Monitoring of the production of monoclonal antibodies by hybridomas. Part II: Characterization and purification of acid proteases present in cell culture supernatant.

An acid proteolytic activity has been found in cell culture supernatants from long-term cultivations of hybridoma cells in hollow fibre bioreactors using serum free medium. The proteolytic activity has now been further characterized and the main results were: (1) the proteolytic activity showed a maximum around pH 3 and declined essentially to zero at pH 8; (2) the activity was specifically inhibited by pepstatin A; (3) the acid proteases consisted of two sets of closely spaced bands with apparent molecular weights of 40-45K and 90-105K, respectively; (4) the protease bands (40-45K and 90-105K) were reactive with anti-human cathepsin D; (5) the IEP values of the acid proteases ranged from pH 4.55-6.5. Furthermore, IgG incubation with the acid proteases isolated from hybridoma cells yielded fragments similar to those found in serum-free hollow fibre cell culture supernatants. These results indicated that the IgG fragments are the result of degradation by cathepsin D like proteases released after cell death or cell lysis.     

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.     

The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants

Extracellular matrix (ECM), prepared from chick embryo fibroblasts, contains fibronectin as the major structural protein along with collagen and other polypeptides as less abundant protein components. When Rous sarcoma virus-transformed chick embryo fibroblasts are cultured on the ECM in the presence of the tumor promoter tetradecanoyl phorbol acetate, the transformed cells lose their characteristic rounded morphology and align on and within the ECM fibrillar network. This restrictive aspect of ECM is only temporary, however, and with time (24-72 h) the transformed cells progressively degrade the ECM fibers and resume their rounded appearance. The matrix degradation can be monitored by employing biosynthetically radiolabeled ECM. The addition of purified chicken plasminogen to the Rous sarcoma virus- transformed chick embryo fibroblast cultures enhances the rate and extent of ECM degradation, due to the elevated levels in the transformed cultures of plasminogen activator. Plasminogen-dependent and -independent degradation of ECM has been characterized with regard to sensitivity to various natural and synthetic protease inhibitors and to the requirement of cell/ECM contact. Plasminogen-dependent degradation of ECM occurs rapidly when ECM and cells are in contact or separated, whereas plasminogen-independent degradation is greatly reduced when ECM and cells are separated, which suggests that cell surface-associated proteolytic enzymes are involved. A possible role in ECM degradation has been indicated for cysteine proteases, metallo enzymes, and plasminogen activator, the latter as both a zymogen activator and a direct catalytic mediator.     

Matrix metalloproteinases MMP-2 and MMP-9 of wound and burn exudates and their effect on extracellular matrix proteins

The dynamics of matrix metalloproteinases (MMP), as well as of fibronectin concentration in wound and burn fluids was traced. The wound fluid proteolytic activity was studied by gelatin zymography method. The data on degradation of fibronectin and various laminin isoforms by wound fluid proteases show that laminin-1, laminin-2/4 and fibronectin were degraded by wound fluid into small fragments. Remodelling of extracellular matrix proteins occurs. Dynamics of MMP-2 and MMP-9 content in wound or burn fluids as well as that of adhesive protein fibronectin content could be used as a base for development of method of controlling the extracellular matrix remodelling process.     

Degradation of insect cuticle by Paecilomyces farinosus proteases

The entomopathogenic fungus Paecilomyces farinosus showed proteolytic activity in both solid and semi-liquid culture with gelatin as sole N and C source. Semi-liquid cultures were used to characterise proteases. Zymography of crude culture filtrates showed several bands of gelatin degradation in electrophoresis gels. Gel filtration chromatography of these filtrates revealed two peaks of proteolytic activity. Ion-exchange absorption eliminated gelatin from culture filtrates while retaining activity and was used to semipurify P. farinosus proteases. Semipurified culture filtrates had basic pH (8.5 approx.) optimum for proteolytic activity. Treatment of these filtrates with effectors revealed that P. farinosus proteases are serine proteases containing sulphydryl groups. Isoelectrofocusing combined with zymography revealed the presence of several active basic isoforms. Larvae of the lepidopteran Galleria mellonella showed cuticle damage and protein release 1h after incubation with semipurified extracts of P. farinosus. These results indicate that proteolytic enzymes could be involved in insect host penetration by P. farinosus.     

