Opossum colonic mucosa contains uroguanylin
`and guanylin peptides
`
`F. KENT HAMRA, WILLIAM J. KRAUSE, SAMMY L. EBER, RONALD H. FREEMAN,
`CHRISTINE E. SMITH, MARK G. CURRIE, AND LEONARD R. FORTE
`The Truman Veterans Affairs Medical Center and Departments of Pharmacology, Anatomy,
`and Physiology, School of Medicine, Missouri University, Columbia 65212;
`and Searle Research and Development, St. Louis, Missouri 63167
`
`Hamra,F. Kent, William J. Krause, SammyL. Eber,
`Ronald H. Freeman, Christine E. Smith, Mark G. Cur-
`rie, and Leonard R. Forte. Opossum colonic mucosa con-
`tains uroguanylin and guanylin peptides. Am. J. Physiol. 270
`(Gastrointest. Liver Physiol. 33): G708—G716, 1996.—Urogua-
`nylin and guanylin are structurally related peptides that
`activate an intestinal form of membrane guanylate cyclase
`(GC-C). Guanylin wasisolated from theintestine, but urogua-
`nylin was isolated from urine,
`thus a tissue source for
`uroguanylin was sought. In these experiments, uroguanylin
`and guanylin were separated and purified independently
`from colonic mucosa and urine of opossums.Colonic, urinary,
`and synthetic formsof uroguanylin had anisoelectric point of
`~3.0, eluted from Cys reverse-phase high-performanceliquid
`chromatography (RP-HPLC) columns at 8-9% acetonitrile,
`elicited greater guanosine3’ ,5’-cyclic monophosphate (cGMP)
`responses in T84 cells at pH 5.5 than pH 8, and were not
`cleaved andinactivated by pretreatment with chymotrypsin.
`In contrast, colonic, urinary, and synthetic guanylin had an
`isoelectric point of ~6.0, eluted at 15-16% acetonitrile on Ci,
`RP-HPLC columns, stimulated greater cGMP responses in
`T84 cells at pH 8 than pH 5.5, and were inactivated by
`chymotrypsin, which hydrolyzed the Phe-Ala or Tyr-Ala
`bonds within guanylin. Uroguanylin joins guanylin as an
`intestinal peptide that mayparticipate in an intrinsic path-
`way for cGMP-mediated regulation of intestinal salt and
`water transport. Moreover, uroguanylin and guanylin in
`urine may be derived from the intestinal mucosa,
`thus
`implicating these peptides in an endocrine mechanism link-
`ing the intestine with the kidney.
`guanylate cyclase; T84 cells; guanosine3’ ,5’-cyclic monophos-
`phate; urine
`$$
`
`UROGUANYLIN AND GUANYLIN are membersof an emerg-
`ing family of peptides that function as the physiological
`ligandsfor an intestinal isoform of membrane guany]l-
`ate cyclase (GC-C) (7, 18; see Ref. 13 for review).
`Uroguanylin and guanylin stimulate GC-C activity,
`resulting in elevations of cellular guanosine 3’,5'-cyclic
`monophosphate (cGMP)(7, 18). All species of mammals
`and birds examined express GC-C-like receptoractivity
`on the apical surface of enterocytes throughout the
`intestine (21, 22). The opossum kidney also expresses
`high levels of GC-C-like receptors located in the apical
`membranes of proximal tubular cells (14). Guanylin
`was first isolated from rat jejunum as a heat-stable,
`15-amino acid peptide that activated GC-C in human
`intestinal T84 cells (7). Guanylin cDNAsencoding 115-
`to 116-amino acid precursors have been isolated from
`rat, human, and mouseintestine (19). Uroguanylin was
`initially purified as 13- to 15-amino acid peptides from
`G708
`
`0193-1857/96 $5.00 Copyright © 1996 the American Physiological Society
`
`opossum urine (18) and was named onthe basis ofits
`structural and functional similarities to guanylin and
`its isolation from urine. Uroguanylin was confirmed as a
`second memberin the guanylin peptide family by the
`subsequentisolation of opossum guanylin (18) and the
`humanandrat formsof uroguanylin (10, 20).
`Before the discovery of guanylin and uroguanylin,
`the only peptide agonists for GC-C were the diarrhea-
`producing, heat-stable enterotoxins (STs) (11, 34). STs
`are producedby different strains of pathogenic, enteric
`microorganisms, including Escherichia coli (25). STs
`act as molecular mimics of guanylin and uroguanylin
`by binding to GC-C and stimulating dramatic increases
`in cellular cGMP accumulation (11, 18, 31). ST-
`stimulated cGMPproduction decreases sodium absorp-
`tion and increases chloride secretion by enterocytes,
`which results in secretory, or “traveler’s,” diarrhea (11,
`25, 31). Similar to STs, uroguanylin and guanylin
`stimulate transepithelial chloride secretion from T84
`cells and intestinal tissues mounted in Ussing cham-
`bers by increasing cellular cGMPproduction (7, 18, 20).
