Search

Some five years ago Keilova (1) reported on low molecular weight substrates and inhibitors of cathepsin D. In the light of the recently introduced technique of affinity chromatography, her data became interesting again. A low molecular weight peptide inhibitor containing a D-amino acid could serve as a ligand in the isolation of cathepsin D and other acid proteinases.

We have investigated the steady state kinetics of the degradation of native fibrillar collagen at pH 3.4 by four collagenolytic cathepsins of rabbit spleen. For each enzyme, the dependence of initial velocity on collagen concentration was well described by the Michaelis-Menten mechanism. Km, expressed as the concentration of triple-helical chains, and kcat values were determined for cathepsins B, L, N and S. The ratio of Kcat to Km suggest that cathepsins L and N are far more effective at collagen solubilization than either cathepsins S or B. Ki values were determined for the inhibition of collagenolytic activity at pH 3.4 using cystatin, a naturally-occurring cysteine proteinase inhibitor. All four cysteine proteinases were inhibited by cystatin in this assay system, although it was found to be a tighter binding inhibitor of cathepsin L, than for cathepsins N and S (approximately 5-fold less), or cathepsin B (approximately 500-fold less).

Cathepsin D, cathepsin L, and cathepsin B activities were detected in homogenates of human gingival tissue from 20 patients with different degrees of periodontal disease and in one patient with juvenile periodontitis. Clinical plaque index (PI), gingival index (GI), and pocket depth were determined. Patients were divided into three groups with regard to the severity of the disease. Averaged values for the activities of all three lysosomal proteinases increased from the group with less damaged tissue to the group with more advanced periodontal disease. When the correlations between the specific activities of the cathepsins and pocket depth in each of 20 patients were considered by the linear regression analysis, the correlation coefficient for cathepsin D was much higher than for the other two cysteine proteinases. Cathepsin D was also extracted from the homogenates by specific immuno-adsorbtion on anticathepsin - D antibody - Sepharose and quantitatively determined; the average amount of enzyme ranged from 118.5 to 184.5 μg per mg of total protein extracted from the tissues and was positively correlated to pocket depths.

Leucocyte cells are known to contain the acid, thiol and many types of neutral proteinases; among the latter also plasminogen activator. During the last five years appeared also many reports on the endogenous inhibitors of proteinases. In the preceeding publications we reported the isolation and characterization of two inhibitors of proteinases, from leucocytes (of thiol proteinases and of serino proteinases).In the present work we present the biochemical characteristics as well as the inactivation (by cathepsin D) of an urokinase inhibitor isolated from pig blood.Leucocytes were isolated from the peripheral pig blood by dextran procedure. Inhibitor of urokinase could be isolated either from cytoplasm (post granular supernatant) or from the extract of nuclei. Further purification of inhibitor was performed by cation exchange chromatography, by gel filtration on Sephadex G-100 and G-150 and by affinity chromatography on urokinase-Sepharose 4B and on antibody (of urokinase inhibitor )-Sepharose 4B. Inhibitor was isolated in the homogenous form, in a single polypeptide chain with molecular weight of 68 000, designated (leucocyte) Inhibitor-3. This inhibitor has an isoelectric point from 4.4-4.5 and is stable in buffer solutions from pH 3-8, and belongs to the type of so called fast reacting inhibitors. We also established that cathepsin D inactivates this inhibitor by hydrolysis of the inhibitor molecule. The conversion of active inhibitor into inactive protein proceeds catalytically. These last inactivation studies suggest a new pathway by which the active urokinase inhibitor in leucocyte cells may be inactivated.

In last decade there has been an intensifying interest in naturally occurring protease inhibitors. Inhibitors which are present in the same cells as the enzymes they inhibit are of great physiological significance. A vast amount of literature1 reports that leucocytes are cells which contain different types of proteinases: neutral, thiol and acid ones. Only for the latter group of enzymes has the endogenous inhibitor not yet been determined, whereas for all other types the specific endogenous inhibitors have already been detected. Many groups of invest a ors have studied the isolation2–6, molecular characterization7–9 and mode of inhibition of these proteins10.

The antiserum was raised in rabbits against intracellular inhibitors I-1, I-2 and I-3 isolated from the soluble phase of disrupted pig peripheral leucocytes. It was demonstrated with double immuno-diffusion and with immunoelectrophoresis that the isolated inhibitors with different biochemical characteristics are three different, specific and unrelated proteins. With the techniques used, it was clearly confirmed that the inhibitors were isolated in a pure form and that they are located in cytoplasm and nucleus. The suppression of inhibitors by antiinhibitors antibodies was also demonstrated.

