Structure and activity of uroguanylin and guanylin
`
`
`
`
`from the intestine and urine of rats
`
`XIAOHUI FAN,1 F. KENT HAMRA,1
`
` ROSLYN M. LONDON,
`2 RONALD H. FREEMAN,2
`SAMMY L. EBER,1,2 WILLIAM J. KRAUSE,
`FORTE1,2
`3 MARK G. CURRIE,3 AND LEONARD R.
`CHRISTINE E. SMITH,
`Pathology and of Pharmacology, 1Truman Veterans Affairs Medical Center and 2Departments
`
`
`
`
`
`Anatomical Sciences and Physiology, School of Medicine, Columbia 65212; and 3Searle Research
`
`
`
`
`
`and Development, St. Louis, Missouri 63167
`
`,2
`
`,2
`
`1,2
`
`
`
`Guanylin was first isolated from the intestine ofrats
`
`
`
`Fan, Xiaohui, F. Kent Hamra, Roslyn M . London,
`
`
`
`as a 15-amino acid peptide containing four cysteines
`Sammy L. Eber, William J. Krause, Ronald
`H . Freeman,
`
`
`with two disulfide bonds that are required for bioactiv­
`
`
`Christine E. Smith, Mark G. Currie, and Leonard R .
`
`
`
`Forte. Structure and activity o f uroguanylin and guanylin
`
`
`ity (4). Then, cDNAs encoding preproguanylins of 115-
`
`
`from the intestine and urine of rats. Am. J. Physiol. 273
`
`
`116 amino acids containing the COOR-terminal gua­
`
`
`(Endocrinol. Metab. 36): E957-E964, 1997.-Uroguanylin
`
`
`
`nylin peptides were isolated (31, 32). Guanylin mRNA
`
`
`
`and guanylin are related peptides that activate common
`
`
`is highly expressed in the ileum and colon, with consid­
`
`
`
`
`
`guanylate cyclase signaling molecules in the intestine and
`erably lower amounts found in the duodenum and
`
`
`kidney. Uroguanylin was isolated from urine and duodenum
`
`
`jejunum (31, 32). The cellular sites ofguanylin produc­
`
`
`but was not detected in extracts from the colon of rats.
`
`
`tion in the intestinal mucosa are reported to include
`
`
`Guanylin was identified in extracts from small and large
`
`goblet cells and absorptive cells (22, 23, 26). Urogua­
`
`
`
`intestine but was not detected in urine. Uroguanylin and
`
`nylin was initially isolated from opossum urine (14). A
`
`
`
`guanylin have distinct biochemical and chromatographic
`search for the tissue source of urinary uroguanylin
`
`
`
`
`properties that facilitated the separation, purification, and
`
`
`
`resulted in the purification of prouroguanylin and
`
`
`identification of these peptides. Northern assays revealed
`
`
`
`uroguanylin from large intestine (13, 15). Recently,
`
`
`that mRNA transcripts for uroguanylin were more abundant
`
`
`
`cDNAs encoding preprouroguanylin were isolated from
`
`
`in small intestine compared with large intestine, whereas
`
`
`
`opossum, human, and rat intestinal cDNA libraries (1,
`
`
`
`
`guanylin mRN A levels were greater in large intestine relative
`
`
`5, 16, 25, 28, 29). The bioactive uroguanylin peptides
`
`
`
`to small intestine. Synthetic rat uroguanylin and guanylin
`
`
`found in urine are located at the COOH terminus of
`
`
`
`
`had similar potencies in the activation of receptors in T84
`prouroguanylin.
`
`
`
`
`
`intestinal cells. Production of uroguanylin and guanylin in
`In the present study, we isolated uroguanylin from
`
`
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`
`
`the mucosa of duodenum is consistent with the postulate that
`
`urine and duodenum ofrats to investigate the structure
`
`
`
`
`both peptides influence the activity of an intracellular guano­
`
`
`
`and biological activity of uroguanylin in this species.
`
`
`
`sine 3 ',5 '-cyclic monophosphate signaling pathway that regu­
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`
`
`Uroguanylin and guanylin were identified by their
`
`
`
`lates the trans epithelial secretion ofchloride and bicarbonate
`
`unique chromatographic properties, by NH2-terminal
`
`in the intestinal epithelium.
`
`
`
`sequence analyses, and by the effects of medium pH on
`
`
`
`
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`guanylate cyclase; guanosine 3 ',5 '-cyclic monophosphate; kid­
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`
`
`
`the relative potencies of the bioactive peptides. The
`
`
`
`ney; heat-stable enterotoxin; human T84 intestinal cells
`
`
`bioactive peptide in the urine is uroguanylin, whereas
`
`
`
`guanylin and uroguanylin were both isolated from the
`
`
`
`duodenum. Only guanylin was purified from the large
`intestine.
