Labelling of Tumour Cells with a Biotinylated Inhibitor of a Cell Surface Protease

Abstract

Our objective has been to prepare a biotinylated affinity probe for the active centre of a protease associated with the surface of tumour cells. We employed three model systems in which easily recognisable tumour cells containing the active protease were uaed as targets for the biotinylated affinity probe. These were: squamous cell carcinoma, leukaemia cells in muscle and outgrowths of prostate carcinoma cells grown in three dimensional collagen gels. The presence of the bound biotinylated affinity probe was demonstrated by its ability to bind Texas-red labelled streptavidin with the results that the tumour cells exhibited red fluorescence. This binding was shown to be competitive with 9-amino acridine, a compound known to bind to the active centre of the target protease. This technique depends upon the affinity of the active centre of an enzyme for a competitive inhibitor and therefore should be applicable to other enzyme systems employing suitable ligands for their activc centres.     

Temporary competitive inhibition of a tumour cell surface protease as a protective mechanism in the preparation of the membrane bound native enzyme in the presence of excess cytoplasmic inhibitors.

Tumour cells possess a cell surface protease which is recognised and inhibited by a cytoplasmic protein extractable from frozen sections of tumour cells. In order to prepare sections with tumour cells carrying cell surface-bound native protease in the absence of this internal inhibitor we have used a reversible competitive inhibition step as a temporary measure to protect the active centre of GB whilst the cytoplasmic inhibitor is extracted from the frozen sections. These sections are described as protected in the sense that the enzyme is native and fully functional now that potential inhibitors have been extracted. The protected cell surface protease immobilised in the cell surface of squamous cell carcinoma cells has been used as the target for inhibition studies and displacement studies. The ability to follow these inhibition and exchange reactions concerning the cell surface protease has been made possible by virtue of the fluorescent probe, 9-amino acridine, which locates the active centre of the protease. Cells with active protease bind 9-amino acridine and fluoresce yellow; cells lacking this protease or having inhibited protease fail to bind 9-amino acridine and do not fluoresce.     

Competitive inhibition of a tumour cell surface protease. A rapid technique for in vitro testing of selective targeting systems

The active centre of a protease on the surface of tumour cells can be located by its affinity for an active site-directed inhibitor, 9-amino acridine. Cells which have uninhibited proteases, bind 9-amino acridine and fluoresce in resin sections. The leukaemic rat was used as a model system to provide tumour cells in a well defined location. Drugs when coupled to a ligand (directed to the active centre of the protease) compete for this binding site with 9-amino acridine. Thus, competitive inhibition of the tumour cell surface protease provides a rapid technique for demonstrating the delivery of liganded molecules to the surface of tumour cells in vitro.     

Inhibition of a cell surface protease after cisplatin chemotherapy.

The epithelial cells in squamous carcinoma and leukoplakia of the oral cavity possess the cell surface protease, guanidinobenzoatase (GB), in an active form. GB is closely similar to plasminogen activator, a protease associated with both transformed cells and tumour cells. The active centre of GB binds the fluorescent probe 9-aminoacridine (9-AA) enabling cells containing active GB to be visualised by fluorescent microscopy. It was observed that chemotherapy with cisplatin resulted in a marked decrease in cell surface GB activity and this decrease was due to the formation of an enzyme-inhibitor complex. One of the results of chemotherapy was shown to be the suppression of a cell surface protease which is known to be associated with migration and malignancy of cells in vivo.     

Binding properties of biotinylated epidermal growth factor to its receptor on cultured cells and tissue sections

A biotinylated derivative of murine epidermal growth factor (EGF) was prepared by covalent attachment of the terminal amino group of EGF to N-biotinyl-epsilon-aminocaproyl-N-hydroxysuccinimide. The stoichiometry of biotin incorporation was in the range of one biotin moiety per EGF molecule. The biotinylated EGF (biotinyl-epsilon-caproyl-EGF, BioEGF) binds to EGF receptors on intact Ehrlich ascites carcinoma (EAC) cells with an affinity similar to that of native EGF and displays the same mitogenic activity as EGF in a soft agar test system with normal rat kidney (NRK) cells. BioEGF was visualized on cultured cells and tissue sections of a head and neck tumour by commercial streptavidin/avidin detection systems. Cytochemical analyses of certain tumour forms can be easily performed using the BioEGF probe.     

