8498
`
`Biochemistry 1998, 3 7, 8498 8507
`
`In Vitro Disulfide-Coupled Folding of Guanylyl Cyclase-Activating Peptide and Its
`Precursor Protein
`
`Yuji Hidaka, *,t Megumu Ohno,! Bahram Henm1asi,U Oliver Hill,11 Wolf-Georg Forssmann,11 and
`Yasutsugu Shimonishit
`
`Institute for Protein Research, Osaka University, Yamada oka 3 2, Suita, Osaka 565 0871, Japan, Institute of Organic
`Chemistry, University of Tubingen, 72076 Tubingen, Gennany, and Lower Saxony Institute for Peptide Research,
`D 30 625 Hannover, Germany
`
`Received December 19, 1997; Revised Manuscript Received April JO, 1998
`
`ABS1RACT: Guanylyl cyclase-activating peptide II (GCAP-11), an endogenous ligand of particulate guanylyl
`cyclase C (GC-C), is processed from the precursor protein and circulates in human blood. GCAP-11
`consists of 24 amino acid residues and contains two disulfide bridges. The correct disulfide paring of
`GCAP-II is an absolute requirement for its biological activity. This study shows that the folding of the
`peptide from the reduced fom1 yields a peptide with the native disulfide paring as a minor product and
`with non-native ones as maj or products, regardless of the presence or absence of reduced and oxidized
`glutathione. The results suggest that GCAP-II does not possess sufficient infomiation to permit the adoption
`of the native confomiation and to effectively fom1 the correct disulfide pairing and, as a result, that GCAP-
`11 is correctly folded by assistance of a factor(s) such as an intra- or intermolecular chaperone. We studied
`whether a peptide in the pro-leader sequence of the precursor protein (proGCAP-II) contains sufficient
`infomiation to facilitate the folding of GCAP-II. For this purpose, we prepared proGCAP-II in Escherichia
`coli by a recombinant technique and exan1ined the disulfide-coupled folding of proGCAP-II from the
`reduced fom1 . proGCAP-11 was quantitatively recovered with the correctly folded structure from the
`reduced fom1 both in the presence and in the absence of reduced and oxidized glutathione. The protein
`contains only disulfide linkages at the same positions as the mature fom1 of proGCAP-11, GCAP-11, and
`the biologically active isomer of GCAP-11 in the molecule. These results provide evidence that the
`propeptide of proGCAP-11 is a critical factor in the fomiation of the correct disulfide paring in the folding
`of the protein.
`
`Guanylin and uroguanylin serve as endogenous ligands
`(1, 2) of particulate guanylyl cyclase C (GC-C) 1 (3). The
`enzyme is localized on the intestinal brush border cell
`membranes and has previously been shown to be a receptor
`protein for heat-stable enterotoxins (STa) produced by enteric
`bacteria (4, 5). This constitutes an important signaling
`system, which functions in the regulation of the level of
`
`* To whom correspondence should be addressed: Institute for Protein
`Research, Osaka University, Yamada-oka 3-2, Sui ta , Osaka 565--0871,
`Japan. Fax: +81--6-879-8(,()3. E-mail: yuji@protein.osaka-u.ac.jp.
`I Osaka University.
`§ University of Tiibingen.
`11 Lower Saxony Institute for Peptide Research.
`1 Abbreviations: GC, guanylyl cyclase; ST a, heat-stable enterotoxin
`of enterotoxigenic E. coli; STp, heat-stable enterotoxin produced by a
`porcine strain of E. coli; STp(4- I 7), derivative containing the amino
`acid sequence of residues 4 -17 of STp; STII, heat-stable enterotoxin
`II; cGMP, cyclic guanosine monophosphate; GCAP-11 , guanylyl
`cyclase-activating peptide II (the plasma form of uroguanylin); pro(cid:173)
`GCAP-11, precursor protein of GCAP-11 with amino acid residues 27-
`112 ofpre-proGCAP-11; Fmoc, 9-fluoromethoxycarbonyl; Acm, acet(cid:173)
`amidomethyl; Trt, trity l; DMF, dimethylformamide; TF A, trifluoroacetic
`acid; HPLC, high-performance liquid chromatography; Tris/HCI, tris(cid:173)
`(hydroxymethyl)aminomethane hydrochloride; PBS, phosphate-buffered
`saline; CD, circular dichroism; OTT, dithiothreitol; IPTG, isopropyl
`/3--0-thiogalactopyranoside; Gu/HCI, guanidine hydrochloride; GSSG
`and GSH, disulfide and thiol forms of glutathione, respectively; SOS,
`sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis;
`PCR, polymerase chain reaction.
