Heat-induced fragmentation of human plasma fibronectin

Human plasma fibronectin was found to undergo fragmentation during heat-denaturation, leading to artifacts in SDS-polyacrylamide gel electrophoretic analyses. Electrophoretic patterns of heated samples showed a progressive decrease in intact fibronectin chains (225 kDa) which coincided with the appearance of increasing amounts of numerous smaller components having molecular weights ranging from 10 000 to 200 000. The fragmentation was temperature-dependent, being undetectable after 2 h at 60 degrees C, but detectable after 30 min at 70 degrees C or as little as 2 min at 100 degrees C. After 2 h at 100 degrees C, the intact monomer was no longer visible. Neither mercaptoethanol nor SDS was required for fragmentation. Sterile filtration or pretreatment with inhibitors of proteolytic enzymes had no effect. Treatment with amines did not diminish the degradation, indicating that the process differs from heat-fragmentation of alpha 2-macroglobulin and complement proteins, which occurs at a reactive internal thiolester bond. Fibronectin fragmentation was highly pH-dependent, being markedly accelerated under acidic conditions, suggesting that autolytic cleavage of the peptide chain at acid-labile aspartyl bonds was responsible for this phenomenon.     

Purification and properties of rabbit liver cathepsin M and cathepsin B

Cathepsins M and B from rabbit liver lysosomes were separated by chromatography on Ultrogel AcA34 at low ionic strength and purified to homogeneity, and their catalytic and molecular properties were compared. Cathepsin M was relatively inactive with synthetic peptide substrates. Thus, it hydrolyzed benzoyl arginine naphthylamide at only one-fifth the rate observed with cathepsin B, and no activity was detected with Gly-Phe naphthylamide which is a relatively good substrate for cathepsin B. On the other hand, cathepsin M exhibited a preference for protein substrates. It was more active than cathepsin B in catalyzing the inactivation of the following enzymes: rabbit muscle or liver fructose-1,6-bisphosphate aldolases, rabbit liver fructose-1,6-bisphosphatase and pyruvate kinase, yeast glucose-6-phosphate dehydrogenase, and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. With glucagon as substrate, both enzymes showed similar peptidyl dipeptidase activities with some minor differences in peptide bond specificity. Cathepsins M and B are similar in size, with apparent molecular weights of 30,200 for cathepsin M and 28,800 for cathepsin B, and in amino acid composition and carbohydrate content. Each contains approximately 2-3 equivalents/mol glucosamine, 3 equivalents/mol mannose, and no fucose or galactosamine. They also show similar microheterogeneity in sodium dodecylsulfate-gel electrophoresis and isoelectric focusing; this microheterogeneity is probably related to differences in glycosylation. Extensive homology in primary structure for the two proteins was indicated by the similar patterns of peptides formed on digestion with trypsin.     

Isolation and characterization of porcine parathyroid cathepsin B.

Cathepsin B was isolated from porcine parathyroid tissue and from liver by a procedure involving acetone precipitation, gel filtration, and carboxymethylcellulose chromatography. The final preparations of each migrated as single bands upon sodium dodecyl sulfate polyacrylamide gels but exhibited several minor active variants upon isoelectric focusing. The parathyroid and liver enzymes were similar to each other and also resembled cathepsin B from other sources. The molecular weights for the porcine enzymes were estimated as 25,000, and the isoelectric point was at pH 4.8. The parathyroid enzyme cleaved benzyloxycarbonyl-Val-Lys-Lys-Arg-(4-methoxy)-2-naphthylamide at pH 5.8 and 37 degrees C with a Km of 0.14 mM and a kcat of 68 s-1. The pH optimum for this reaction was pH 6 to 7. The enzyme was unstable above pH 7.5 and below pH 4.5. It was strongly inhibited by HgCl2, ZnSO4, iodoacetate, iodoacetamide, and N-ethylmaleimide which indicated that it is a thiol protease, and by leupeptin, a strong inhibitor of cathepsin B from other sources. Antibodies to the parathyroid enzyme were elicited in rabbits. The antisera formed single precipitin bands upon double diffusion in agar gels against both the parathyroid and liver enzymes. Precipitin bands were formed at both pH 6 and pH 8.5 which indicated that the antisera recognized both native and denatured forms of the enzymes.     