`
`GUANYLIN AND UROGUANYLIN are small peptides that
`
`
`activate membrane receptor-guanylate cyclase signal­
`
`
`ing molecules in the intestine, kidney, and other epithe­
`
`
`
`lia (reviewed in Ref. 8). These receptors are localized to
`Purification of uroguanylin from urine. Three batches of
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`apical membranes of cells lining the gastrointestinal
`
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`urine (2-3 liters) pooled from 12 male Sprague-Dawley rats
`
`
`tract (6, 20, 23, 30) and renal proximal tubules (9, 10,
`
`
`were used to isolate and identify uroguanylin with chromato­
`
`
`
`graphic methods that have been previously described ( l 3-
`
`
`
`21). Heat-stable enterotoxin (ST) peptides secreted by
`
`
`15). Briefly, urine was collected daily from rats housed in
`
`
`
`enteric bacteria that cause traveler's diarrhea act as
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`
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`metabolic cages, pooled, and stored at -20°C. After thawing,
`
`
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`molecular mimics ofuroguanylin and guanylin (4, 6, 8,
`
`the urine was centrifuged at I 0,000 g for 20 min. Trifluoroace­
`
`
`14). Intracellular accumulation of the second messen­
`
`tic acid (TFA), 0.1 %, was added to the supernatant, and the
`
`
`
`ger guanosine 3',5'-cyclicmonophosphate (cGMP)influ­
`
`sample was then applied to C18 Sep-Pak cartridges and eluted
`
`
`ences the phosphorylation state and putative chloride
`
`
`with 40% acetonitrile and 0.1 % TFA. The eluted polypeptides
`
`
`
`
`(Cl-) channel activity of the cystic fibrosis transmem­
`
`were dried and resuspended in 50 mM ammonium acetate
`
`and then chromatographed using a 2.5 X 90-cm column of
`
`
`
`brane conductance regulator protein, which may serve
`
`
`Sephadex G-25 gel. Fractions eluted from the G-25 column
`
`as an efflux pathway for c1-secretion from the intesti­
`
`
`and in subsequent purification steps were bioassayed using
`
`
`
`nal mucosa (8). The net effect of receptor activation in
`
`
`T84 cells by measurement of cGMP accumulation as previ­
`
`
`
`the intestine is to stimulate the transepithelial secre­
`
`
`
`
`ously described (8, 9). The active fractions were pooled, dried
`
`tion of c1-and HCO3, thus increasing fluid secretion
`
`
`in a Speed-Vac, resuspended in 0.1 % TFA, and loaded onto C18
`
`
`and modulating the intraluminal pH (4, 6, 11, 14, 17).
`
`
`
`Sep-Pak cartridges. The peptides were eluted with a gradient
`MSN Exhibit 1016 - Page 1 of 8
`MSN v. Bausch - IPR2023-00016
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`MATERIALS AND METHODS
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`E957
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`Downloaded fromjoumals.physiology.org/joumal/ajpendo (098.109.055.010) on January 14, 2021.
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`

`

`E958
`
`
`
`STRUCTURE AND ACTIVITY OF UROGUANYLIN AND GUANYLIN
`
`erously provided by Dr. Roger Weigand (Monsanto, St.
`
`
`
`
`
`
`
`of 10%, followed by 30% and then 60% acetonitrile solutions
`Louis, MO).
`
`
`containing 0.1 % TFA. The bioactive peptides were eluted with
`Cell culture. T84 cells were obtained from Dr. Jim McRob­
`
`
`
`
`the 30% acetonitrile-0.1 % TFA solution, and this fraction was
`
`
`
`erts (Harbor-University of California, Los Angeles, CA) at
`
`
`dried, resuspended in 10% acetonitrile and 0.1% TFA, and
`
`
`
`
`passage 21. Cells were cultured in Dulbecco's modified Eagle's
`
`
`liquid applied to a C18 semipreparative high-performance
`
`medium (DMEM) and Ham's F-12 medium (1:1), supple­
`
`chromatography (HPLC) column (Waters semipreparative
`
`
`mented with 5% fetal bovine serum, 60 mg penicillin/ml, and
`
`
`
`
`µBondapak, 7 .8 mm X 30 cm). The peptides were eluted with
`100 mg streptomycin/ml.
`
`
`a gradient of 10% acetonitrile-0.1 % TFA to 30% acetonitrile-
`cGMP bioassay in T84 cells. T84 cells were cultured in
`
`
`
`
`0.1 % TFA over a period of 180 min. The peaks ofbioactive
`
`
`
`
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`24-well plastic dishes, and cellular cGMP levels were mea­
`
`
`
`peptides were pooled, dried, resuspended in H20 with 0.8%
`
`
`sured in control and agonist-stimulated cells by radioimmuno­
`
`
`ampholytes [pH range 3-10 (Bio-Rad)], and subjected to
`
`
`assay (12-15). Briefly, column fractions of the synthetic
`
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`
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`preparative isoelectric focusing (Rotorfor, Bio-Rad). The frac­
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`
`
`
`peptides, uroguanylin and guanylin, were suspended in 200
`
`
`
`
`tions containing bioactivity were combined, applied to a C18
`
`µl ofDMEM containing 20 mM N-(2-hydroxyethyl)piperazine­
`
`
`HPLC column (Waters analytic µBondapak, 3.9 mm X 30 cm),
`
`
`N'-(2-ethanesulfonic acid) (HEPES), pH 7.4 or 5.5 buffer,
`
`
`
`and eluted with a gradient of 5% acetonitrile-10 mM ammo­
`
`consisting of DMEM, 20 mM 2-(N-morpholino)-ethanesul­
`
`
`mM ammonium nium acetate (pH 6.2) to 25% acetonitrile-10
`fonic acid (MES, pH 5.5), and 1 mM isobutylmethylxanthine
`
`
`
`peptides peak ofbioactive acetate (pH 6.2) over 180 min. The
`
`
`
`
`werepeptides (IBMX). The solutions containing bioactive
`
`
`was subjected to a second purification procedure with the
`
`
`then added to cultured cells and incubated at 37°C for 40 min.
