rapid communication
`
`Characterization of human uroguanylin: a member
`of the guanylin peptide family
`
`TOSHIHIRO KITA, CHRISTINE E. SMITH, KAM F. FOK, KEVIN L. DUFFIN,
`WILLIAM M. MOORE, PETER J. KARABATSOS, JAMES F. KACHUR, F. KENT HAMRA,
`NYKOLAI V. PIDHORODECKYJ, LEONARD R. FORTE, AND MARK G. CURRIE
`Monsanto Corporate Research, Monsanto, St. Louis 63167; Department of Pharmacology,
`Missouri University, and Truman Veterans Affairs Hospital, Columbia, Missouri 65212;
`and Department of Inflammatory Disease Research, G. D. Searle, Skokie, Illinois 60077
`
`Kita, Toshihiro, Christine E. Smith, Kam F. Fok,
`Kevin L. Duffin, William M. Moore, Peter J. Karabatsos,
`James F. Kachur, F. Kent Hamra, Nykolai V. Pidhoro(cid:173)
`deckyj, Leonard R. Forte, and Mark G. Currie. Character(cid:173)
`ization of human uroguanylin: a member of the guanylin
`peptide family. Am. J. Physiol. 266 (Renal Fluid Electrolyte
`Physiol. 35): F342-F348, 1994.-Guanylin, a peptide homo(cid:173)
`logue of the bacterial heat-stable enterotoxins (ST), is an
`endogenous activator of guanylate cyclase C (GC-C). We have
`initiated a search for other members of the guanylin peptide
`family and in the current study describe a "guanylin-like
`peptide" from human urine. Bioactivity was monitored by
`determining the effect of urine extracts on T84 cell guanosine
`3' ,5' -cyclic monophosphate (cGMP) levels. Purification yielded
`two bioactive peaks of peptides that, when sequenced by
`NHrterminal analysis, possessed 15 and 16 amino acids. The
`sequence of the smaller peptide represented an NH2-terminal
`truncation of the larger peptide. We have termed the larger
`peptide human uroguanylin; it has the following amino acid
`sequence: NDDCELCVNVACTGCL. Human uroguanylin
`shares amino acid sequence homology with guanylin and ST.
`Synthetic uroguanylin increased cGMP levels in T84 cells,
`competed with 1251-labeled ST for receptors, and stimulated
`Cl- secretion as reflected by an increased short-circuit current.
`Thus we report the isolation from human urine of a unique
`peptide, uroguanylin, that behaves in a manner similar to
`guanylin and appears to be a new member of this peptide
`family.
`guanosine 3' ,5' -cyclic monophosphate; guanylate cyclase; chlo(cid:173)
`ride secretion; T84 cell; rat colon
`
`THE MEMBRANE-BOUND FORMS of guanylate cyclase have
`been found to serve as receptors for specific peptide
`ligands and are thought to participate in the regulation
`of blood pressure as well as water and electrolyte
`homeostasis (8, 22). These receptors share several struc(cid:173)
`tural motifs, including an extracellular ligand-binding
`site, a transmembrane region, an intracellular ATP
`regulatory domain, and an intracellular catalytic site for
`the generation ofguanosine 3',5'-cyclic monophosphate
`(cGMP) (3, 8). The natriuretic peptide family of recep(cid:173)
`tors with guanylate cyclase activity has been particu(cid:173)
`larly well characterized. These receptors include guanyl(cid:173)
`ate cyclases A and B (GC-A and GC-B, respectively), also
`
`known as natriuretic peptide receptors A and B (8, 22).
`The two subtypes of natriuretic peptide receptor show
`different tissue distributions and exhibit selective affini(cid:173)
`ties for the three different members of the natriuretic
`peptide family (18). GC-A has been shown to be quite
`sensitive to stimulation by atrial natriuretic peptide
`(ANP) and brain natriuretic peptide (BNP) but is very
`insensitive to stimulation by C-type natriuretic peptide
`(CNP) (8, 13, 18). On the other hand, GC-B is potently
`stimulated by CNP and only slightly responsive to either
`ANP or BNP (13).
