Isolation and characterization of a thermolabile beta-2 macroglycoprotein ('thermolabile substance' or 'Hakata antigen') detected by precipitating (auto) antibody in sera of patients with systemic lupus erythematosus.

A novel thermolabile beta-2 macroglycoprotein ('thermolabile substance' (TLS) or 'Hakata antigen' (HA], which was detected by the precipitating (auto) antibodies of patients with systemic lupus erythematosus, was isolated and characterized. The purification procedure entailed the following steps: isoelectric precipitation in the range between pH 5.2-6.1, hydroxyapatite absorption chromatography, 35% saturated ammonium sulfate precipitation, Sephadex G-200 gel filtration, Pevikon block electrophoresis, lentil lectin affinity chromatography and immobilized rabbit anti-human whole serum IgG column chromatography. Utilizing these procedures, 0.1 mg of HA was purified from 3 1 of pooled human serum. The molecular mass of HA was determined as 650 kDa by Sepharose 4B gel filtration. On SDS-PAGE analysis, HA showed a single band at 35 kDa under reduced conditions and numerous ladder bands between 35 kDa to more than 300 kDa under nonreduced conditions. On analytical ultracentrifugation, HA gave a molecular mass of 520 kDa with a single meniscus and a sedimentation constant of 12.0. The amino acid and carbohydrate analysis of reduced and S-pyridylethylated HA revealed that it contained five residues of hydroxyproline and an N-linked type sugar chain.

Hypertension-induced cerebral atherosclerosis in the cholesterol-fed rabbit.

Foam cell lesions were found in cholesterol-fed rabbits with induced hypertension, particularly in intimal cushions at branching sites, where permeability to horseradish peroxidase was enhanced. Permeability to horseradish peroxidase was enhanced at the edge of intimal cushions without foam cell accumulation. This finding suggests that permeability is increased before foam cell infiltration. No foam cell lesions were observed in the intima of cerebral arteries distant from branching sites, but insudation of plasma constituents here caused endothelial cells to separate from the subendothelial matrices. Foam cell lesions were absent from the cerebral arteries in normotensive cholesterol-fed rabbits.

Effects of hypertension and hypercholesteremia on the permeability of fibrinogen and low density lipoprotein in the coronary artery of rabbits. Immunoelectron-microscopic study.

In an attempt to elucidate the effects of hypertension and/or hypercholesteremia on atherogenesis, with special reference to permeation and deposition of fibrinogen and low density lipoprotein (LDL) in the coronary artery, we studied electron-microscopically the localization of fibrinogen and LDL. In the untreated control rabbits, fibrinogen was localized in the caveolae and vesicles of the endothelial cells and in very small amounts in the subendothelial spaces of the coronary artery. Hypertension or hypercholesteremia was related to an enhanced insudation of fibrinogen into the subendothelial spaces of the coronary artery. The insudation of fibrinogen seemed to have occurred by way of vesicular transport and, to some extent, by junctional transport. LDL was localized only in the caveolae and vesicles of the endothelial cells of the coronary artery in the untreated control rabbits. LDL was deposited in the subendothelial space of the hypercholesteremic rabbits, with or without hypertension. Despite the lack of clear-cut and direct evidence, the insudation of LDL into the intima appeared to be enhanced by way of vesicular transport.

Hakata antigen, a new member of the ficolin/opsonin p35 family, is a novel human lectin secreted into bronchus/alveolus and bile.

Hakata 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.

Proteolytic cleavage sites of band 3 protein in alkali-treated membranes: fidelity of hydropathy prediction for band 3 protein.

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.

Abnormal lipoprotein appearing in plasma of patients who received a ten percent soybean oil emulsion infusion.

