The Hakata antigen is a novel, thermolabile ␤ 2 -macroglycoprotein that reacts with sera from patients suffering from systemic lupus erythematosus. In this study we present the structure and the function of the Hakata antigen. We have identified cDNA clones encoding the Hakata antigen and analyzed its function. The cDNA included a possible open reading frame of 897 nucleotides, encoding 299 amino acids. The Hakata antigen consisted of a collagen-like domain in the middle section and a fibrinogen-like domain in the COOH terminus, both of which are homologous to human ficolin-1 and opsonin P35, indicating that these three molecules form a distinct family. The molecular mass of the Hakata antigen expressed in transfected cells was 35 kDa under reduced conditions, and it formed ladder bands under nonreducing conditions compatible with the previous result that the Hakata antigen exists in serum as homopolymers. Purified Hakata antigen sustained lectin activity, showing affinity with GalNAc, GlcNAc, D-fucose as mono/oligosaccharide, and lipopolysaccharides from Salmonella typhimurium and Salmonella minnesota. These results suggest that the Hakata antigen, a new member of the ficolin/opsonin P35 family, plays a role in the serum exerting lectin activity under physiological conditions. Inaba and Okochi (1) reported that sera from patients with systemic lupus erythematosus (SLE) 1 contained an antibody that reacted with normal sera. The antibody was shown to react against a novel thermolabile ␤ 2 -macroglycoprotein, designated the "Hakata antigen" (2). A similar thermolabile substance had been reported by Epstein and Tan (3), but it was not known whether the two proteins are the same. The molecular mass of the Hakata antigen in serum was 650 kDa as determined by gel filtration. The antigen was thermolabile because it lost antigenicity upon heating to 56°C for 1 min. The Hakata antigen was separated as a single band of 35 kDa by SDS-PAGE under reducing conditions. However, under nonreducing conditions it separated as ladder bands from 35 kDa to nearly the top of the gel, suggesting that the Hakata antigen exists in serum as homopolymers consisting of the 35 kDa subunit (2). All sera from 10,050 Japanese healthy blood donors, 99.99% of 751,352 Japanese patients' sera, and 99.98% of 41,430 Swedish patients' sera contained the Hakata antigen (4), thus implying that the Hakata antigen is a normal serum protein. The reference range of the Hakata antigen was 7-23 g/ml (2). The antibody against the Hakata antigen was possessed by 4.3% of 349 SLE patients and 0.3% of 703 patients with other autoimmune diseases (4). Among patients with other autoimmune diseases who possessed the antibody against the Hakata antigen, one patient was found among those with chronic glomerulonephritis and another in the group with primary biliary cirrhosis.In this study, we have cloned and characterized cDNA clones encoding the Hakata antigen revealing that the Hakata antigen is a novel serum protein that has Ca 2ϩ -independent lectin activity. The pri...