`
`cGMP as a second messenger in intestinal or kidney cells,
`that is, the regulation of chloride transport in electrolytes by
`means of a paracrine interaction (6). The accumulation of
`epithelial cGMP induces the activation of a cystic fibrosis
`transmembrane conductance regulator via cross-talk with
`protein kinase A in the apical membranes, which, in tum,
`results in chloride and water secretion from the inside of a
`cell to the outside (7). Guanylin and uroguanylin were
`originally isolated from intestinal mucosa and urine, respec
`tively (1 , 2, 8). Northern blot analysis indicates that guanylin
`occurs not only in intestinal tissue but also in a variety of
`other tissues, such as kidney, airway epithelia, pancreas, and
`liver, while uroguanylin occurs in the intestine, atrium, and
`ventricle (9). Recently, another endogenous ligand of GC-C
`has been isolated from human blood and identified as a form
`ofuroguanylin which is extended at the N terminus (Figure
`1) . This ligand has been referred to as guanylyl cyclase
`activating peptide II (GCAP-II) (10). cDNA cloning of the
`peptides showed that uroguanylin and GCAP-Il are produced
`from the same precursor protein (prepro form, Figure l a)
`and that a peptide in the presequence (amino acid residues
`1 26) of the protein functions as a signal peptide for the
`secretion of these peptides (11 , 12). After secretion,
`prouroguanylin ancVorproGCAP-Il (amino acid residues 27
`112) are further processed to produce the mature forms of
`
`S0006-2960(97)03124-3 CCC: $15.00 © 1998 American Chemical Society
`Published on Web 05/22/1998
`
`Bausch Health Ireland Exhibit 2008, Page 1 of 10
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`
`

`

`Folding of Guanylyl Cyclase Activating Peptide
`a
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`IGCRAUGILLl'GVAVVLLLLLQS'l"QSVYIQrQC]FRVQLBSllltKf;
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`(a) Primaiy structure of pre proGCAP II (or pre
`FIGURE 1:
`prouroguanylin), a precursor ofGCAP II and uroguanylin . (b) The
`disulfide linkages in guanylin, uroguanylin, GCAP II, and heat
`stable enterotoxins are shown by solid lines.
`
`uroguanylin or GCAP II. It is noteworthy that both pro
`GCAP II and GCAP II are found in opossum plasma (12) .
`Thus, uroguanylin in opossum urine is thought to be derived
`from GCAP II in the plasma via glomerular filtration, since
`no uroguanylin mRNA is observed in kidney (12) .
`Guanylin and GCAP II consist of 15 24 amino acid
`residues and are homologous to STa in primary structure,
`as shown in Figure I b. These peptides contain four cysteine
`residues, which are localized at the same relative positions
`and are linked by disulfide bridges at the same positions as
`those in STa, as shown in Figure I b. The disulfide parings
`of guanylinand GCAP II play a critical role in the expression
`of biological activity (J, 2, 13). However, little is known
`concerning the folding pathway of the peptides, more
`specifically, the process by which the correct disulfide paring
`is achieved. In general, in eukaryotes, peptides having
`multiple disulfide linkages undergo a disulfide coupled
`folding during and/or after the elongation of peptide chains
`(14, 15). Moreover, non covalent interactions and a redox
`voltage thermodynamically or kinetically control folding and
`disulfide bond formation in vivo, resulting in the correct
`formation of the tertiary structures of peptides. Recently,
`the disulfide coupled folding of several peptides and proteins
`has been examined, and the results suggest that, in some
`cases, mature peptides do not possess sufficient information
`to allow for correct folding, and that a peptide in the pro
`leader sequence assists in the folding of the domain of the
`mature peptide or protein (14, 16 20). Preliminary experi
`ments in this laboratory regarding the folding mechanism
`of GCAP II showed that the mature form of proGCAP II,
`GCAP II, cannot correctly fold spontaneously, suggesting
`the possibility that folding and disulfide bond formation in
`GCAP II are controlled by a peptide in the pro leader
`sequence (amino acid residues 27 88) of proGCAP II.