Affinity chromatography with an immobilized substrate proved to be a successful tool in the preparation of pure and undegraded cathepsin D (1). Immobilized reversible inhibitors are even more suitable for the isolation because the enzyme remains inactive during the affinity chromatography procedure. Pepstatin is known as a strong inhibitor of carboxyl proteases (2) and we wanted to make use of its inhibitory property for the isolation of cathepsin D. In this report, a method for isolation of cathepsin D is described using affinity chromatography on pepstatin-agarose.

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.

The amino acid sequence of papaya proteinase IV (PPIV), a major proteinase from the latex of Carica papaya [(1989) Biochem. J. 261, 469-476] is described. The enzyme has a high degree of sequence identity with papaya proteinase III, chymopapain and papain (81, 70 and 67%, respectively), and is clearly a member of the papain superfamily of cysteine proteinases. Nevertheless, the sequence shows substitution of certain residues conserved in all other known members of the superfamily. It is suggested that some of these substitutions may account for the unusual specificity of PPIV.

The shorter-chain form of chicken egg white cystatin has been crystallized in 1.6 M-phosphate buffer at pH 4.0 by vapour diffusion. The crystals are of trigonal space group P3121 (or P3221), have cell constants a = b = 47.9 A, c = 87.5 A, alpha = beta = 90 degrees, gamma = 120 degrees, and contain one molecule of 12,191 molecular weight per asymmetric unit. They diffract well to about 2.0 A resolution and are suitable for X-ray crystal structure analysis.

Although cathepsin E is present in trace amounts in spleen from several species, it was found in large amounts in rat spleen. This observation can be correlated with the fact that spleen in the rat is an important organ in haemopoeisis.

A procedure for classifying proteins of known sequence into structurally similar groups was developed on the basis of the Argos parametric approach. It is shown that stefins and cystatins constitute two structurally well resolved, but homologous groups of proteins. Furthermore, it is very probable that segments of secondary structures within each family are conserved, although significant differences between stefins and cystatins are indicated at the level of secondary structure. Next, secondary structures of all sequenced stefins and cystatins were predicted and used in the construction of secondary structures of the "typical stefin" and the "typical cystatin". Results were interpreted in the light of evolution and inhibition mechanism: Alignment of the "typical stefin" versus the "typical cystatin" secondary structure segments suggests that the divergence of stefin and cystatin families did not occur by a gene fusion event, but only by a mechanism of substitution, insertion and/or deletion. The central region of low-molecular mass cystatins, which is assumed to interact with cysteine proteinases, is predicted to be in a beta-sheet conformation. This resembles the beta-sheet in the active site of "standard mechanism" serine proteinases inhibitors.

The intracellular proteinase, cathepsin D, has been isolated from bovine spleen and thymus, in times as short as several hours, by affinity chromatography of partially purified and unpurified tissue extracts on haemoglobin-agarose resin. After subsequent separation from an inactive higher-molecular-weight protein by gel permeation chromatography, the enzyme from both tissues shows three dominant proteolytically active bands on gel electrophoresis at pH 4.3 and 9.5: this proteolytic activity is completely inhibited by the acid-proteinase inhibitor, pepstatin. These enzyme electrophoretic patterns were approximately constant with variation in isolation time and with various preliminary purification procedures. The enzyme shows only traces of polypeptides other than that with an apparent molecular weight of 42000 on dodecylsulphate electrophoresis, in contrast to the enzyme prepared by conventional methods, which contains considerable amounts of smaller polypeptides. This difference in polypeptide composition is shown to be the result of degradation of the enzyme in vitro during isolation by the previously published methods.

The at present known leucocyte and spleen endogenous inhibitors of proteinases are described. These proteins are located in nuclei and cytosol and they differ in molecular weight, isoelectric point, specificity against the tested proteinases and immunologically. Inhibition effects of inhibitors are reported: 1) on isolated proteinases, 2) on in vitro colony forming ability of cells, 3) on in vivo tumour growth and metastases formation. Some of these inhibitors could be degraded by cathepsin D, by hydrolysis of the inhibitor molecule.

A DNA containing the coding sequence for the human cysteine proteinase inhibitor stefin A was obtained by enzymic ligation of chemically synthesized deoxyoligonucleotides, using the Khorana ligation method. The 306-bp synthetic gene carries signals for the initiation and termination of its translation. The gene was expressed in E. coli using a cytoplasmic expression vector and stefin A was secreted under the control of the E. coli alkaline phosphatase signal sequence, respectively. The secreted hybrid protein was shown to exhibit biological properties similar to the native protein isolated from human plasma.