Characterization of fibronectin from fetal human plasma in comparison with adult plasma fibronectin

Peptides containing fewer than 50 amino acids show little ordered structure under physiological conditions. In this paper it is shown that in the receptor environment, secondary structure could be induced in small peptides that involves 87% of all the amino acid residues. The statistical methods of Chou and Fasman are used to predict the conformation of 41 peptide hormones or neuromodulators in the proteinaceous environment of the receptor, and four distinct conformational groupings are elucidated. beta-bend, beta-structure and alpha-helical conformation are possible for distinct groups of linear peptides, and disulfide bridge containing peptides show a common beta-bend beta-structure conformation at the receptor. In the predicted receptor conformation, the peptides show hydrophobic and hydrophilic domains that must reflect the distribution of corresponding regions in the ligand-binding site of the receptor. The predicted ligand conformation should allow a more rational approach to interpreting existing structure activity studies and the design of new analogs of pharmacological interest.     

Rat liver thiol proteinases: cathepsin B, cathepsin H and cathepsin L

Data on following points of lysosomal thiol proteinases (cathepsins B, H and L) from rat liver are described in this paper: Partial amino acid sequence of cathepsin B, substrate specificity of cathepsin L, immunological studies of cathepsin B and H and effectiveness of E-64, specific thiol proteinase inhibitor in vivo.     

Novel carbohydrate structures of cathepsin B from porcine spleen

Two 13-residue glycopeptides were isolated from the digestion of purified porcine spleen cathepsin B by Staphylococcus aureus protease using high performance liquid chromatography. The major peptide, which is about 73% of the total, had the amino acid sequence His-His-Val-Asn(CH2O)-Gly-Ser-Arg-Pro-Pro-Cys-Thr-Gly-Glu. This peptide contains only a single N-acetylglucosamine residue linked to asparagine at the fourth residue by a beta-linkage. The minor peptide had a single amino acid replacement in a sequence otherwise identical to that of the major peptide. A serine was found at residue 10 instead of a half-cystine. The minor peptide also contains different carbohydrates, which were determined using proton NMR to be Man alpha 1----6 Man beta 1----4 GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAc beta 1----n Asn. These results suggest that the cathepsin B carbohydrates are processed in vivo by enzymic systems specific to each isozyme.     

Hydrophobic properties of porcine fibronectin and its functional domains

The relations between surface hydrophobicities and binding properties of the functional domains of porcine plasma fibronectin were investigated. Porcine plasma fibronectin as well as human plasma fibronectin was adsorbed on a hydrophobic column with butyl or phenyl ligands in the presence of 0.5 M ammonium sulfate, and recovered in a single peak by decreasing the concentration of ammonium sulfate to 0 M, indicating that both fibronectins have very high surface hydrophobicities. On digestion with thermolysin, porcine plasma fibronectin yielded five fragments (140-150, 43, 25, 17, and 14 kDa) similar to those reported for human fibronectin, although porcine fibronectin was more resistant to the digestion than human fibronectin. The three heparin-binding fragments were found to have a wide range of surface hydrophobicities, the 140-150 kDa fragment having the lowest, the 25 kDa fragment a higher, and the 14 kDa fragment the highest among all the fragments. The 43 kDa collagen-binding and 17 kDa fragments had surface hydrophobicities as high as that of fibronectin. It is noteworthy that the 43 kDa collagen-binding fragment contributes to the high surface hydrophobicity of intact fibronectin in spite of the high content of carbohydrates.     

Proteolysis of liver acetyl coenzyme A carboxylase by cathepsin B

Proteolysis of acetyl-CoA carboxylase was examined with cathepsin B. When chicken liver acetyl-CoA carboxylase was incubated with cathepsin B at pH 6.3, the native 220-kDa polypeptide was primarily cleaved into two polypeptides of 125 and 115 kDa, and further degraded to polypeptides of 100-50 kDa.     

