Eur. J. Biochem. 2/9, 727-736 (1994)
`© FEBS 1994
`
`Characterization and partial purification of the human receptor
`for the heat-stable enterotoxin
`
`Sandhya S. VISWESWARIAH, Vasanthi RAMACHANDRAN,Surabhi RAMAMOHAN,Goutam DASand Janakiraman RAMACHANDRAN
`
`Astra Research Centre, Malleswaram, Bangalore, India
`
`(Received August 2/October 20, 1993) ~- EJB 93 1152/1
`
`The receptor for the Escherichia coli heat-stable enterotoxin has been characterized and partially
`purified from the T84 humancolonic cell line. Using a novel mutant heat-stable enterotoxin peptide
`as a radioligand (the C-terminal tyrosine residue is replaced by phenylalanine in the mutant), a
`single class of high-affinity receptor sites was detected in T84 cells, with a K, of 0.1 nM, similar in
`affinity to the receptor described in human intestinal tissue. The receptor was solubilised from T84
`cell membranes andaffinity cross-linking of the solubilised preparation indicated that a single spe-
`cies of M, 160000 served as the receptor. Freshly solubilised preparations of the receptor retained
`heat-stable enterotoxin-activable guanylyl cyclase activity. Purification of the receptor was achieved
`through sequential affinity chromatography on GTP~epoxy-Sepharose and wheat-germ-agglutinin
`columns resulting in purification of the receptor by 3000 fold. The heat-stable enterotoxin-binding
`characteristics of the receptor were unchanged during the purification and silver staining of the
`purified receptor preparation indicated a band of M, 160000, which wasspecifically cross-linked to
`the '*°I-labeled mutant peptide. The purified receptor retained guanylyl cyclase activity, but the
`activity was not stimulated on addition of human heat-stable enterotoxin, suggesting that accessory
`structural factors may be involved in the activation of the guanylyl cyclase/receptor.
`
`The heat-stable enterotoxins (ST) are a family of low-
`molecular-mass peptide toxins, and are one of the major
`causes of watery secretory diarrhoea all over the world [1,
`2}. Various forms of the toxins, differing in amino acid se-
`quence, are produced by a number of pathogenic bacteria (3,
`4], and all these peptides contain a cysteine-rich core essen-
`tial for full biological activity [5, 6]. ST peptides bind to
`a receptor on intestinal cells and activate membrane-bound
`guanylyl cyclase [7, 8]. Increased levels of cyclic GMP
`(cGMP) within the cell are hypothesised to lead to enhanced
`Cl secretion from the intestinal cell by as yet undefined
`mechanisms, resulting in fluid loss and subsequent diarrhoea
`[9].
`
`Early biochemical studies had postulated that in rat intes-
`tinal membranes the ST-binding and guanylyl cyclase activi-
`ties were located on separate molecules [10]. However, the
`cloning and expression of the rat and human intestinal ST
`receptor [11-13] suggested that the ST receptor wasin fact
`a high-molecular-mass protein and a memberof the guanylyl
`cyclase family of receptors, described earlier for atrial natri-
`uretic factor and the sea urchin egg peptides [14]. These ob-
`servations implied that the ST-binding and guanylyl cyclase
`activities were present in the same receptor molecule. How-
`Correspondence to S. S. Visweswariah, Astra Research Centre,
`India, P. O. Box 359, Malleswaram, Bangalore, India 560 003
`Fax: +91 80 3340449.
`Abbreviations. cGMP, cyclic GMP; ST, heat-stable entero-
`toxin(s); ST,, human heat-stable enterotoxin(s); ST,, porcine heat-
`stable enterotoxin; ST Y72F,heat-stable enterotoxin with C-terminal
`tyrosine residue replaced by phenylalanine.
`Enzyme. Guanylyl cyclase (EC 4.6.1.2).
`
`ever, there is no biochemical evidence using purified receptor
`preparations to confirm that the ST receptor does in fact exist
`in the cell as suggested for the recombinant protein. Hugues
`et al. reported the purification of the rat ST receptor from
`intestinal membranes using ST—ligand affinity chromatogra-
`phy and suggested that a protein of M, 70000 was the recep-
`tor [15]. However, the purified protein did not possess gua-
`nylyl cyclase activity, and this was attributed to the instability
`of the guanylyl cyclase activity from rat intestinal mem-
`branes during purification procedures. However, the major
`protein purified in these studies was of a lower molecular
`mass than that predicted from the DNA sequence of the
`cloned receptor and hence may not have represented the
`functional ST receptor [15].