`same C18 analytic HPLC column, but with the acetonitrile
`
`
`
`After incubation, the reaction medium was aspirated and 200
`
`
`gradient containing 0.1% TFAinstead of ammonium acetate.
`
`
`µl of 3.3% perchloric acid were added per well to stop the
`
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`
`
`The bioactive peptides were then applied to a C8 microbore
`
`
`
`reaction and extract cGMP. The extract was adjusted to pH
`
`column and eluted with a gradient of 0.33% of acetonitrile
`
`7.0 with KOH and centrifuged, and 50 µl of the supernatant
`
`
`and 0.1 % TFA per minute as previously described (4, 14, 15).
`were used to measure cGMP.
`
`
`
`
`The purified peptides were subjected to automated Edman
`
`
`
`(4, 14, 15). as previously described NH2-terminal sequencing,
`
`
`
`Purification of peptides from the mucosa of colon and
`RESULTS
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`
`
`duodenum. The mucosa (100 g wet weight) was scraped from
`Purification of bioactive uroguanylin from urine.
`
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`
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`colons by use of a glass microscope slide and then boiled in 10
`
`
`Uroguanylin was purified from rat urine using C18
`
`volumes of 1 M acetic acid for 10 min, homogenized, and
`
`
`Sep-Pak and gel filtration chromatography, prepara­
`
`centrifuged at 10,000 g for 20 min. The supernatant was
`
`
`
`
`tive isoelectric focusing, and a series of reverse phase
`
`followed by Sephadex extracted with C18 Sep-Pak cartridges
`
`
`(RP)-HPLC steps (4, 13-15).
`
`
`G-25 column fractionation, as described above. The bioactive
`
`peptide fractions from the gel column were combined and
`After the isolation of bioactive peptides with C18
`
`
`
`
`fractionated a second time with C18 Sep-Pak cartridges. The
`
`
`
`cartridges, a second chromatographic step with a Sepha­
`
`
`
`peptides were eluted using 5, 10, 15, 20, 25, 40, and 60%
`
`
`dex G-25 column yielded a single peak ofpeptides that
`
`
`
`
`acetonitrile solutions containing 0.1 % TFA. The bioactive
`
`
`stimulated cGMP accumulation in T84 cells (data not
`
`
`
`peptide fractions (i.e., 25% acetonitrile) were pooled and
`
`
`
`shown). This peak of bioactive peptides eluted at a
`
`
`
`
`subjected to isoelectric focusing as described above. The final
`
`
`
`position identical to that previously found for opossum
`
`
`
`
`purification of the active peptides was accomplished by HPLC
`
`
`uroguanylin (14, 15). Preparative isoelectric focusing
`
`by use ofa series ofC18 columns as we have described.
`
`
`separates the more highly acidic uroguanylin from
`
`
`Fifty-five grams wet weight of mucosa were scraped from
`
`
`
`guanylin (14, 15). The bioactive peptides eluting from
`
`the duodenum (proximal one-third of the small intestine),
`
`
`
`Sephadex G-25 columns were subjected to preparative
`
`
`
`and the bioactive peptides were purified using the same
`
`
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`isoelectric focusing, and the active peptides migrated to
`
`
`
`
`methods as described above for the peptides isolated from
`
`
`
`colonic mucosa, except that the isoelectric focusing step was
`
`
`
`the most acidic region, eluting at pH values of2.4-3.7
`not used.
`
`
`
`in fractions 1-3 (Fig. 1). This peptide fraction from
`
`Northern assays ofuroguanylin and guanylin mRNA. Total
`
`
`
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`urine stimulated cGMP accumulation in the T84 cells
`
`
`RNA was prepared (RN easy kit, Qiagen) from the mucosa of
`
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`to a greater magnitude when the medium pH was 5.5
`
`
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`individual intestinal segments, and 20 µg of each RNA
`
`
`
`compared with the stimulation at pH 7.4. The profile of
`
`
`
`preparation were subjected to electrophoresis in formalde­
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`pH dependence for agonist activity in T84 cells is
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`hyde-agarose gels and then transferred to nylon membranes
`
`
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`consistent with this urine peptide being uroguanylin
`
`
`(Bio-Rad). The blots were hybridized with rat uroguanylin
`
`
`(12). This peptide fraction was then combined and
`
`
`
`
`and [3-actin cDNAs or rat guanylin plus [3-actin cDNAs (27).
`
`subjected to RP-HPLC by use of C18 columns and a
`
`
`
`Prehybridization was for 1 h with QuickHyb (Stratagene, La
`
`
`
`gradient of5-25% acetonitrile containing 10 mM ammo­
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`
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`hybridization for 2 Jolla, CA) at 68°C, which was followed by
`
`nium acetate, pH 6.2 (13-15). Under these RP-HPLC
`
`h at 68°C with each cDNAprobe labeled by random priming
`
`
`
`(Boehringer Mannheim). The blots were washed twice with
`
`
`conditions, guanylin elut es at 16-18% a cetonitrile,
`
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`2 X standard sodium citrate (SSC) and 0.1 % sodium dodecyl
`
`whereas uroguanylin elutes at 10-11% acetonitrile.