`Thusprevious studies of the mechanismsby which STs
`exert their pathological effects may provide insights
`into the physiological roles of uroguanylin and gua-
`nylin.
`*Guanylin mRNAis found throughout the intestine,
`with the highest levels of expression in the ileum and
`colon (20). Guanylin and/or its mRNA has been re-
`ported to occur in a heterogeneouspopulationofintesti-
`nal cell types, including absorptive enterocytes (27),
`Panethcells (9), enterochromaffin cells (4), and goblet
`cells (6). Northern analysis has also demonstrated
`lower levels of guanylin mRNAin the kidney, adrenal
`gland, and the uterus/oviduct (33). Moreover, progua-
`nylin has been shownto circulate in the plasma of
`humans, demonstrating that guanylin may potentially
`regulate GC-C or other target receptors via an endo-
`crine mechanism (23, 24, 30). Presently, uroguanylin
`has only been isolated from urine, andlittle is known
`regarding the tissues that may produce this peptide
`(10, 18, 20). The high levels of uroguanylin found in the
`urine of opossums, humans,andrats could be derived
`from the kidney and/or from othertissuesvia filtration
`of uroguanylin from the circulation. In the current
`study, we have isolated uroguanylin and guanylin
`peptides from the colonic mucosa of opossums. Several
`independent analytical techniques were used to iden-
`tify the bioactive peptides. Thus intestinal mucosais a
`potential tissue source for uroguanylin and guanylin
`foundin urine (10, 18, 20).
`
`MSNExhibit 1019 - Page 1 of 9
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`

`

`UROGUANYLIN: AN INTESTINAL PEPTIDE
`
`G709
`
`MATERIALS AND METHODS
`
`Purification of colon peptides. Full-length colons, including
`cecums,were removedfrom adult opossums (Didelphis virgin-
`tana), and the mucosae (150 g wet wt) were scraped from
`colonic muscle with the use of a glass microscopeslide. Only
`healthy opossums with hard stools were used in these stud-
`ies. The mucosaeweredivided into two batches, which were
`processed separately (75 g wet wt/batch). Each batch was
`suspended in 10 vol of 1 M acetic acid, heated at 100°C for 10
`min with constant stirring, homogenized, and stored at
`—20°C. The homogenate was thawed and centrifuged at
`10,000 g for 20 min, and the supernatant was made to 0.1%
`trifluoroacetic acid (TFA). The supernatants were processed
`with Waters Sep-Pak cartridges [octadecylsilane cartridges
`(Cjg)] as described previously (7, 18). Eluted fractions of the
`colon extract from Cj, cartridges were dried in a Speed-Vac,
`resuspended in 10 ml of 25 mM ammoniumacetate, pH 5.0,
`and centrifuged at 500 g for 10 min. From eachbatch, 8.0 ml
`of supernatant were applied to a 2.5 X 90 cm Sephadex G-25
`column, and 10-mlfractions werecollected for two successive
`runs. Fractions from this step and subsequent purification
`steps were assayed for bioactivity as described in cGMP
`accumulation assay in T84 cells. Bioactive fractions from
`each batch were combined after the Sephadex G-25 step.
`Final purification by isoelectric focusing on a Rotofor appara-
`tus (Bio-Rad) and reverse-phase high-performance liquid
`chromatography (RP-HPLC)of individual uroguanylin-like
`or guanylin-like bioactive peptides was achieved using the
`samepurification schemeas described previously for isolation
`of intestinal peptides from the opossum andrat (7, 18). An
`ampholyte range of pH 3.0 to 10.0 (Bio-Rad) was used for
`isoelectric focusing.
`Purification of urine peptides. Uroguanylin and guanylin
`peptides were extracted from 4.5 liters of opossum urine and
`purified as previously described, except that an isoelectric
`focusing step was addedafter the semipreparative HPLC step
`(18). Active fractions were identified using the bioassay as
`described below. Separate columns and Rotofor apparatus
`materials were used during purification of urine peptides
`than were usedfor purification of the colon peptides.
`Cell culture. T84 cells (passage 21 obtained from Jim
`McRoberts, Harbor-University of California Los Angeles Medi-
`cal Center, Torrance, CA) were cultured in Dulbecco’s modi-
`fied Eagle’s medium (DMEM)-Ham’s F-12 medium (1:1) con-
`taining 5% fetal bovine serum, 60 yg penicillin, and 100 pg
`streptomycin per milliliter as previously described (18).