`Earlier studies from this laboratory have demonstrated
`[17] that application of ST and other forms of ST peptides,
`produced by a variety of bacteria, to T84 cells, a cell line
`derived from a human colonic carcinoma [18], resulted in
`enhanced cGMPproduction bythe cells, strongly suggesting
`that these peptides all bind to the same receptor. Interestingly,
`the response of T84 cells to various analogues of ST led to
`the differential production of cGMP [19], suggesting altered
`interaction of the ST peptides with the human receptor on
`T84 cells. The T84 cell line thus served as a model system
`to study ST/human receptor interactions in detail. In this
`study, the characterization and purification of the ST receptor
`from T84 cells are described. We report the partial purifica-
`tion of a high-molecular-mass protein that retains both ST-
`binding and guanyly} cyclase activities, and presumably acts
`as a functional ST receptor.
`
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`

`728
`
`MATERIALS AND METHODS
`
`Culture and maintenance of T84 cells
`
`T84 cells were cultured and maintained as described
`earlier
`[18]. Cells were either grown in 24-well dishes
`(Nunc) to a density of 5X10° cells/well, at which time they
`were used directly for monitoring the activation of guanylyl
`cyclase by ST [19], or grown to confluency in bottles and
`harvested for the preparation of membranes.
`
`Production of mutant ST peptide
`
`Mutagenesis was performed on the cloned ST gene [16],
`essentially according to the method of Kunkel [20]. The se-
`quence of the mutagenic primer was 5’ CCGGGTGCTTTT-
`AATAATAT3’, and the sequence of the product mutant
`gene was confirmed by DNA sequencing [21]. The mutant
`gene wascloned into the pET7 vector as a BamHI—Hindlll
`fragment to yield the vector pARC 0730, which was trans-
`formed into the strain BL21 (DE3) as described earlier [16].
`The mutant ST peptide was overexpressed under identical
`conditions described for the human heat-stable enterotoxin
`(ST,) gene [16].
`
`Purification of ST,, and mutant ST peptide
`from the culture fluid of the overexpressing strains
`
`ST,, peptide was purified according to the method re-
`ported earlier [16]. The purification procedure for the ST
`Y72F mutant peptide (tyrosine at position 72 is replaced by
`phenylalanine) was similar to that employed for ST,, except
`for a few modifications. Following adsorption of the peptide
`to Amberlite XAD-2, elution and concentration by lyophili-
`sation of the bound peptide, ST Y72F was purified by re-
`verse-phase HPLC, using an Applied Biosystems 150 series
`HPLC system on an Aquapore RP 300 column (7-um particle
`size, 220 mmX4.6 mm, Applied Biosystems). Gradient elu-
`tion of the bound peptide was achieved using a gradient of
`20—100% B over 30 min at a flow rate of 1 ml/min. Buffer
`A consisted of 0.05% trifluoroacetic acid in water, and buffer
`B consisted of 70% acetonitrile in 0.05 % trifluoroacetic acid/
`water. ST Y72F peptide was eluted with a retention time of
`16-17 min in this gradient. Both ST, and ST Y72F were
`purified again through the gradient system described above
`to ensure homogeneity of the preparations.
`Peptides were quantified by amino acid analysis [22], and
`the analysis confirmed the sequence of the two peptides.
`
`Radio-iodination of ST Y72F and purification
`of the radiolabelled peptide
`Purified ST Y72F was radiolabelled with carrier-free
`Na‘*°I (ICN Radiochemicals) by the lactoperoxidase method.
`Lactoperoxidase beads (BioRad) were reacted with 5 pg ST
`Y72F and 1 mCi carrier-free Na’in 50 pl 100 mM sodium
`phosphate, pH 7.5, containing 40 pg of f-p-glucose, at 4°C
`for 1h. The reaction mixture was applied to a Sep-Pak car-
`tridge (Millipore) previously equilibrated with water. The
`cartridge was washed with water to remove free Na’™I and
`the bound peptide was eluted with 50% acetonitrile in water.
`The fractions containing radiolabelled peptide were concen-
`trated to remove the acetonitrile, and subjected to reverse-
`phase HPLC to purify the radiolabelled peptide. Reverse-
`phase HPLC was performed on a wBondapak C,, (column
`4-um particle size, 2 mm*X30 mm, Waters Associates). The
`
`radiolabelled peptide was eluted using a gradient from 10%
`acetonitrile in water to 30% acetonitrile in water in 90 min
`at a flow rate of 0.3 ml/min. Fractions were collected every
`2 min, and the radiolabelled peptide eluted with a retention
`time of 70—75 min in this gradient. Under identical condi-
`tions, unlabelled ST Y72F eluted with a retention time of
`60-65 min. The specific activity of the radiolabelled ST
`Y72F was 2000Ci/mmol, equivalent to the specific activity
`of the Na’. Radiolabelled ST Y72F was stored at 4°C in
`the presence of 20% acetonitrile, under which conditionsit
`was stable for more than a month.