`To further elucidate the folding mechanism of GCAP II,
`we report in this paper an investigation of the disulfide
`coupled folding of GCAP II in the presence or absence of
`reduced and oxidized glutathione. In addition, we examined
`the in vitro folding ofproGCAP II produced in Escherichia
`coli by a recombinant technique and determined the disulfide
`parings of proGCAP II to elucidate the role of a peptide in
`the pro leader sequence of GCAP II in the folding mecha
`nism. The data obtained shed light on our understanding of
`
`..
`l:ifllil~·1·I
`
`pro
`
`Biochemistry, Vol. 37, No. 23, 1998 8499
`
`the folding mechanism of small peptides, such as GCAP II
`and/or uroguanylin.
`
`MATERIALS AND METHODS
`
`T4 DNA ligase and restriction enzymes were purchased
`from Takara Shuzo Co. (Kyoto, Japan) and New England
`Biolabs, Inc. (Bervely, MA), respectively. Amino acid
`derivatives for peptide syntheses were obtained from Japan
`PerSeptive Biosystems, Inc. (Tokyo, Japan). Reagents were
`purchased from Sigma Chemical Co. (St. Louis, MO). All
`other chemicals and solvents were reagent grade. Continuous
`flow solid phase peptide synthesis was carried out using a
`MilliGen 9050 peptide synthesizer (Bedford, MA).
`Peptide Synthesis. The protected peptide (GCAP II),
`corresponding to the 24 C terminal amino acid residues of
`proGCAP II, was synthesized by the Fmoc solid phase
`method on a MilliGen peptide synthesizer. Two different
`types of protecting groups, the Acm group (residues 103 and
`111) and the Trt group (residues I 00 and 108), were
`employed for blocking cysteine residues, to selectively form
`the disulfide paring of GCAP II essentially as described in
`an earlier report (21) . Coupling reactions were carried out
`with O (7 azabenzotriazol I yl) 1, 1,3,3 tetramethyluronium
`hexafluorophosphate and diisopropylethylamine in DMF. The
`peptide resin was treated with 20% piperidine/DMF contain
`ing 2% diazabicycloundecene to remove the Fmoc group at
`the N terminus and then with TFA in the presence of
`ethanedithiol, thioanisole, and m cresol to release the peptide
`from the resin. The resulting peptide, carrying Acm groups
`on Cys residues (103 and 111) , was air oxidized to form
`the first disulfide bond between Cys100 and Cys108 and
`purified by HPLC. The Acm group of the peptide was
`removed by iodine, which then permitted the formation of
`the second disulfide bond. Finally, the resulting peptide was
`purified by HPLC and characterized by mass spectrometry
`and amino acid analysis. STp( 4 17) was synthesized
`essentially as described earlier ( 21).
`Reversed Phase High Perfonnance Liquid Chromatogra
`phy. The HPLC apparatus was comprised of a Waters 600
`multisolvent delivery system (Bedford, MA) equipped with
`a Hitachi L 3000 photodiode array detector and a D 2000
`chromato integrator (Tokyo, Japan). Synthesized peptides
`were purified by HPLC using a Develosil UG 5 column
`(ODS, 4.6 x 150 mm; Nomura Chemicals, Aichi, Japan).
`The peptides were eluted using a linear gradient of CH3CN
`in 0.05% TFA at a flow rate of I mUmin increasing at a
`rate of 1%/min from solvent A (0.05% TFA/H2O) to solvent
`B (0.05% TFA/CH3CN) . The separated peptides were
`rechromatographed using 10 mM AcONH4 (pH 5.7) in place
`of 0.05% TFA.
`Binding Activity of GCAP II to GC C £-cpressed on 293T
`Mammalian Cells. 293T mammalian cells, which express
`porcine GC C, were prepared as reported (2 2). The cells (3
`x 106) were suspended in 50 mM Tris/HCl (pH 8.0, 200
`,uL) containing 0.5 M NaCl and I mM phenylmethanesulfo
`nyl fluoride and sonicated. STp(4 17) was radioiodinated
`with chloramine T and Na125I and purified by HPLC to
`approximately 2000 Ci/mmol as described previously (22).