Purification and characterization of chicken liver cathepsin B

Cathepsin B was purified, 400-fold, to homogeneity from chicken liver. The enzyme comprised a mixture of two kinetically indistinguishable forms (approximately 1:1), which were separated on concanavalin A (Con A)-Sepharose; one consisting of Mr 25,500 and 5,000 polypeptide chains bound to Con A-Sepharose but the other, composed of Mr 24,500 and 5,000 polypeptide chains, did not. N-terminal amino acid sequence analyses of a mixture of the Mr 25,500 and 24,500 polypeptide chains, and of the Mr 5,000 polypeptide chain revealed single amino acid sequences, respectively. These amino acid sequences were homologous to those of the heavy and light chains of mammalian enzymes, respectively. The chicken liver and mammalian cathepsin B were similar in structure and properties.     

Monoclonal antibodies to rabbit liver cathepsin B

Three stable hybridoma cell lines (AF8, BC11, CE2) have been produced that secrete antibodies specific for cathepsin B. These have been characterized by ELISA, SDS-PAGE immunostaining, immunoprecipitation and immunofluorescent staining. CE2 immunoprecipitated native cathepsin B with retention of enzymic activity, but failed to cross-react with the alkali-denatured enzyme. BC11 bound only to the denatured form of cathepsin B and AF8 cross-reacted with both native and denatured cathepsin B. However, unlike CE2-immunoprecipitated enzyme, activity could be detected only after dissociation of the antigen-AF8 antibody complex. No cross reaction was found with any lysosomal protein includihg the cysteine proteinases, catbepsins H and L.Abbreviations ELISA Enzyme-linked immunoadsorbent assay - EIP Enzyme immunoprecipitation - PAGE polyacrylamide gel electrophoresis - Ep-475 L-trans-epoxysuccinyl-leucylamido (-methyl) butane - Z benzyloxycarbonyl - NMec N-methylcoumarin - PEB phosphate-EDTA-Brij 35 - IAA iodoacetic acid - PBS phosphate-buffered saline - DMEM Dulbecco's Minimal Essential Medium - FITC fluorescein isothiocyanate     

The degradation of proparathormone and parathormone by parathyroid and liver cathepsin B.

Purified cathepsin B from porcine parathyroid glands was allowed to act upon radioactive bovine parathormone and proparathormone at various ratios of enzyme to substrate and for different times. The reaction products were isolated by ion exchange chromatography and analyzed by gel electrophoresis, amino acid composition, sequence analysis, and bioassay. The enzyme cleaved parathormone between residues 36 and 37 yielding a major carboxyl and amino fragment and appeared to cleave proparathormone at the same locus. The amino fragments were degraded further by removal of small peptides (possibly, di- or tripeptides) from their COOH termini. In contrast there was little if any degradation of the carboxyl fragment (residues 37 to 84). Despite the ease with which the enzyme cleaved the arginyl bond in the synthetic substrate benzyloxycarbonyl-Val-Lys-Lys-Arg-(4-methoxy)-2-naphthylamide, it did not remove the near homologous NH2-terminal hexapeptide extension of proparathormone (Lys-Ser-Val-Lys-Lys-Arg-R)--a reaction that would lead to the formation of parathormone from proparathormone. Purified liver cathepsin B cleaved the hormonal substrates in a fashion identical with that of the parathyroid enzyme.     

Potent slow-binding inhibition of cathepsin B by its propeptide.

A peptide (PCB1) corresponding to the proregion of the rat cysteine protease cathepsin B was synthesized and its ability to inhibit cathepsin B activity investigated. PCB1 was found to be a potent inhibitor of mature cathepsin B at pH 6.0, yielding a Ki = 0.4 nM. This inhibition obeyed slow-binding kinetics and occurred as a one-step process with a k1 = 5.2 x 10(5) M-1 s-1 and a k2 = 2.2 x 10(-4) s-1. On dropping from pH 6.0 to 4.7, Ki increased markedly, and whereas k1 remained essentially unchanged, k2 increased to 4.5 x 10(-3) s-1. Thus, the increase in Ki at lower pH is due primarily to an increased dissociation rate for the cathepsin B/PCB1 complex. At pH 4.0, the inhibition was 160-fold weaker (Ki = 64 nM) than at pH 6.0, and the propeptide appeared to behave as a classical competitive inhibitor rather than a slow-binding inhibitor. Incubation of cathepsin B with a 10-fold excess of PCB1 overnight at pH 4.0 resulted in extensive cleavage of the propetide whereas no cleavage occurred at pH 6.0, consistent with the formation of a tight complex between cathepsin B and PCB1 at the higher pH. The synthetic propeptide of cathepsin B was found to be a much weaker inhibitor of papain, a structurally similar cysteine protease, and no pH dependence was observed. Inhibition constants of 2.8 and 5.6 microM were obtained for papain inhibition by PCB1 at pH 4.0 and 6.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of human liver cathepsin B