`An additional form of membrane guanylate cyclase is
`GC-C (intestinal guanylate cyclase), which is an appar(cid:173)
`ent receptor for guanylin and bacterial heat-stable en(cid:173)
`terotoxins (ST) (2, 3, 17). This receptor is abundant in
`intestinal epithelial cells, where it serves as a target for
`ST to cause secretory diarrhea (16, 17). Recently, gua(cid:173)
`nylin, a peptide composed of 15 amino acids, has been
`identified as an endogenous ligand for GC-C (2, 23, 24).
`Guanylin was initially purified from rat jejunum and
`proposed as a modulator of intestinal fluid and electro(cid:173)
`lyte homeostasis (2). Subsequent cloning of a guanylin
`cDNA from rat (16, 23) and human (4, 24) intestinal
`cDNA libraries has determined that this peptide is
`synthesized as part of an 115-amino acid precursor. The
`COOR-terminal region is highly conserved between
`human, rat, and mouse species, and it is this portion of
`the prohormone that contains the guanylin peptide
`sequence (12). In these studies, it was found that
`proguanylin was relatively inactive and required the
`cleavage of the COOR-terminal bioactive region (4, 16).
`Guanylin mRNA, as has been previously described for
`GC-C mRNA, is most abundant in the intestinal tract,
`with the ileum and colon possessing the greatest level of
`expression (12, 23, 24). However, guanylin expression
`has also been detected in extra-intestinal sites, including
`the kidney, uterus, and adrenal gland (16).
`Because of the similarities of the natriuretic peptide
`guanylate cyclase system with the guanylin GC-C sys(cid:173)
`tem (8), we have initiated a search for guanylin and
`potential members of this peptide family in sites other
`than the intestine. Previous evidence suggested that the
`kidney may be a fruitful site for such a search. Rat
`kidney has a modest amount of guanylin-like bioactivity
`
`F342
`
`0363-6127 /94 $3.00 Copyright
`
`1994 the American Physiological Society
`
`Bausch Health Ireland Exhibit 2065, Page 1 of 7
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`BIOLOGICAL AND STRUCTURAL CHARACTERIZATION OF HUMAN UROGUANYLIN
`
`F343
`
`on T84 cell cGMP levels (2) and detectable levels of
`guanylin mRNA (16). ST binding sites, which have also
`been reported to be localized in the proximal tubules of
`opossum kidney and ST, caused large increases in cGMP
`levels in opossum renal tissue (6). Thus we hypothesized
`that endogenous ligand(s) for GC-C may be produced by
`the kidney and excreted in the urine. Efforts were
`initiated to isolate endogenous peptide activators of
`GC-C from both human and opossum urine. The find(cid:173)
`ings with the opossum are the subject of a separate
`manuscript (10). In the current study, we report the
`isolation and characterization of a novel 16-amino acid
`peptide from human urine with biological activity and
`structural similarities to guanylin. However, it is dis(cid:173)
`tinct from guanylin, and we contend that uroguanylin
`represents a new member of the guanylin family. We
`have designated this peptide as uroguanylin because
`urine was the source and because of its sequence
`similarity with guanylin, including the four conserved
`cysteine residues.
`
`MATERIALS AND METHODS
`
`Cell culture. T84 cells were obtained from the American
`Type Culture Collection at passage 52. Cells were grown to
`confluence in 24-well culture plates with a 1:1 mixture of
`Ham's F-12 medium and Dulbecco's modified Eagle's medium
`(DMEM) supplemented with 10% fetal bovine serum, 100 U
`penicillin/ml, and 100 µg streptomycin/ml. Cells were used at
`passages 56-58.
`cGMP determination. Confluent monolayers of T84 cells in
`24-well plates were washed twice with 250 µl of DMEM
`containing 50 mM N-2-hydroxyethylpiperazine-N' -2-ethanesul(cid:173)
`fonic acid (HEPES) (pH 7.4), preincubated at 37°C for 10 min
`with 250 µl of DMEM containing 50 mM HEPES and 1 mM
`isobutylmethylxanthine, and incubated with agents or frac(cid:173)
`tions for 30 min. The medium was aspirated, and the reaction
`was terminated by the addition of 0.5 ml of ice-cold 0.1 M HCl.