`293T cell membranes, which expressed the recombinant GC
`C, and [125I)STp( 4 17)(6 x 104 cpm) were incubated at 37
`°C for I h in a final volume of 60 ,uL of PBS( ) in the
`
`Bausch Health Ireland Exhibit 2008, Page 2 of 10
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`
`

`

`8500 Biochemistry, Vol. 37, No. 23, 1998
`presence or absence of 10 6 M synthetic GCAP II. [1251]
`STp( 4 17) which bound to the membranes was separated
`from free [125I]STp(4 l 7) by filtration on GF/C glass filters
`(Whatman) which had been pretreated with poly(vinylpyr
`rolidone) as described previously (23). The radioactivities
`of the membranes were measured using a y well counter.
`The binding assays were performed in duplicate.
`cGMP Assay. The cyclic GMP assay was carried out
`using 293T cells expressing porcine GC C, as reported
`previously (22). 293T cells (105 cells) were incubated with
`synthetic GCAP II (10 5 M) at 37 °C for 15 min in 20 mM
`Tris/HCl (pH 7.6, 300 µL) containing 0.1 mM 3 isobutyl
`1 methylxanthine. The reaction was stopped by the addition
`of 3 M sodium acetate (pH 4.6), followed by boiling. The
`amounts of cGMP in the samples were assayed in duplicate
`using a cGMP radioimmunoassay kit according to the
`specifications provided by the manufacturer (Yamasa Shoyu
`Inc., Tokyo, Japan).
`Circular Dichroism Measurement. Far UV CD spectra
`(190 260 nm) of each of the synthetic peptides (10 nmol)
`in PBS(
`) (0.5 mL) were measured at 25 °C in a cuvette
`with a 0.2 cm path length using a model J 700 spectropo
`larimeter (Jasco, Tokyo, Japan).
`Reduction and Reoxidation of GCAP II. Fully reduced
`GCAP II was prepared by incubating GCAP II (100 nmol)
`with 20 equiv ofDTT in 50 mM Tris/HCl (pH 8.0, 500 µL)
`under a N2 atmosphere at 50 °C for 1 h. Reduced GCAP II
`was purified by HPLC and lyophilized. Reduced GCAP II
`(10 nmol) was dissolved in 0.05% TFA (20 µL) and treated
`with 9 volumes each of the buffers described in Figure 6 at
`25 °C for 2 days. The reaction mixture was subjected to
`HPLC, and the recovery of the reoxidized peptide was
`estimated from the HPLC peak area. All solutions used for
`the experiment were flushed with N2, and the reaction was
`carried out in a sealed vial under a N2 atmosphere.
`Construction of Expression Vectors of proGCAP II. The
`cDNA encoding human proGCAP II (residues 27 112) was
`first subcloned into the expression vector pIN III ompA 1
`(24), following introduction by PCR of an EcoRI site at its
`5' end and a BamHI site at its 3' end using pEX2 containing
`a full cDNA gene of pre proGCAP II (25) as a template.
`Thus, three amino acid residues, Ala Asn Ser, were intro
`duced at the N terminus of proGCAP II. The resulting
`construct for the expression of proGCAP II is named pIN
`III ompA PU. pIN III ompA 1 contained the lpp promoter,
`the lac promoter, the Shine Dalgarno sequence, and the
`sequence encoding an OmpA signal peptide. Next, the
`cDNA encoding the OmpA signal peptide and proGCAP II
`was subcloned into the pET l 7b expression vector (Novagen),
`following introduction by PCR of an Ndel site at its 5' end
`and a BamHI site at its 3' end. The resulting expression
`vector, referred to as pET l 7b APU, contained the cDNA
`sequence which encoded the signal peptide of OmpA and
`Ala Asn Ser proGCAP II. The DNA sequences of the
`vectors thus constructed in this study were confirmed by
`analysis using an Applied Biosystems 373A sequencing
`system.
`Expression of proGCAP II. E. coli BL2 l(DE3) cells
`transformed with pETl 7b APU were grown at 3 7 °C in Luria
`broth medium (1 L) supplemented with ampicillin (50 µgl
`mL). The expression of proGCAP II was induced by the
`addition of 1 mM IPTG at the midlog phase of cell growth.
`
`Hidaka et al.
`
`After incubation at 37 °C for 3 h, E. coli cells were harvested
`and washed with 50 mM sodium phosphate (pH 7.8)
`containing 0.3 M NaCl and 1 mM phenylmethanesulfonyl
`fluoride. The cells were resuspended in the buffer (20 mL)
`and treated with lysozyme (1 mg/mL) on ice for 15 min,
`followed by sonication. The mixture was centrifuged
`(10000g for 20 min), and proGCAP II was obtained as an
`insoluble material. The yield of proGCAP II was ap
`proximately 3 5 mg from 1 L of the culture medium.
