Alpha 2-macroglobulin used to isolate intracellular endopeptidases from mammalian cells in culture.

Extracts of cell cultures labelled with [3H]leucine were incubated with human alpha 2-macroglobulin (alpha 2M), a plasma proteinase inhibitor. The proteinase-alpha 2M complexes were then precipitated with immobilized monoclonal antibodies to alpha 2M and analysed by SDS/polyacrylamide-gel electrophoresis. Parallel experiments were done with methylamine-inactivated alpha 2M to check for unspecific binding of cell proteins to alpha 2M. Several 3H-labelled cell proteins bound to active, but not to inactivated, alpha 2M. Such proteins are likely to be proteinases. Putative endopeptidases of subunit Mr 112000, 78,000, 53,000, and in some experiments 88,000 and 16,000, were trapped by alpha 2M in supernatant fractions from IMR90 human fibroblasts, EBTr bovine fibroblasts and HeLa human carcinoma cells. No additional proteins were trapped in the presence of ATP. The Mr-78,000 endopeptidase was identified as calpain II by immunoblotting. At pH 5.3 putative endopeptidases of subunit Mr 80,000, 53,000 and 28,000-32,000 were trapped from IMR90-fibroblast extracts. Immunoblotting showed that both cathepsin B and cathepsin D were present in the Mr-28,000-32,000 electrophoretic bands. The use of alpha 2M and immobilized antibody to alpha 2M thus allows a rapid enrichment of endopeptidases from cell extracts. Some potentials and limitations of the method are discussed.     

Structural and functional characterization of the inhibition of urokinase by alpha 2-macroglobulin

We have investigated the interaction of alpha 2-macroglobulin (alpha 2M) with the serine proteinase urokinase, an activator of plasminogen. Urokinase formed sodium dodecyl sulfate stable complexes with purified alpha 2M and with alpha 2M in plasma. These complexes could be visualized after polyacrylamide gel electrophoresis by protein blots using 125I-labeled anti-urokinase antibody or by fibrin autography, a measure of fibrinolytic activity. According to gel electrophoretic analyses under reducing conditions, urokinase cleaved alpha 2M subunits and formed apparently covalent complexes with alpha 2M. Urokinase cleaved only about 60% of the alpha 2M subunits maximally at a mole ratio of 2:1 (urokinase: alpha 2M). Binding of urokinase to alpha 2M protected the urokinase active site from inhibition by antithrombin III-heparin and inhibited, to a significant extent, plasminogen activation by urokinase. Reaction of urokinase with alpha 2M caused an increase in intrinsic protein fluorescence and, thus, induced the conformational change in alpha 2M that is characteristic of its interactions with active proteinases. Our results indicate that both in plasma and in a purified system the alpha 2M-urokinase reaction is functionally significant.     

Proteolytic activity of HtpX, a membrane-bound and stress-controlled protease from Escherichia coli

Escherichia coli HtpX is a putative membrane-bound zinc metalloprotease that has been suggested to participate in the proteolytic quality control of membrane proteins in conjunction with FtsH, a membrane-bound and ATP-dependent protease. Here, we biochemically characterized HtpX and confirmed its proteolytic activities against membrane and soluble proteins. HtpX underwent self-degradation upon cell disruption or membrane solubilization. Consequently, we purified HtpX under denaturing conditions and then refolded it in the presence of a zinc chelator. When supplemented with Zn2+, the purified enzyme exhibited self-cleavage activity. In the presence of zinc, it also degraded casein and cleaved a solubilized membrane protein, SecY. We verified its ability to cleave SecY in vivo by overproducing both HtpX and SecY. These results showed that HtpX is a zinc-dependent endoprotease member of the membrane-localized proteolytic system in E. coli.     