`Aliquots (150 µl total) were evaporated under a hot-air dryer
`and resuspended in 5 mM sodium acetate buffer, pH 6.4. The
`samples were subsequently measured for cGMP by radioimmu(cid:173)
`noassay as described previously (19).
`Purification of uroguanylin. Five separate batches of adult
`male human urine samples, 5 1 each, were collected and
`immediately placed on ice. The urine samples were applied to
`C18 Sep-Pak columns (Waters). The columns were washed
`with 10% acetonitrile/0.1 % trifluoracetic acid (TFA)/H20 and
`eluted with 40% acetonitrile/0.1 % TFA/H20. The eluted
`peptide fraction was lyophilized, resuspended in 7 ml of
`distilled H20, and centrifuged at 20,000 g for 20 min at 4°C.
`The resulting supernatant was separated by gel filtration
`chromatography (Sephadex G-25, superfine, 2.6 x 94 cm). The
`fractions were bioassayed, and the active fraction was lyophi(cid:173)
`lized. The sample was resuspended in 1 ml of 10% acetonitrile/
`0.1% TFA/H20 and applied to a C18 semipreparative high(cid:173)
`performance liquid chromatography (HPLC) column (Vydac,
`Hesperia, CA). The column was developed with the following
`linear gradient: 10% acetonitrile/0.1 % TFA/H20 to 40% aceto(cid:173)
`nitrile/0.1 % TFA/1120 in 120 min at a flow rate of 3 ml/min.
`The active fraction was lyophilized and resuspended in 1 ml of
`10% acetonitrile/0.1 % TFA/H20. The sample was applied to a
`C18 analytical HPLC column (Vydac), and active peptides were
`eluted using the above gradient over 180 min at a flow rate of 1
`ml/min. Two active fractions were separately lyophilized and
`reconstituted in 0.05 ml of 0.1 % TFA 1120. The samples were
`then separately applied to a C8 microbore column (Applied
`
`Biosystems, Foster City, CA) eluted with an increasing gradi(cid:173)
`ent of 0.33% acetonitrile/min in 0.1% TFA/H20. Two sepa(cid:173)
`rately purified peptides of each batch were then subjected to
`sequence analyses.
`NHTterminal protein sequence analysis. Automated Edman
`degradation chemistry was used to determine the NHr
`terminal protein sequence. An applied Biosystems model 4 70A
`gas-phase sequencer was employed for the degradations (11)
`using the standard sequencer cycle 03RPTH. The respective
`phenylthiohydantoin (PTH)-amino acid derivatives were iden(cid:173)
`tified by reverse-phase HPLC analysis in an on-line fashion
`employing an Applied Biosystems model 120A PTH analyzer
`fitted with a Brownlee PTH-C18 column. Reduction and pyri(cid:173)
`dylethylation for cysteine residue identification were per(cid:173)
`formed directly on the filter.
`Electrospray mass spectrometry. The molecular weights of
`uroguanylin samples were determined by a previously de(cid:173)
`scribed technique (2). Briefly, a triple quadrupole mass spec(cid:173)
`trometer equipped with an atmospheric pressure ion source
`was used to sample positive ions produced from an electro(cid:173)
`spray interface. Mass analysis of sample ions was accom(cid:173)
`plished by scanning the first quadrupole in increments of 1
`atomic mass unit from 1,000 to 2,400 atomic mass units in "'3
`s and passing mass-selected ions through the second and third
`quadrupoles operated in the radio frequency-only mode to the
`multiplier. For maximum sensitivity, the mass resolution of
`the quadrupole mass analyzer was set so that ion signals were
`z 2 atomic mass units wide at one-half peak height but the
`centroid of the ion signal still represented the correct mass of
`the ion.
`Radioligand binding assay. 1251-labclcd ST-(5-18) was
`prepared by the lodo-Gen method as previously described (7).