`Renaturation of proGCAP II from an Inclusion Body.
`Recombinant proGCAP II (100 µg), obtained as an insoluble
`material, was dissolved in 50 mM Tris/HCl (pH 8.0, 1 mL)
`containing 6 M Gu/HCl, 0.6 M Na2SO3, and 0.1 M Na2S4O6
`and kept at 50 °C for 1 h. After centrifugation, the
`supernatant was dialyzed against 50 mM Tris/HCl (pH 8.0,
`100 mL) at room temperature with two changes of buffer
`for 24 h. The supernatant, containing sulfonated proGCAP
`II, was subsequently dialyzed against 50 mM Tris/HCI (pH
`8.0, 100 mL) in the presence of2 mM GSH and 1 mM GSSG
`under a N2 atmosphere at 25 °C for 24 h. Renatured
`proGCAP II was purified by HPLC and characterized by
`mass spectrometry, amino acid analysis, and Edman degra
`dation.
`Refolding of proGCAP II. Renatured proGCAP II (2
`nmol) was dissolved in 0.1 M Tris/HCl (pH 8.0, 500 µL)
`containing 0.1 M DTT in the presence of 6 M Gu/HCl and
`kept at 37 °C for 30 min. Reduced proGCAP II was purifed
`by HPLC and lyophilized. The resulting material was
`redissolved in 0.1 M Tris/HCl (pH 8.0, 500 µL) containing
`10 mM DTT and 6 M Gu/HCl and dialyzed against 50 mM
`Tris/HCl (pH 8.0, 100 mL) in the presence or absence of 2
`mM GSH and 1 mM GSSG with three changes of buffer at
`25 °C for 3 days. Refolded proGCAP II was recovered in
`94 and 87% yields, estimated by the HPLC peak area, in
`the presence or absence of GSH and GSSG, respectively.
`Endoproteinase Arg C Digestion of proGCAP II. Re
`folded proGCAP II (3 nmol) was incubated with Arg C (30
`pmol) in 0.1 M Tris/HCI (pH 8.0, 200 µL) at 37 °C for 12
`h. The digest was then subjected to HPLC. The separated
`peptides were analyzed by mass spectrometry and amino acid
`analysis. The Arg C digest of proGCAP II (1.5 nmol) was
`dissolved in 0.1 M Tris/HCl (pH 8.0, 200 µL) containing
`20 mM CaCh and the mixture incubated at room temperature
`for 30 min with anhydrotrypsin agarose (Takara Shuzo),
`which had been equilibrated with buffer beforehand. The
`mixture was centrifuged (5000g for 5 min) and the super
`natant subjected to HPLC. The recovery of the C terminal
`fragment was determined by amino acid analysis.
`
`RESULTS
`
`Chemical Synthesis of GCAP II. GCAP II was synthe
`sized by the Fmoc solid phase method followed by the
`formation of disulfide linkages in a stepwise manner using
`two types of selectively removable thiol protecting groups
`(Trt for Cys 100 and Cys 108 and Acm for Cys 103 and Cys111)
`for the four Cys residues. Removal of the Trt group and air
`oxidization gave a peptide with one disulfide bond between
`Cys100 and Cys 108
`. Cleavage of the Acm groups by treatment
`with iodine then yielded GCAP II with the second disulfide
`linkage between Cys 103 and Cys111. These peptides with one
`or two disulfide bonds were purified by HPLC, as shown in
`
`Bausch Health Ireland Exhibit 2008, Page 3 of 10
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`□□
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`Biochemistry, Vol. 37, No. 23, 1998 8501
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`Retention time
`FIGURE 3: HPLC profiles of (a) GCAP 11 N and (b) GCAP 11 N'
`separated from Figure 2b and kept in 0. 1 M Tris/HCI (pH 8.0) at
`25 °C for 2 days and (c) GCAP 11 N and (d) GCAP 11 N' kept in
`0. 1 M Tris/HCI (pH 8.0) containing 6 M Gu/HCI at 25 °C for 2
`days.
`
`consisted of two topological isomers, similar to uroguanylin
`and GCAP II, and showed characteristics similar to those
`observed for GCAP II N and GCAP II N' (data not shown).