`
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`and once with sulfate (SDS) for 15 min at room temperature
`
`
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`Fractions 1-3 from the isoelectric focusing purification
`
`0.2X SSC and 0.1 % SDS for 15 min at 60°C. The exposure to
`
`step (Fig. 1) were combined for RP-HPLC under these
`
`
`Rat film was for 24 h at -80°C with intensifying screens.
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`conditions. A single peak ofbioactive peptides eluted at
`
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`uroguanylin cDNA (nucleotides 117-292) was produced by
`
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`10% acetonitrile and 10 mM ammonium acetate, an
`
`
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`polymerase chain reaction (PCR) amplification from intesti­
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`
`
`
`
`elution pattern consistent with this peptide being uro­
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`nal mRNA-cDNA (1, 27). This cDNA was isolated and se­
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`
`
`guanylin (Fig. 2). This peak of bioactive peptides was
`
`
`quenced to confirm that it matched the uroguanylin ex­
`
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`purified further using the same C18 column by RP­
`
`
`pressed sequence tag (EST) of rat uroguanylin with 100%
`
`
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`HPLC with an acetonitrile gradient containing 0.1 %
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`identity. A rat guanylin cDNA (nucleotides 1-531) was gen-
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`MSN Exhibit 1016 - Page 2 of 8
`MSN v. Bausch - IPR2023-00016
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`Downloaded fromjoumals.physiology.org/joumal/ajpendo (098.109.055.010) on January 14, 2021.
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`

`

`STRUCTURE AND ACTIVITY OF UROGUANYLIN AND GUANYLIN
`
`E959
`
`= 3�-------------�
`
`Q) 3i: 2.5
`
`E/DXXELXINVAXTGX
`
`... Q)
`C. 2
`
`C. :!E 1.5
`(!) (.) 1
`� 0.5
`3 5 7 9 11 13 15 17 19
`Q. 0..LLILIL.L.!LUL!A.l.L..IU.IL.!L..LIIL.L.J.11:lJL!A-lLJ.IU.II.L
`
`10
`
`20
`
`Fraction
`
`minute
`
`
`Fig. 3. Purification ofuroguanylin by RP-HP LC from urine. Ultravio­
`
`let (UV) absorbance of the last RP-HP LC step using a C8 microbore
`Fig. 1. Isolation ofuroguanylin from rat urine by isoelectric focusing.
`
`
`
`
`
`
`
`
`column. Arbitrary units for UV absorbance are used. Peak 4 (shaded
`
`
`Rat urine was first chromatographed with C18 Sep-Pak cartridges
`
`
`
`
`
`area) contains the bioactive peptides eluted; this fraction was sub­
`
`
`
`followed by gel filtration chromatography with Sephadex G-25, as
`
`
`
`
`jected to sequence analysis. A residue of either glutamate or aspar­
`
`
`
`
`described in MATERIALS AND METHODS. Active fractions from the G-25
`
`
`
`tate was observed at the first position, and the second position was
`
`
`
`
`column were pooled, lyophilized, and subjected to isoelectric focusing.
`
`
`
`not determined. The other 4 positions marked as X correspond to the
`
`
`
`Fractions were assayed using the T84 cell guanosine 3',5'-cyclic
`
`
`
`
`conserved cysteine residues within this family of peptides. The
`
`
`
`monophosphate (cGMP) accumulation bioassay under conditions of
`
`
`
`
`partial amino acid sequence that was obtained is shown at top.
`MES, DMEM at pH 5.5 (open bars), or HEPES and DMEM at pH 7.4
`
`(solid bars).
`
`residues found in opossum uroguanylin. Taken to­
`
`
`TFA(20, 21). The bioactive peptides were eluted at 21 %
`
`
`
`
`
`
`gether, these findings suggest that uroguanylin is the
`
`
`
`acetonitrile and were combined for microbore RP­
`
`
`major bioactive peptide appearing in the urine of rats.