`T84 cell monolayers were washed two times with 1.0 ml/well
`of ice-cold binding assay buffer (Earle's medium containing 25
`mM 2-N-morpholino ethanesulfonic acid, pH 5.5). The cells
`were incubated for 30 min at 37°C in 0.5 ml/well of binding
`assay buffer with 1251-ST [100,000 counts/min (cpm) per wellJ
`and unlabeled peptides. Then the cells were washed four times
`with 1 ml of ice-cold binding assay buffer and solubilized with
`0.5 ml of 1 M NaOH per well. This volume was transferred to
`tubes and assayed for radioactivity by a multigamma counter.
`Results are expressed as the percentage of cells specifically
`bound.
`Measurement of Isc in T84 monolayers. Analysis of the effect
`of human uroguanylin on Isc in T84 cells was performed as
`previously described (5). Briefly, T84 cells raised on permeable
`filters were mounted in a custom-made Ussing chamber for
`measurement of Cl secretion. The buffer was a Krebs-Ringer
`bicarbonate solution, pH 7.4, containing 10 mM glucose. Both
`reservoir buffer solutions were mixed and oxygenated by
`bubbling 95% Or5% CO2 through the medium. Isc was mea(cid:173)
`sured continuously, and the potential difference across the
`epithelium was measured intermittently.
`Measurement of Isc in rat colon. Rat proximal colon tissue,
`consisting of only mucosa and submucosa, was mounted
`between two U ssing half-chambers and bathed on both sides in
`a manner similar to that previously reported (21). Electrical
`measurements were monitored with an automatic voltage
`clamp, and direct-connecting voltage- and current-passing
`electrodes were used to measure transepithelial potential
`difference and Isc· Tissues were equilibrated under short(cid:173)
`circuit conditions until J50 had stabilized.
`Chemical synthesis of uroguanylin. Uroguanylin was synthe(cid:173)
`sized by the solid-phase method (20) on an Applied Biosystems
`model 430A peptide synthesizer and purified by reverse-phase
`C18 chromatography. The purity and the structure were
`
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`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`F344
`
`BIOLOGICAL AND STRUCTURAL CHARACTERIZATION OF HUMAN UROGUANYLIN
`
`Fig. 1. Purification ofuroguanylin from human urine by
`gel filtration chromatography. Extract of 5 I of human
`urine was applied to a 2.6 x 94-cm Sephadex G-25
`(superfine) gel filtration column. Isocratic 50 mM ammo(cid:173)
`nium acetate was used to elute peptides at a rate of 0.5
`ml/min, and 5 ml of fractions were collected after 100 ml
`of initial elution. Molecular weight standards were sepa(cid:173)
`rately assessed lvoid volume (V0 ): blue dextran 200,
`insulin (mo! wt 5,750), atriopeptin III (APIII; mo! wt
`2,550), rat guanylin (mo! wt 1,516)]. All fractions were
`assessed in T84 cell cGMP accumulation bioassay.
`
`1000 ~
`
`Q)
`
`~
`0
`
`E -._..
`
`800 ~
`
`600 ~
`
`400 •
`
`200 •
`
`0
`
`insulin
`
`APIII
`
`guanylin
`
`Vo
`
`.J.
`
`,1,11.,■ulld1JI
`
`I
`
`20
`
`I 11,1 l,1,l,■1■,■1■ 1,l 1,1 ■,I I ■1111,11
`.
`.
`
`I
`
`30
`
`40
`
`50
`
`60
`
`Fraction number
`
`verified by analytical HPLC, amino acid composition analysis,
`mass spectroscopy, and sequence analysis.
`
`RESULTS
`
`In initial experiments, the peptide fraction of human
`urine samples resulting from C18 Sep-Pak extraction
`was assayed for activity to increase cGMP levels in T84
`cells. These preliminary experiments strongly suggested
`the presence of GC-C stimulatory activity. The urine
`extract was subjected to fractionation by gel filtration
`and a series of reverse-phase HPLC steps to produce a
`sufficiently pure preparation for the purpose of struc(cid:173)
`tural determination. Fractionation by G-25 gel filtration
`chromatography yielded a single major bioactive frac(cid:173)
`tion that migrated on the column with an apparent size
`of 5,000 Da (Fig. 1). Subsequently, this active fraction
`was further purified by reverse-phase HPLC using a
`semipreparative C18 column, and the bioactivity was
`determined to reside in only one fraction eluting at
`27.8% acetonitrile/0.1% TFA/H20 (data not shown).