The design of fluorescent probes which bind to the active centre of guanidinobenzoatase. Application to the location of cells possessing this enzyme

Cells possessing a known enzymic activity may be located by fluorescent probes designed to act as competitive inhibitors of this enzyme. We have prepared a series of dansyl N-substituted guanidino derivatives which bind to the active centre of guanidinobenzoatase. 9-Aminoacridine also acts as a competitive inhibitor and behaves similarly to these guanidino derivatives. These fluorescent probes have been used to locate tumour cells possessing this enzyme in thin sections of fixed tissue by employing fluorescent microscopy.     

Fluorescent location of human colonic tumour cells by means of an enzyme-inhibitor complex

We studied the enzymic status of the tumour cell surface protease, guanidinobenzoatase (GB) in frozen sections of a human colonic tumour grown in nude mice and also in human colons. Active enzyme was demonstrated by the binding of a synthetic fluorescent probe for the active centre of guanidinobenzoatase (GB). It was observed that tissue derived inhibitors of GB blocked the binding of this fluorescent probe and that enzyme inhibitor complex formation could be controlled by lowering the pH of the medium with lactic acid. The presence of an inhibitor of GB in the mouse tumour extract was taken advantage of by making two fluorescent derivatives of this inhibitor; both of which located GB on colonic tumour cells in frozen sections of human colon.     

Fluorescent Location of Human Colonic Tumour Cells by Means of an Enzyme-Inhibitor Complex

Abstract

We studied the enzymic status of the tumour cell surface protease, guanidinobenzoatase (GB) in frozen sections of a human colonic tumour grown in nude mice and also in human colons. Active enzyme was demonstrated by the binding of a synthetic fluorescent probe for the active centre of guanidinobenzoatase (GB). It was observed that tissue derived inhibitors of GB blocked the binding of this fluorescent probe and that enzyme inhibitor complex formation could be controlled by lowering the pH of the medium with lactic acid. The presence of an inhibitor of GB in the mouse tumour extract was taken advantage of by making two fluorescent derivatives of this inhibitor; both of which located GB on colonic tumour cells in frozen sections of human colon.     

Direct evidence for the cell surface location of a protease-inhibitor complex on intact leukaemia cells

The interaction of a protease with two fluorescent inhibitors has been studied using intact fixed leukaemia cells as the source of the membrane bound enzyme. Fresh rat leukaemia cells were disrupted and the cytosol collected; this extract was known to contain a protein inhibitor of guanidinobenzoatase (GB) associated with leukaemia cells. All the cytosolic proteins were derivatised with Texas red acid chloride. Leukaemia cells with latent GB failed to bind the Texas red inhibitor protein but did so after activation of GB. Competition experiments with 9-amino acridine (a fluorescent marker for the active site of GB) demonstrated that the Texas red-inhibitor protein could only bind to intact leukaemia cells when the active centre of GB was not already occupied by 9-amino acridine. This competition between these two fluorescent inhibitors demonstrated their specificity for GB. The use of intact leukaemia cells and the high molecular weight of the inhibitor protein precludes the possibility of any interaction between GB and inhibitor within the cells. It is concluded that GB and the GB-inhibitor complex of latent GB are located on the external surface of intact leukaemia cells.     

The effect of avidin-biotin interactions in detection systems for in situ hybridization.

The effect of avidin-biotin interactions in several detection systems for the non-radioactive in situ hybridization (ISH) technique was studied in a model system using a transitional cell carcinoma line and a biotinylated DNA probe. We performed fluorescence ISH to unravel the individual steps in a sensitive and frequently used amplification method which makes use of the alternating cytochemical detection layers of fluorescein isothiocyanate-conjugated avidin (AvFITC) and biotinylated goat anti-avidin (BioGAA) antibodies to detect the hybridized and biotinylated probe. Our experiments revealed that BioGAA antibodies bind with their antigen binding sites and not with their biotin moieties to avidin molecules that have already interacted with the DNA probe. The probable working mechanism of this amplification method is presented in a model. Furthermore, we used a peroxidase staining technique to compare with each other the sensitivity of several other detection systems in which avidin-biotin interactions play an important role, e.g., the avidin-biotinylated peroxidase complex (ABC) system. The experiments show that avidin molecules can not be efficiently used to interconnect two biotinylated molecular layers, since their introduction leads to firmly closed cytochemical networks. Such a closed network is already formed between the hybridized and biotinylated DNA probe and a first detection layer of avidin molecules, as appears from the finding that biotinylated molecules could hardly be coupled to these avidin molecules in a following detection layer. Therefore, the results presented here provide us with new insight into the molecular basis of cytochemical network formation. This will enable us to choose the proper procedures for increasing the sensitivity of ISH detection systems.     