`
`
`
`HPLC (data not shown). After further purification with
`
`
`
`Purification of uroguanylin and guanylin from intes­
`
`
`(Fig. 3), the RP-HPLC with a C8 microbore column
`
`
`
`tine. Isolation ofuroguanylin and prouroguanylin from
`
`
`bioactive peptides in the shaded portion ofthe ultravio­
`
`
`colon and small intestine and uroguanylin mRNA
`
`
`
`
`let absorbance tracing were combined and subjected to
`
`
`expression in the intestinal mucosa of other species
`
`
`
`
`NHrterminal sequence analysis (5, 20). A partial se­
`
`
`suggests that the intestine of rats may be a source of
`
`
`quence of E/DXXELXINVAXTGX (X is unknown) was
`
`
`uroguanylin in urine (5, 13, 15, 16). To investigate this
`
`
`
`
`obtained because oft he low quantity of peptides remain­
`
`
`
`
`possibility, we isolated bioactive peptides from the
`
`
`ing at this stage ofpurification. The partial amino acid
`
`
`mucosa of colon and duodenum from rats. Extracts of
`
`
`
`sequence obtained for the rat urine peptide is similar to
`
`
`colonic mucosa were prepared and purified by C18
`
`
`
`the corresponding residues reported for opossum and
`
`
`
`chromatography, followed by Sephadex G-25 chromatog­
`
`human forms of bioactive uroguanylin isolated from
`
`raphy as described above. A single peak of bioactive
`
`urine (14, 18) and identical to the deduced sequence
`
`
`
`
`peptides was observed eluting from the Sephadex G-25
`
`
`from a uroguanylin EST cDNA isolated from rat intes­
`
`
`
`
`column (data not shown). The active peptide peak was
`
`tine (1). An acidic residue of either glutamate or
`
`
`
`
`combined and subjected to preparative isoelectric focus­
`
`
`
`aspartate was observed at the position where gluta­
`
`
`
`ing. The bioactive peptides eluted in fractions 1-3 with
`
`mate is found in opossum uroguanylin and where
`
`pH values of2.6-3.5 (Fig. 4). At this stage of purifica­
`
`
`aspartate occurs in human uroguanylin (14, 18). The
`
`
`
`tion, the peptide components from rat colon exhibited a
`
`
`amino acids identified by sequence analysis consisting
`
`
`
`
`property similar to that of guanylin, because the colon
`
`
`of ELXINVAXTGX are identical to the corresponding
`
`
`
`peptides stimulated cGMP accumulation in T84 cells to
`
`
`a greater level at the medium pH of7.4 compared with
`30
`
`
`the cGMP responses at pH 5.5 (12). When the active
`cii 25
`20
`C. 20
`15
`10
`Q. 5
`0
`
`Fig. 2. Purification ofuroguanylin from rat urine by reverse-phase
`
`
`
`
`
`high-performance liquid chromatography (RP-HPLC). Active frac­
`
`
`
`tions from the isoelectric focusing step were combined and subjected
`
`
`
`were eluted with a to RP-HP LC using a C18 analytic column. Peptides
`Fig. 4. Isolation of guanylin from colonic mucosa by isoelectric
`
`
`
`
`
`
`gradient from 5% acetonitrile containing 10 mM ammonium acetate
`
`
`
`
`focusing. Extracts of colonic mucosa were chromatographed with C18
`
`
`Sep-Pak cartridges and then fractionated on a Sephadex G-25
`
`
`
`to 25% acetonitrile containing 10 mM ammonium acetate over a
`
`
`period of 180 min. Bioassay was conducted with T84 cells in HEPES
`
`
`
`
`column before bioactive peptides were subjected to isoelectric focus­
`
`
`and DMEM at pH 7.4. Bioactive peptides eluted from this column at
`
`ing. Each fraction was assayed with T84 cells in MES, DMEM at pH
`I 0% acetonitrile.
`
`5.5 (open bars), or HEPES and DMEM at pH 7.4 (solid bars).
`
`MSN Exhibit 1016 - Page 3 of 8
`MSN v. Bausch - IPR2023-00016
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`10 13 16 19
`
`Fraction
`
`3:
`
`2
`C,
`(.)
`
`0
`E
`
`38
`
`54
`
`70
`
`89
`
`Fraction
`
`1 8
`
`"cf.
`
`1 6
`
`)>
`1 4 (')
`CD
`
`0
`-
`1 2 :::,
`::;:
`:::!.
`1 0 ;-
`
`=
`
`cij 15
`C. :i:10
`5
`
`C,
`
`0
`E
`Q.
`
`
`
`Downloaded fromjoumals.physiology.org/joumal/ajpendo (098.109.055.010) on January 14, 2021.
`
`
`
`
`
`
`
`

`

`E960
`
`STRUCTURE AND ACTIVITY OF UROGUANYLIN AND GUANYLIN
`
`-
`
`� 3
`
`2 !2_
`1·6 G>
`�
`
`(')
`
`::: I\ :::
`
`18
`
`17
`::R. 0
`16 )>
`
`15 S'
`:::s
`
`C,
`
`r
`
`)>
`
`1 4
`12 £
`0
`1 0
`=·
`s:
`8 ci>
`� 6
`
`fractions were combined and subjected to RP-HPLC
`
`
`
`0
`
`
`peptides with a C18 analytic column, the bioactive
`::C 2 5 +-...L......J....---L..---L----1-L-..___,___.__.__._--+
`=
`
`eluted at 15.5% acetonitrile (Fig. 5). This characteristic
`Peak 2
`c.
`
`
`
`
`elution profile for rat guanylin (4) indicates that the
`- 20 Peak 1
`
`
`
`
`active peptides isolated from the colonic mucosa of rats
`-
`j
`
`
`are predominantly guanylin. This peak ofguanylin-like
`
`
`
`peptides was purified further by use of C18 RP-HPLC
`
`
`
`with an acetonitrile gradient containing 0.1% TFAand
`:iii:
`8 column as
`
`
`finally by microbore RP-HPLC with a C
`I �
`�
`
`
`
`described above. This peptide fraction was then sub­
`0.4
`�
`� 5
`•-c.,'\;••..-1,,•o. .. c:,,•C"-<>"o•<> S'
`
`
`
`jected to NH2-terminal sequence analysis, and the
`u•o•-l
`::c
`0
`E o -l-a=t=o1:::t=:1::1::C___::;._�.;::»:""'9"""'"""'"""'=---I-
`o
`
`
`15-residue peptide PNTCEICAYAACTGC was ob­
`18 22 26 30 34 38
`C.