`Further purification by reverse-phase HPLC using a C18
`analytical column yielded two active fractions that ap(cid:173)
`peared to elute with peaks of substances that absorbed
`at 220 nm (Fig. 2). These two fractions were separately
`subjected to further characterization by microbore HPLC
`(C8 column), and each fraction exhibited a single bioac(cid:173)
`tive peak that absorbed at 220 nm (data not shown). The
`amino acid sequences of the two peaks were indepen(cid:173)
`dently determined by the Edman degradation proce(cid:173)
`dure. The sequences are NDDCELCVNV ACTGCL and
`DDCELCVNVACTGCL for peaks 1 and 2, respectively.
`These two peptides are identical except that the peptide
`contained in peak 1 possesses an additional amino acid
`(asparagine) at the NHrterminus. It is likely that peak 2
`is a degradation product of peak 1, probably a result of
`aminopeptidase action. Electrospray mass spectromet(cid:173)
`ric analysis of the two fractions yielded observed molecu(cid:173)
`lar masses of 1,666.6 and 1,552.6 atomic mass units,
`
`respectively, for the peptides contained in peaks 1 and 2.
`These molecular weights correspond to the theoretical
`molecular weights derived from the sequences if two
`disulfide bonds link the four cysteines, and therefore
`indicate that the full sequences of these peptides were
`determined by NHrterminal protein sequence analysis.
`Comparison of the sequence of peak 1 with other
`proteins in the GenBank, National Biomedical Research
`Foundation, and SwissProt databases by computer(cid:173)
`based search indicates that this sequence is a unique
`sequence. This search did reveal that human urogua(cid:173)
`nylin shares homology with guanylin and ST. The
`comparison between human uroguanylin, opossum uro(cid:173)
`guanylin, Escherichia coli ST, and human guanylin is
`shown in Fig. 3. The comparisons indicate that the four
`cysteine residues and the ACTGC COOR-terminal amino
`acid region of all four peptides are conserved. A unique
`feature of uroguanylin is the more acidic nature of the
`NHrterminal region, because the human peptide pos(cid:173)
`sesses two adjacent aspartic acid residues and the
`opossum peptide has a glutamate and an aspartate
`residue at these positions. Thus human uroguanylin
`appears to be a member of the guanylin/ST family of
`peptides.
`Chemical synthesis of bioactive human uroguanylin
`(the 16-amino acid-containing peptide) was accom(cid:173)
`plished by directed folding of the peptide. The synthetic
`bioactive peptide possesses disulfide-linked bridges be(cid:173)
`tween the 4-12 and 7-15 amino acid positions as
`previously suggested for the disulfide links of guanylin
`(2, 24). Analysis of the biological activity of human
`uroguanylin was assessed by determining its effect on
`T84 cGMP levels, competition-binding studies with
`1251-ST as the radioligand in T84 cells, and stimulation
`of Cl- secretion as reflected by increases in lsc using T84
`cells and rat colon.
`Synthetic human uroguanylin caused a concentration(cid:173)
`dependent increase in T84 cell cGMP (Fig. 4A). Human
`
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`
`

`

`BIOLOGICAL AND STRUCTURAL CHARACTERIZATION OF HUMAN UROGUANYLIN
`
`F345
`
`--------------
`
`---------
`
`-------------------------
`
`--------
`
`~ ' '
`30 ..!.-
`z
`25 U
`M :c
`20 u
`~ 0
`
`Fig. 2. Purification ofuroguanylin from human urine by
`reverse-phase high-performance liquid chromatography
`(HPLC). Five liters of human urine extract was purified
`through the semipreparative reverse-phase HPLC, and
`active fraction was fractionated on an analytical C1s
`column (Vydac). A linear gradient of 10-40% acetoni(cid:173)
`trile, 0.1% TFA was developed at 1.0 ml/min over 3 h.
`One-minute fractions were collected and assayed for
`activity in T84 cell cGMP bioassay. This figure shows the
`biologically active region, with two peaks associated with
`changes in ultraviolet absorbance.
