THE JOURNAL ()f B10LOGWAL CHEMJSTR\'
`© 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
`
`VoL 268, No. 30, Issue of October 25, pp. 22397-22401, 1993
`Printed in U.S.A.
`
`Processing and Characterization of Human Proguanylin Expressed
`in Escherichia coli*
`
`(Received for publication, June 30, 1993)
`
`K. Christopher Garcia:1:§§, Frederic J. de Sauvage:I:, Martin Struble§, William Henzel,i.
`Dorothea Reillyll, and David V. Goeddel:f:**
`From tfu! Departments of §§Protein Engineering, Wolecular Biology, §Bioorganic Chemistry, 'l]Protein Chemistry, and
`!Process Development, Genentech Inc., South San Francisco, California 94080
`
`Guanylin is a 15-amino acid peptide hormone that was
`originally isolated from the jejunum of the rat small in•
`testine and shown to be an endogenous activator of the
`intestinal heat-stable enterotoxin receptor-guanylyl cy(cid:173)
`clase. Guanylin is synthesized as a 115-amino acid pro•
`hormone, proguanylin, which is processed at a site yet to
`be determined, into a C-terminal bioactive fragment(s).
`In order to examine the processing of proguanylin in
`vitro, we have generated large quantities of the properly
`folded prohormone by constructing an expression vec(cid:173)
`tor that directs its secretion into the periplasmic space
`of E•cherichia coli. The bacterially expressed human
`proguanylin was then processed to smaller C-terminal
`fragments by protease digestion. Digestion with trypsin
`or lysine-C generated C-terminal peptides of different
`length, which have been purified and characterized.
`Guanylin-22 and guanylin-32 have binding aff'mities and
`biological activities similar to guanylin-15, while gua(cid:173)
`nylin-68 and the entire proguanylin have only minimal
`bioactivity. Circular dichroism spectroscopy reveals
`that proguanylin is a stably folded protein containing
`mostly f:3-sheet and f:3-turn structure.
`
`called guanylin (Currie et al., 1992}. Guanylin shares struc(cid:173)
`tural similarities to STa, including 4 cysteine residues. Gua(cid:173)
`nylin is also able to compete for 1251-STa binding and stimulate
`cGMP production in cell lines expressing the STaR (Currie et
`al,, 1992; de Sauvage et al., 1992a). Recent cDNA cloning ex(cid:173)
`periments showed that guanylin is synthesized as a lO•kDa
`precursor called proguanylin (de Sauvage et al., 1992a; Wie(cid:173)
`gand et al., 1992; Schulz et al., 1992) (Fig. 4). Upon trypsin or
`acid treatment of proguanylin, a C-terminal fragment is re·
`leased that binds to and activates the STaR (de Sauvage et al.,
`1992a). While the site(s) of physiological processing is un(cid:173)
`known, there are several basic residues in proguanylin that
`could potentially fill this role.
`Functional studies aimed at determining the size of a gua(cid:173)
`nylin molecule with maximal activity require large quantities
`of the prohormone. Correct disulfide pairing of the 4 cysteines
`in the C-terminal fragment of proguanylin may require the
`membrane translocation of the entire molecule through the
`endoplasmic reticulum. Therefore, we have secreted the entire
`prohormone into the periplasm of E. coli. The soluble progua(cid:173)
`nylin has been purified and processed enzymatically and
`chemically to yield fully active mature hormone. Additionally,
`the high level expression has enabled us to begin structural
`characterization of both proguanylin and guanylin.
