Evidence for the selective release of lysosomal proteinases in fasted rabbits.

The enzyme responsible for the conversion of "neutral" to "alkaline" fructose 1,6-bisphosphatase (EC 3.1.3.11) by removal of a 7000 dalton peptide (converting enzyme, Proteinase I) has been shown to be localized in rat liverlysosomes. Lysosomes also contain a specific proteinase (Proteinase II) that catalyzes the release of a small peptide from the NH2-terminus of the native subunits. In fasted rabbits Proteinase II is released into the cytoplasm, together with Cathepsin A, but Proteinase I remains associated with the lysosomal fraction. Increased osmotic fragility of liver lysosomes in fasted rabbits has also been observed, but this increased fragility does not result in the release of Proteinase I. The appearance of Proteinase II in the cytoplasm may be due either to its selective release from the lysosomes, without release of Proteinase I, or its localization in a different lysosomal fraction. Changes in lysosomal structure induced by fasting may play a dual role in : 1) the mobilization of amino acids for gluconeogenesis and 2) the modulation of activity of gluconeogenic enzymes.     

The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan structure and its susceptibility to proteolysis.

1. Proteoglycan was obtained from bovine nasal cartilage by a procedure involving sequential extraction with a low-ionic-strength KCl solution, then a high-ionic-strength CaCl2 solution. Purification was by CsCl-density-gradient centrifugation. 2. The CaCl2- extracted proteoglycan was subjected to proteolytic degradation by papain, trypsin, cathepsin D, cathepsin B, lysosomal elastase or cathepsin G. Degradation was allowed to proceed until no further decrease in viscosity was detectable. 3. The size and chemical composition of the final degradation products varied with the different proteinases. Cathepsin D and cathepsin G produced glycosaminoglycan-peptides of largest average size, and papain produced the smallest product. 4. The KCl-extracted proteoglycan was intermediate in molecular size and composition between the CaCl2-extracted proteoglycan and the largest final degradation products, and may have been formed by limited proteolysis during the extraction procedure. 5. It is postulated that the glycosaminoglycan chains are arranged in groups along the proteoglycan core protein. Proteolytic cleavage between the groups may be common to the majority of proteinases, whereas clevage within the groups is dependent on the specificity of each individual proteinase.     

Evidence for hemiketals as intermediates in the inactivation of serine proteinases with halomethyl ketones

The mechanism of inactivation of serine proteinases by peptide halomethyl ketone inhibitors was studied through the inhibition of trypsin with a series of model peptide ketones (Lys-Ala-LysCH2X). In this series, X is a poor leaving group with increasing electron-withdrawing capacity (X = H, CH2CO2CH3, COCH3, OCOCH3, and F), and as expected, the peptide ketones are reversible, competitive inhibitors of trypsin. The strength of binding of these inhibitors to trypsin increases with the electron-withdrawing ability of X, indicating that the inhibition constant Ki obtained is a measure of reversible hemiketal formation between the inhibitor ketone carbonyl group and the hydroxyl group of the active site serine. A Hammett plot of -log Ki vs. sigma I, the inductive substituent constant of X, reveals a linear relationship between the free energy of binding and the electron-withdrawing power of X. The reversible binding constant obtained for the corresponding chloromethyl ketone Lys-Ala-LysCH2Cl falls on this line, indicating that the reversible binding involves hemiketal formation, which is followed by alkylation of the enzyme.     

Evidence for the involvement of proteinases in the differentiation of haploid cells ofPhysarum flavicomum

When haploid cells ofPhysarum flavicomum reach the end of the growth phase in liquid semidefined medium they differentiate to dormant microcysts. Several compounds, effective as proteinase inhibitors in cell extracts, either blocked or delayed microcyst formation in semidefined medium. In addition, the compounds (except chloroquine) affected the conversion of swarm cells to myxamoebae.N-α-p-Tosyl-l-lysine chloromethyl ketone blocked microcyst formation and the vast majority of the cells existed as swarm cells. Chloroquine was an effective inhibitor in that the cells quickly became inactive; only about one-third of the cells converted to microcysts while the remaining cells were abnormally small. Both antipain and leupeptin inhibited normal microcyst formation and those microcysts produced were morphologically aberrant. Microcysts of normal appearance were produced in the presence of pepstatin and hemin but the process was significantly delayed.     

The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan heterogeneity and the pathway of proteolytic degradation.

