Structural analysis of the sugar chains of human urinary thrombomodulin.

The sugar chains of human urinary thrombomodulin were studied. N- and O-linked sugar chains were simultaneously liberated by hydrazinolysis followed by N-acetylation and were tagged with 2-aminopyridine. Then the structures of the N- and O-linked pyridylamino (PA-) sugar chains were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion. The major N-linked sugar chains of human urinary thrombomodulin were found to be monosialo- and disialofucosylbiantennary chains, while the major O-linked sugar chain was +/-Siaalpha2-3Galbeta1-3(+/-Siaalpha2-6)GalNAc. Thrombomodulin also contained the reported structure SO4-3GlcAbeta1-3Galbeta1-3(+/-Siaalpha2-6)Galbeta1-4Xyl [H. Wakabayashi, S. Natsuka, T. Mega, N. Otsuki, M. Isaji, M. Naotsuka, S. Koyama, T. Kanamori, K. Sakai, and S. Hase (1999) J. Biol. Chem. 274, 5436-5442]. In addition to these sugar chains, a single Glc was linked to Ser 287.

The cleavage site specificity of human prostate specific antigen for insulin-like growth factor binding protein-3.

The cleavage site of human insulin-like growth factor binding protein-3 by urinary prostate specific antigen was examined. Human insulin-like growth factor binding protein-3 was incubated with urinary prostate specific antigen at 37 degrees C and its proteolyzed fragments were separated by a reversed phase HPLC followed by N-terminal amino acid sequence analysis, demonstrating that the cleavage mainly occurred at Tyr-159. The synthetic peptide including Tyr-159 was also cleaved at the same site, although its reaction rate was relatively low. These results indicate that human insulin-like growth factor binding protein-3 is specifically cleaved at Tyr-159 by prostate specific antigen. Human insulin-like growth factor binding protein-3 was previously reported to be cleaved at five sites including Arg-97, Arg-132, Tyr-159, Phe-173 and Arg-179 by another group, however, prostate specific antigen preparation is possibly contaminated by trypsin-like protease. In contrast, our purified urinary prostate specific antigen had only a chymotrypsin-like activity, demonstrating that prostate specific antigen has the high substrate specificity for human insulin-like growth factor binding protein-3.

Purification and characterization of prostate specific antigen from human urine.

Prostate specific antigen (uPSA) was purified to homogeneity from human urine using SuperQ-Toyopearl, Sulfate-Cellulofine, Phenyl-Toyopearl, CM-Sepharose, anti-urokinase IgG Sepharose and Sephadex G-100. The purified uPSA gave a major band at 32.9 kDa on SDS-PAGE under the reduced condition. However, it shows multiple bands on native PAGE. Substrate specificity of purified uPSA is identical with that of PSA from human seminal plasma and uPSA shows the kallikrein and chymotrypsin-like activities. On the analysis of N-terminal amino acid, two amino acid residues at N-terminal position of uPSA were detected and other amino acid sequence of uPSA was identical with that of sPSA. In addition, we isolated the multiple components of uPSA using anion-exchange chromatography. They were almost the same in amino acid composition and N-terminal amino acid sequences and showed differences in lectin-blotting pattern.

Increased stability of PEG-PPG conjugated human urokinase against autolysis.

Human urokinase (UK) was easily degraded during the incubation at 37 degrees C in a time-dependent manner. The degradation was also observed in the presence of trypsin inhibitors, suggesting that UK was not degraded by exogeneous trypsin-like serine protease but by autolysis. In this cases, the A-chain of UK was selectively degraded. Polyethylene glycol-polypropylene glycol conjugated urokinase (PEG-PPG-UK) was not degraded after prolonged incubation at 37 degrees C. These results demonstrated that PEG-PPG modification completely blocked the degradation of UK by autolysis.

Sulfatide, a specific sugar ligand for L-selectin, blocks CCl4-induced liver inflammation in rats.

The effects of sulfatide, which is a specific sugar ligand for L-selectin (LECAM-1), on CCl4-induced liver inflammation was studied in rats. Intramuscular pretreatment with sulfatide suppressed the levels of serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) that were increased by CCl4 injection, but galactosylceramide, a desulfated form of sulfatide, did not. A light-microscopic analysis found that the extent and the severity of lesions of the liver cells induced by CCl4 injection were significantly less in the rats treated with sulfatide. These results show that sulfatide suppresses the CCl4-induced liver inflammation by inhibiting the attachment of L-selectin expressing lymphocytes to their native sugar ligands.

Amino acid sequence of natural tumor necrosis factor alpha inhibitor purified from human urine.

Tumor necrosis factor alpha inhibitor (TNF-INH) was purified from human urine and it was composed of 161 amino acid residues. The complete amino acid sequence of TNF-INH found by sequence analysis agreed with that predicted from the cDNA structure for the extracellular domain (1-161 portion) of 55-kDa TNF receptor and its processing site at the C-terminal was Asn-161.

Physicochemical characterization of PEG-PPG conjugated human urokinase.

Human urokinase (UK) was conjugated with polyethylene glycol-polypropylene glycol (PEG-PPG) and its physicochemical properties were examined. PEG-PPG modification decreased the activity for plasminogen activation, but increased the half-life of this protein when injected intravenously in rabbits. Kinetic analysis of PEG-PPG conjugated UK (PEG-PPG-UK) revealed that the kcat for plasminogen activation decreased 1/5-fold with the increase of Km in comparison with that of UK, although these parameters for cleavage of synthetic substrate (S-2444) did not change. However, the inhibitor constant of PEG-PPG-UK for plasminogen activator inhibitor 1 (PAI 1) was equal to that of UK. Peptide mapping analysis revealed that PEG-PPG binding sites were mainly determined to be Lys 35, 46, 61, 98, 120 and 135 in A-chain and Lys 211, 300, 318, 338, 348, 383 and 404 in B-chain. In addition, the modification rates of A and B-chain were 37.8% and 19.8% on average, respectively.