The complete amino acid sequence of cathepsin B (EC 3.4.22.1) from human liver was determined. The 252-residue sequence was obtained by automated solid-phase Edman degradation of the light and heavy chain resulting from limited proteolysis of the single-chain enzyme and of fragments produced by cyanogen bromide and enzymatic cleavage of the heavy chain. Human liver cathepsin B has 83.7% identical residues with the corresponding enzyme from rat liver. Comparison of both mammalian cathepsin B sequences with the sequence of papain provides further evidence that lysosomal and plant cysteine proteinases have evolved from a common ancestor and share a similar catalytic mechanism.     

Further characterization of porcine plasma fibronectin which contains fucosylated carbohydrate chains

Porcine plasma fibronectin and its functional four fragments produced by cathepsin B digestion were examined for biological, immunochemical and biochemical properties. Native fibronectin, 150-kDa and 130-kDa fragments exhibited similar cell attachment-promoting activity to each other. In an Ouchterlony double immunodiffusion system, these three polypeptides formed a precipitin line with anti-fibronectin antiserum, while the 50-kDA and 30-kDa fragments did not. The 150-kDa and 130-kDa fragments contained free sulfhydryl(s). The glycopeptide fractions were prepared by pronase digestion of porcine and human plasma fibronectin, and radiolabeled with [¹⁴C]acetic anhydride. The results of affinity chromatography with concanavalin A and lentil lectin immobilized on agarose indicated that the porcine glycopeptide fraction was different from the human fraction in that a larger part (58%) of the former was bound to lentil lectin. About 90% of this lentil lectin-reactive glycopeptides lost this reactivity upon α-L-fucosidase digestion. The glycopeptide fractions were also prepared from three carbohydrate-containing domains. Less than 30% of the radioactivity of the glycopeptide fractions of 150-kDa and 130-kDa fragments was retained on the lentil lectin-agarose, while about 90% of that from the 50-kDa fragment was retained. These results indicate that porcine plasma fibronectin has characteristics very similar to those of human plasma fibronectin and others, but is unique in that it contains fucosylated carbohydrate chains which unevenly distribute through functional domains.     

Characterization and expression of cathepsin B2 in Fasciola gigantica

Fasciola gigantica cathepsin B belongs to a family of cysteine proteases which is involved in invasion of host tissues. In this study, the recombinant cathepsin B2 (rFgCatB2), synthesized in Pichia pastoris, showed enzymatic activity on a fluorometric substrate Z-Phe-Arg-AMC and gelatin. Furthermore, this recombinant enzyme could degrade IgG and type I collagen. Mouse antiserum against rFgCatB2 reacted with the native FgCatB2 in whole body (WB) extracts of metacercariae (MET), newly excysted juveniles (NEJ) and 2 week-old juveniles, but not in 3, 4 week-old juveniles and adult flukes. Immunolocalization showed the presence of cathepsin B2 only in the caecal epithelium of MET, NEJ and 2 week-old juveniles. Co-localization of FgCatB2 and a prominent antigen of NEJ, FgCatB3, revealed that these proteins were expressed at the same regions in the caecal epithelium. Anti-rFgCatB2 showed no cross reaction with the other parasites’ antigens by Western blotting. These findings suggest that CatB2 is expressed only in early stages of the parasite and may be involved in digestion of host connective tissues and evasion of the host immune system during their penetration and migration. Thus, CatB2 could be considered as an immunodiagnostic and vaccine candidate for fasciolosis.     

Characterization of acidic glycolipids in porcine plasma

Acidic glycosphingolipids including two sulfatides and five gangliosides were isolated from porcine plasma. They were characterized by NMR spectrometry as galactosylceramide-I³-sulfate and lactosylceramide-II³-sulfate, gangliosides GM3, GD3, GM1, GD1a and fucosyl GM1.