1. CaCl2-extracted proteoglycan from bovine nasal cartilage was degraded by four tissue proteinases till no further decrease in hydroynamic size was obtained. The proteoglycan and its final degradation products were then fractionated by Sepharose 2B chromatography. 2. The average size of the degradation products was least for cathepsin B and lysosomal elastase, and greatest for cathepsin D and cathepsin G. The latter two proteinases also produced degradation products that showed the widest range of sizes. 3. The structure of the degradation products ranged from peptides containing a single glycosaminoglycan chain to those containing twelve or more chains. Of the four proteinases, only cathepsin B produced peptides that contained a single chondroitin sulphate chain. 4. The proteoglycan was very heterogeneous with respect to size and chemical composition. Its behaviour on electrophoresis suggested that at least two genetically distinct core proteins might exist. 5. Irrespective of their structural variations, all proteoglycan molecules were able to interact with hyaluronic acid. In contrast, none of the degradation products were capable of this type of interaction. 6. A pathway for the proteolytic degradation of proteoglycans is postulated in which the sites of initial cleavage may be common to the majority of proteinases, whereas the production of the final clusters is dependent on the specificity of the proteinase. Only those proteinases of broadest specificity can produce single-chain chondroitin sulphate-peptides.     

Lysozyme in preosseous cartilage VII. Evidence for physiological role of losozyme in normal endochondral calcification

Previous work demonstrated that micropuncture aspirates from rat epiphysical plate cartilage contain a nucleating agent for Ca₃(PO₄)₂ mineral growth, and that the nucleation is inhibited by proteoglycan aggregates. In this report data are described which show that mammalian lysozyme inactivates the inhibition. When micropuncture aspirates are incubated in vitro with mammalian lysozyme, a rapid, spontaneous initiation of mineral growth occurs. Incubation of proteoglycan aggregate preparations in the presence of cartilagea lysozyme, but not hen egg white lysozyme, causes a marked decrease of the sedimentation coefficients of the proteoglycans, usually to values close to those obtained with proteoglycan monomer preparations. The inhibition of this effect of mammalian lysozyme by a specific inhibitor of the enzyme tri(N-acetyl-D-glucosamine) suggests that it may be enzymatic in nature.     

Role of the subchondral vascular system in endochondral ossification: endothelial cell-derived proteinases derepress late cartilage differentiation in vitro.

Endochondral ossification in growth plates proceeds through several consecutive steps of late cartilage differentiation leading to chondrocyte hypertrophy, vascular invasion, and, eventually, to replacement of the tissue by bone. The subchondral vascular system is essential for this process and late chondrocyte differentiation is subject to negative control at several checkpoints. Endothelial cells of subchondral blood vessels not only are the source of vascular invasion accompanying the transition of hypertrophic cartilage to bone but also produce factors overruling autocrine barriers against late chondrocyte differentiation. Here, we have determined that the action of proteases secreted by endothelial cells were sufficient to derepress the production of the hypertrophy-markers collagen X and alkaline phosphatase in arrested populations of chicken chondrocytes. Signalling by thyroid hormones was also necessary but endothelial factors other than proteinases were not. Negative signalling by PTH/PTHrP- or TGF-beta-receptors remained unaffected by the endothelial proteases whereas signalling by FGF-2 did not suppress, but rather activated late chondrocyte differentiation under these conditions. A finely tuned balance between chondrocyte-derived signals repressing cartilage maturation and endothelial signals promoting late differentiation of chondrocytes is essential for normal endochondral ossification during development, growth, and repair of bone. A dysregulation of this balance in permanent joint cartilage also may be responsible for the initiation of pathological cartilage degeneration in joint diseases.     

Biochemical analysis of synoviocytes from normal and arthritic rats. Evidence for an activated state associated with adjuvant polyarthritis

Adjuvant-induced polyarthritis in rats is a common model system used for the study of the synovitis that occurs in rheumatoid arthritis. Synoviocytes A, the major cell type covering the internal surface of the joint, could be involved in the pathogenesis of rheumatoid arthritis because of their increased proliferation and the intraarticular manifestations of the disease. So far only a few molecular studies have been reported on synoviocytes upon arthritis induction. We report here changes in polypeptides, between control and arthritic synoviocytes, by using two different radiolabeling methods and two-dimensional gel electrophoresis analysis. Major differences were found using metabolic labeling on regions of tropomyosins, cyclin, tubulins and vimentin. In addition, external surface labeling of the cells with lactoperoxidase showed clear differences between control and arthritic synoviocytes in the region of 77-100-kDa proteins. Some of these differences can be reproduced by certain macrophage activators such as phorbol myristate acetate and lipopolysaccharide acting on synoviocytes in vitro and in vivo respectively. These results exclude the possibility that the changes observed were due to a possible infiltration of other cell types in the arthritic synovia and strongly support the existence of an activated state of synoviocytes associated with arthritis induction.     