`
`0.04
`
`0.03
`
`0.02
`
`0.01
`
`0
`
`E
`s:::
`
`0
`N
`N
`i;
`
`Q)
`(J
`s:::
`111 .c
`...
`0
`Ul .c
`<
`
`Q)
`
`15000
`~
`0
`10000
`
`E --
`
`C.
`:E
`(!)
`u
`
`5000
`
`0
`
`90
`
`100
`
`110
`
`120
`
`130
`
`Time
`
`(min)
`
`uroguanylin appeared to be more potent than human
`guanylin but less potent than ST for activation of GC-C
`in T84 cells. A similar profile of relative affinity was
`obtained using the competitive binding assay with 1251-
`ST-(5-18) as the radioligand (Fig. 4B), but it should be
`noted that all of the peptides acted in a more potent
`manner in causing 1251-ST-(5-18) displacement than in
`increasing cGMP levels. This difference in potencies for
`receptor binding and guanylate cyclase activation has
`been noted for the other members of the particulate
`guanylate cyclase family (8) and has recently been
`observed for cells transfected with human GC-C (9). The
`data indicate that these peptides all possess the ability to
`
`Human Uroguanylin
`
`Human Guanylin
`
`PGTCEICAYAACTGC
`
`stimulate GC-C and share similar binding sites with
`varying degrees of relative affinities for the receptors in
`T84 cells.
`To assess the effect of human uroguanylin on well(cid:173)
`characterized ST- and guanylin-sensitive transport func(cid:173)
`tions, we assessed the effects of the peptide on Isc of T84
`cells and proximal rat colon. In these experiments, the
`measurement of I sc is used as an indicator of transepithe(cid:173)
`lial chloride secretion. Previous studies in these prepara(cid:173)
`tions have indicated that the change in /sc elicited by ST
`and guanylin is mostly accounted for by an increase in
`chloride secretion (5). Human uroguanylin added to the
`apical reservoir of T84 cells mounted in Ussing cham(cid:173)
`bers caused a marked and rapid increase in lsc (Fig. 5A).
`Similarly, human uroguanylin added to the mucosal
`reservoir of rat colon mounted in an U ssing chamber
`also caused a sustained rise in lsc (Fig. 5B).
`
`Opossum Uroguanylin
`
`QEDCE C INVACTGC
`
`DISCUSSION
`
`E.coli ST
`
`Fig. 3. Comparison of the structures of human uroguanylin, human
`guanylin, opossum uroguanylin, and E. coli heat-stable enterotoxin
`(ST). Identical amino acids are boxed. Human uroguanylin (position
`1-15) is 53%, 80%, and 73% identical to human guanylin, opossum
`uroguanylin, and ST, respectively.
`
`The present study describes the purification and
`structural identification of a new peptide, isolated from
`human urine, that activates GC-C. The finding of a
`bioactive peptide in human urine extends our prior
`efforts to isolate and identify peptides in the opossum
`(10). We have termed this peptide human uroguanylin
`
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`
`

`

`F346
`
`BIOLOGICAL AND STRUCTURAL CHARACTERIZATION OF HUMAN UROGUANYLIN
`
`10 6 A
`---o-- STa
`-
`Human Uroguanylin
`---o-- Human Guany/in
`
`:::-
`'ii 105
`~
`
`(I)
`Q)
`0
`
`E 10 4 -~
`
`D.
`:l:
`C,
`()
`
`10 3
`
`10 2
`
`18
`
`110
`
`0
`
`-1 0
`
`- 9
`
`-8
`
`- 7
`
`- 6
`
`0
`Ill
`0 90
`~ 0
`,:i 70
`C
`:::l
`0
`
`Ill .,
`I-
`~ 30
`.,
`« - 10
`
`50
`
`-10
`
`0
`
`-1 0
`
`-9
`
`-8
`
`-7
`
`-6
`
`- 5
`
`[Peptide], log (M)
`
`Fig. 4. A: concentration-response effect of synthetic human urogua(cid:173)
`nylin, human guanylin, and E.coli ST-(5~18) (STa) on cGMP levelR in
`T84 cells. Cells were incubated with various concentrations of ligands
`for 30 min. Values are means ± SE (n = 4). B: displacement of
`1251-STa-specific binding from T84 cells by human uroguanylin,
`human guanylin, and STa. Cells were incubated for 30 min at 37°C
`with labeled STa and indicated concentrations of ligands. Specific
`binding(%) was determined by dividing specifically bound 1251-STa at
`each ligand concentration by specifically bound 1251-STa in absence of
`ligands. Each determination represents the mean of 4 wells examined.