`U1
`
`
`
`tained. This is the same amino acid sequence as that
`Fraction
`.e
`
`
`obtained when guanylin was originally isolated from
`
`
`Fig. 6. Separation of uroguanylin-like and guanylin-like peptides
`
`the jejunum of rats (4).
`
`
`from duodenum by gel filtration chromatography. Mucosa from
`
`The duodenum may produce uroguanylin, because
`
`
`
`duodenum was heated at I 00°C in I M acetic acid; then extracts were
`
`
`the content of guanylin mRNA in duodenum ofrats is
`
`
`to a Sephadex G-25 fractionated with C18 Sep-Pak before application
`
`
`
`considerably lower than the mRNAlevels of colon, and
`
`column. Fractions were assayed using the T84 cell cGMP accumula­
`
`tion bioassay in MES and DMEM at pH 5.0 (dashed line) and in
`
`
`the duodenum has substantial cGMP responses to
`
`HEPES and DMEM adjusted to pH 8.0 with 50 mM sodium bicarbon­
`
`
`
`these peptides (20, 21, 31). Bioactive peptides were
`
`ate (solid line).
`
`
`isolated from the mucosa of rat duodenum, and two
`
`
`
`separate peaks of peptide bioactivity eluted at different
`matographic elution profile using RP-HPLC and the
`
`
`
`
`
`
`positions within the internal volume of Sephadex G-25
`
`
`
`pH dependency for activation of receptor guanylate
`
`columns (Fig. 6). When these fractions were bioassayed
`
`
`cyclases (GCs) of this peptide from the duodenum
`
`
`using T84 cells, we found that peak 1 stimulated cGMP
`
`
`mucosa are characteristic properties of uroguanylin.
`
`
`accumulation greater at pH 5.0 than at pH 8.0 (urogua­
`
`
`An insufficient quantity of the purified uroguanylin­
`
`
`nylin-like) and that peak 2 stimulated cGMP accumula­
`
`
`
`like peptide was available for NHrterminal sequence
`
`tion greater at pH 8.0 than at pH 5.0 (guanylin-like).
`
`
`
`analysis; thus confirmation of these findings by elucida­
`
`
`The very low stimulation of cGMP accumulation ob­
`
`tion of the peptide's sequence was not possible.
`
`served for the peak 1 aliquot at pH 8.0 and the
`
`
`A partial cDNA EST encoding the COOR-terminal
`
`
`correspondingly low stimulation for the peak 2 aliquot
`
`
`
`portion ofprouroguanylin was isolated from the duode­
`
`
`at pH 5.0 may be explained by the relatively low
`
`
`num of zinc-deficient rats (1). This information facili­
`
`
`
`concentrations of these peptides in the aliquots from
`
`
`tated the production of a uroguanylin cDNA by use of
`
`
`
`the columns that were bioassayed. Peak 1 (urogua­
`
`reverse transcription of RNA from rat duodenum and
`
`
`
`nylin) was pooled and further purified by C18 RP-HP LC
`
`
`the PCR to amplify this form ofuroguanylin cDNA. The
`
`
`by use of a 5-25% acetonitrile gradient containing
`
`
`uroguanylin cDNA was cloned and sequenced to con­
`
`
`
`ammonium acetate. The bioactive peptides eluted at
`
`firm its identity and then used as a cDNA probe in
`
`
`
`11 % acetonitrile, which is consistent with this peptide
`
`
`
`Northern assays to assess the relative abundance of
`
`
`being uroguanylin (Fig. 7). Moreover, this peptide
`
`
`
`uroguanylin mRNA compared with guanylin mRNA in
`
`
`
`stimulated cGMP accumulation greater at the medium
`
`
`
`the intestine. Uroguanylin transcripts of ~0.75 kilo-
`
`
`pH of5.0 than at pH 8.0, which is also a property found
`
`
`
`in the uroguanylin peptides. To summarize, the chro-
`3
`G)
`:i= 2.5
`cii
`C. 2
`fl.
`:iii: 1 .5
`(.) 1
`0
`E o.5
`C.
`0
`
`50
`
`Q) 40
`�
`fl.
`:iii: 30
`
`C,
`
`(.) 20
`0
`E 10
`C.
`
`14
`s.
`ci>
`1 3
`
`7
`
`55
`
`39
`23
`71
`Fraction
`70 74 78 82 86 91 95 99 103 107 112
`
`Fig. 7. Isolation ofuroguanylin from duodenum by RP-HP LC. Peak 1
`
`
`
`from the Sephadex G-25 gel filtration column step was combined and
`Fraction
`
`
`
`with a RP-HPLC and fractionated subjected to C18 semipreparative
`
`
`
`Fig. 5. Purification of guanylin from colonic mucosa by RP-HPLC.