The amino acid sequence of stem bromelain, the major cysteine proteinase from pineapple stem is described. It shows that the enzyme is a member of the papain superfamily of cysteine proteinases, but is not very closely related to any other known member of this group. The sequence shows mutation or deletion of several residues that have been conserved in cysteine proteinases examined previously, including Asn-175 (papain). We suggest that some of these changes have the effect of altering the active-site geometry of stem bromelain, and that this accounts for the resistance of the enzyme to inhibition by cystatins and E-64 [L-3-carboxy-2,3-trans-epoxypropionylleucylamido(4-guanidino)butane].

Human high- and low-Mr kininogens were shown to be potent inhibitors of cysteine proteinases such as cathepsin L and papain (Ki = 17-48 pM). A strong immunological cross-reaction between the kininogens and low-Mr alpha-cysteine proteinase inhibitor from human plasma was found. Comparison of partial amino acid sequences from high- and low-Mr kininogen and low-Mr alpha-cysteine proteinase inhibitor demonstrated sequence identity for all segments analyzed. These findings suggest that the kininogens and the alpha-cysteine proteinase inhibitors from human plasma are identical proteins.

An alkaline and a trypsin-like serine proteinase were purified from the culture filtrate of Streptomyces rimosus by ultrafiltration, acetone fractionation, ion exchange chromatography, gel filtration and affinity chromatography on Kunitz soybean trypsin inhibitor – Sepharose. Both enzymes were isolated in electrophoretically pure form and characterized.

There are many reports in the literature (1) on the purification and properties of plasma proteinase inhibitors (i.e. alpha 1-antitrypsin, alpha 2-macroglobulin) and on attempts to make antisera, but so far there have been very few adequate reports on the isolation of intracellular leukocyte proteinase inhibitors and there have been no adequate reports on a specific anti-(LNPI) serum.

Antiviral activities of known protease inhibitors were assayed in virus-infected cell cultures. Some members of the cystatin superfamily, in particular chicken cystatin, were able to block virus replication. In a binding assay, using purified components, chicken and human cystatin were able to bind poliovirus protease with affinities which were reflected in their relative anitviral potencies. Prospects for application of protease inhibitors in clinical viral infections are discussed.

Cathepsin D is found in the cell in two forms, one a single polypeptide chain (Mr 44 000) and the other a non-covalent complex of two peptides of Mr 14 000 and 30 000. These correspond to the N-terminal and C-terminal regions of the single chain from which they originate. It has been shown that the two forms of the enzyme are closely similar in secondary-structure content, in aromatic amino acid environment and in denaturation behaviour. The two-chain enzyme has half the specific activity of the single-chain form. The denaturation and renaturation of the single-chain cathepsin D has now been studied by c.d., fluorescence and enzyme activity. Activity is lost irreversibly on unfolding, but the loss of backbone ellipticity and of folded aromatic environment is 75% reversible. The enzyme unfolds in two main stages, and the kinetics of these transitions indicate the existence of at least two intermediate forms between the native and the fully unfolded states. A further form of the enzyme exists in 0.5 M-guanidinium chloride. It is characterized by having an activity 40% greater than that of the native state. This increase is not reversed on removing the denaturant. The similarities between cathepsin D and pepsin are discussed.

Properties of cathepsin L from rat liver lysosomes were compared with those of a similar enzyme, cathepsin S from beef spleen. Major characteristics of cathepsin L are the high activity against Z-Phe-Arg-methylcoumarylamide and sensitivity to the fast reacting irreversible inhibitor Z-Phe-Phe-diazomethane. In contrast, cathepsin S hydrolyzes Z-Phe-Arg-methylcoumarylamide only slowly and Z-Phe-Phe-diazomethane cannot be regarded as a potent inhibitor of this enzyme. The differences in the substrate specificity of cathepsin L from rat liver and cathepsin S from beef spleen are discussed in comparison with the substrate specificity of cathepsin B from rat and human liver and beef spleen.

Publisher Summary This chapter focuses on the purification, characterization, and structural studies of bovine spleen cathepsins D and S. It is known that lysosomal proteases play an important role in the normal turnover of tissue proteins. The chapter presents a modified purification procedure for bovine spleen cathepsin S together with some of its basic properties. Cathepsin B activity was determined using α- N -benzoyl-DL-arginine-2-naphthyl-amide (BANA) as substrate. Cathepsin S belongs to the thiol endopeptidases. This enzyme, originally isolated from calf lymph nodes, can be found in similar quantity also in bovine spleen. The specificity of cathepsin S was studied using different protein substrates. Preliminary CD spectra of cathepsin S in the near ultraviolet region show entirely different patterns compared to cathepsin D, which is in accord with its inability to bind pepstin.