`
`Guanylyl cyclase assays on T84 monolayers
`Assays were performed as described earlier [19]. Pep-
`tides were added in serum-free media and intracellular cGMP
`was measured by radioimmunoassay after 15 min following
`application of the toxin. Radioimmunoassay was performed
`using '*°I-labelled cGMPas reported earlier [19]. All cGMP
`levels were measured without acetylation of either the sam-
`ple or the standards.
`
`Preparation of T84 membranes
`Confluent monolayers of T84 cells were harvested by
`scraping into 50mM Hepes, pH 7.5, containing 0.25M
`sucrose, 1 g/ml leupeptin, 1 g/ml aprotinin, 2 mM phenyl-
`methylsuiphonyl fluoride, 100 mM NaCl, 1 mM dithiothrei-
`tol and 5 mM EDTA (108 cells/10 ml buffer). Cells were ho-
`mogenised in this buffer and the broken cell suspension was
`subjected to centrifugation at 100000 g for 1h. The mem-
`brane pellet thus obtained was suspended at a concentration
`of 5—10 mg protein/ml in 50 mM Hepes, pH 7.5, 10 pg/ml
`leupeptin and 10 g/ml aprotinin. Protein was estimated by
`the method of Bradford [23].
`
`In vitro activation of guanylyl cyclase
`In vitro guanylyl cyclase assays were performed essen-
`tially according to Hugueset al. [15]. T84 membranefrac-
`tions, or solubilised or purified preparations of the ST recep-
`tor, were suspended in 60 mM Tris/HCl, pH. 7.6, containing
`10 mM theophylline, 7.5 mM creatine phosphate, and 20 pg
`creatine phosphokinase. ST peptides, at the indicated concen-
`trations were added to the mixture, and the assay wasiniti-
`ated by addition of a Mg-GTPsolution to a final concentra-
`tion of 1 mM GTP and 4mM MgCl. The assay mixture
`(final volume 100 11) was incubated at 37°C for 10 min and
`the reaction was stopped by the addition of 0.4 ml 50 mM
`sodium acetate, pH 4.0, and boiling of the reaction mixture
`for 10 min. Samples were centrifuged at 10000 g for 10 min
`and analysed for cGMPby radioimmunoassay. For monitor-
`ing guanylyl cyclase activity following the binding of ST to
`T84 membranes at different pH, ST, (1 WM) was added to
`membrane preparations at the pH indicated, and the incuba-
`tion was continued at 37°C for 10 min. Samples werecentri-
`fuged, the membranes suspended in buffer containing com-
`ponents for the in vitro guanylyl cyclase assay and the assay
`wasstarted by the addition of Mg-GTP. The assay wasper-
`formed at 37°C for 10 min and cGMP was measured as
`described above.
`
`Binding assays
`Binding to T84 membranes was performed using 20—
`50 ug T84 membrane protein,
`in 50mM Hepes, pH 7.5,
`
`MSN Exhibit 1032 - Page 2 of 10
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`
`

`

`4mM MgCl, 0.1% bovine serum albumin, and 10 pg/ml
`leupeptin, at 37°C for 60 min. The total assay volume was
`100 jl and contained 0.1 nM **J-labelled ST Y72F. Non-spe-
`cific binding was determined in the presence of unlabelled
`ST Y72F, ST,,, or atrial natriuretic factor. Following incuba-
`tion, samples were filtered through GF/B filters (Whatman)
`and washed three times with 3 ml chilled 10mM sodium
`phosphate, pH 7.2, containing 0.9% NaCl and 0.2% bovine
`serum albumin. Filters were measured for radioactivity using
`a scintillation counter (LKB Clini Gamma).
`Binding assays at different pH were performed in either
`50 mM sodium acetate, pH 5.0, or 100 mM sodium carbon-
`ate/sodium bicarbonate buffer, pH 10.7. Washing of the fil-
`ters was performed as above.