Characterization of cathepsin L secreted by Sf21 insect cells

Sf21 cells, derived from the Spodoptera frugiperda pupa, are commonly used for the heterologous expression of proteins. While purifying recombinant proteins from this system we encountered a protease, secreted at high levels by Sf21 cells, that readily degraded recombinant proteins and also tended to co-purify with histidine-tagged proteins from Ni(2+) affinity columns. Purification and characterization of the protease revealed that it has many properties consistent with cysteine proteases of the papain family, including autoactivation under reducing conditions and acidic pH, and inhibition by E-64. Amino acid sequence analysis showed that the Sf21 enzyme may be identical to a putative insect procathepsin L cloned from the cotton bollworm. The subsite specificity of the Sf21 cathepsin and its inhibition profile by cystatins are consistent with the protease being an insect homologue of cathepsin L. Monoclonal antibodies useful for the detection and purification of the insect cathepsin L were developed.     

Characterization of new fluorogenic substrates for the rapid and sensitive assay of cathepsin E and cathepsin D.

Cathepsin E and cathepsin D are two major intracellular aspartic proteinases implicated in the physiological and pathological degradation of intra- and extracellular proteins. In this study, we designed and constructed highly sensitive synthetic decapeptide substrates for assays of cathepsins E and D based on the known sequence specificities of their cleavage sites. These substrates contain a highly fluorescent (7-methoxycoumarin-4-yl)acetyl (MOCAc) moiety and a quenching 2,4-dinitrophenyl (Dnp) group. When the Phe-Phe bond is cleaved, the fluorescence at an excitation wavelength of 328 nm and emission wavelength of 393 increases due to diminished quenching resulting from the separation of the fluorescent and quenching moieties. The first substrate, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Le u-Lys(Dnp)gamma-NH2, in which the Lys-Pro combination at positions P5 and P4 was designed for specific interaction with cathepsin E, is hydrolyzed equally well by cathepsins E and D (kcat/Km = 10.9 microM(-1) x s(-1) for cathepsin E and 15.6 microM(-1) x s(-1) for cathepsin D). A very acidic pH optimum o was obtained for both enzymes. The second substrate, MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Le u-Lys(Dnp)gamma-NH2, in which the isoleucine residue at position P2 was meant to increase the specificity for cathepsin E, is also hydrolyzed equally by both enzymes (kcat/Km = 12.2 microM(-1) x s(-1) for cathepsin E and 16.3 microM(-1) x s(-1) for cathepsin D). The kcat/Km values for both substrates are greater than those for the best substrates for cathepsins E and D described so far. Unfortunately, each substrate shows little discrimination between cathepsin E and cathepsin D, suggesting that amino acids at positions far from the cleavage site are important for discrimination between the two enzymes. However, in combination with aspartic proteinase inhibitors, such as pepstatin A and Ascaris pepsin inhibitor, these substrates enable a rapid and sensitive determination of the precise levels of cathepsins E and D in crude cell extracts of various tissues and cells. Thus these substrates represent a potentially valuable tool for routine assays and for mechanistic studies on cathepsins E and D.     

Interaction and complex-formation between the eosinophil cationic protein and alpha 2-macroglobulin.

The interaction between the highly basic and cytotoxic eosinophil cationic protein (ECP) and human plasma proteins is described. The major plasma protein responsible for complex-formation with ECP was shown to be the 'fast' form of alpha 2-macroglobulin (alpha 2M). Large amounts of complexes were observed in a serum obtained from a patient with hypereosinophilic syndrome. The amount of complexes that could be generated in vitro in normal fresh serum was rather low and was even less in fresh citrated plasma. Complex-formation between the non-proteolytic ECP and alpha 2M was augmented in the presence of methylamine. Binding of ECP to alpha 2M was also induced by the proteinases cathepsin G and thrombin, and the binding was competitive with cathepsin G. Methylamine and the proteinases seem to share a common mechanism in inducing binding of ECP. The nature of the ECP-alpha 2M interaction is non-covalent, but withstands high salt concentrations. The interaction with alpha 2M may reflect a mechanism by which the organism protects itself against the deleterious effects of the highly cytotoxic protein ECP.     

