Human erythrocytes contain a membrane protein, MACIF, which inhibits the formation of a membrane attack complex (MAC) of complement. We have cloned and sequenced the complementary DNA of MACIF messenger RNA. The amino acid sequence predicted from its nucleotide sequence consists of 128 amino acids. The amino-terminal 25 residues may correspond to a signal peptide. The carboxy-terminal sequence confirmed that MACIF is a glycosylphosphatidylinositol (GPI)-anchored protein. The amino acid sequence of MACIF was partially determined by established techniques for protein chemistry and the resultant sequence was consistent with that predicted from the nucleotide sequence. The results of sequence analyses also suggested that asparagine at the 18th position was N-glycosylated. When mRNA obtained from the MACIF cDNA clone with SP6 RNA polymerase was microinjected into Xenopus oocytes, the oocytes synthesized a product which exhibited MACIF activity and reacted with anti-MACIF antibody. Comparison of the predicted sequence revealed significant homology with mouse Ly-6 antigens.
Plasmapheresis with a dextran sulfate column is a treatment for patients with hypercholesteremia. When proteins bound to the column during the treatment were fractionated to prepare some known proteins, we found a 57 kDa glycoprotein designated GP57 which showed a new N-terminal amino acid sequence. Western-blot analysis of human plasma revealed that only a 120 kDa protein, GP120, reacted with anti-GP57 antibody. Since GP120 and GP57 had an identical N-terminal amino acid sequence, GP120 is probably the intact form of GP57. The isoelectric point of GP120 was 6.8. N-Glycanase treatment decreased the molecular weight of GP120 by 15 kDa. Neuraminidase and O-glycanase, however, did not affect the molecular weight. Amino acid sequence analyses of the lysylendopeptidase digest of GP120 revealed significant homology to the heavy chains of inter-alpha-trypsin inhibitor (ITI) family. Since GP120 showed no bikunin sequence, and chondroitinase treatment and alkaline treatment of GP120 did not affect its molecular weight, we concluded that GP120 was not a complex with bikunin. We designated GP120 as IHRP (ITI heavy chain-related protein).
MACIF (CD59) 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 chymotryptic 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-Cys13, Cys19-Cys39, Cys45-Cys63 (or 64), and Cys63 (or 64)-Cys69.
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