`
`
`
`gradient of acetonitrile containing 10 mM ammonium acetate, as
`
`
`
`Bioactive fractions from the isoelectric focusing step were applied to a
`
`
`
`described in Fig. 2. Eluted fractions were assayed by T84 cell cGMP
`
`
`ile/ammo­C18 RP-HPLC analytic column and eluted with acetonitr
`
`
`
`stimulation bioassay in MES and DMEM at pH 5.0 (dashed line) and
`
`
`
`
`nium acetate, as described in Fig. 2. Fractions were bioassayed using
`
`
`in HEPES and DMEM adjusted to pH 8.0 with 50 mM sodium
`
`T84 cells in HEPES and DMEM adjusted to pH 8.0 with 50 mM
`
`
`
`
`
`bicarbonate (solid line). Major peak ofbioactive peptides eluted from
`
`
`
`sodium bicarbonate. Peak of bioactive peptides eluted from this
`
`
`this column at 11 % acetonitrile, consistent with the chromatographic
`
`
`
`column at 15.5% acetonitrile is similar to that of authentic guanylin.
`properties ofuroguanylin.
`
`0
`
`1 2
`
`MSN Exhibit 1016 - Page 4 of 8
`MSN v. Bausch - IPR2023-00016
`
`
`
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`Downloaded fromjoumals.physiology.org/joumal/ajpendo (098.109.055.010) on January 14, 2021.
`
`

`

`
`
`S TRU CT URE AND ACTIVITY OF UROGUANYLIN AND GUANYLIN
`
`E961
`
`base (kb) were detected throughout the intestinal tract,
`
`
`
`substantially less potent (Fig. 9). These data indicate
`
`
`
`
`but the highest levels were found in the duodenum and
`
`
`that the NHrterminal residues found in the bioactive
`
`
`
`
`uroguanylin peptide consisting of TDE increase the
`
`
`jejunum of small intestine (Fig. 8). Lower levels of
`
`
`uroguanylin mRNA were observed in ileum and the
`
`
`
`
`potencies ofthis peptide agonist for activation ofrecep­
`
`cecum and colon compared with duodenum and jeju­
`
`
`tor GCs on T84 cells compared with the potency of the
`
`
`
`num. Guanylin mRNA transcripts of ~0.6 kb were
`
`
`
`truncated 12-residue form of uroguanylin. However,
`
`
`
`detected throughout the intestinal tract, with the high­
`
`
`the 12 amino acids in the truncated uroguanylin analog
`
`
`
`est mRNAlevels observed in cecum and colon compared
`
`
`containing the peptide domain between the first and
`
`
`with the levels in small intestine. The lowest guanylin
`
`
`last cysteine residues with two intramolecular disulfide
`
`
`mRNA levels were found in the duodenum relative to
`
`
`bonds represent a core structure that is required for
`
`
`
`
`other segments of intestine. Progressively greater lev­
`
`
`biological activity in this assay.
`
`els of guanylin mRNA were found along the longitudi­
`
`
`nal axis oft he small intestine from duodenum to ileum,
`DISCUSSION
`
`
`
`with the greatest mRNA levels observed in the cecum
`Uroguanylin was isolated from both urine and duode­
`
`
`and colon.
`
`num mucosa of rats and identified by its unique bio­
`
`
`
`
`Analysis ofthe EST for uroguanylin derived from rat
`
`
`
`chemical and pharmacological properties (12-15). Uro­
`
`
`
`intestine (1) confirmed that the partial amino acid
`
`
`
`guanylin is present in the urine ofrats, as it is in the
`
`
`
`sequence obtained for the urinary peptide was consis­
`
`urine of the opossum and human species (14, 18).
`
`
`tent with the sequence predicted by the uroguanylin
`
`
`Guanylin was isolated from mucosa of both the duode­
`
`
`
`EST. Thus a synthetic peptide was prepared on the
`
`
`num and large intestine, but active guanylin peptides
`
`basis of the amino acid sequence TDECELCINVAC­
`
`
`
`
`were not detected in urine. Sequence analysis ofurogua­
`
`
`
`TGC, and the potency of this peptide was compared
`
`
`
`with the potencies ofrat guanylin and a truncated form
`
`nylin from rat urine revealed that the eight residues
`
`
`obtained were identical to those found in opossum
`
`
`of uroguanylin, CELCINVACTGC, by use of the T84
`
`
`
`uroguanylin. One oft he two NH2-terminal acidic amino
`
`cell bioassay (4, 14). Uroguanylin and guanylin had
`
`
`
`
`similar potencies in the activation of receptor GCs in
`
`
`acids unique to uroguanylin was not clearly defined
`
`
`
`T84 cells, but the truncated form of uroguanylin was
`
`(Glu or Asp), and the other acidic amino acid was not
`
`A
`
`.... ....
`00
`00
`
`....
`00
`�
`"O j
`e ::§
`�
`Q
`
`e
`= 0
`= �
`"S
`<II
`u
`u
`
`13-Actin
`...
`
`Uroguanylin
`...
`
`B
`
`...
`13-Actin
`
`...