`Binding assays to solubilised preparations of the receptor
`or purified fractions were performed under identical condi-
`tions in a total volume of 100 yl. However, the separation of
`bound ligand from free peptide was through precipitation
`using poly(ethylene glycol) 6000. Following binding, 200
`30% poly(ethylene glycol) in 50 mM Tris/HCl, 1 mM EDTA,
`pH 7.5, and 20 ul 3 mg/ml bovine immunoglobulin G was
`added to the samples and they were incubated at 4°C for
`10 min. Samples were filtered through GF/B filters pre-
`viously soaked in 0.2% bovine serum albumin, and filters
`were washedthrice with 3 ml chilled 10% poly(ethylene gly-
`col) in 50 mM Tris/HCl, pH 7.5, containing 1mM EDTA.
`Filters were dried and the radioactivity was measured as be-
`fore.
`In all binding assays, non-specific binding contributed to
`less than 10% of the total binding observed. Equilibrium
`binding data were analysed by the LUNDON 1 programme
`supplied by LUNDONSoftware Inc.
`
`Association and dissociation kinetics for the binding
`of '*I-labelled ST Y72F
`
`Association kinetics of '*°I-labelled ST Y72F were deter-
`mined at an ST Y72F concentration of 0.1 nM in the pres-
`ence or absence of 100 nM unlabelled ST Y72F. Binding was
`performed as described above and aliquots of the reaction
`mixture were removedat the times indicated andfiltered. For
`dissociation experiments, membranes were incubated with
`25]-labelled ST Y72F (1 nM)for 1 h followed by centrifuga-
`tion at 25000 g for 10 min at 4°C. Membranes were sus-
`pended in the original binding buffer in the absence of la-
`belled ligand. Aliquots were removed at the times indicated
`and samples werefiltered. The kinetic data for ligand associ-
`ation and dissociation were subjected to the analysis of Wei-
`land and Molinoff
`[24]. The dissociation rate constant
`(k_,) was determined directly from a first-order plot of ligand
`dissociation versus time. The rate of ligand association (k,)
`was determined from the. equation k, =k. (ILR]./{(L]
`[LR]max}), where [L] is the ligand concentration, [LR], is the
`concentration of the complex at equilibrium, [LR]. is the
`maximum number of receptors present and k,,, is the slope
`of the pseudo-first-order plot of In ((LR]./{{LR].—[LR].})
`versus time.
`
`Solubilisation and cross-linking or the ST receptor
`from T84 membranes
`
`T84 membrane suspensions (5 mg/ml) were adjusted to
`0.3% Lubrol PX (Sigma) and 0.5 M NaCl. The suspension
`was gently homogenised and kept stirring at 4°C for 1 h. The
`
`729
`
`sample was centrifuged at 100000 g for 1 h and the superna-
`tant was used as the soluble receptor preparation.
`For cross-linking of '**I-labelled ST Y72Fto the receptor,
`100 ug solubilised receptor preparation was incubated with
`1 nM **I-labelled ST Y72F,either in the presence or absence
`of 100 nM unlabelled ST,,, at 37°C for 1h. The cross-linker
`dithiobis-(succinimidyl propionate), obtained from Pierce,
`was added to a concentration of 2 mM in a minimal volume
`of dimethyl sulphoxide, and the incubation was continued
`at 25°C for 30 min. Samples were subjected to SDS/PAGE
`according to the method of Laemmli [25]. Samples were
`boiled either in the presence or absence of 2-mercaptoethanol
`and electrophoresed in 7.5% acrylamide gels. Following
`electrophoresis, the gels were dried and subjected to autora-
`diography.
`
`Affinity cross-linking of the ST receptor
`from humanintestinal tissue
`
`Biopsy tissue from regions of the human small intestine
`were obtained from a local hospital, and scrapings of the
`mucosal membranetissue were prepared. The scrapings were
`suspended in 50 mM Hepes, pH 7.5, containing 1 pg/ml leu-
`peptin, 100 mM NaCl and 2 mM dithiothreitol and homogen-
`ised. The suspension was centrifuged at 100000 g for 30 min,
`and the membrane fraction suspended in 50 mM Hepes,
`pH 7.5, containing 10 pg/ml leupeptin, 10 pg/ml aprotinin
`and 4 mM MgCl, to a concentration of 20 mg membranepro-
`tein/ml. The suspension was adjusted to 0.3% Lubrol PX and
`0.5 M NaCl, and stirred at 4°C for 1h. The supernatant of
`this suspension, obtained after centrifugation at 100000 g for
`1h, was used for cross-linking analysis in an identical man-
`ner to that employed for the T84 cells, except that 500 ug
`total protein was used for cross-linking.