`cGMPaccumulation assay in T84 cells. T84 cells were
`cultured in 24-well plastic dishes, and the cGMPlevels were
`measuredin control and agonist-stimulated cells by radioim-
`munoassay (18). Aliquots of column fractions and vehicle
`were suspendedin 200ul of each of two assay buffers: pH 8.0
`buffer, consisting of DMEM, 20 mM N-2-hydroxyethylpipera-
`zine-N’-2-ethanesulfonic acid (HEPES), 50 mM sodiumbicar-
`bonate, pH 8.0, and 1 mM 3-isobutyl-1-methylxanthine
`(IBMX); and pH 5.5 buffer, consisting of DMEM, 20 mM
`2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5, and 1
`mM IBMX. Ammonium acetate and TFA were removed from
`test samples by drying fraction aliquots in a Speed-Vac before
`suspension in assay buffers. This was doneto avoid changes
`in pH causedby the column reagents. T84 cells were washed
`twice with 200 yl of the respective pH 8.0 and pH 5.5 buffers
`before addition of reagents. These solutions of medium plus
`bioactive peptides were then addedto T84 cells and incubated
`at 37°C for 40 min. After incubation,the reaction medium was
`aspirated, and 200 yl of 3.8% perchloric acid was added per
`well to stop the reaction and extract cGMP. The extract was
`
`adjusted to pH 7.0 with potassium hydroxide and centrifuged,
`and 50 nl of the extract was used to measure cGMP. For pH
`titration studies, DMEM, 20 mM HEPES, 1 mM IBMX was
`adjusted to pH 7.0, 7.5, 8.0, and 8.5 with NaOH, and DMEM,
`20 mM MES,1 mM IBMX wasadjusted to pH 5.0, 5.5, 6.0,
`and 6.5 with NaOH.
`Peptide-agonist concentration-response curves were ana-
`lyzed with the computer program Prism (GraphPad Software,
`San Diego, CA). The concentrations at which peptide agonists
`elicited 50% of the maximal cGMP accumulation response
`(ECs9) were obtained by nonlinear regression of agonist-
`stimulated cGMPaccumulation data.
`Synthesis of uroguanylin, guanylin, and ST peptides. Syn-
`thetic uroguanylin-(2—15), EDCELCINVACTGC, and syn-
`thetic guanylin-(1—15), SHTCEICAFAACAGC,weresynthe-
`sized by the solid-phase method with an Applied Biosystems
`431A peptide synthesizer. N-(9-fluorenylmethoxycarbonyl)
`(FMOC)-protected amino acids activated with 2-(1H-benzo-
`triazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
`were added to FMOC-Cys-(trityl)-Wang resin (Nova Bio-
`chem). Couplingefficiencies were monitored by the ultravio-
`let absorbance of the released FMOC groups. The peptides
`were cleaved from the resin, and the side chains were
`deprotected, except for the acetamidomethyl groups on Cys®
`and Cys!!, by incubation in TFA, ethanedithiol, and water
`(95:2.5:2.5, vol/vol) for 2 h at room temperature. The peptides
`were cyclized with the use of air oxidation. The acetamido-
`methyl groups on Cys? and Cys!! were removed with iodine.
`The peptides were desalted with a 12-ml Whatman ODS-3
`solid-phase extraction device and purified to a single peak by
`Cyg RP-HPLC (acetonitrile-ammonium acetate). The se-
`quences of uroguanylin and guanylin were confirmed by
`protein sequencing on an Applied Biosystems 470A gas-phase
`protein sequencer. Amino acid composition analysis was
`performed to estimate peptide mass, after RP-HPLC purifica-
`tion.
`E. coli ST-(5—17), CCELCCNPACAGC,wasprepared as
`previously described by the solid-phase method with an
`Applied Biosystems 430A peptide synthesizer on Cys-(4-
`CH3Bzl)-OCH2-Pam resin, using double coupling cycles to
`ensure complete coupling at each step (6). The peptide was
`cyclized with the use of dimethyl sulfoxide, and its structure
`wasverified by electrospray mass spectrometry, gas-phase
`sequenceanalysis, and amino acid composition analysis(7).
`Synthesis, purification, and composition analysis of rat
`guanylin-(101—115), rat guanylin-(93—115), and human uro-
`guanylin were performedby our previously described meth-
`ods(7, 20).
`Chymotrypsin digestion ofpeptides. Vehicle, synthetic pep-
`tides, or purified peptides were separated into aliquots in
`Microfuge tubes and dried in a Speed-Vac. Digestion reac-
`tions were started by resuspendingthe dried aliquots in 100
`pl of 10 mM HEPES,pH 8.0, containing 0.15 U (3 pgsolid) of
`bovine pancreatic a-chymotrypsin (Sigma, St. Louis, MO),
`either with or without 100 pM chymostatin (Sigma). Reac-
`tions were incubated at 34°C in a waterbath for 1 h. After 1h,
`the reaction tubes were placed on ice, 100 pM chymostatin
`was addedto the reactions that had been incubated in the
`absence of chymostatin, and all the tubes were frozen at
`—80°C. After freezing, the reaction mixtures were dried in a
`Speed-Vac and then resuspendedin 200 pl of DMEM contain-
`ing 50 mM sodium bicarbonate (pH 8.0) and 1 mM IBMX for
`analysis in the T84 cell cGMP accumulation bioassay. The
`sameexperimentswere repeated and confirmed with sequenc-
`ing-grade bovine pancreatic chymotrypsin from a second
`source (Boehringer-Mannheim,Indianapolis, IN).