Inhibition of a protease associated with tumour cells and the probable mechanism of regulation of this interaction in vivo

The regulation of activity of a protease on the surface of human colonic tumour cells implanted in nude mice has been studied. A synthetic fluorescent probe was used to locate cells possessing active guanidinobenzoatase in frozen sections of tumour bearing tissues. These studies demonstrated the presence of intracellular protein inhibitors of guanidinobenzoatase which could be extracted in isotonic saline and transferred to the outer surface of the tumour cells. This study showed that the inhibition of guanidinobenzoatase was pH dependent and that the tumour cell may regulate the activity of its surface guanidinobenzoatase by exporting lactic acid.     

Further inhibition studies on guanidinobenzoatase, a trypsin-like enzyme associated with tumour cells

Guanidinobenzoatase is a proteolytic enzyme capable of degrading fibronectin and is a tumour associated enzyme. Guanidinobenzoatase has been shown to be an arginine selective protease and is distinct from trypsin, plasminogen activator, plasmin, thrombin and a newly described tumour associated enzyme specific for guanidino phenylalanine residues. These conclusions have been derived from inhibition studies employing 4-methyl-p-guanidinobenzoate as substrate. Three active site titrants for trypsin have been shown to be good substrates for guanidinobenzoatase. A new active site titrant for trypsin, rhodamine bisguanidinobenzoate, can also be used to assay guanidinobenzoatase in a stoichiometric manner. This active site titrant can be employed to label guanidinobenzoate on the surface of leukaemia cells.     

Affinity purification of the HIV-1 protease

An inhibitor of the HIV-1 protease has been employed in the generation of a resin which allows the rapid purification of this enzyme. A peptide substrate analogue, H2N-Ser-Gln-Asn-(Phe-psi[CH2N]-Pro)-Ile-Val-Gln-OH, was coupled to agarose resin. The HIV-1 protease was expressed in E. coli and the supernatant from lysed cells was passed through the affinity resin. Active HIV-1 protease was then eluted with a buffer change to pH 10 and 2 M NaCl. Final purification to a homogeneous preparation, capable of crystallization, was achieved with hydrophobic interaction chromatography. Solutions containing HIV-1 protease bound to competitive inhibitors do not bind to the column.     

Affinity cleavage and targeted catalysis of proteins using the avidin-biotin system

The avidin-biotin system was used in order to target enzymes to their substrates in complex mixtures of proteins in solution. The approach described here thus mimics natural systems in which enzymes usually act in selective fashion, due, perhaps, to proximity effects. For affinity cleavage studies, biotinyl transferrin was used as a model target substrate. Avidin or streptavidin was then employed to bridge between the biotinylated target protein and a biotinyl protease. Bovine serum albumin was included in the reaction mixtures to assess the level of nonspecific cleavage. In the case of an unbiotinylated target protein, avidin could be used to inhibit the hydrolytic action of the biotinyl protease. In some systems, a biotinyl antibody could be used to direct the avidin-bridged biotinyl protease to an unbiotinylated target antigen. The data support the contention that preferential cleavage reflects two separate phenomena: (i) avidin confers a conformational alteration of the biotinylated target protein, and (ii) the biotinyl protease is targeted (via the avidin bridge) to the proximity of the biotinylated target protein, thereby promoting cleavage of the conformationally altered molecule. This is the first report in which a proteolytic enzyme could be selectively targeted to specifically hydrolyze a defined protein substrate in solutions containing a complex mixture of other proteins. The approach appears to be a general phenomenon for "targeted catalysis", appropriate for other applications, particularly for affinity cleavage and targeted catalysis of cell-based macromolecules.     

A scintillation proximity active site binding assay for the hepatitis C virus serine protease

A binding assay suitable for the identification of active site-directed inhibitors of the hepatitis C virus serine protease NS3 was developed. A C-terminal extension of 13 residues that is specifically recognized by the Escherichia coli biotin holoenzyme synthetase (Bir A) was fused to a truncated NS3 protease domain, allowing the efficient production of in vivo biotinylated protease. This enzyme was purified and shown to have the same properties as its wild-type counterpart concerning substrate binding and turnover, interaction with a cofactor peptide, and inhibition by three different classes of inhibitors. Immobilization of the biotinylated protease, using streptavidin-coated scintillation proximity beads, allowed detection, by scintillation counting, of its interaction with a tritiated active site ligand spanning the whole substrate binding site of the protease from P6 to P4('). Immobilization did not measurably affect accessibility to either the active site or the cofactor binding site of the protease as judged by the unchanged affinities for a cofactor peptide and for two active site binders. Using the displacement of the radioligand as readout, we were able to set up a rapid, robust, and fully automated assay, suitable for the selective identification of novel active site ligands of the NS3 protease.     