`This observation indicates that the N terminal region of
`GCAP II has no effect on the interconversion of the two
`topological isomers. The Leu residue at the C terminus of
`GCAP II, therefore, may contribute to the interconversion
`of the two isomers as has been reported for uroguanylin (26).
`The two isomers of GCAP II or Thr Ile Ala uroguanylin
`were incubated in the same environment in the absence of
`Arg C, like that for the digestion ofGCAP II with Arg C at
`37 °C, to estimate the interconversion of the isomers during
`the digestion of GCAP II, as described below. These two
`isomers interconverted at a rate of approximately 2% per
`24 h. On the basis of these observations, we were able to
`estimate the quantities of the two topological isomers
`(GCAP II N and GCAP II N') in proGCAP II, if they are
`latent within the molecule.
`Biological Activities of Two Topological Isomers of
`GCAP II. Two topological isomers (GCAP II N and GCAP
`II N') of synthetic GCAP II were assayed with respect to
`binding to GC C in 293T cell membranes and the generation
`of cGMP in 293T cells using procedures described previously
`(22). The biological activities of the two isomers of synthetic
`GCAP II were estimated by comparison with those of STp.
`In this experiment, a shorter analogue [STp(4 l 7)] which
`had the same level of biological activity as STp was used
`(23). GCAP II N was less active than STp(4 l 7) and
`showed approximately 30% of the binding activity of STp
`( 4 17) for binding to GC C, while GCAP II N' showed no
`significant activity, as shown in Figure 4a. The same results
`were obtained in the assay for cGMP production (Figure 4b).
`These results suggest that GCAP II N represents the biologi
`cally active form and the major form of GCAP II in
`circulation in human blood.
`Circular Dichroism. To obtain further evidence that
`shows that GCAP II N is the active form of GCAP II, CD
`spectra were measured in the far UV region. As shown in
`Figure 5, the spectrum of GCAP II N was nearly the same
`as that of STp(4 l 7), which is consistent with a previous
`report that showed that the active form of guanylin contains
`a backbone structure similar to STa (29, 30). In addition,
`
`Bausch Health Ireland Exhibit 2008, Page 4 of 10
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`-
`
`0
`
`20
`10
`30
`Retention time (min)
`
`40
`
`0
`
`30
`40
`10
`20
`Retention time (min)
`
`FIGURE 2: HPLC profiles of synthetic GCAP 11 analogues: (a)
`[Cys103(Acm),Cys111(Acm)]GCAP II with a disulfide bond between
`Cys100 and Cys108 and (b) [Cys103(Acm),Cys111(Acm)]GCAP II
`oxidized by iodine. The peak with an asterisk is derived from an
`impurity, produced during the Fmoc solid phase synthesis. The inset
`shows an expansion in the range of the 26 32 min retention time.
`N and N' represent two isomers of GCAP 11: GCAP 11 N and
`GCAP 11 N', respectively.
`
`
`
`panels a and b of Figure 2, respectively. The peptide
`(GCAP II) with two disulfide linkages which should be at
`the same positions was isolated as two peak fractions
`(GCAP II N and GCAP II N', labeled as N and N', respec
`tively, in Figure 2b). Chino et al. (26) reported that
`uroguanylin, a form which was secreted into urine and an
`analogue lacking eight amino acid residues at the N terminus
`of GCAP II (Figure lb), consisted of two topological
`isomers, when synthesized via a procedure similar to that
`used for the synthesis of GCAP II. The ratio (N:N'
`7:3)
`of two peak fractions in the synthetic GCAP II (Figure 2b)
`was nearly the same as that for the synthetic uroguanylin.
`This indicates that the N terminal peptide in GCAP II has
`no effect on either the formation of or the ratio of two
`topological isomers of uroguanylin or GCAP II.
`Interconversion of the Two Topological Isomers ofGCAP
`II. GCAP II exists as two topological isomers both in human
`plasma (27) and in the synthetic material (in this experiment),
`as well as in synthetic uroguanylin (26). Recently, Klodt et
`al. (28) reported that the maximum half life for the conver
`sion to one topological isomer of guanylin was approximately
`90 min. We examined the interconversion of each of the
`two isomers (GCAP II N and GCAP II N') of GCAP II.