Caspase-2 promotes cytoskeleton protein degradation during apoptotic cell death

The caspase family of proteases cleaves large number of proteins resulting in major morphological and biochemical changes during apoptosis. Yet, only a few of these proteins have been reported to selectively cleaved by caspase-2. Numerous observations link caspase-2 to the disruption of the cytoskeleton, although it remains elusive whether any of the cytoskeleton proteins serve as bona fide substrates for caspase-2. Here, we undertook an unbiased proteomic approach to address this question. By differential proteome analysis using two-dimensional gel electrophoresis, we identified four cytoskeleton proteins that were degraded upon treatment with active recombinant caspase-2 in vitro. These proteins were degraded in a caspase-2-dependent manner during apoptosis induced by DNA damage, cytoskeleton disruption or endoplasmic reticulum stress. Hence, degradation of these cytoskeleton proteins was blunted by siRNA targeting of caspase-2 and when caspase-2 activity was pharmacologically inhibited. However, none of these proteins was cleaved directly by caspase-2. Instead, we provide evidence that in cells exposed to apoptotic stimuli, caspase-2 probed these proteins for proteasomal degradation. Taken together, our results depict a new role for caspase-2 in the regulation of the level of cytoskeleton proteins during apoptosis.     

Disulfide bridge-mediated folding of Sindbis virus glycoproteins.

The Sindbis virus envelope is composed of 80 E1-E2 (envelope glycoprotein) heterotrimers organized into an icosahedral protein lattice with T=4 symmetry. The structural integrity of the envelope protein lattice is maintained by E1-E1 interactions which are stabilized by intramolecular disulfide bonds. Structural domains of the envelope proteins sustain the envelope's icosahedral lattice, while functional domains are responsible for virus attachment and membrane fusion. We have previously shown that within the mature Sindbis virus particle, the structural domains of the envelope proteins are significantly more resistant to the membrane-permeative, sulfhydryl-reducing agent dithiothreitol (DTT) than are the functional domains (R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330-336, 1992). We have used DTT to probe the accessibility of intramolecular disulfides within PE2 (the precursor to E2) and E1, as these proteins fold and are assembled into the spike heterotrimer. We have determined through pulse-chase analysis that intramolecular disulfide bonds within PE2 are always sensitive to DTT when the glycoproteins are in the endoplasmic reticulum. The reduction of these disulfides results in the disruption of PE2-E1 associations. E1 acquires increased resistance to DTT as it folds through a series of disulfide intermediates (E1alpha, -beta, and -gamma) prior to assuming its native and most compact conformation (E1epsilon). The transition from a DTT-sensitive form into a form which exhibits increased resistance to DTT occurs after E1 has folded into its E1beta conformation and correlates temporally with the dissociation of BiP-E1 complexes and the formation of PE2-E1 heterotrimers. We propose that the disulfide bonds within E1 which stabilize the protein domains required for maintaining the structural integrity of the envelope protein lattice form early within the folding pathway of E1 and become inaccessible to DTT once the heterotrimer has formed.     

Monoclonal antibodies specific for neutrophil proteinase 4. Production and use for isolation of the enzyme

Four stable hybridoma cell lines producing monoclonal antibodies specific for neutrophil proteinase 4 (NP4) were established and one monoclonal antibody was chosen to produce an immunoaffinity-resin for the purification of NP4. In a precipitation assay system these antibodies bound NP4 in a dose-dependent manner, but did so neither with neutrophil elastase nor with cathepsin G. NP4 was purified and electrophoresis of the affinity-purified enzyme in sodium dodecyl sulfate polyacrylamide gels resulted in a single Mr = 30,000 polypeptide. The purified enzyme digested fibrin but not elastin and it cleaved Boc-Ala-ONp readily (Km = 0.47mM) at neutral pH, but had no effect on Suc-[Ala]3 Nan and N-Suc-[Ala]2-Pro-Phe-pNA. The proteolytic activity was inhibited by DFP, alpha 1 PI and alpha 2 M with a Ki of 10(-9)M for the NP4-alpha 1 PI complex. The NH2-terminal sequence and the amino-acid composition of NP4 were distinct from those of elastase and cathepsin G. Neutrophils contain large amounts of NP4 as judged by the comparable amounts of elastase- and NP4-alpha 1 PI complexes present in inflammatory exudates.     

