Sequence‐specific

Abstract

CD59 is a recently discovered cell-surface glycoprotein that restricts lysis by homologous complement and has limited sequence similarity to snake venom neurotoxins. This paper describes the first results of a two-dimensional NMR study of CD59 prepared from human urine. Nearly complete 'H-NMR assignments were obtained for the 77 amino acid residues and partial assignments for the N-glycan and the glycosylphosphatidylinositol (GPI) anchor. These results together confirm that the C-terminal residue of the mature protein is Asn 77 and that the urine-derived form retains the nonlipid part of the GPI anchor. The data further indicate that the GPI anchor and possibly the N-glycan are structurally inhomogeneous and suggest that the phospholipid present in the intact GPI anchor was removed by phosphatidylinositol-specific phospholipase-D. The folding topology of the protein was determined from NOE enhancements and slowly exchanging backbone amide protons and consists primarily of five extended strands (denoted p l -f l 5 in sequence order), arranged into separate two-stranded (PI and &) and three-stranded (&&) antiparallel P-sheets. The same folding topology is found in all of the snake venom neurotoxins whose structures have been determined. The region between the & and & strands has helical character, a feature that is not present in the neurotoxins but that is seen in the topologically similar wheat germ agglutinin.

Ligand Interaction between Urokinase-Type Plasminogen Activator and Its Receptor Probed with 8-Anilino-1-naphthalenesulfonate. Evidence for a Hydrophobic Binding Site Exposed Only on the Intact Receptor

“…Recently, the disulfide bond connectivity of the NH2-terminal domain of uPAR has been solved (Ploug et al, 1993) and has been found to be homologous to that of the nonglycosylated snake venom a-neurotoxins, suggesting that the individual uPAR domains adopt the same overall structural topology as these toxins. Recent 'H-NMR assignments for MIRL/CD59 are consistent with a model in which this protein, homologous to the uPAR domains, has a gross folding topology similar to that of the snake a-neurotoxins (Fletcher et al, 1993).…”

Identity of the Residues Responsible for the Species-restricted Complement Inhibitory Function of Human CD59

“…The amino acid sequence and solution structure of CD59 conform to those of the Ly6/cardiotoxin protein superfamily in which five internal disulfide bonds stabilize the polypeptide into a discoid structure consisting of a three-stranded ␤-sheet apposed to a two-stranded ␤-finger (14 -17). In CD59, the GPI anchor is attached to the C-terminal Asn 77 , and Asn 18 provides a single site of N-linked glycosylation (14,15,18). Whereas the carbohydrate attached to Asn 18 occludes most of one face of the discoid surface of the protein, these glycosidic residues do not contribute to the MAC inhibitory properties of CD59 (14, 15, 18 -20).…”

Elimination of Potential Sites of Glycosylation Fails to Abrogate Complement Regulatory Function of Cell Surface CD59

“…Nevertheless, because the level of cell surface expression of each recombinant protein (mutant or wild type) was not reported, these data do not permit definitive conclusion as to the relative MAC inhibitory function of the Ser 20 3 Ala mutant. Furthermore, this study did not resolve whether this mutation resulted in glycosylation at CD59 residue Asn 8 , a second potential site for N-glycosylation that is normally not glycosylated in wild type CD59.…”

“…Based upon sequence and solution structure, CD59 belongs to the snake venom/LY6 protein superfamily, whose members include cardiotoxins, erabutoxins, and the murine lymphocyte LY6 antigen (7)(8)(9). These proteins contain a characteristic disulfide-bonded polypeptide core comprised of a four-stranded ␤-sheet apposed to a two-strand ␤-finger.…”

“…The solution structure of CD59 suggests that this carbohydrate occludes one face of the disc-like structure formed by the core polypeptide. In human erythrocyte CD59, a second potential motif for N-linked glycosylation (Asn 8 ) is not utilized. The deduced amino acid sequences of CD59 homologues identified in Old and New World primates as well as in rat reveal that the motif for N-linked glycosylation at the residue corresponding to Asn 18 of human CD59 is invariably conserved, despite considerable sequence divergence elsewhere in the protein (11)(12)(13)(14).…”