Expression of transformation-associated protease(s) that degrade fibronectin at cell contact sites

Virus-transformed fibroblasts show an increased production of proteases as well as loss of extracellular adhesive proteins. To determine whether these transformation-associated events are related, we investigated the capacity of Rous sarcoma virus-transformed cells (embryonic chick fibroblasts and mouse BALB/c 3T3) to degrade fibronectin by using a novel cross-linked protein substratum: fluorescence-labeled or radiolabeled fibronectin covalently linked to the surface of a fixed gelatin film. In serum-containing medium, the coupled fibronectin was not released when incubated without cells, and only a small amount was released when incubated with nontransformed cells. However, when transformed cells were seeded on the radiolabeled fibronectin-coupled substratum, there was a threefold increase in the time-dependent release of radioactivity into the medium. The released material was characterized as peptides with molecular sizes of less than 30,000 daltons. Correspondingly, growth of transformed cells on the rhodamine-fibronectin substratum resulted in the appearance of discrete negative fluorescent spots beneath the cells and along their migratory paths, whereas a uniform fluorescent carpet was detected with nontransformed cells. The release of radioactivity was partially inhibited by protease inhibitors, including alpha 2-macroglobulin, leupeptin, and benzamidine, but the negative fluorescent spots appeared unaffected by any of these inhibitors. However, both the release of radiolabeled peptides and the appearance of fluorescence-negative spots were inhibited by 1,10-phenanthroline at concentrations that did not affect cellular attachment and protein synthesis, thus supporting a role for proteases in localized degradation of fibronectin substratum. These fluorescence-negative spots coincided with sites of fibronectin disappearance as judged by indirect labeling with antibodies to cellular fibronectin. In addition, immunofluorescent analyses showed a correlation between vinculin localization and the negative fibronectin spots found under transformed cells, indicating that degradation occurs at cell substratum contact sites. These results can be correlated with other transformation-associated phenotypic changes, and are discussed in terms of the invasion of tumor cells into the extracellular matrix.     

Identification and partial characterisation of metalloproteases secreted by a Mesanophrys-like ciliate parasite of the Norway lobster Nephrops norvegicus

A ciliate parasite, tentatively identified as Mesanophrys sp. of Norway lobsters Nephrops norvegicus, is demonstrated to secrete several proteases into the culture medium (modified Nephrops saline). Analyses using substrate-impregnated sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed 12 activity bands differing greatly in mobility in the gels. The complete inhibition of proteolytic activity by 1,10-phenanthroline indicated that the proteases are of the metallo class. The proteases were active at the physiological temperature (8 degrees C) and haemolymph pH (7.8) of the host. The proteases were selective in the degradation of several host proteins, including the myosin heavy chain, which is a major structural component of lobster muscle. Consequently, these proteases may have important roles in several aspects of the host-parasite interaction including invasion, nutrient uptake by the ciliate, and pathogenesis.     

Supernatant proteolytic activities of High-Five insect cells grown in serum-free culture

The proteolytic activity of High-Five insect cell culture supernatants was analysed using substrate gel electrophoresis (zymography). During growth in serum-free media, High-Five cells constitutively expressed and secreted proteases that were active on casein gel but not on gelatin or bovine serum albumin gels. Two main protease bands were visible at about 41–42 kDa and 32–33 kDa. By addition of various protease inhibitors in the incubation buffer, the proteases were identified as metalloproteases as complete and specific inhibition of the proteolytic activities was only obtained by 1,10-phenanthroline.     

Reelin is a serine protease of the extracellular matrix.

Reelin is an extracellular matrix protein that plays a pivotal role in development of the central nervous system. Reelin is also expressed in the adult brain, notably in the cerebral cortex, where it might play a role in synaptic plasticity. The mechanism of action of reelin at the molecular level has been the subject of several hypotheses. Here we show that reelin is a serine protease and that proteolytic activity is relevant to its function, since (i) Reelin expression in HEK 293T cells impairs their ability to adhere to fibronectin-coated surfaces, and adhesion to fibronectin is restored by micromolar concentrations of diisopropyl phosphorofluoridate, a serine hydrolase inhibitor; (ii) purified Reelin binds FP-Peg-biotin, a trap probe which irreversibly binds to serine residues located in active catalytic sites of serine hydrolases; (iii) purified Reelin rapidly degrades fibronectin and laminin, while collagen IV is degraded at a much slower rate; fibronectin degradation is inhibited by inhibitors of serine proteases, and by monoclonal antibody CR-50, an antibody known to block the function of Reelin both in vitro and in vivo. The proteolytic activity of Reelin on adhesion molecules of the extracellular matrix and/or receptors on neurons may explain how Reelin regulates neuronal migration and synaptic plasticity.     