In 42 of 43 surgical patients who received a 10% soybeam oil emulsion (Intralipid), abnormal lipoprotein was detected in their plasma 1 to 2 days after the initial Intralipid infusion. This abnormal lipoprotein was proven to appear as a result of the infusion of soybean oil emulsion regardless of the patient's original diseases, age, sex, liver function, or concomitantly administered solutions. In addition, this abnormal -ipoprotein was found to have various similarities to lipoprotein-X (LP-X) which is found in plasma from patients with obstructive jaundice or familial lecithin:cholesterol acyltransferase deficiency. Therefore, this abnormal lipoprotein was tentatively named LP-X--like substance (LP-X-LS). A comparison of the properties of LP-X and LP-X-LS was performed and the following results were obtained: (1) LP-X-LS migrated toward the cathode on Bacto-Agar gel electrophoresis similarly to LP-X; (2) the stability of LP-X and LP-X-LS against heating and freezing were almost equal under various conditions; (3) LP-X-LS could be absorbed by anti--LP-X serum; (4) LP-X-LS existed in low density fraction (d = 1.063) separated by ultracentrifugation from plasma; (5) electron microscopic study of low-density lipoprotein particles from LP-X-LS positive plasma revealed that LP-X-LS had a similar ultrastructure to LP-X. From these results it is suggested that LP-X-LS is an abnormal lipoprotein quite similar to LP-X.

Serological studies of an SLE-associated antigen-antibody system discovered as a precipitation reaction in agarose gel: the HAKATA antigen-antibody system.

Current population studies indicate that the HAKATA antigen is one of the normal plasma proteins not yet completely characterized. The frequency of Japanese donor, patients and Swedish patients was 100%, 99.99% and 99.98%, respectively. Anti-HAKATA antibody production was found in three patients, all with systemic lupus erythematosus (SLE). Transient HAKATA antigen deficiency was found in 13 patients and appeared to be strongly associated with SLE (11 out of 13). None of the 14 SLE patients had a history of transfusion. It is therefore concluded that anti-HAKATA antibody is produced as one of the autoantibodies in SLE.

Permeation and deposition of fibrinogen and low-density lipoprotein in the aorta and cerebral artery of rabbits--immuno-electron microscopic study.

The localization of fibrinogen and low-density lipoprotein (LDL) in the arterial wall has been studied to determine whether they mediate the effects of hypertension and/or hypercholesteraemia on atherogenesis. In untreated control rabbits, fibrinogen was localized in the caveolae and vesicles of the endothelial cells and in the subendothelial spaces of the aorta. No fibrinogen was found in the subendothelial spaces of the cerebral artery. Hypertension or hypercholesteraemia was accompanied by enhanced insudation of fibrinogen into the subendothelial spaces of the aorta and cerebral artery, and fibrinogen deposition was most prominent in the hypercholesteraemic rabbits with induced renovascular hypertension. The insudation of fibrinogen appeared to occur by way of vesicular transport, and to some extent by junctional transport. In the untreated control rabbits, LDL was localized only in the caveolae and vesicles of endothelial cells in both aorta and cerebral artery. LDL was deposited in the subendothelial space of the aorta of hypercholesteraemic rabbits with or without hypertension, and in the cerebral artery of hypercholesteraemic rabbits with hypertension. These findings suggest that fibrinogen insudates into the intima of the aorta and cerebral artery both during hypertension and hypercholesteraemia, and that LDL insudation into the intima of the aorta in hypercholesteraemia is accentuated by hypertension. LDL insudated into the intima of the cerebral artery in the presence of hypercholesteraemia linked to hypertension. Thus, hypertension plays a significant role in the pathogenesis of cerebral atherosclerosis.

Clinical significance of lipoprotein-X in congenital biliary atresia.

Quantitative lipoprotein-X (Lp-X) was measured for diagnostic and postoperative examination of congenital biliary atresia (CBA). There was no significant difference in the levels in children with CBA (n = 23) or neonatal hepatitis (NH) (n = 14). However, a value over 200 mg/dl was found in 7 of CBA but not NH patients. As to 13 postoperative non-icteric patients with CBA, Lp-X was absent in only 2 patients. Thus, postoperative patients without jaundice do not always have a normal biliary secretion. From those results, estimation of Lp-X is both useful for diagnosis and postoperative evaluation of CBA.

An abnormal fibrinogen Fukuoka II (Gly-B beta 15-->Cys) characterized by defective fibrin lateral association and mixed disulfide formation.

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.

Cloning and characterization of the Hakata antigen, a member of the ficolin/opsonin p35 lectin family.

The Hakata antigen is a novel, thermolabile beta2-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.