`
`Purification of the ST receptor from T84 cells
`
`Solubilised receptor preparations were dialysed against
`50 mM Hepes, pH 7.5, containing 0.1% Lubrol PX, 2 mM
`NaN;, 4mM MgCl,, 1 pg/ml leupeptin,
`1 pg/ml aprotinin
`and 20% glycerol. Removal of NaCl in the solubilised prepa-
`rations was essential prior to subjecting the preparation to
`GTP—epoxy-Sepharose affinity chromatography. The GTP—
`epoxy-Sepharose affinity matrix was prepared by coupling
`GTP to epoxy-activated Sepharose (Pharmacia) following the
`manufacturer’s instructions, which resulted in the coupling
`of 8-15 mol GTP/ml agarose. The dialysed receptor prepa-
`ration was mixed gently with the affinity matrix at 4°C for
`16h after which the beads were washed with the dialysis
`buffer to remove unboundproteins. The receptor was eluted
`with the same buffer without MgCl, but containing 10 mM
`EDTA.Fractions were collected and tested for their ability to
`bind radioactive ST Y72F. Fractions containing ST-binding
`activity were pooled, adjusted to 0.5 M NaCl and directly
`applied to a column of wheat-germ lectin coupled to Sephar-
`ose 6B. Unbound proteins were removed by washing with
`the buffer used for dialysis containing 0.5 M NaC} and bound
`proteins were eluted by incorporating 0.5M N-acetylglu-
`cosamine in the buffer. Approximately 35% of the ST-bind-
`ing activity could be recovered in the eluted fractions. Sam-
`ples were subjected to electrophoresis and silver staining
`[26]. Cross-linking of the purified receptor preparations and
`guanylyl cyclase activity were determined as described
`above.
`
`MSN Exhibit 1032 - Page 3 of 10
`MSNv. Bausch - IPR2023-00016
`
`

`

`0.3
`
`0.2
`
`1
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`0.
`
`E
`E
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`
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`
`40
`
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`
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`
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`
`CG
`
`4
`
`8
`
`12
`
`16
`
`20
`
`24
`
`Time (min )
`Fig. 1. Reverse-phase HPLC profile of purified ST Y72F peptide.
`The peptide was purified from the culture supernatants of the strain
`overexpressing the ST Y72F peptide and chromatographed for a
`second time using the gradient described in the Materials and Meth-
`ods section.
`
`RESULTS
`
`Production and characterization of mutant ST peptide
`for radioligand-binding studies
`The ST, form of the toxin has two tyrosine residues
`which can incorporate iodine during preparation of a radio-
`ligand; the presence of an iodine atom in the C-terminal tyro-
`sine residue prevents binding of the radioligand to intestinal
`cells in the porcine heat-stable enterotoxin form (ST,) of the
`toxin [27]. We therefore chose to change the tyrosine residue
`at position 19 of ST, to phenylalanine, thereby retaining the
`hydrophobic nature of the C-terminus but preventing the
`incorporation of iodine into the C-terminal residue during
`radioiodination. Incorporation of iodine at position 5 of ST,
`has been shown not to affect the biological activity of the
`toxin [13].
`The mutant ST peptide, ST Y72F, was purified from the
`culture medium of the overexpressing strain [16], and the
`purity of the peptide was confirmed by reverse-phase HPLC
`(Fig. 1). When similar amounts of purified ST, and ST Y72F
`were applied to the T84 monolayer, a comparable activation
`of guanylyl cyclase was observed, since intracellular concen-
`trations of cGMP were similar at different concentrations of
`the toxin (Fig. 2). This confirmed the equipotency of ST, and
`ST Y72Fin the T84 cell line, and their near identical interac-
`tion with the receptor on T84 cells. The in vivo activity of
`ST Y72F was also confirmed by the suckling mouse assay
`(data not shown), and 10 ng peptide induced fluid accumula-
`tion in the intestine of the suckling mouse, which was a con-
`centration identical to that observed for ST,, [19].
`
`Binding of '*I-labelled ST Y72F to T84 membranes
`
`[ST] (uM)
`Fig. 2. Activation of guanylyl cyclase following application of ST,
`and ST Y72F to T84 monolayers. Purified ST, and ST Y72F were
`applied to T84 cells at the concentrations indicated and the intracel-
`lular concentrations of cGMP were monitored by radioimmunoassay
`as described earlier [19].
`
`Table 1. Activation of guanylyl cyclase in T84 membranes. Pep-
`tides were added to T84 membranes and cGMP was measured by
`radioimmunoassay. The values represent the mean + SE of duplicate
`experiments. ANF,atrial natriuretic factor.