Leukocyte proteinases are postulated to be involved in variety of physiological and pathological events (7,9). It has also been proposed that proteinases are involved in the process of malignant invasion and that they could be responsible for the altered growth control in tumor cells (14). In this context the so-called neutral proteinases — histonases are of interest. It has been suggested that the histone hydrolyzing proteinases remove histones, which are known to act as gene repressors by binding to the DNA double helix, and thus may cause the derepression which is followed by DNA synthesis and cell division (6). It has already been found that leukocyte, as well as spleen elastases and chymotrypsinlike neutral proteinases have the ability to catalyze the hydrolysis of histones (11). Relevant to this ability of neutral proteinases, proteinase inhibitors are of particular interest and many studies have appeared dealing with the effect of synthetic and protein inhibitors on neutral proteinases.

Cystatin, the protein inhibitor of cysteine proteinases from chicken egg white was purified by a new method. The two major forms with pI 6.5 (Peak I) and 5.6 (Peak II) were separated. Molecular masses of both forms are approx. 12700 Da as determined by gel chromatography; Form A from Peak I has a molecular mass of 12191 Da as calculated from its amino-acid sequence. The complete amino-acid sequence of Form A was determined by automated solid-phase Edman degradation of the whole inhibitor and its cyanogen bromide fragments. It contains 108 amino-acid residues. Form B from Peak II represents an elongation of Form A by 8 amino-acid residues at the N-terminus. Cystatin contains four cysteine residues, presumably forming two disulphide bridges. Comparison of the amino-acid sequences and near ultraviolet circular dichroism spectra of stefin, the cysteine proteinase inhibitor from human granulocytes, and cystatin shows that the two proteins are entirely different. According to the primary structures, probably neither proteinase inhibitor is involved in a thiol-disulphide exchange mechanism in the interaction with its target enzyme.

The proteolytic activity in homogenates and extracts of subcellular fractions prepared from subcutaneous Lewis lung carcinoma was determined using proteins and synthetic peptides as substrates. The presence of cathepsin D, plasminogen activator, cathepsin B-, cathepsin G-and elastase-like enzymes was observed. No difference was revealed between the proteolytic activity in homogenates of Lewis lung carcinoma, at the growth stage examined, and in homogenates of normal lung. High specific activities were found in the lysosomal extract, whereas decreasing activities were found in the nuclear extract, the homogenate and the post-lysosomal mitochondrial supernatant; no active or trypsin-activatable collagenase activity was detected. The presence in the tumor tissue of these enzymatic activities is in agreement with their proposed role in the process of metastasis. The lack of differences between homogenates of tumor and normal lung tissue suggests that the use of whole cells is required to selectively study tumor proteinases specifically involved in tumor malignancy.

Acid-induced denaturation of recombinant human stefin B was followed using circular dichroism (CD) and fluorimetry. By comparing different spectroscopic probes, a number of equilibrium intermediates were detected. In pH denaturation at very low salt concentration (0.03 M NaCl) four states can be distinguished: N - I(N) - I1 - U, where N is the native state, I(N) is a native-like intermediate, I1 is an acid intermediate state with properties of a molten globule and U is the unfolded state. State 1, exhibits no near-ultraviolet CD but has some residual far-ultraviolet CD. It differs from U in its ability to increase fluorescence of 1-anilino-naphthalene 8-sulfonate (ANS). In 0.42 M salt, the pH denaturation is three-state between the dimeric native state N2 and intermediates I(N2) and I2, which are also dimeric according to size-exclusion chromatography. The acid intermediate I2 is more structured than I1: it binds ANS to a lower extent an I1, its Tyr residues are protected from the solvent, it shows some near-ultraviolet CD and its far-ultraviolet CD is even more intense than that for the native state. 1H-NMR spectra confirmed the overall structural features of the acid intermediates. To obtain the enthalpies of unfolding, microcalorimetric measurements were performed under conditions where the acid intermediates are maximally populated (18 degrees C): state I(N) from pH 5.0 to 4.6, 0.03 M salt: state I1 below pH 3.8, 0.42 M salt; and state I1 in equilibrium with I(N) at pH 4.05, 0.03 M salt. Enthalpies of unfolding for states I(N) and I1 were comparable to those of the native state. The enthalpy of unfolding for state I1 could not be determined.