Proteolytic enzymes in normal and transformed cells.

Virally transformed cells show an increased production of proteolytic enzymes. These might be involved in transformation-dependent alterations of cell surface glycoproteins. The possibility arises that some of these proteases might be membrane-bound. To investigate this possibility, we have undertaken a comparative study of the reactivity of intact normal and transformed cells with the tritium labelled protease inhibitor diisopropylfluorophosphate, in parallel with fibrinolytic assays. Using these two approaches in concert, it was possible to identify and localize in the transformed cells several proteases which were present in the particulate cell fraction and were probably membrane bound. In particular, a diisopropylfluorophosphate-reactive polypeptide of 62,000 was increased 5--8-fold on transformation. It comigrated with a fibrinolytic activity. Other particle-bound activities were also detected. While diisopropylfluorophosphate-labelling can be useful for detecting proteases inside cells, it does not appear to be specific for surface proteases.     

Proteolytic enzymes in normal and transformed cells

Virally transformed cells show an increased production of proteolytic enzymes. These might be involved in transformation-dependent alterations of cell surface glycoproteins. The possibility arises that some of these proteases might be membrane-bound. To investigate this possibility, we have undertaken a comparative study of the reactivity of intact normal and transformed cells with the tritium labelled protease inhibitor diisopropylfluorophosphate, in parallel with fibrinolytic assays. Using these two approaches in concert, it was possible to identify and localize in the transformed cells several proteases which were present in the particulate cell fraction and were probably membrane bound. In particular, a diisopropylfluorophosphate-reactive polypeptide of 62 000 was increased 5–8-fold on transformation. It comigrated with a fibrinolytic activity. Other particle-bound activities were also detected. While diisopropylfluorophosphate-labelling can be useful for detecting proteases inside cells, it does not appear to be specific for surface proteases.     

Balamuthia mandrillaris exhibits metalloprotease activities

Balamuthia mandrillaris is a recently identified protozoan pathogen that can cause fatal granulomatous encephalitis. However, the pathogenesis and pathophysiology of B. mandrillaris encephalitis remain unclear. Because proteases may play a role in the central nervous system (CNS) pathology, we used spectrophotometric, cytopathic and zymographic assays to assess protease activities of B. mandrillaris. Using two clinical isolates of B. mandrillaris (from human and baboon), we observed that B. mandrillaris exhibits protease activities. Zymographic assays revealed major protease bands of approximate molecular weights in the region of 40-50 kDa on sodium dodecyl sulfate-polyacrylamide gels using gelatin as substrate. The protease bands were inhibited with 1,10-phenanthroline, suggesting metallo-type proteases. The proteolytic activities were observed over a pH range of 5-11 with maximum activity at neutral pH and at 42 degrees C. Balamuthia mandrillaris proteases exhibit properties to degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue. This is shown by degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as other substrates such as casein and gelatin but not haemoglobin. However, these proteases exhibited a minimal role in B. mandrillaris-mediated host cell death in vitro using human brain microvascular endothelial cells (HBMECs). This was shown using broad-spectrum matrix metalloprotease inhibitors, GM 6001 and GM 1489, which had no effect on B. mandrillaris-mediated HBMEC cytotoxicity. This is the first demonstration that B. mandrillaris exhibits metalloproteases, which may play important role(s) in the ECM degradation and thus in CNS pathology.     