`MSN Exhibit 1019 - Page 2 of 9
`MSNv.Bausch - IPR2023-00016
`
`

`

`G710
`
`UROGUANYLIN: AN INTESTINAL PEPTIDE
`
`For NH2-terminal sequence analysis of digested peptides,
`400 pmol of synthetic opossum uroguanylin, synthetic opos-
`sum guanylin, and synthetic rat guanylin-(101—115) were
`incubated for 10 h at 34°C in a Microfuge tube containing 50
`ul of 10 mM HEPES,pH 8.0, with either 20 or 200 pmol of
`bovine pancreatic a-chymotrypsin (sequencing grade, Boeh-
`ringer-Mannheim).
`
`RESULTS
`
`Selective bioassay for uroguanylin and guanylin pep-
`tides. During the initial isolation of uroguanylin and
`guanylin peptides, we observed that a reduction in
`medium pH reduced the cGMP response elicited by
`guanylin in T84 cells. In contrast, uroguanylin wasless
`sensitive to changes in pH.To further investigate these
`observations, the effects of medium pH on the cGMP
`responseselicited by 30 nM of the synthetic forms of
`opossum uroguanylin and guanylin in T84 cells were
`examined (Fig. 1). Uroguanylin caused a greater in-
`crease in cellular cGMP levels when assayed at pH 5
`compared with pH 8. In contrast, guanylin caused only
`a doubling in cGMP accumulation above basallevels at
`pH 5, with the cGMPresponse increasing to 13-fold at
`pH 8 (Fig. 1). Results obtained with 3 nM E£.coli
`ST-(5—17) were similar to those obtained with 30 nM
`uroguanylin (data not shown). Synthetic rat guanylin,
`like opossum guanylin, elicited greater cGMP re-
`sponses in T84 cells at pH 8 than at pH 5.5 (Fig. 2).
`Moreover, the synthetic form of human uroguanylin
`waslike opossum uroguanylin and E.coli ST-(5—17) in
`that each peptide stimulated greater levels of cGMP
`accumulation in T84 cells at acidic relative to alkaline
`pH (Fig. 2). These experiments defined pH conditions
`that were used to estimate the potencies of these
`
`120
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`
`
`pmolcGMPperwell
`
`opGn
`1¢Gn
` opUGn huUGn
`ST
`Basal
`Fig. 2. Agonist-stimulated cGMP accumulation in T84cells at pH 5.5
`(open bars) and pH 8.0 (solid bars). Peptides and vehicle were
`suspended in HEPES and Dulbecco’s modified Eagle’s medium
`(DMEM)containing 50 mM sodium bicarbonate (pH 8.0), or 2-(N-
`morpholino)ethanesulfonic acid (MES) and DMEMat pH 5.5 (pH 5.5)
`for analysis in the T84 cell cGMP accumulation bioassay. Basal,
`vehicle control; ST, synthetic E. coli ST-(5—17); opGn, synthetic
`opossum guanylin; opUGn,synthetic opossum uroguanylin; huUGn,
`synthetic human uroguanylin; rGn, synthetic rat guanylin-(101—
`115). All peptides were tested at 30 nM except for EF. coli ST-(5—17),
`which wastested at 3 nM.Errorbars indicate standarderrorof the
`meanfor 3 experiments.
`
`peptide agonists. Uroguanylin was more potent at pH
`5.5 (ECs, 200 + 50 nM, n = 5) than at pH 8 (ECs,
`1,900 + 100 nM, n = 5)(Fig. 3). In contrast, guanylin
`wasless potent at pH 5.5 (ECs, 10.6 + 4.2 pM, n = 3)
`than at pH 8 (ECso, 0.68 + 0.1 pM, n= 3). ST-(5—17)
`appearedsimilar in potency at both pH 5.5 (ECs, 40 +
`10 nM, n = 3) and pH 8 (ECgo, 130 + 60 nM, n = 38).
`Interestingly, the rank orderof potency of ST > urogua-
`nylin > guanylin at pH 5.5 changed to ST > guanylin >
`uroguanylin at pH 8 in the T84 cell bioassay. These
`results demonstrate that guanylin-selective and urogua-
`nylin-selective pH conditions can be used to identify
`these peptides.