`
`Conditions
`
`Guanylyl cyclase activity
`
`T84 membranes(basal)
`+ST, (1 uM)
`+ ANF(1 1M)
`+ST,, (1 WM) + ANF (1 pM)
`
`pmol cGMP/min/mg protein
`7.9413
`14.9 + 2.6
`6.6+12
`13.2 + 1.7
`
`Table 2. Specificity of binding of '*I-labelled ST Y72F analog.
`T84 membranes were incubated with '*I-labelled ST Y72F (0.1 nM)
`for 1 h at 37°C in the absence or presence of peptides as indicated.
`Samples were filtered and the radioactivity bound to individual fil-
`ters was measured. The values represent the mean + SE ofduplicate
`experiments. ANF,atrial natriuretic factor.
`
`Displacing ligand
`
`None
`ST, (0.2 g/ml)
`ST Y72F (0.2 pg/ml)
`ANF(0.2 j1g/ml)
`
`Amount of bound
`25]labelled ST Y72F
`
`cpm/20 pg protein
`8604 + 260
`1152 + 146
`1063 + 216
`8312 + 320
`
`Membranes were prepared from monolayer cultures of
`T84 cells. On addition of ST, or ST Y72F to the membrane
`preparations of T84 cells, the accumulation of cGMP through
`activation of membrane-bound guanylyl cyclase was ob-
`served (Table 1). Atrial natriuretic factor was unable to in-
`duce cGMP accumulation. These membrane preparations
`were used to monitor the binding of ‘*I-labelled ST Y72F to
`the receptor (Table 2). Specific binding was detected which
`could be efficiently displaced by 100 nM ofeither ST,, or ST
`Y72F, but not atrial natriuretic factor, confirming the earlier
`
`observation [19] that there was no interaction betweenatrial
`natriuretic factor and the ST Y72F receptor.
`Scatchard analysis of the equilibrium binding data of '**I-
`labelled ST Y72F to T84 membranes (Fig. 3) indicated that
`a single class of binding sites of affinity 0.37 nM (+0.03)
`was present on T84 cells at concentrations of 200 fmol/mg
`membrane protein. A similar affinity was detected when
`binding of ’*I-labelled ST Y72F to T84 monolayers was
`studied, indicating no gross changesin affinity during prepa-
`ration of the membranes (data not shown.) Using a truncated
`
`MSN Exhibit 1032 - Page 4 of 10
`MSNv. Bausch - IPR2023-00016
`
`O ST,
`ST Y72F
`
`300
`
`200
`
`100
`
`
`
`cGMP(pmol/10®
`
`celts)
`
`

`

`
`
`
`
`[*1-tabeltedSTY72F]boundlM)
`
`200
`1890
`160
`140
`120
`100
`a0
`
`40
`20
`
`8.0
`5.5
`
`x10°
`
`45
`4.0
`35
`2.0
`2.5
`20
`+5
`to
`
`
`
`(STY72F]tree os
`
`(STY72F]bound
`
`00
`
`00
`
`50
`
`100
`
`150
`
`200
`
`[STY 72F]youna ( #M)
`Fig. 3. Equilibrium binding of ST Y72F to T84 membranes. (A)
`1257_labelled ST Y72F (0.1 nM) was incubated with 50 ug T84 mem-
`brane protein at 37°C for the times indicated, the suspensions were
`filtered and the radioactivity of the filters was measured. Non-spe-
`cific binding was determined at each point by incubation in the
`presence of 100 nM ST,,. The values represent the mean oftriplicate
`determinations in two independent experiments.
`(B) Scatchard
`analysis of the equilibrium binding data. The data presented in
`Fig. 3A were subjected to an analysis using the LUNDON1 pro-
`gramme to determine the K, and the B,,,, values. The K, was found
`to be 0.3 nM and B,,,, was 200 fmol/mg protein.
`
`analog of ST [29], the cloned human receptor showed a Ky
`of 0.1 nM,indicating that the radioligand we have used for
`our studies shows properties similar to ST,. We were unable
`to detect either the presence of a low-affinity binding site
`coupled to the activation of guanylyl cyclase, as has been
`described in rat intestinal membranes, or a second class of
`high-affinity binding sites, as has been reported forrat intes-
`tinal membranesin the presence of NaCl [30].
`Competition binding analysis was performed using equal
`concentrations of ST,and ST Y72F to inhibit the binding of
`'25].labelled ST Y72F to T84 membranes (Fig. 4). The IC.,
`value of ST, (the concentration giving half-maximal binding)
`was similar to ST Y72F indicating that the binding affinities
`of ST, and ST Y72F were not significantly different.