`
`because of its source, human urine, and because of its
`strong structural similarity to guanylin and opossum
`uroguanylin. Uroguanylin shares many biological prop(cid:173)
`erties with guanylin and ST. Uroguanylin stimulated
`cGMP accumulation and competed with 125I-ST-(5-18)
`for binding sites on T84 cells. The more potent effect of
`uroguanylin on binding displacement than on the activa(cid:173)
`tion of GC-C is an unexplained observation, but it is
`characteristic of particulate guanylate cyclases, includ(cid:173)
`ing GC-C (8, 9). In the T84 cell, this discrepancy may in
`part be explained by the presence of multiple receptors
`with different binding affinities. Indeed, our recent
`detailed characterization of ST and guanylin binding in
`T84 cells was consistent with a model predicting two
`receptors with different affinities for the peptide ligands.
`Uroguanylin also stimulated an incr~ase in lsc when
`added to the apical surface of both T84 cell monolayers
`and isolated proximal rat colon. These properties make
`uroguanylin an ideal candidate to serve as a paracrine/
`
`endocrine regulator of fluid and electrolyte homeostasis
`in tissues that express GC-C.
`The observation that human urine contains urogua(cid:173)
`nylin but little or perhaps no guanylin is different from
`our results with opossum urine. We isolated both urogua(cid:173)
`nylin and a second peptide that is a putative opossum
`homologue of guanylin (10) from opossum urine. Spe(cid:173)
`cies differences between human and opossum may ac(cid:173)
`count for this difference. It is also possible that guanylin
`was secreted in an inactive form. Recently, it was
`reported that the plasma form of guanylin from human
`renal patients is a much larger precursor that may have
`less intrinsic activity (14). We could find no evidence for
`the presence of a guanylin precursor. Attempts were
`made to activate the crude extract and larger-molecular(cid:173)
`weight fractions from the G-25 column by acetic acid
`treatment, with no success (data not shown). This
`treatment has been shown to activate the guanylin
`precursor because of cleavage at an acid-labile bond (4,
`16). These data strongly indicate that the major and
`perhaps the sole source of GC-C stimulatory activity in
`human urine is uroguanylin.
`The finding of another member of the guanylin pep(cid:173)
`tide family has parallels with other peptides and is
`particularly relevant to the natriuretic peptides. Both
`the guanylin peptides and the natriuretic peptides acti(cid:173)
`vate specific forms of particulate guanylate cyclase. ANP
`and BNP both potently activate GC-A, whereas CNP
`activates GC-B (13). These peptides appear to play an
`important role in the regulation of blood pressure and
`
`14 A
`12
`
`"'
`!310
`' < ... 8
`u
`Ill
`1-t 6
`
`4
`
`20
`
`40
`
`B
`
`60
`
`80
`TIME MIN
`
`100
`
`120
`
`140
`
`t
`
`Uroguanylln [0.2 µM]
`
`S minutes
`
`Fig. 5. Effect of synthetic human uroguanylin on short-circuit current
`Uscl of T84 cells or rat colon. A: stimulation of transepithelial Cl(cid:173)
`secretion in T84 cells by human uroguanylin. T84 cells cultured on
`collagen-coated filters were mounted in a modified Ussing chamber at
`time 0. After a steady baseline was achieved, 1 µM human uroguanylin
`was added to the apical reservoir. B: effect of human uroguanylin (0.2
`µM) on l,c across rat proximal colon after a mucosa! addition.
`Response in both A and B is characteristic of results from 3 other
`experiments.