SUMMARYHakata antigen was first reported as a serum protein that reacted with an autoantibody from patients with systemic lupus erythematosus. Recently, it has been found that Hakata antigen is a new member of the ficolin/opsonin p35 family, which is a distinct lectin family, on the basis of homology of structures and the common characteristic of possessing lectin activity. In this study we analyzed the tissue distribution of Hakata antigen. Hakata antigen mRNA and protein were generated in the lung and liver. In the lung, Hakata antigen was produced by both ciliated bronchial epithelial cells and Type II alveolar epithelial cells and was secreted into the bronchus and alveolus. In the liver, Hakata antigen was produced by bile duct epithelial cells and hepatocytes and was also secreted into the bile duct. These results demonstrate that Hakata antigen is a unique lectin protein that exists not only in serum but also in bronchus/alveolus and bile, and indicate that Hakata antigen plays a role in bronchus/alveolus and bile under physiological conditions. H akata antigen was first reported by Inaba and Okochi (1978) as a serum protein that reacted with an autoantibody from patients with systemic lupus erythematosus (SLE). Since then, it has been demonstrated that Hakata antigen is a novel thermolabile ␤ 2-macroglycoprotein that exists as a monomer of 35 kD in reducing conditions and as a huge homopolymer of 650 kD in serum or in nonreducing conditions (Yae et al. 1991). All sera from 10,050 healthy Japanese donors, 99.99% of 751,352 Japanese outpatients, and 99.98% of 41,430 Swedish outpatients contained Hakata antigen, showing that Hakata antigen is a normal serum constituent (Inaba et al. 1990). The range of Hakata antigen in serum was 7-23 g/ ml (Yae et al. 1991).We have recently identified the cDNA encoding Hakata antigen, revealing that Hakata antigen consists of a collagen-like domain in the middle section and a fibrinogen-like domain in the C terminus, both of which are homologous to two lectin activity-possessing proteins, human ficolin-1 and opsonin p35 (Sugimoto et al. 1998). Homologies between Hakata antigen and either human ficolin-1 or opsonin P35 were both 48% overall, higher in the collagen-like domain (48%, 54%) and in the fibrinogen-like domain (52%, 53%), respectively (Sugimoto et al. 1998). We have further demonstrated that purified Hakata antigen sustains lectin activity, showing affinity with GalNac, GlcNac, and d -fucose as mono/oligosaccharide, and LPSs from Salmonella typhimurium and S. minnesota (Sugimoto et al. 1998). These three molecules apparently form a distinct human lectin family. However, the physiological roles of these related molecules have remained unresolved. To date, porcine and mouse homologues of human ficolin-1 and opsonin p35 have been identified, whereas that of Hakata antigen has not been found (Ichijo et al.

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To assess the fidelity of hydropathy prediction for band 3 protein, we determined the cleavage sites of the protein and the portions of the protein tightly bound to the membrane lipid bilayer by means of in situ proteolytic digestion. For the removal of all anticipated hydrophilic connector loops from membranes, we had to denature the band 3 protein molecule in situ by alkali treatment. When the alkali-treated membranes were digested with trypsin, chymotrypsin, and pepsin, the majority of the anticipated transmembrane portions remained in the membrane fraction. However, five anticipated transmembrane portions were released into the supernatant fraction. Thus, the first, second, third, sixth and tenth anticipated transmembrane portions, in accordance with the hydropathy prediction, were released into the supernatant with the proteolytic digestion method. This indicates that these anticipated transmembrane portions are not bound with the boundary lipids although the hydrophobicity of these portions is comparable to that of the portions experimentally remaining in the membrane fraction. It is conceivable that the membrane peptide portions of band 3 protein could be classified into at least two categories, i.e. one bound to the boundary lipids and the other free from the boundary lipids. Approximately 90% of the transmembrane domain of the band 3 protein are recovered in either the supernatant fraction or the membrane fraction. The fidelity of hydropathy prediction for polytopic membrane proteins and the nature of the membrane embedded peptide portions are discussed.

A dysfibrinogenemia was attributable to a single amino acid substitution from glycine to cysteine at residue 15 of the B beta chain in a fibrinogen molecule designated as fibrinogen Fukuoka II. The fibrinogen Fukuoka II showed prolonged thrombin and reptilase times and impaired fibrinopeptide B release by thrombin, resulting in abolition of fibrin monomer repolymerization under physiological conditions. Repolymerization of the des-(B beta 1-42)-fibrin monomers, however, was not distinguished from the normal pattern of des-(B beta 1-42)-fibrin monomers, suggesting that no other abnormality existed in fibrinogen Fukuoka II. Although an additional cysteine was substituted at residue 15 of the B beta chain, fibrinogen Fukuoka II had no free sulfhydryl group within the molecule. Instead, fibrinogen Fukuoka II formed a disulfide bond with cysteine, albumin, another mutated B beta chain within the same molecule, or intermolecular dimeric fibrinogen Fukuoka II. The mutation in fibrinogen Fukuoka II was the same as that in fibrinogen Ise published previously (Yoshida, N., Wada, H., Morita, K., Hirata, H., Matsuda, M., Yamazumi, K., Asakura, S., and Shirakawa, S. (1991) Blood 77, 1958-1963). Fibrinogen Ise, however, has been described as having prolonged thrombin time but normal reptilase time. Reasons for the discrepancy were not clear. Analysis of the B beta 1-42 fragment showed that fibrinogen was heterogeneous at position 31 of the B beta chain with respect to proline or hydroxyproline.