Age-related increase in a cathepsin D like protease that degrades brain microtubule-associated proteins

In microtubules isolated from brains of very old rats, two of the major microtubule-associated proteins, MAP1 and MAP2, are found only in degraded form. MAP1 is present as a piece whose molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is circa 50,000 smaller than the native protein, and MAP2 is extensively fragmented. The native forms of both proteins are present in tissue homogenates but are rapidly degraded during microtubule isolation. The proteolytic activity responsible for this degradation is cathepsin D like, being more active at acid pH than neutral and being completely blocked by pepstatin at 10(-7) M. Fractionation of aged brain supernatant by gel permeation chromatography showed that the MAP1 and MAP2 degrading activity elutes with a single peak of cathepsin D like activity. MAP1 and MAP2 are known to promote microtubule assembly, and their degradation by a protease whose levels increase with age could be related to defective microtubule assembly which is known to occur in age-related degenerative conditions such as Alzheimer's disease.     

Modification of the substrate specificity of porcine pepsin for the enzymatic production of bovine hide gelatin

The substrate specificity of porcine pepsin has been altered by site-directed mutagenesis in an attempt to selectively cleave bovine hide collagen at only a few sites, similar to cathepsin D, for the production of high quality gelatin. Kinetic parameters were determined using chromogenic peptide substrates based on the sequence Lys-Pro-Xaa-Yaa-Phe*Nph-Arg-Leu (where Xaa is Ile or Pro, Yaa is Glu. Leu, Gln or Lys, Nph is p-nitrophenylalanine, and * is the site of cleavage). Substitution of Thr222 and Glu287 within the S2 subsite of pepsin by Val and Met, respectively, produced a double mutant with a two- to fourfold higher kcat/Km, compared with wild-type pepsin, for the chromogenic peptides with residues Leu, Gln, and Glu at position P2 (Yaa). The results suggest that the functional group of the P2 side chain may be exposed to solvent, while the aliphatic portion interacts with hydrophobic residues comprising S2. Wild-type pepsin cleaved a peptide corresponding to the carboxy-terminal telopeptide region of bovine type I collagen alpha1 chain, SGGYDLSFLPQPPQE, predominantly at three sites (Asp-Leu, Leu-Ser, and Phe-Leu) and at a significantly lower rate at Ser-Phe. However, Thr222Val/Glu287Met cleaved site Ser-Phe at a rate 20-fold higher than the wild-type. Significantly, enzymes containing the double substitution Phe111Thr/Leu112Phe cleaved this peptide predominantly at one site Leu-Ser (similar to cathepsin D) and at a rate 23-fold higher than the wild-type. These mutants can potentially enhance the rate of solubilization of bovine hide collagen under conditions mild enough to maintain the triple helix structure and hence minimize the rate of subsequent denaturation and proteolytic cleavage.     

Cathepsin E deficiency induces a novel form of lysosomal storage disorder showing the accumulation of lysosomal membrane sialoglycoproteins and the elevation of lysosomal pH in macrophages

Cathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE(-/-)) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE(-/-) macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H(+)-ATPase activity in both cell types, the elevated lysosomal pH in CatE(-/-) macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder.     

Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli

Lon protease from Escherichia coli degraded lambda N protein in a reaction mixture consisting of the two homogeneous proteins, ATP, and MgCl2 in 50 mM Tris, Ph 8.0. Genetic and biochemical data had previously indicated that N protein is a substrate for Lon protease in vivo (Gottesman, S., Gottesman, M., Shaw, J. E., and Pearson, M. L. (1981) Cell 24, 225-233). Under conditions used for N protein degradation, several lambda and E. coli proteins, including native proteins, oxidatively modified proteins, and cloned fragments of native proteins, were not degraded by Lon protease. Degradation of N protein occurred with catalytic amounts of Lon protease and required the presence of ATP or an analog of ATP. This is the first demonstration of the selective degradation of a physiological substrate by Lon protease in vitro. The turnover number for N protein degradation was approximately 60 +/- 10 min-1 at pH 8.0 in 50 mM Tris/HCl, 25 mM MgCl2 and 4 mM ATP. By comparison the turnover number for oxidized insulin B chain was 20 min-1 under these conditions. Kinetic studies suggest that N protein (S0.5 = 13 +/- 5 microM) is intermediate between oxidized insulin B chain (S0.5 = 160 +/- 10 microM) and methylated casein (S0.5 = 2.5 +/- 1 microM) in affinity for Lon protease. N protein was extensively degraded by Lon protease with an average of approximately six bonds cleaved per molecule. In N protein, as well as in oxidized insulin B chain and glucagon, Lon protease preferentially cut at bonds at which the carboxy group was contributed by an amino acid with an aliphatic side chain (leucine or alanine). However, not all such bonds of the substrates were cleaved, indicating that sequence or conformational determinants beyond the cleavage site affect the ability of Lon protease to degrade a protein.     

Membrane-bound tubulin: Resistance to cathepsin D and susceptibility to thrombin

In recent studies we found that cytoplasmic tubulin from brain was rapidly split by brain cathepsin D. Two pools could be established; the major portion was split at 18%/h, a minor portion at 2%/h, under our experimental circumstances. In the present work these experiments were extended to membrane-bound tubulin from brain. The membrane-bound form, in contrast to the cytoplasmic tubulin, was not degraded by cerebral cathepsin D under similar experimental conditions. This was not due to the presence of an inhibitory protein since added cytoplasmic tubulin was degraded. Several other protein components of membrane fractions (synaptosomal, mitochondrial) were degraded by cathepsin D, as measured on two-dimensional electropherograms. Thrombin degraded cytoplasmic tubulin, but the degradation products differed from those of cathepsin D degradation. Thrombin also hydrolyzed membrane-bound tubulin, but at a lower rate than the cytoplasmic form. Our results indicate great differences in the breakdown rate of a protein, which depend on its localization, in accord with the differences found in in vivo turnover rates.     

Proteolysis of Microtubule-Associated Protein 2 and Tubulin by Cathepsin D

The in vitro degradation of microtubule-associated protein 2 (MAP-2) and tubulin by the lysosomal aspartyl endopeptidase cathepsin D was studied. MAP-2 was very sensitive to cathepsin D-induced hydrolysis in a relatively broad, acidic pH range (3.0-5.0). However, at a pH value of 5.5, cathepsin D-mediated hydrolysis of MAP-2 was significantly reduced and at pH 6.0 only a small amount of MAP-2 was degraded at 60 min. Interestingly, the two electrophoretic forms of MAP-2 showed different sensitivities to cathepsin D-induced degradation, with MAP-2b being significantly more resistant to hydrolysis than MAP-2a. To our knowledge, this is the first clear demonstration that MAP-2 is a substrate in vitro for cathepsin D. In contrast to MAP-2, tubulin was relatively resistant to cathepsin D-induced hydrolysis. At pH 3.5 and an enzyme-to-substrate ratio of 1: 20, only 35% of the tubulin was degraded by cathepsin D at 60 min. The cathepsin D-mediated hydrolysis of tubulin was optimal only at pH 4.5. These results demonstrate that MAP-2 and tubulin are unequally susceptible to degradation by cathepsin D. These data also imply a potential for rapid degradation of MAP-2 in vivo by cathepsin D either in lysosomes or perhaps autophagic vacuoles of the neuron.     