Evidence for non-competitive inhibition between two calcium-dependent activated neutral proteinases and their specific inhibitor

Two muscle thiol proteinases causing partial degradation of myofibrillar constituents were isolated and purified from skeletal muscle. The two proteinases that differ significantly in calcium requirements were designated respectively high- and low-Ca2+-requiring proteinase. Both are inhibited, in vitro, by a specific inhibitor which is a protein also isolated from skeletal muscle. Experiments using carboxymethylated monomeric proteinases and inhibitor-conjugated Sepharose were carried out in order to understand the mechanism of control of the proteinases by the inhibitor. The results using increasing inhibitor concentrations show a non-competitive inhibition for both enzymes. The Ki value for the low-Ca2+-requiring form was 0.3 microM, while the Ki value for the high-Ca2+-requiring form was 0.9 microM. Likewise, the low-Ca2+-requiring form needs about 3-fold more inhibitor than the high-Ca2+-requiring form for the same per cent inhibition.     

Evidence for a differential distribution of reducing equivalents from NADPH in cartilage

It has been suggested that the NADPH, generated from the activity of glucose 6-phosphate dehydrogenase, may be utilized by two different routes, namely either for biosynthesis purposes (type 2) or for microsomal respiration (type 1). This concept has been tested in the rabbit ear model in which an injection of papain into the rabbit causes loss of proteoglycans of the auricular matrix followed by its restoration over the following eight days. It is shown that whereas the type 1 pathway was either unaltered or diminished, the doubling of the glucose 6-phosphate dehydrogenase activity was related solely to the type 2 pathway.     

Circular dichroism of cysteine proteinases from papaya latex. Evidence of differences in the folding of their polypeptide chains.

Two forms of proteinase omega were isolated from a commercial preparation of chymopapain (EC 3.4.22.6) by means of cation-exchange liquid chromatography. Their circular dichroism (CD) spectra in the 182-320 nm region indicated that the two forms possess closely related structures. For comparison, we also recorded the CD spectra of chromatographically purified samples of papain (EC 3.422.2) and the most abundant form of chymopapain. According to the qualitative criteria proposed by Manavalan and Johnson (1983) Nature 305, 831-832), the spectral characteristics of papain correctly indicate that this protein belongs to the alpha + beta class. Proteinase omega is also placed in the alpha + beta category, while chymopapain seems to be an alpha/beta protein. Quantitative estimation of secondary structures yielded contents of helices and parallel beta-sheet that were higher in the case of chymopapain. Thus, the results of this work suggest that there are some differences in the folding pattern of chymopapain with respect to the other two proteinases. This proposal seems unexpected when the high amino acid sequence identity among these enzymes is considered.     

Evidence linking chondrocyte lipid peroxidation to cartilage matrix protein degradation. Possible role in cartilage aging and the pathogenesis of osteoarthritis

Reactive oxygen species (ROS) are implicated in both cartilage aging and the pathogenesis of osteoarthritis. We developed an in vitro model to study the role of chondrocyte-derived ROS in cartilage matrix protein degradation. Matrix proteins in cultured primary articular chondrocytes were labeled with [(3)H]proline, and the washed cell matrix was returned to a serum-free balanced salt solution. Exposure to hydrogen peroxide resulted in oxidative damage to the cell matrix as established by monitoring the release of labeled material into the medium. Calcium ionophore treatment of chondrocytes, in a dose-dependent manner, significantly enhanced the release of labeled matrix, suggesting a chondrocyte-dependent mechanism of matrix degradation. Antioxidant enzymes such as catalase or superoxide dismutase did not influence matrix release by the calcium ionophore-activated chondrocytes. However, vitamin E, at physiological concentrations, significantly diminished the release of labeled matrix by activated chondrocytes. The fact that vitamin E is a chain-breaking antioxidant indicates that the mechanism of matrix degradation and release is mediated by the lipid peroxidation process. Lipid peroxidation was measured in chondrocytes loaded with cis-parinaric acid. Both resting and activated cells showed constitutive and enhanced levels of lipid peroxidation activity, which were significantly reduced in the presence of vitamin E. In an immunoblot analysis, malondialdehyde and hydroxynonenal adducts were observed in chondrocyte-matrix extracts, and the amount of adducts increased with calcium ionophore treatment. Furthermore, vitamin E diminished aldehyde-protein adduct formation in activated extracts, which suggests that vitamin E has an antioxidant role in preventing protein oxidation. This study provides in vitro evidence linking chondrocyte lipid peroxidation to cartilage matrix protein (collagen) oxidation and degradation and suggests that vitamin E has a preventive role. These observations indicate that chondrocyte lipid peroxidation may have a role in the pathogenesis of cartilage aging and osteoarthritis.     