`Guanylin
`
`Fig. 8. Distribution of uroguanylin and gua­
`
`
`
`nylin mRNAin the intestine. Total RNAof20 µg
`
`from mucosa of rat proximal small intestine
`
`(Prox. SI), middle small intestine (Mid. SI),
`
`
`distal small intestine (Dist. SI), cecum, and
`
`colon were loaded on each lane. A: arrows mark
`
`single transcripts for [3-actin mRNA of 1.9 kilo­
`
`
`base (kb) and uroguanylin mRNAof0.75 kb; B:
`
`
`arrows indicate single transcripts for [3-actin
`
`mRNAof 1.9 kb and guanylin mRNAof0.6 kb.
`
`MSN Exhibit 1016 - Page 5 of 8
`MSN v. Bausch - IPR2023-00016
`
`
`
`
`
`
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`Downloaded fromjoumals.physiology.org/joumal/ajpendo (098.109.055.010) on January 14, 2021.
`
`

`

`E962
`
`STRUCTURE AND ACTIVITY OF UROGUANYLIN AND GUANYLIN
`
`1000.0
`
`100.0
`
`10.0
`
`1.0
`
`0.1
`
`sodium diets downregulate the guanylin receptor-Ge
`
`
`
`
`
`signaling pathway in the rat colon suggests that the
`
`
`
`guanylin signaling pathway may participate in the
`
`
`maintenance of salt and water homeostasis (24).
`
`In rats, guanylin mRNA levels appear to be most
`
`
`abundant in colon and ileum, with intermediate mRNA
`levels in jejunum and the lowest mRNA levels in
`
`duodenum (25, 27, 31). In the present experiments,
`
`
`
`
`bioactive guanylin was isolated from colonic mucosa
`
`
`
`but bioactive uroguanylin was not detected. This find­
`
`
`
`
`ing is consistent with the high levels ofguanylin mRNA
`
`
`that were detected using Northern assays with total
`
`RNA from colon and cecum in this study compared with
`
`
`
`the lower uroguanylin mRNA levels oflarge intestine.
`
`
`The lower abundance of uroguanylin mRNAs in the
`
`
`colon and cecum provides one explanation for our
`Fig. 9. Bioactivity of synthetic uroguanylin and guanylin in T84
`
`
`
`
`
`
`
`
`inability to detect bioactive uroguanylin in extracts of
`
`
`
`
`cells. Values are representative of 3 experiments conducted with
`
`
`
`large intestine. Isolation of uroguanylin and guanylin
`
`
`cultured T84 cells and are means of duplicate assays at each peptide
`
`from duodenum in this study suggests that both pep­
`■, Rat guanylin
`concentration.
`
`(PNTCEICAYAACTGC); .&, rat uro­
`
`
`
`tides are present and may regulate the activity of
`
`
`
`
`guanylin (TDECELCINVACTGC); 0, 12-residue portion of urogua­
`
`
`
`receptor-Ge signaling molecules in this segment.
`
`
`
`nylin (CELCINVACTGC). Disulfide bonds in these synthetic peptides
`
`
`occur between 1st to 3rd and 2nd to 4th cysteine residues. Medium is
`
`
`Whereas Northern assays suggest that uroguanylin
`DMEM at pH 7.4 for this assay.
`
`
`
`mRNA expression is greater than guanylin mRNA
`
`
`
`expression in the duodenum, both peptides are present
`
`in the mucosa of the duodenum in concentrations
`determined. The amino acid sequence of rat urogua­
`
`
`
`
`
`sufficient for purification and identification of this
`
`
`
`nylin has been recently elucidated by the isolation of
`
`
`
`bioactive peptide (25, 27, 29). Other studies have also
`
`
`
`cDNAclones encoding preprouroguanylin and by purifi­
`
`
`
`
`
`found a similar pattern of expression of guanylin and
`
`cation of uroguanylin from duodenum and NH2-
`
`
`
`uroguanylin mRNAlevels along the longitudinal axis of
`
`
`
`terminal sequence analysis (1, 26, 29). These studies
`
`the intestinal tract ofrats (25, 27, 29).
`
`
`revealed that the sequence of the 15 COOR-terminal
`Uroguanylin and guanylin markedly stimulate the
`
`
`
`
`
`
`residues for rat uroguanylin is TDECELCINVACTGC,
`
`
`transepithelial secretion of both c1-and HCO3 anions
`
`
`which agrees with the partial sequence that we ob­
`
`
`in the duodenum (11, 17). Exposure of the apical
`
`
`
`tained in the present study. A synthetic peptide pre­
`
`
`
`
`surface of duodenum to these peptides elicits a stimula­
`
`
`
`pared according to this sequence activated the T84 cell
`
`
`
`tion of short-circuit current consisting of both c1-and
`
`
`
`
`receptor GC with potency and efficacy similar to the
`
`
`
`activation elicited by synthetic rat guanylin.
`
`
`
`may be Both peptides HCO3 transport components.
`
`
`
`released from enterocytes into the luminal microdo­
`
`
`The reason bioactive guanylin is not found in rat or
`
`
`
`main at the surface of this epithelium where binding of
`
`human urine is unclear, but it may be the susceptibility
`
`
`
`the peptides to receptor-GCs occurs, thus activating
`
`
`
`
`of guanylin in the tubular filtrate to cleavage and
`
`
`
`inactivation by proteases within renal tubules. Gua­
`
`
`
`these signaling molecules and regulating anion secre­
`
`
`
`nylin is inactivated by chymotryps