`
`Bausch Health Ireland Exhibit 2065, Page 5 of 7
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`BIOLOGICAL AND STRUCTURAL CHARACTERIZATION OF HUMAN UROGUANYLIN
`
`F347
`
`fluid and electrolyte homeostasis through cGMP(cid:173)
`mediated processes (8, 22). Similarly, guanylin and
`uroguanylin activate GC-C and possess the characteris(cid:173)
`tics of paracrine regulators of electrolyte transport
`through cGMP-mediated processes. Guanylin, urogua(cid:173)
`nylin, and other potential members of this peptide
`family may play a critical role in the regulation of
`epithelial cell function by acting on GC-C, proteins
`homologous to GC-C, and perhaps non-guanylate cy(cid:173)
`clase receptors. Future exploration of this area may
`reveal an intricate paracrine/endocrine network for the
`regulation of epithelial cell function through the manipu(cid:173)
`lation of multiple guanylin-like peptides and their recep(cid:173)
`tors.
`The role of uroguanylin in the control of kidney
`function is unknown at present. However, ST has been
`shown to cause a marked increase of sodium excretion in
`the perfused rat kidney (15). It has been proposed that
`guanylin is secreted into the lumen of the intestine and
`subsequently activates the apically located GC-C (2, 23,
`24). Activation of GC-C results in an increase in intracel(cid:173)
`lular cGMP levels, a decrease in sodium and water
`absorption, and an increase in chloride secretion (2, 5).
`Uroguanylin could act in a similar manner in the
`kidney. It may be secreted or filtered into the lumen of
`the nephron to act on local or distal sites in the nephron
`to regulate the action of apical receptors and to influence
`fluid and electrolyte homeostasis. Because the rat kid(cid:173)
`ney responds to ST with an increase of sodium excre(cid:173)
`tion, a receptor for guanylin/uroguanylin is likely pre(cid:173)
`sent in renal tissue. ST binding has been demonstrated
`on the apical surface of the opossum proximal tubule,
`and ST increased cGMP levels in opossum renal tissue
`(6). However, attempts to demonstrate the presence of
`GC-C mRNA in rat renal tissue have been unfruitful
`(16, 17). A potential molecular target for cGMP / cGMP(cid:173)
`dependent protein kinase resulting from uroguanylin
`stimulation is the cystic fibrosis transmembrane conduc(cid:173)
`tance regulator (CFTR). CFTR has been localized to the
`apical surface of proximal and distal human renal
`tubules (1). Further studies examining the role of
`guanylin/uroguanylin and GC-C in the regulation of
`CFTR appear to be warranted.
`Determination of the tissue and cellular source of
`uroguanylin will provide useful information regarding
`the potential physiological role of this peptide. The
`kidney is a likely source of uroguanylin because the
`urine levels appear to be relatively high, but it is possible
`that uroguanylin is made by another organ and is
`filtered by the kidney into the urine. To rule out the
`possibility that the source of uroguanylin in males was
`the prostate gland or other male accessory sex glands,
`we purified the source of bioactivity from urine obtained
`from female subjects. The bioactivity found in female
`urine was similar in quantity to that found in male urine
`and eluted with similar retention times using gel filtra(cid:173)
`tion and reverse-phase HPLC (data not shown). Thus
`future studies targeted towarcl determining the exact
`tissue and cellular sources of this peptide are critical to
`our understanding of the physiological actions of urogua(cid:173)
`nylin.
`
`In summary, the present study describes the discov(cid:173)
`ery of a novel bioactive peptide, uroguanylin, from
`human urine with GC-C stimulatory activity. Urogua(cid:173)
`nylin appears to belong to the guanylin/ST peptide
`family. Human urine contains a relatively large amount
`of uroguanylin but an undetectable level of guanylin.
`This observation indicates that at least two independent
`regulatory systems for GC-C may exist. It is possible
`that renal electrolyte balance is under the control of
`uroguanylin and that guanylin acts predominantly in
`the intestine. Thus the findings of this study should
`provide an impetus to elucidate the role of uroguanylin
`in the control of renal function and to further examine
`the molecular features of the guanylin/ guanylate cy(cid:173)
`clase system.
`
`Address for reprint requests: M. G. Currie, Mail Zone T3P,
`Monsanto Corporate Research, 800 N. Lindbergh Blvd., St. Louis, MO
`63167.
`Received 29 September 1993; accepted in final form 12 November
`1993.
`
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