`Comparison of cGMP responses to endogenous and
`synthetic peptides. To demonstrate that the selective
`bioassay conditions using synthetic uroguanylin and
`guanylin could be extended to endogenous uroguanylin
`and guanylin,we isolated bioactive peptides from opos-
`sum urine, which contains greater levels of urogua-
`nylin than guanylin (10, 18, 20). At each stage of
`purification before the isoelectric focusing step, the
`urine extracts elicited much larger cGMPresponsesin
`T84 cells at pH 5.5 compared with the cGMPresponses
`at pH 8 (data not shown). The bioactivity profile
`obtained using preparative isoelectric focusing of the
`partially purified urine extract revealed a dominant
`peak of bioactivity migrating with a pI of about 3.0
`(peak 1, Fig. 4). This pI is similar to that of the
`uroguanylin peptides previously isolated from urine,
`which contained twoor three acidic amino acids and no
`basic residues (18). The bioactive components in peak 1
`also elicited greater cGMP responses in T84 cells when
`assayed at pH 5.5 than at pH 8. A small amount of
`
`MSN Exhibit 1019 - Page 3 of 9
`MSNv. Bausch - IPR2023-00016
`
`100
`
`a oO
`
`
`
`(%MaximalResponse) po°°o
`
`
`
`cGMPAccumulation
`
`ny o
`
`e
`
`5.0
`
`5.5
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`pH
`Fig. 1. Effects of medium pH on uroguanylin (O) and guanylin
`(@)-stimulated guanosine3’ ,5'-cyclic monophosphate (cGMP) accumu-
`lation in T84 cells. Vehicle, 30 nM synthetic opossum uroguanylin,
`and 30 nM synthetic opossum guanylin were suspendedin buffered
`assay medium previously adjusted to pH values indicated, as de-
`scribed in MATERIALS AND METHODS.Levels ofT84 cell cGMP accumula-
`tion (pmol/well, average of 3 wells) elicited by vehicle and peptides in
`this experiment whentested at pH 5.0 and pH8.0, respectively, were
`as follows: basal(vehicle control) = 0.45 and 0.78, uroguanylin = 43.9
`and 17.5, and guanylin = 0.85 and 10.0. Data are representative of 4
`experimentswith similarresults.
`
`

`

`UROGUANYLIN: AN INTESTINAL PEPTIDE
`
`G711
`
`1000
`
`100
`
`10
`
`1000
`
`100
`
`10
`
`1000
`
`100
`
`
`
`cGMP(pmol/well)
`
`10 U3
`
`00 9
`
`8
`
`7
`
`6
`
`5
`
`4
`
`-log Peptide [M]
`Fig. 3. Concentration-response curvesfor stimulation of cGMP accu-
`mulation in T84 cells by synthetic opossum uroguanylin (A), syn-
`thetic opossum guanylin (B), and synthetic E. coli ST-(5—17) (C).
`Peptides were suspended in MES, DMEMat pH 5.5 (pH 5.5) and in
`HEPES, DMEMadjusted to pH 8.0 with 50 mM sodiumbicarbonate
`(pH 8.0). Data are representative of 3-5 experiments with each
`agonist at both pH 5.5 (O) and pH 8.0 (@).
`
`guanylin-like activity was also observed migrating
`with a pl of 5.2 (peak 2, Fig. 4), similar to the pI of
`opossum guanylin, which contains onehistidine (18).
`The guanylin-like peptide stimulated a fourfold in-
`crease in cellular cGMP whenassayed at pH 8, with no
`detectable increase in cGMPat pH 5.5. These experi-
`ments confirmed that endogenous uroguanylin and
`guanylin peptides respond similarly to the synthetic
`forms of uroguanylin and guanylin whenassayed for
`the ability to elicit cGMP responses at acidic vs. alka-
`line pH in T84 cells.
`Isolation of uroguanylin and guanylin from colonic
`mucosa. Preliminary examination of extracts from co-
`lonic mucosa,full-length small intestinal mucosa,kid-
`ney, and plasmaof the opossum revealed the presence
`of both uroguanylin-like and guanylin-like peptides in
`each of the extracts (data not shown). Because the
`amounts of extracted uroguanylin-like bioactivity in
`colonic mucosa appeared to be the greatest per gram
`wet weight of tissue, we further characterized the
`peptides from this tissue. Bioactive peptides were
`extracted from 150 g of opossum colonic mucosa and
`fractionated by gel-filtration chromatography. The pro-
`file of bioactivity obtained when columnfractions were
`
`assayed at pH 5.5 revealed two distinct peaks of
`bioactivity that eluted within the internal volume of
`the Sephadex G-25 gel column (Fig. 5). When the
`column fractions were assayed at pH 8, a single peak of
`bioactivity was observed that was coincident with the
`peak 2 that was found whenthe assay was conducted at
`pH 5.5. The uroguanylin-like peptides in peak 1 elicited
`27-fold increases in cellular cGMP accumulation when
`assayedat pH 5.5 and onlyfivefold increasesin cellular
`cGMPat pH 8. In contrast, the guanylin-like peptides
`in peak 2 stimulated greater levels of cGMP at pH 8
`(100-fold) than when assayedat pH 5.5 (28-fold).