`GCAP II N converted to GCAP II N' at a rate of 0.6% per
`24 h at 25 °C and vice versa, as shown in panels a and b of
`Figure 3, respectively, under the conditions described in the
`legend of Figure 3. The two isomers of GCAP II also
`interconverted at a similar rate, regardless of the presence
`or absence of a strong denaturant (6 M Gu/HCI), as shown
`in panels c and d of Figure 3. These results suggest that the
`two isomers of GCAP II interconvert rather slowly, com
`pared to guanylin, and that they are not constrained by
`intramolecular hydrogen bonds but, rather, that their inter
`conversion is mainly controlled by steric factors.
`To better understand the role of steric effects on the
`interconversion of the two topological isomers ofGCAP II,
`we examined the behavior ofThr Ile Ala uroguanylin. This
`peptide corresponds to the C terminal fragment ofGCAP II
`or proGCAP II prepared by Arg C digestion, lacks five
`amino acid residues at the N terminus of GCAP II, but carries
`an additional three amino acid residues at the N terminus of
`the urine form of uroguanylin. Thr Ile Ala uroguanylin
`
`Folding of Guanylyl Cyclase Activating Peptide
`
`a
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`8502 Biochemistry, Vol. 37, No. 23, 1998
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`FIGURE 4: Biochemical activities of two isomers ofGCAP II. (a)
`Activities of binding to GC C expressed in 2 93T cells of two
`topological isomers (GCAP II N and GCAP II N') and STp( 4
`17) . The nonspecific binding was less than 1 (J>/o of the specific
`binding. (b) The amount of cGMP in 2 93T cells stimu lated b y
`GCAP II N, GCAP II N', and STp( 4 17) . Both sets of data were
`estimated as the ratios ofGCAP Il N and GCAP II N' to STp(4
`17) . Experimental data are represented as the average of two data
`sets. A control experiment was carried out using 2 93T cells not
`transfected with pCG pSTaR (22).
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`Retenticn time (min)
`
`FIGURE 6: Folding of GCAP II. GCAP II was oxidatively folded
`in (a) 0.1 M Tris/HCl (pH 8.0), (b) 0 .1 M Tris/HCl (pH 8.0)
`containing 2 mM GSH and 1 mM GSSG, and (c) 0.1 M Tris/HCl
`(pH 8 .0) containing 6 M Gu/HCI. The 1, 2 , N, and N' represent
`isomers 1 and 2 and GCAP II N and GCAP II N' , respectively.
`were separated by HPLC, as shown in Figure 6. Of the four
`peaks, two were assigned to the topological isomers (GCAP
`II N and GCAP II N') of GCAP II and the remaining two
`to the disulfide isomers l and 2 (shown later), not only by
`mass spectrometric and amino acid analyses but also by
`comparison with the HPLC retention times of authentic
`peptides. These peptides had relatively the same intensity
`of molar absorbance on HPLC, indicating that the yield of
`each peptide can be estimated by HPLC peak area measure
`men ts. Indeed, the summation of each peak area of the four
`peaks (Figure 6) was almost equivalent to that of reduced
`GCAP II. Two isomers (GCAP II N and GCAP II N') of
`GCAP II were recovered as minor products regardless of
`the presence or absence of GSH and GSSG (6% for GCAP
`II N and 8% for GCAP II N' in the absence of GSH and
`GSSG), while isomers l and 2 (disulfide isomers) with
`disulfide linkages different from those of GCAP II N and
`GCAP II N' were the major components (56 and 30% for
`isomers l and 2, respectively). The same phenomenon was
`observed in the reoxidation of reduced GCAP II in PBS( )
`(data not shown). Interestingly, the ratio ofGCAP II N to
`GCAP II N' increased in the presence of GSH and GSSG
`in comparison with the ratios in their absence (Figure 6a,b ),
`implying that GCAP II N is more thermodynamically stable
`than GCAP II N'. When the refolding of reduced GCAP II
`was carried out in the presence of 10 mM GSH and l mM
`GSSG, isomers l and 2 were the major products as shown
`by HPLC, which was similar to that observed above (data
`not shown). Moreover, the folding efficiency of GCAP II
`was not affected by a denaturant (6 M Gu/HCl), as shown
`in Figure 6c. The experiment regarding the reductive
`unfolding of GCAP II in the presence of 2 mM GSH and l
`mM GSSG gave the same HPLC profile as that in Figure
`6b. These results suggest that correctly folded GCAP II is
`less thermodynamically stable than isomers l and 2 and that
`an additional factor(s) must be involved in the efficient
`folding of GCAP II from the reduced structure to the
`oxidized structure.