Relationship between the ligand structure of copper and the stability of superoxide dismutase.

The ligand structure of chelated copper in recombinant human Cu(2)Zn(2)SOD (r-hSOD) and its metal-substituted SODs was examined. In r-hSOD the configuration of the copper binding site was axially symmetrical and that of metal-substituted SODs had a tendency to change towards a tetrahedral type compared with r-hSOD except for Cu(2)(VO)(2)SOD. Cu(2)(VO)(2)SOD was not tetrahedral and in this case the ligand atom of copper was inclined to convert from nitrogen towards oxygen. In Cu(2)E(2)SOD, Ag(2)Cu(2)SOD, and Cu(2)(VO)(2)SOD the specific activities were lower but in CU(2)Cu(2)SOD and Cu(2)Co(2)SOD they were nearly identical with that of r-hSOD. These results suggested the ligand structure of chelated copper was not related to the specific activity of SOD. However, these SODs were less stable than r-hSOD against heat treatment and denaturing reagent. Further they were less stable against attack by an inactivator (hydrogen peroxide) except for Cu(2)(VO)(2)SOD. In this case the decreased stability of these SODs was associated with the change of the ligand structure of copper from that of r-hSOD. These results suggested that the presence of zinc contributed highly to the stable formation of the ligand structure of copper and the enzymatic stability.

Comparison of properties between human recombinant and placental copper-zinc SOD.

The physicochemical properties of purified recombinant human copper-zinc superoxide dismutase (r-hSOD) were compared with those of human placental copper-zinc superoxide dismutase (h-SOD). No differences were found in specific activity, metal contents, amino acid composition, and tryptic peptide map. The spectrophotometric properties including UV, ESR, and CD spectra were also similar. The result of isoelectric gel electrophoresis showed that the difference in isoelectric point (pI) was derived from acetylation of the N-terminal amino acid (alanine) in h-SOD. In SDS-polyacrylamide gel electrophoresis, both SODs showed the same behavior and enzymic activity was retained only under non-reducing conditions. ESR analysis of the denatured enzyme suggested that the high stability was derived from the structure of the active site around copper. Experiments using other metal-substituted SODs (Cu, Co in place of zinc) suggested that zinc contributed to the stability and the unique electrophoretic behavior of the enzyme.

Relation between ESR-detectable Cu(II) and superoxide dismutase activity.

The relation between ESR-detectable Cu(II) and Cu,Zn-superoxide dismutase activity was examined. The Cu(II) spin numbers per one unit of SOD were 6.26 X 10(12) (+/- 0.51 X 10(12] spins in several preparations of recombinant human Cu,Zn-SOD, native placental, and erythrocyte SOD. Measurement could be performed over a wide range of pH (4.0-10.0), preferably at temperatures below -40 degrees C. The data obtained by this method correlated well to the results obtained by the method of Fridovich et al. using the xanthine-xanthine oxidase system (correlation coefficient 0.995). The specific activity of SOD was proportional to the Cu(II) content measured by ESR, but not to the total Cu content measured by atomic absorption. This indicates that it is important to measure the Cu(II) content for determining Cu,Zn-SOD activity.

Physicochemical properties of charge isomers of recombinant human superoxide dismutase.

Recombinant human Cu2Zn2SOD expressed in Escherichia coli consisted of mainly three isomers with isoelectric points of 5.14 (A), 5.06 (B), and 4.99 (C). Each isomer was isolated by DEAE-Toyopearl chromatography and the physiochemical properties were investigated. No significant differences in chemical and spectrophotometric properties, such as specific activity, metal contents, amino acid composition, and UV and ESR spectra, were found. The result of labeling of free cysteine residues with ABD-F showed the disulfide bond to be formed between 57Cys and 146Cys in every isomer. A few differences were found in the CD spectrum around 260 nm and in the elution patterns on reverse-phase HPLC. The isoelectric points of the three isomers became the same after treatment by reduction and carboxymethylation and even after reduction only, pI of isomers tended to be at the value of component (A). These results suggest that the three isomers are identical in primary structure but slightly different in secondary or tertiary structure. These differences are probably derived from structural alterations around 111Cys.

Molecular cloning and expression in Escherichia coli of the cDNA coding for rabbit tumor necrosis factor.

cDNA clones containing the entire sequence of the precursor of rabbit tumor necrosis factor (TNF) were identified from a cDNA library prepared using poly(A)+RNA of lipopolysaccharide-induced rabbit alveolar cells. Synthetic oligodeoxynucleotides based on the amino acid sequence of rabbit TNF were selected by RNA blot hybridization and used as probes to screen this library. The DNA sequence and the amino acid sequence deduced from it showed very high homology to the reported DNA and amino acid sequences of human TNF. A plasmid containing the tac promoter and cDNA sequence coding for the 154-amino-acid sequence of mature rabbit TNF protein was constructed and expressed in Escherichia coli. The polypeptide produced had the characteristics of purified rabbit serum TNF and caused a necrotic response in a transplanted syngeneic tumor in mice.