In vitro characterization of the presenilin-dependent gamma-secretase complex using a novel affinity ligand

Gamma-secretase is the enzyme activity releasing the amyloid-beta peptide from membrane-bound processing intermediates derived from the beta-amyloid precursor protein. Cellular release and subsequent aggregation of the amyloid-beta peptide is thought to be causative for the pathogenesis of Alzheimer's disease. Gamma-secretase performs an unusual intramembranous cleavage and has been closely linked to a macromolecular complex containing presenilins. To generate a molecular probe for gamma-secretase, we have developed a novel biotinylated affinity ligand which is based on a specific inhibitor containing a hydroxyethylene dipeptide isostere, known to serve as a transition state analogue for aspartic proteinases. Using this probe we confirmed the presence of the presenilin heterodimer and mature nicastrin in the active enzyme complex and, furthermore, that substrate binding site(s) and active center(s) are spatially separated. Affinity precipitations suggest that only a discrete fraction of cellular presenilin is present in the active gamma-secretase complex and that both gamma(40)- and gamma(42)-activities are mediated by the same molecular entity. This was also reflected by a co-distribution of both enzyme activities in subcellular fractions enriched for trans-Golgi network membranes.     

Inhibition of guanidinobenzoatase by a substrate for trypsin-like enzymes

Guanidinobenzoatase is a proteolytic enzyme capable of degrading fibronectin and is a tumour associated enzyme. Guanidinobenzoatase has been shown to be an arginine selective protease and is distinct from trypsin, plasmin and thrombin, the latter enzymes can be assayed with bis(carbobenzyloxycarbonyl-L-argininamido)-Rhodamine or BZAR. Guanidinobenzoatase is inhibited by BZAR when the enzyme is assayed in free solution and when the enzyme is cell-bound in frozen sections of tumour containing tissues. It is proposed that BZAR and its analogues may be of value in inhibiting tumour cell invasion in vivo and also that the selectivity of BZAR may be used to direct cytotoxic drugs to tumour cells possessing active guanidinobenzoatase.     

Fluorescent inhibitors of a cell surface protease used to locate leukaemia cells in kidney sections

Guanidinobenzoatase is a trypsin-like protease on the surface of cells capable of migration, for example leukaemia cells. We have used a number of fluorescent probes that are competitive inhibitors of guanidinobenzoatase to locate leukaemia cells in resin sections of kidney tissue obtained from leukaemic rats. We have demonstrated how this competitive inhibition system can be used to direct desired molecules (such as cytotoxic drugs) to these cells and to monitor the arrival of such compounds at the active site of guanidinobenzoatase. The principles developed in this study could equally well be applied to other enzymes on other cells provided suitable competitive inhibitors were designed. The presence of an enzyme on the surface of a cell can be used to direct molecules to that cell provided that these molecules contain a functional group that acts as an inhibitor for the chosen enzyme.     

Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease

Drug resistance in HIV-1 protease, a barrier to effective treatment, is generally caused by mutations in the enzyme that disrupt inhibitor binding but still allow for substrate processing. Structural studies with mutant, inactive enzyme, have provided detailed information regarding how the substrates bind to the protease yet avoid resistance mutations; insights obtained inform the development of next generation therapeutics. Although structures have been obtained of complexes between substrate peptide and inactivated (D25N) protease, thermodynamic studies of peptide binding have been challenging due to low affinity. Peptides that bind tighter to the inactivated protease than the natural substrates would be valuable for thermodynamic studies as well as to explore whether the structural envelope observed for substrate peptides is a function of weak binding. Here, two computational methods-namely, charge optimization and protein design-were applied to identify peptide sequences predicted to have higher binding affinity to the inactivated protease, starting from an RT-RH derived substrate peptide. Of the candidate designed peptides, three were tested for binding with isothermal titration calorimetry, with one, containing a single threonine to valine substitution, measured to have more than a 10-fold improvement over the tightest binding natural substrate. Crystal structures were also obtained for the same three designed peptide complexes; they show good agreement with computational prediction. Thermodynamic studies show that binding is entropically driven, more so for designed affinity enhanced variants than for the starting substrate. Structural studies show strong similarities between natural and tighter-binding designed peptide complexes, which may have implications in understanding the molecular mechanisms of drug resistance in HIV-1 protease.