Fasciola hepatica: parasite-secreted proteinases degrade all human IgG subclasses: determination of the specific cleavage sites and identification of the immunoglobulin fragments produced

The study was focused on the relationship of Fasciola hepatica-secreted proteinases and human IgG subclasses. Each IgG was incubated at different pH values and lengths of time with either the adult parasite excretion-secretion products or the purified cysteinyl proteinases cathepsin L1 and cathepsin L2. The Ig fragments produced were isolated and characterized by Western blot analysis, and the specific cleavage sites were determined by amino acid sequence analysis. Parasite excretion-secretion products and both cathepsins L produced similar degradation patterns and cleaved all human IgG subclasses at the hinge region, yielding at pH 7.3 and 37 degrees C Fab and Fc fragments in the case of IgG1 and IgG3 or Fab(2) and Fc in IgG2 and IgG4. While IgG1 and IgG3 were readily degraded by E/S products either in the presence or in the absence of reducing agents, IgG2 and IgG4 were resistant to proteolysis and were only digested in the presence of 0.1 M dithiothreitol. The cathepsins L needed the presence of dithiothreitol to digest IgG1, IgG2, and IgG4 whereas IgG3 was identically cleaved under both reducing and nonreducing conditions. The main cleavage sites produced by E/S products, CL1, or CL2 were located at the positions peptide bonds: His237-Thr238, Glu237-Cys239, Gly233-Asp234, and Ser241-Cys242 for gamma1, gamma2, gamma3, or gamma4, respectively. The enzymes gave additional splitting sites on the middle hinge of IgG3 to produce shorter Fc fragments and also produce Fd degradation of the IgG4. No cleavage specificity differences were found between CL1 and CL2, but they differed in the kinetics of IgG3 degradation. By lowering the pH, only the E/S products produced concomitant destruction of the Fc while preserving the Fab portion. Under all the conditions assayed the enzymes produced an Fc'-like fragment of 14-15 kDa corresponding to the whole CH3 domain of the immunoglobulin. Contrary to the extensive degradation produced by cathepsins on digested proteins, its actions on IgG subclasses were specific and restricted; thus, all the fragments produced could be potentially involved in the mechanisms used by the parasite to evade the host immune response.     

Cell surface proteolysis and down-regulation of the hepatic insulin receptor. Evidence for selective sorting of intact and degraded receptors after internalization

Insulin binding to rat liver plasma membranes promotes proteolysis of the Mr 135,000 alpha subunit of the insulin receptor to a fragment of Mr 120,000 (Lipson, K. E., Yamada, K., Kolhatkar, A. A., and Donner, D. B. (1986) J. Biol. Chem. 261, 10833-10838). The enzyme that catalyzes this degradation copurifies with plasma membranes and cannot be identified in any other cellular organelle or in cytosol. The proteinase has optimal activity above pH 7 and is an integral protein based upon its resistance to extraction with 2 M NaCl. After affinity labeling, degraded insulin receptors were identified in plasma membranes isolated from a liver perfused with 1 nM 125I-insulin for 10 min at 37 degrees C, indicating that proteolysis occurs in the hepatocyte cell membrane under physiological conditions. Microsomes do not contain the receptor degrading activity or a detectable amount of degraded receptors under basal conditions. After perfusion of a liver with 125I-insulin, Mr 135,000 and Mr 120,000 complexes were detected in microsomes, suggesting that both intact and degraded receptors can be internalized. The initial absence of degraded receptors in plasma membranes suggests that, following internalization, such sites do not recycle. Thus, hormone-induced proteolysis of the insulin receptor begins at the surface of the rat hepatocyte and can lead to loss of receptors from the plasma membrane.     