`To further characterize the uroguanylin-like pep-
`tides,
`the components of peak 1 were purified as
`previously described (7, 18). After each purification
`step, a dominant activity peak was observed that
`elicited greater cGMPresponsesat pH 5.5 than at pH 8.
`In addition, a pI of 3.0 was observed whenthebioactive
`components from peak 1 were fractionated by prepara-
`tive isoelectric focusing (data not shown), consistent
`with peak 1 containing an acidic, uroguanylin-like
`peptide (10, 18, 20). The chromatographic properties of
`peak I were characterized further with the use of
`RP-HPLC. The peak fraction of bioactive peptides
`eluting from a C,;g, RP-HPLC column stimulated a
`23-fold increase in cGMP when assayed at pH 5.5,
`compared with only an eightfold increase in cGMP at
`pH 8 (Fig. 6). Moreover, a characteristic uroguanylin
`elution profile (18) was observed, with the uroguanylin-
`like peptide(s) eluting at 8% acetonitrile from this Cj,
`RP-HPLC column (Fig. 6). This elution pattern is
`
`cGMPperwell
`pmol
`
`3
`
`6
`
`9
`
`12
`
`15
`
`18
`
`Fraction
`
`Fig. 4. Isoelectric focusing of peptides extracted from opossum urine.
`Peptides were extracted from 4.5 liters of opossum urine using Cj
`cartridges and purified as described in MATERIALS AND METHODS.
`Purified peptides were suspended in 50 ml water containing 0.8%
`ampholytes (pH range 3-10, Bio-Rad) and then fractionated on a
`preparative isoelectric focusing cell (Rotofor, Bio-Rad). Fractions
`(0.2% of fraction volume) were next assayedfor the ability to elicit a
`cGMPresponse in T84 cells after suspension in MES, DMEM at pH
`5.5 (O), and in HEPES, DMEM adjusted to pH 8.0 with 50 mM
`sodium bicarbonate (™). Right y-axis represents pH (#) of Rotofor
`fractions obtained after isoelectric focusing of partially purified
`peptides. pH of the Rotofor fraction containing peakof bioactivity, as
`determined by T84 cell cGMP accumulation bioassay, was estimated
`as the isoelectric point(pI).
`MSNExhibit 1019 - Page 4 of 9
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`

`G712
`
`UROGUANYLIN: AN INTESTINAL PEPTIDE
`
`Peak #2
`
`Peak #1
`
`cGMPperwell
`pmol
`
`19
`
`23
`
`31
`27
`Fraction
`
`35
`
`39
`
`Fig. 5. Sephadex G-25, gel-filtration chromatographyelution profile
`of uroguanylin-like and guanylin-like peptides extracted from colonic
`mucosa. Peptides were extracted from colonic mucosa using Cig
`cartridges and processed as described in MATERIALS AND METHODSfor
`application to a 2.5 X 90 cm Sephadex G-25 gel column.Fractions (10
`ml) werecollected, and 1.0% of each fraction was assayed in MES,
`DMEMat pH 5.5 (©), and in HEPES, DMEMadjusted to pH 8.0 with
`50 mM sodium bicarbonate (™) for stimulation ofcGMP accumulation
`in T84cells. Illustrated bioactivity profile is a representative experi-
`mentfrom 1 of 2 Sephadex G-25 column runs.
`
`consistent with that previously observedfor the 14- and
`15-aminoacid forms of opossum uroguanylin that were
`isolated from urine (18). When the putative urogua-
`nylin peptide from colon was subjected to NH2-terminal
`
`
`
`pmolcGMPperwell
`
`%BNWUOoy
`
`29
`
`33
`31
`Fraction
`
`35
`
`Fig. 6. T84 cell cGMP accumulation responseselicited by urogua-
`nylin purified from opossum colonic mucosa. Fractions were bioas-
`sayed using MES, DMEMat pH 5.5 (©), and HEPES, DMEM,50 mM
`sodium bicarbonate at pH 8.0 (™). Uroguanylin-like peptides (includ-
`ing the fractions of peak 1 shown in Fig. 5) from 2 successive
`Sephadex G-25 columnrunsof colonic mucosal extracts were further
`purified as previously described (18) by a preparative isoelectric
`focusing step and 3 reverse-phase high-performanceliquid chromatog-
`raphy (RP-HPLC) steps. Purified uroguanylin-like bioactivity shown
`is from fractions obtained after the third RP-HPLCstep (thin solid
`line), which used acetonitrile and 10 mM ammoniumacetate, pH 6.2,
`to purify peptides, using a 4.9 x 250 mm Cig column,as previously
`described (18).
`
`sequence analysis, no signal for amino acids was ob-
`served, indicating that this peptide may be blocked at
`the NH,-terminal end, a phenomenon previously en-
`countered when uroguanylin was purified from urine.