`
`Correlation of ST binding and guanylyl! cyclase activation
`
`to T84
`Application of varying concentrations of ST,
`membranes exhibited a dose-dependent increase in cGMP
`production which correlated with an increase in binding of
`ST to the membranes (Fig. 5). Thus, half-maximal binding
`and half-maximal activation of guanylyl cyclase occurred at
`
`731
`
`bound(%total) a
`
`10
`
`100
`
`1
`
`0.0
`
`02
`
`04
`06
`08
`10
`["i-labelled STY72F},,..( 1M)
`
`12
`
`Leb
`
`[ST](nM)
`
`Fig. 4. Inhibition of binding of '**I-labelled ST Y72F by ST,, and
`ST Y72F. Purified ST,, and ST Y72Fat the concentrations indicated
`were incubated with 0.1 nM ‘*I-labelled ST Y72F and 50 pg T84
`membrane protein for 1h at 37°C, Samples were filtered and the
`radioactivity of the filters was measured. The values represent the
`mean of duplicate determinations of two independent experiments.
`ST, (@); ST Y72F (A).
`
`©
`2
`8
`yO
`Mm
`c
`oO
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`20
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`
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`[ST] (nM)
`
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`6o =
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`we
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`o
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`a.
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`100
`
`Fig. 5. Correlation of ST binding and guanylyl cyclase activation.
`Increasing concentrations of ST, were added to 50 pg of T84 mem-
`brane protein and guanylyl cyclase activity was determined as de-
`scribed in the Materials and Methodssection: In a parallel experi-
`ment, 0.1 nM ‘*I-labelled ST Y72F was incubated with varying con-
`centrations of unlabelled ST,, and the amount of ST,, bound to the
`membranes at each concentration was determined from the binding
`data obtained. The values represent the mean of duplicate determin-
`ations of two independent experiments.
`
`5 nM ST,,indicating that the binding of ST, was functionally
`coupled to the activation of guanylyl cyclase.
`The binding of ST to the human receptor showed a
`marked pH dependency in that the binding of '*I-labelled
`ST Y72F was doubled at pH 5 (Table 3). Further analysis
`revealed no change in the affinity of the receptor to the li-
`gand, but a doubling in the capacity of the binding sites.
`Similar observations were reported earlier using monolayer
`cultures of T84 cells [28], and the cloned human receptor
`expressed in 293 cells [29]. We could show howeverthat the
`increase in binding at pH 5.0 was correlated with an increase
`in cGMPproduction by T84 membranes, indicating a func-
`tional coupling of binding and activation at
`low pH
`(Table 3). A higher basal activity of guanylyl cyclase was
`observed following prior incubation at lower pH, but there
`was a concomitant increase in activation of guanylyl cyclase
`
`MSNExhibit 1032 - Page 5 of 10
`MSNv. Bausch - IPR2023-00016
`
`

`

`10°x"1-labelledSTY72Fbound(cpm) 5 0
`
`
`
`In(Be/Be-Bt)
`
`20
`
`30
`
`40
`
`$o
`
`60
`
`70
`
`
`50
`
`80
`
`Time (min }
`
`Time (min)
`
`Fig.6. Association kinetics of the binding of ™I-labelled ST
`Y72¥F to T84 membranes. (A) '”I-labelled ST Y72F (0.1 nM) was
`incubated at 37°C with T84 membranesfor the times and pH values
`indicated. Samples werefiltered and the radioactivity boundto indi-
`vidual filters was measured. Non-specific binding was determined
`in the presence of 100 nM unlabelled ST,, and has been subtracted
`from the values shown. The data represent the mean oftriplicate
`determinations. (B) A semi-logarithmic representation of the pseu-
`do-first-order association reaction at pH 7.5, where B, and B, repre-
`sent ligand bound at equilibrium and at time ¢, respectively.
`
`732
`
`Table 3. pH dependenceof the binding and activation of guany-
`lyl cyclase by ST. Equilibrium binding of '*I-labelied ST Y72F to
`T84 membranes was performed at the pH indicated and the data
`were subjected to Scatchard Analysis using the LUNDON1 pro-
`gramme. Guanylyl cyclase assays were conducted in the absence of
`peptide to monitor the basal activity present in T84 membranes, or
`in the presence of 1M ST,. The values represent the mean of
`duplicate experiments.