Acidic ribosomal proteins of Dictyostelium discoideum that show electrophoretic similarity to Escherichia coli proteins L7 and L12

This study documents the presence of three acidic proteins, A1 (pI 4.95), A2 (pI 4.85), and A3 (pI 4.70), in Dictyostelium discoideum ribosomes. All three proteins showed an apparent molecular mass of 13,000 by two-dimensional, sodium dodecyl sulfate gel electrophoresis. They were selectively released by treatment of ribosomes with 50% ethanol -1 M NH4Cl. The amino acid composition of A1, A2, and A3 were identical and indicated a predominant amount of alanine. All the above properties are shared by Escherichia coli proteins L7 and L12 and acidic ribosomal proteins in many eukaryotes. Unlike other eukaryotic systems, the acidic proteins of D. discoideum were found associated with the 40S rather than the 60S ribosomal subunit. Acidic proteins analogous in size and electrophoretic mobility to those of D. discoideum were also detected in several other cellular slime mold strains. Not one of the cellular slime mold acidic proteins reacted with antibodies to E. coli proteins L7 and L12 in immunodiffusion tests. In D. discoideum, the distribution of acidic proteins was altered during development. Amoebae contained all three proteins. In spores, A1 was absent and the relative amounts of A2 and A3 were lower than in amoebae. In addition, nine other acidic ribosomal proteins exhibited differences between vegetative amoebae and spores.     

Resistance of cathepsin L compared to elastase to proteolysis when complexed with the serpin endopin 2C, and recovery of cathepsin L activity

This study demonstrates unique differences in the conformational nature of cathepsin L compared to elastase when complexed with the serpin endopin 2C, assessed by susceptibilities of protease/endopin 2C complexes to proteolysis by trypsin. Complexed and uncomplexed cathepsin L were resistant to degradation by trypsin, which indicated that trypsin cleavage sites within cathepsin L remain inaccessible when this cysteine protease is complexed with the endopin 2C serpin. In contrast, elastase in complexes with endopin 2C was degraded by trypsin, but uncomplexed elastase was not degraded. These results demonstrate a change in the conformational properties of trypsin cleavage sites within elastase when it is complexed with endopin 2C, compared to uncomplexed elastase. Cathepsin L complexes with endopin 2C were short-lived, but elastase complexes were stable. Furthermore, cathepsin L dissociated from complexes demonstrated recovery of cathepsin L activity, and reducing conditions provided optimum recovery of cathepsin L activity. These findings suggest that cathepsin L, when complexed with endopin 2C, maintains its general conformation in a manner that allows recovery of cathepsin L activity upon dissociation from endopin 2C. These results demonstrate differences in the relative conformational properties of the cysteine protease cathepsin L, compared to the serine protease elastase, in complexes with the serpin endopin 2C.     

Recombinant cryptic human fibronectinase cleaves actin and myosin: substrate specificity and possible role in muscular dystrophy

The N-terminal heparin/fibrin binding domain of human plasma fibronectin (pFN) contains a cryptic proteinase. The enzyme could be generated and activated in the presence of Ca2+ from the purified 70 kDa pFN fragment produced by cathepsin D digestion of pFN. In this work we cloned and expressed the serine proteinase, designated fibronectinase (Fnase), in E. coli. The recombinant pFN protein fragment was isolated from inclusion bodies, subjected to folding and autocatalytic degradation in the presence of Ca2+, and yielded an active enzyme capable of digesting fibronectin. Cleavage of pFN and the synthetic peptides Ac-I-E-G-K-pNA and Bz-I-E-G-R-pNA demonstrated identical specificity of the recombinant and the isolated fibronectinase. Further investigations of the substrate specificity revealed for the first time the muscle proteins actin and myosin as being substrates of fibronectinase. The enzyme can be inhibited by alpha1-proteinase inhibitor. In the context of induced cathepsin D release, e. g. from granulocytes under inflammatory conditions, these results indicate an increase in specific proteolytic potential against muscular proteins in dystrophic diseases by the release of cryptic fibronectinase.