`To evaluate the bioactive components contained in
`peak 2 (Fig. 5), the same purification methods were
`utilized. When the componentsofpeak 2 were further
`purified by isoelectric focusing, both guanylin-like and
`uroguanylin-like peptides were resolved (peaks 2A and
`2B,Fig. 7). Peak 2A migrated with a pI of 3.0, similar to
`the pI of uroguanylin. Peak 2A also caused a greater
`cGMPresponsein T84 cells at pH 5.5 than it did at pH
`8, which is consistent with this peptide being urogua-
`nylin. It is likely that uroguanylin was not completely
`separated from guanylin in peak 2 (Fig. 5) during
`Sephadex G-25 chromatography. This uroguanylin-like
`activity ofpeak 2A exhibited the same chromatographic
`properties as authentic uroguanylin when subjected to
`further analysis by RP-HPLC using C3 columns (data
`not shown). An insufficient quantity of this purified
`peptide prevented its identification by NH»-terminal
`sequence analysis. The guanylin-like bioactivity peak
`that was resolved byisoelectric focusing migrated with
`a pl of 6.0, elicited 27-fold increases in T84 cell cGMP
`levels when assayed at pH 8, and caused only twofold
`increases in cGMPat pH 5.5 (peak 2B, Fig. 7). The pl of
`6.0 is similar to the pI estimated for opossum guanylin
`(~5.2), which contains a histidine residue (18). Peak 2B
`was further purified by a series of C;g RP-HPLC steps
`(7, 18). The guanylin-like activity eluted at about 15%
`acetonitrile (Fig. 8), which is a characteristic property
`of guanylin (18). One percent of this peak elicited a
`15-fold increase in cGMP accumulation when assayed
`at pH 8, but caused no detectable increase in cGMP
`levels in T84 cells at pH 5.5. Further purification by
`RP-HPLCfollowed by NH,-terminal sequence analysis
`
`15
`
`_ nD
`
`o
`
`ao
`
`
`
`pmolcGMPperwell
`
`2
`
`Peak #2B
`
`panne
`
`3
`
`6
`
`12
`9
`Fraction
`
`15
`
`18
`
`Fig. 7. Isoelectric focusing of peptides extracted from colonic mucosa.
`Fractions displaying predominately guanylin-like activity (including
`fractions comprising peak 2 in Fig. 5) were pooled from 2 separate
`Sephadex G-25 column runsandfractionated by isoelectric focusing
`on the Rotofor apparatus, as described in Fig. 4 legend. Fractions
`(1.0% of fraction volume) were bioassayed for cGMP accumulation in
`T84 cells using pH 5.5 (O) and pH 8.0 (™) medium,as described in
`Fig. 4 legend. @, pH of Rotofor fractions.
`
`MSN Exhibit 1019 - Page 5 of 9
`MSNv. Bausch - IPR2023-00016
`
`

`

`UROGUANYLIN: AN INTESTINAL PEPTIDE
`
`G713
`
`
`
`pmolcGMPperwell
`
`%ILWUoOsIy 14.5
`
`95
`
`101
`
`but not opossum uroguanylin. Highly purified, sequenc-
`ing-grade chymotrypsin was used to digest the syn-
`thetic peptides for these studies, which wasfollowed by
`five cycles of NH»-terminal sequence analysis. Two
`amino acid sequences wereobtained from chymotrypsin-
`digested opossum guanylin, SHTCE and AACAG,which
`wereidentical to the first five amino acids of the NH»,
`
`40
`
`L1Control
`bcs
`eT
`mcT+cs
`
`bY
`bed
`=
`Px
`a
`S
`Bs
`330,
`rd
`RS
`5
`hg
`RS
`a
`‘en
`KO
`be
`2
`2
`i
`ky
`¢
`bey
`Ke
`© 20
`bed
`KI
`6
`99
`97
`be
`ke
`=
`Se
`KI
`°
`Fraction
`i
`Po
`E
`
`
`* Bom|(8 :
`Fig. 8. Purification of guanylin from opossum colonic mucosa by
`bs
`RS
`10
`RB
`RP-HPLC. Guanylin-like peptides were purified from colonic mucosal
`R
`iM
`RB
`RY
`RY
`<
`extracts and bioassayed at pH 5.5 (©) and pH 8.0 (™) for stimulation
`BS
`Ry
`<<
`P
`<]
`x
`KI
`of cGMP accumulation in T84 cells as described in Fig. 6 legend.
`se
`bx
`&
`
`lee Ml|IB...RS bes
`
`
`
`Purified guanylin shownhere was obtainedafter the third RP-HPLC
`Opossum Rat Guanylin Rat Guanylin
`Opossum
`Basal
`ST-(5-17)
`step, which used acetonitrile (thin solid line) and 10 mM ammonium
`(101-115)
`Uroguanylin
`Guanylin
`(93-115)
`acetate, pH 6.2, to purify peptides using a 4.9 X 250 mm Cj, column,
`as previously described (18).