`To determine the positions of the disulfide linkages of
`isomers l and 2, we synthesized the disulfide isomers of
`GCAP II, which possess non native disulfide paring, on the
`basis of procedures similar to those used for the synthesis
`of GCAP II. Coelution experiments on HPLC of the
`synthetic isomers and the isomers l or 2 (in Figure 6a)
`indicated that the disulfide linkages of isomer l were between
`Cys 103 and Cys 108 and between Cys 100 and Cys 111 and those
`
`..25-1---~- - - -------'
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`2IO 220 238 240 251 2'0
`
`190 20)
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`Wavek nglh (nm)
`
`FIGURE 5: CD spectra of the two topological isomers of GCAP II
`and STp( 4 17) . Far lN CD spectra of GCAP II N, GCAP II N',
`and STp( 4 17) were measured at a peptide concentration of 20
`nmoVmL in PBS( ) at room temperature. (0 ) is the mean residue
`ellipiticity: (thin line) GCAP II N', (medium line) GCAP II N , and
`(thick line) STp(4 17).
`
`GCAP II N' showed a much smaller ellipticity at around 200
`run, compared to that of GCAP II N or STp(4 17). In a
`previous paper (31), we concluded that STp consists of three
`f3 tum structures: type I /3 turns in the N terminal and central
`regions and a type II /3 tum in the C terminal portion. The
`large ellipticity of STp(4 17) in the far UV region appears
`to reflect these f3 tum structures, since reduced or reduced
`and carboxymethylated STp had much smaller ellipticities
`than STp (data not shown). Furthermore, Skelton et al. (29)
`reported that the active form of guanylin is composed of
`three rigid /3 tum structures and that the inactive one assumes
`a flexible structure. Taken together, these studies provide
`evidence that GCAP II N represents the active form of
`GCAP II and has a conformation different from GCAP II
`N'. GCAP II has a much larger ellipticity at around 200
`run than that reported for guanylin (30). This may be due
`to the fact that the CD spectrum of guanylin was measured
`for a mixture of two topological isomers. These data also
`imply that GCAP II N contains the f3 tum structure which
`is similar to STp.
`Refolding of Reduced GCAP II. To study the folding of
`GCAP II, GCAP II was reduced by DTT and reoxidized in
`the presence or absence of 2 mM GSH and l mM GSSG in
`0.1 M Tris/HC! (pH 8.0) at 25 °C for 2 days. Four peaks
`
`Bausch Health Ireland Exhibit 2008, Page 5 of 10
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Folding of Guanylyl Cyclase Activating Peptide
`
`Biochemistry, Vol. 37, No. 23, 1998 8503
`
`- ◄ proGCAP-II
`
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`FIGURE 7: SOS PAGE of proGCAP II. Proteins were separated
`on a 15% (w/v) polyaCI)'lamide gel and visualized by staining with
`Coomassie brilliant blue: lane 1, total proteins expressed in£. coli
`cells without induction by IPTG; lane 2, total proteins in E. coli
`cells after induction by IPTG; lane 3, the supernatant of the cell
`lysates in lane2; lane 4, th e precipitates of the cell lysates in lane
`2; lane 5, the supernatant from the sulfonation and dialysis of the
`precipitates in lane 4; and lane 6, the supernatant in lane 5 refolded
`in the presence of GSH and GSSG.
`
`of isomer 2 were between Cys100 and Cys103 and between
`Cys108 and Cys1".
`Expression of proGCAP II in E. coli. To examine the
`effect of the pro region of proGCAP II on the formation of
`disulfide linkages and the tertiary structure of GCAP II,
`proGCAP II was biosynthetically prepared by a recombinant
`technique.
`In a previous report (30), proguanylin was
`expressed as a soluble material in£. coli cells using the pAP
`vector, which carries an alkaline phosphatase promoter and
`a signal sequence of STII. In this study, we first employed
`a pIN ill ompA I vector with the signal sequence of OmpA
`(24). However, no proGCAP II was expressed in £ . coli
`cells transformed with pIN ill ompA PU after induction by
`IPTG. To improve the expression efficiency ofproGCAP
`II, the cDNA encoding the signal se