Similarities and differences between the fibronectins of normal and transformed hamster cells

Fibronectins from normal and virally transformed hamster cells were compared by several criteria. The fibronectin from transformed cells was similar to that from normal cells in being an intact dimeric glycoprotein with the ability to bind to gelatin, activated thiol-Sepharose, and cells. No evidence was found for proteolytic cleavages or abnormalities in disulfide bonding of transformed cell fibronectin. This fibronectin was also shown to be active in promoting cell attachment, elongation, and alignment. Therefore, the fibronectin produced by transformed cells is not defective. However, it was shown that the transformed cells were partially deficient in their capacity to bind fibronectins from either normal or transformed cells. This deficiency has implications for the significance of the loss of fibronectin on oncogenic transformation. Partial proteolysis of the fibronectins from normal and transformed cells gave rise to the same fragments. However, the glycosylated fragments from transformed cell fibronectin appeared somewhat larger than those from normal cell fibronectin. Analysis of fibronectin glycopeptides showed that transformation leads both to more branches per core and to a higher sialylation of the asparagine-linked complex carbohydrate side chains.     

Degradation of connective tissue proteins by serine proteases from Streptococcus pneumoniae.

The proteolytic activity of pneumococcal culture supernatants was investigated. Phenylmethylsulfonyl fluoride and diisopropylfluorophosphate inhibited the proteolytic activity by 94% indicating that the enzymes are serine proteases. Zymogram analysis with inhibitors utilizing a non-denaturing gelatin substrate gel revealed two classes of serine proteases; one sensitive to calcium chelators and one resistant. Enzymes from the culture supernatant cleaved fibronectin, fibrinogen, elastin, and laminin; whereas bovine albumin, and the human immunoglobulins, IgG, IgM, and IgA, were not cleaved. These results indicate that pneumococci produce previously unrecognized serine proteases that degrade several tissue and blood proteins.     

Granule-associated serine neutral proteases of the mouse bone marrow-derived mast cell that degrade fibronectin: their increase after sodium butyrate treatment of the cells

Mouse bone marrow-derived mast cells, differentiated in vitro with concanavalin A splenocyte-conditioned medium and sensitized with monoclonal IgE, release neutral serine proteases after activation with specific antigen. Sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis of the supernatants from immunologically activated mast cells revealed the presence of four prominent proteins of 27,000, 29,000, 30,000 and 31,000 m.w. When the supernatants and sonicated residual cells from antigen-challenged or nonactivated IgE-sensitized mast cells were incubated with [3H]diisopropylfluorophosphate ([3H]DFP) and the proteins were subjected to SDS-PAGE followed by autoradiography, proteins of 27,000 to 31,000 m.w. were labeled with [3H]DFP. The antigen-dependent release of labeled proteins was accompanied by a corresponding depletion of similarly sized [3H]DFP-labeled proteins from these cell pellets relative to unactivated cells. The SDS gels were also stained with Coomassie Blue and were sectioned to separate the individual proteins for measurement of their incorporated radioactivity; the net percent antigen-dependent release of all four [3H]DFP-labeled proteins ranged from 64 to 68% and was comparable to that of the secretory granule markers, beta-hexosaminidase and histamine. That the [3H]DFP-labeled proteins were derived from the secretory granules of the cells was supported by studies in which mast cells were cultured for 4 days in the presence of 1 mM sodium butyrate. This treatment produced a differential increase in their cellular content of histamine (10-fold), [3H]DFP binding proteins (two- to fourfold), and beta-hexosaminidase (minimally), while the net percent antigen-dependent release of each of these constituents was unchanged. After sensitization and antigen activation, the net percent release of histamine, beta-hexosaminidase, and the four [3H]DFP-labeled proteins was 51, 59, and 53 to 61%, respectively, for sodium butyrate-treated cells, and 53, 60, and 64 to 68%, respectively, for cells not exposed to sodium butyrate. Human plasma fibronectin was used as a substrate to demonstrate that the exocytosed proteins possessed proteolytic activity. As assessed by optical density scanning of stained SDS-PAGE gels of the substrate, the proteases present in the supernatants of antigen-activated cells, but not of sensitized unchallenged cells, rapidly degraded native fibronectin at pH 7.0. This degradation was prevented by pretreatment of the exocytosed proteins from immunologically activated cells for 90 min at 37 degrees C with 2 mM DFP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhanced degradation of oxidized glutamine synthetase in vitro and after microinjection into hepatoma cells