Circular dichroism of cysteine proteinases from papaya latex. Evidence of differences in the folding of their polypeptide chains

Two forms of proteinase Ω were isolated from a commercial preparation of chymopapain (EC 3.4.22.6) by means of cation-exchange liquid chromatography. Their circular dichroism (CD) spectra in the 182–320 nm region indicated that the two forms possess closely related structures. For comparison, we also recorded the CD spectra of chromatographically purified samples of papain (EC 3.4.22.2) and the most abundant form of chymopapain. According to the qualitative criteria proposed by Manavalan and Johnson ((1983) Nature 305, 831–832), the spectral characteristics of papain correctly indicate that this protein belongs to the α + β class. Proteinase Ω is also placed in the α + β category, while chymopapain seems to be an α/β protein. Qualitative estimation of secondary structures yielded contents of helices and parallel ß-sheet that were higher in the case of chymopapain. Thus, the results of this work suggest that there are some differences in the folding pattern of chymopapain with respect to the other two proteinases. This proposal seems unexpected when the high amino acid sequence identity among these enzymes is considered.     

Histochemical analysis of insulin-like growth factor-1 binding sites in mouse normal and expermmentally induced arthritic articular cartilage

Summary Insulin-like growth factor-1 (IGF-1) plays a key role in regulation of chondrocyte metabolism. We examined the localization of IGF-1 binding sites on chondrocytes in cartilage from normal and experimentally induced arthritic mouse knee joints. Cryostat sections from patellar cartilage were incubated either with IGF-1 receptor antibody or biotinylated IGF-1. Subsequently confocal laser scanning microscopy was applied to compare the two staining procedures qualitatively and quantitatively. This approach allowed detailed analysis of membrane-associated and intracellular staining. Using IGF-1 receptor antibody, IGF-1 receptors were found on the cell membrane of chondrocytes in the middle and deeper cartilage zones, whereas intracellular staining was highest in chondrocytes of superficial zones. After incubation with biotinylated IGF-1, distinct membrane staining was not present and fluorescence was localized homogeneously in themiddle and deeper zones but not in superficial zones. In cartilage from inflamed knee joints staining with the use of IGF-1 receptor antibody did not change significantly, whereas a pronounced increase in staining was noted with biotinylated IGF-1 in chondrocytes of the middle and deeper zones of cartilage. It is concluded that the staining patterns obtained with the use of IGF-1 receptor antibody and biotinylated IGF-1 are remarkably different, suggesting that the latter also detects IGF-binding proteins. The results suggest that joint inflammation has no consistent effect on IGF-1 receptor expression but may induce a significant upregulation of IGF-binding proteins in chondrocytes of the middle and deeper zones of cartilage.     

Hydrolysis of histones by proteinases.

Hydrolysis of histones by proteinases from rat liver, skin and other sources was studied by using a rat thymus histone preparation as the substrate and polyacrylamide-gel electrophoresis and densitometric analysis as the methods to detect histone subtypes and their hydrolysis. The rat mast-cell proteinase I effectively hydrolysed histones except type H4. Thrombin hydrolysed effectively histones H1 and H2A, whereas plasmin hydrolysed all types of histones. Cathepsin D hydrolysed especially histone H2A. Cathepsins B and L hydrolysed all histones more slowly, and cathepsin H hydrolysed them extremely slowly. Epidermal aminoendopeptidase did not hydrolyse histones. Trypsin and chymotrypsin were used as reference enzymes, which hydrolysed all types of histones in very low concentrations. This study suggests that a variety of proteinases could play a role in histone hydrolysis. Hydrolysis of a specific subtype of histones, such as histone H2A at pH 6 by cathepsin D, may be directly involved in regulation of epidermal-cell differentiation.     

Cancer biology: extracellular proteinases in malignancy.

Secreted, matrix-degrading proteinases have been viewed as contributing to tumor metastasis. A recent study indicates that the gene for one of these enzymes, the matrix metalloproteinase stromelysin-1, can actually cause cancer when expressed in transgenic mice.