`
`pH
`
`Amount of
`ST bound
`
`fmol/mg protein
`
`15
`5.0
`
`200
`450
`
`Ks
`
`nM
`
`0.31
`0.4
`
`cGMP
`
`—ST,
`
`+ST,,
`
`pmol/min/mg protein —
`
`8.8
`50
`
`29
`110
`
`by ST, corresponding to an enhanced binding of the peptide
`at pH 5.0.
`At a pH greater than 10.0, ST failed to bind to the recep-
`tor in T84 membranes, but this lack of binding was not due
`to inactivation of the receptor, since binding was effectively
`and quantitatively restored on lowering the pH to 7.5 during
`the binding assay (data not shown).
`The association and dissociation kinetics of the binding
`of ST Y72F to T84 membranes are shown in Figs 6 and 7.
`A calculation of the K, from the kinetic data, indicated a
`value of 0.4 nM in close agreement with that estimated from
`a Scatchard analysis. Dissociation of the ligand at pH 5.0
`was slower than that at pH 7.5, despite the similarity in bind-
`ing affinity to that observed at pH 7.5 as estimated from a
`Scatchard analysis (Fig. 3).
`Dissociation of bound ***I-labelled ST Y72F was rapid
`at pH 10.7 (Fig. 7), and binding was eventually abolished,
`suggesting that any local changes in pH in the intestine may
`regulate ST-induced diarrheal responses. A study does indeed
`indicate an increase in alkalinity of the intestinal mucosae
`following application of ST, and this may play a role in the
`transient nature of ST-mediated diarrhoea [31].
`
`Solubilization and affinity cross-linking of radiolabelled
`ST Y72F to the receptor on T84 cells
`
`Affinity cross-linking of ST Y72F
`to the solubilised receptor
`
`Optimum solubilization of the ST receptor from T84
`membranes was achieved using 0.3% Lubrol and 0.5M
`The molecular mass of the ST receptor in T84 cells was
`NaCl. This combination of a non-ionic detergent and NaC]
`determined by affinity cross-linking using the cross-linker
`dithio-bis(succinimidy] propionate). Cross-linking of the so-
`was recommended previously for efficient solubilization of
`lubilised receptor using 0.10M ’J-labelled ST Y72F and
`the rat intestinal membrane receptor [32] and, in the case of
`2mM_dithio-bis(succinimidyl propionate) followed by SDS/
`the humanreceptor in T84 cells, the presence of 0.5 M NaCl
`during solubilization was essential. The affinity of the recep-
`PAGEandautoradiography (Fig. 8) revealed a single radio-
`tor was not significantly changed on solubilisation (Table 4),
`active band of M, 160000, which was abolished when cross-
`and approximately 70% of the receptor could be solubilised
`linking was performed in the presence of 100 nM ST,,. The
`under these conditions. Some amount of ST-binding activity
`M,of the cross-linked protein was similar to the calculated
`was howeverassociated with the insoluble cytoskeletal frac-
`M, based on the predicted sequence of the recombinantpro-
`tion, which also retained guanylyl cyclase activity.
`tein {12, 13]. Treatment of the cross-linked sample with
`Guanylyl cyclase activity was, markedly enhanced during
`100 mM 2-mercaptoethanol resulted in the disappearance of
`solubilisation, and this detergent-induced activation has been
`the cross-linked band on autoradiography, since the thiol-
`reported for all membrane-associated guanylyl cyclases to
`cleavable cross-linker dissociates the ligand from the recep-
`date [33, 34]. On addition of 100 nM ST, to the solubilised
`tor under reducing conditions. No low-M, forms ofthe recep-
`preparation, an increase over the basal activity was observed
`tor were observed after SDS/PAGE,in contrast to the variety
`especially in freshly solubilised preparations (Table 4), but
`of low-molecular-mass receptors that were identified in the
`rat intestinal membranes by cross-linking [35], as well as
`this property was lost on storage (data not shown).
`
`MSN Exhibit 1032 - Page 6 of 10
`MSNv. Bausch - IPR2023-00016
`
`

`

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`60
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`70
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`
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`
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`Time (min)
`
`Fig. 7. Dissociation kinetics of ’*I-labelled ST Y72F binding to
`T84 membranes.
`'**I-labelled ST Y72F (0.1 nM) was incubated
`with T84 membranes for 1 h at 37°C at the pH indicated and the
`sample was centrifuged. Membranepellets were suspended in buffer
`at the pH used during binding, but without labelled ST Y72F and
`aliquots were removed at the times indicated and filtered. Values
`represent the mean oftriplicate determinations of two independent
`experiments. (B) A semi-logarithmic representation ofthe first-order
`dissociation reac