Mixed-function oxidation of Escherichia coli glutamine synthetase has previously been suggested to mark the enzyme for intracellular degradation, and in vitro studies have demonstrated that oxidation renders the enzyme susceptible to proteolytic attack. In this study, the susceptibility of glutamine synthetase to degradation by purified proteases has been compared with the rate of degradation after microinjection into hepatoma cells. Upon exposure to an ascorbate mixed-function oxidation system the enzyme rapidly loses most of its activity, but further oxidation is required to cause susceptibility to extensive proteolytic attack either by a high-molecular-weight liver cysteine proteinase or by trypsin. The rate of degradation of biosynthetically 14C-labeled native and oxidized glutamine synthetase preparations after injection into hepatoma cells parallels their susceptibility to proteolysis in vitro. Native enzyme preparations and enzyme oxidatively inactivated, but not susceptible to extensive degradation by purified proteases, had similar intracellular half-lives; however, oxidized enzyme preparations that were susceptible to proteolytic breakdown in vitro were degraded almost ten times faster than the native enzyme within the growing hepatoma cells. These results suggest that the same features of the oxidized enzyme that render it susceptible to proteolysis in vitro are also recognized by the intracellular degradation system. In addition, they show that loss of enzyme activity does not necessarily imply decreased metabolic stability.     

Effect of Aeromonas proteases on the binding of Aeromonas hydrophila strains to connective tissue proteins

125I-labelled connective tissue protein binding to cells of Aeromonas hydrophila, A. caviae, and A. sobria strains isolated from diseased fish, was correlated with the Aeromonas protease degradation of 125I-labelled collagen types I and IV, fibronectin and laminin, immobilized on tissue culture microtitre plates. An inverse relation between 125I-labelled connective tissue protein binding to cells of Aeromonas strains and proteolytic degradation of immobilized connective tissue proteins by Aeromonas proteases was established. Inhibition of the Aeromonas proteolytic activity by protease inhibitors enhances the 125I-labelled connective tissue protein binding to cells of Aeromonas hydrophila strains. Culture conditions were found to influence both expression of proteolytic activity and binding properties.     

Glycosaminoglycans enhance phorbol ester-induced proteolytic activity and angiogenesis in vitro

Summary It has been reported that endothelial cells suspended in three-dimensional type I collagen gels can be induced to undergo tube formation by 12-o-tetradecanoyl phorbol 13-acetate (TPA). In this report, we show that TPA-induced endothelial cell tube formation can be further enhanced by the addition of other matrix components in the collagen gels. In the presence of TPA, both high molecular weight hyaluronate and chondroitin sulfate elicit a dose-dependent stimulation of tube formation. The enhanced tube formation appears to be due to an increase in the number of cells undergoing morphogenesis as the average length per tube is not obviously increased. Concomitant with the increased cell morphogenesis, there is an increase in proteolytic activity secreted by the cells. Treatment of cells with cycloheximide suppresses hyaluronate- and chondroitin sulfate-enhanced cell morphogenesis and proteolytic activity suggesting that new protein synthesis, perhaps proteases, is necessary for endothelial cell morphogenesis. The possible role of the production of proteolytic activity in endothelial cell tube formation is discussed.     

Toxocara canis: proteolytic enzymes secreted by the infective larvae in vitro

Second-stage larvae of the dog nematode Toxocara canis are infective to man and cause the syndromes of visceral larva migrans and ocular toxocariasis. Larvae cultured in vitro secrete proteases which degrade components of a model of extracellular matrix and basement membranes. These enzymes have been characterized using a variety of techniques. Multiple enzyme activities were demonstrated by substrate gel electrophoresis, associated with proteins of molecular weights of 120 and 32 kDa. The enzyme activity was inhibited both in substrate gels and in a radiogelatin microplate assay by phenylmethylsulfonyl fluoride. Optimal activity occurred at pH 9, with minor activities apparent at pH 5 and 7; the relationship between these proteolytic activities is currently under investigation.     

Calcium-Dependent Proteases in Neuroblastoma Cells

Abstract: Extracts of neuroblastoma cells contained calcium-dependent proteolytic activity. This activity was accounted for by two distinct proteases. These proteases were separated by ion-exchange chromatography. Although each was totally dependent on calcium, the calcium requirements of the enzymes were different; one enzyme required approximately 40 μM Ca²⁺ for half-maximal activity and the other enzyme required approximately 150 μM Ca²⁺ for half-maximal activity.