when its glycan population is viewed in the context of the complete structure of the molecule. In previous work, the three-dimensional structure of the protein component of CD59 (17,18) and the location of the active site of the molecule 2 have been determined. The aim of the present study was to determine the structure of both the GPI anchor and the N-and O-linked glycans of CD59 in order to complete the initial structural analysis and to provide a context for considering the function of the glycosylation of the molecule.…”

“…Further, the N-and O-linked glycans covalently attached to human erythrocyte CD59 were characterized, and the full HPLC glycosylation profile compared with that of human platelet CD59 to explore the extent of cellspecific glycosylation. Molecular modeling, based on the solution structure of the protein (18) was used to explore the spatial relationships between the lipid bilayer, the protein, and the N-and O-linked glycans. Several possible roles for the heterogeneous array of glycans attached to CD59 were proposed, including orientation of the active site, protection from protease cleavage, and limiting interactions with other CD59 molecules and with the cell surface.…”

Removal of domain D2 or D3 of the human urokinase receptor does not affect ligand affinity

“…Each domain (D1, D2 and D3) is related to a family of single-repeat, GPI-anchored surface proteins that include CD59, the Ly-6 antigens as well as snake neurotoxins [15,16]. The disulfide bond pattern of D1 conforms to the general structure of the Ly-6/neurotoxins protein family and has suggested overall structural homology to CD59 and snake a-bungarotoxin for which a tertiary structural analysis is available [17][18][19]. The purified amino terminal domain (D1) of uPAR possesses independent ligand-binding activity, although with an affinity 1,500 fold lower than the wild type [1]; the purified carboxy-terminal fragment (D2D3) appears to be devoid of binding activity [1,15].…”

Activity after Site-Directed Mutagenesis of CD59 on Complement-Mediated Cytolysis

“…The precursor of human CD59 is a single peptide composed of 128 amino acids deduced from its cDNA sequence (6,7). The mature protein consists of 77 amino acids arranging in a single cysteine-rich domain composed of two antiparallel β-sheets, 5 protruding surface loops and a short helix (8)(9)(10).…”