`
`
`
`
`100:
`
`NNCT
`HMCT+cs
`
`ESs
`Ex 75
`
`© =x a
`
`=©o
`
`50
`
`25:
`
`Synthetic
`
`Colon
`
`Uroguanylin
`
`Synthetic
`Guanylin
`
`Fig. 9. cGMP responseselicited by chymotrypsin-treated synthetic
`peptides (A) and purified colonic peptides (B). Vehicle or peptides
`were incubated for 1h at 34°C in 100 pl of 10 mM HEPES,pH 8.0,
`under the following conditions: minus both chymotrypsin and chy-
`mostatin (control), minus chymotrypsin and plus chymostatin (CS),
`plus chymotrypsin and minus chymostatin (CT), and plus both
`chymotrypsin and chymostatin (CT + CS). Chymotrypsin (0.15 units)
`and chymostatin (100 pM) were used in reactions where indicated.
`After 1-h incubation, 100 »M chymostatin was addedto CTreactions.
`Reaction mixtures were processed and analyzed in the T84 cell
`bioassay as described in MATERIALS AND METHODS. A: ST-(5—17) = 3
`nM;opossum guanylin,rat guanylin-(101—115), and opossum urogua-
`nylin = 30 nM;and rat guanylin-(93—-115) = 100 nM.Error bars
`indicate standarderror of the meanfor 3 experiments. B: %maximal
`cGMPresponseselicited by purified colonic forms of opossum urogua-
`nylin (Fig. 6) and guanylin (Fig. 8) are shown in comparison to cGMP
`responseselicited by 10 nM of synthetic opossum uroguanylin and
`guanylin after treatmentsas described for reactions CT and CT + CS.
`cGMPvalues (pmol/well), representing average of 2 experiments,
`minusbasal values of cGMP (~0.2 pmol/well), are as follows: colonic
`uroguanylin, CT = 4.2, CT + CS = 5.7; synthetic uroguanylin, CT =
`2.16, CT + CS = 2.91; colonic guanylin, CT = 0.86, CT + CS = 45.3;
`synthetic guanylin, CT = 0.25, CT + CS = 18.7.
`
`MSN Exhibit 1019 - Page 6 of 9
`MSNv. Bausch - IPR2023-00016
`
`of the guanylin-like peptide confirmed that this bioac-
`tive substance was guanylin (SHTCEICAFAACAGC)(18).
`Inactivation of guanylin by chymotrypsin. As an
`additional test that the uroguanylin-like peptide identi-
`fied in opossum colon was not guanylin, we examined
`the guanylin-like and uroguanylin-like peptides that
`were isolated from colonic mucosa for sensitivity to
`inactivation by chymotrypsin in vitro. It has recently
`been reported that treatment of guanylin with chymo-
`trypsin resulted in the inactivation of rat guanylin-
`(101—115), but not a rat guanylin analogue containing
`an asparagine in place of Tyr!(3). Because urogua-
`nylin has an asparagine instead of Tyr’, we postu-
`lated that uroguanylin would be resistant to chymotryp-
`sin. Figure 9A demonstrates that chymotrypsin
`treatment did not reduce the bioactivity of synthetic E.
`coli ST-(5—17) and synthetic opossum uroguanylin
`underconditions that completely inactivated the syn-
`thetic forms of opossum guanylin, rat guanylin-(101—
`115), and rat guanylin-(93—115). The chymotrypsin
`inhibitor, chymostatin, blocked the inactivation of syn-
`thetic guanylin peptides by chymotrypsin, as deter-
`mined by the T84 cell cGMP bioassay. The T84 cell
`cGMPresponseselicited by purified colonic urogua-
`nylin and synthetic opossum uroguanylin were reduced
`by only 25% after treatment with chymotrypsin (Fig.
`9B). In contrast, the cGMPresponseselicited by puri-
`fied colonic guanylin and synthetic opossum guanylin
`were reduced by >98% when pretreated with chymo-
`trypsin (Fig. 9B). Thus colonic mucosa contains chymo-
`trypsin-sensitive guanylin peptides, as well as chymo-
`trypsin-resistant uroguanylin peptides.
`NH,-terminal sequence analysis of synthetic pep-
`tides treated with chymotrypsin confirmed that this
`enzymecleaved the rat and opossum formsof guanylin,
`
`

`

`G714
`
`UROGUANYLIN: AN INTESTINAL PEPTIDE
`
`terminus of opossum guanylin andthefirst five amino
`acids following the phenylalanine residue in opossum
`guanylin, respectively (18). Moreover, NH.-terminal
`sequences were obtained from chymotrypsin-digested
`rat guanylin, PNTCE and AACTG,which were identi-
`cal to thefirst five amino acids on the NH, terminusof
`rat guanylin andthefirst five amino acids following the
`tyrosineresiduein rat guanylin, respectively (7). These
`results confirmed that chymotrypsin cleaves opossum
`guanylin between its Phe-Ala bond and cleaves rat
`guanylin between its Tyr-Ala bond. In these experi-
`ments, r