Determination of Carboxyl-Terminal Residue and Disulfide Bonds of MACIF (CD59), a Glycosyl-Phosphatidylinositol-Anchored Membrane Protein

Abstract

MACIF (CD 59) is a glycosyl-phosphatidylinositol (GPI)-anchored membrane glycoprotein which inhibits the formation of membrane attack complex of human complement. MACIF prepared from human erythrocyte membranes was digested with pronase. When the digest was subjected to two-phase partition with butanol and 0.1 N HCl, the carboxyl-terminal peptide was recovered in the butanol phase because of the attachment of the highly hydrophobic GPI. The amino acid sequence of the peptide was determined to be Asn72 at its amino-terminus and up to Glu76, while the presence of Asn77 was ambiguous. To allow unequivocal determination of the carboxyl-terminus, a soluble form of MACIF was prepared from human urine on a large scale. The carboxyl-terminal peptide from the soluble form was prepared by tryptic digestion followed by reversed-phase HPLC. The sequence and composition of the peptide unequivocally revealed Asn77 as the carboxyl-terminus. The pattern of disulfide bonds of MACIF was also determined with the membrane form as well as the soluble form. Cystine-containing peptides were prepared by chymo-tryptic and tryptic digestion, purified by HPLC, and their amino acid sequences were determined. The results indicated that disulfide bonds were formed at Cys3-Cys26, Cys6-Cysl3, Cys19-Cys39, Cys45-Cys63 (or 64), and Cys63 (or 64)-Cys69.