`
`Heat-stable enterotoxins (STa) 1 are small peptides of 18 or
`19 amino acids that are secreted into the intestine by entero(cid:173)
`toxigenic strains of Escherichia coli (Chan and Giannella,
`1981). The 13 amino acids necessary for the toxic activity of the
`peptide include 6 cysteines that form three disulfide bridges
`(Yoshimura et al., 1985). STa exerts its actions by binding and
`activating a member of the receptor-guanylyl cyclase family
`that is preferentially expressed in the intestine and is called
`the heat-stable enterotoxin receptor (STaR) (Schulz et al., 1990;
`de Sauvage et al., 1991). Binding of STa to STaR induces a
`dramatic increase of the cyclic guanine monophosphate (cGMP)
`content of the cell (Field et al., 1978; Hughes et al., 1978). The
`cGMP increase inhibits salt absorption and stimulates chloride
`secretion into the gut. The imbalance of ions is accompanied by
`a massive accumulation of water in the gut that gives rise to
`diarrhea and dehydration characteristic of enterotoxin activity.
`A search for an endogenous activator of STaR resulted in the
`isolation from the rat small intestine of a 15-amino acid peptide
`
`* The costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby marked
`uaduertisement" in accordance with 18 U.S.C. Section 1734 solely to
`indicate this fact.
`"" To whom correspondence should be addressed: Genentech Inc., 460
`Point San Bruno Blvd., South San Francisco, CA 94080. Tel.: 415-225-
`1081; Fax: 415-225-6127.
`1 The abbreviations used are: STa, heat-stable enterotoxin; STaR,
`heat-stable enterotoxin receptor; HPLC, high performance liquid chro(cid:173)
`matography; RP-HPLC, reverse-phase HPLC; PAGE, polyacrylamide
`gel electrophoresis; LC-MS, liquid chromatrography-mass spectrom(cid:173)
`etry; HIC, hydrophobic interaction chromatography.
`
`MATERIALS AND METHODS
`Vector Construction and Expression-The cD.NA encoding the 99-
`amino acid human proguanylin (de Sauvage et al., 1992a) was sub(cid:173)
`cloned into the expression vector pAK19 (Carter et al., 1992) following
`introduction by polymerase chain reaction of an Mlul site at its 5' end
`and an SphI site at its 3' end. pAK19 contains the E. coli alkaline
`phosphatase promoter, the heat-stable enterotoxin II (STII) Shine-Dal(cid:173)
`garno sequence, and sequence that encodes STU signal peptide. The 5'
`Mlul site places the proguanylin cDNA insert, minus its signal peptide,
`in frame with amino acid 23 of the STU signal peptide (Carter et al.,
`1992). The resulting expression construct, named pHPG2, was trans(cid:173)
`formed into competent E. coli strain W3110 ton A and grown overnight
`in Luria broth (LB) media supplemented with 50 µg/ml carbenicillin. At
`this point, induction media (final concentrations: 16.4 mM K2HPO4, 9.2
`mM NaH 2PO 4 , 47.4 mM (NH 4)zSO 4 , 3.7 mM sodium citrate, 22 mM KCl,
`7.7 mM MgSO4, 11 g/liter casein hydrolysate, and 11 g/liter yeast ex(cid:173)
`tract) was inoculated with the saturated culture at a ratio of 1 volume
`of saturated culture to 100 volumes of induction media and grown at
`37 °C for up to 32 h. Expression was checked by osmotically shocking
`the cell pellet contained in 1 ml of a 1 O.D. (A600) E. coli culture with 0.1
`volume of ice-cold H 2O, and analyzing protein content of the superna(cid:173)
`tant by 18% SDS-polyacrylamide gel electrophoresis. Fermentations
`were carried out in 10-liter Biolaffitte fermentors.
`Proguanylin Purification-The media from the induced culture con(cid:173)
`tained substantial amounts of the expressed proguanylin, as did the
`periplasmic space of the induced E. coli. Tu purify the proguanylin in the
`periplasmic fraction, 100 g of cell paste was osmotically shocked by the
`addition of 500 ml of ice-cold distilled H 20 containing 10 mM EDTA
`(Carter et al., 1992). The suspension was stirred at 4 •c for 1 h, centri(cid:173)
`fuged at 5000 x g for 30 min, and the supernatant from the centrifu(cid:173)
`gation clarified by filtration through a 0.2-µm filter. The clarified su-
`22397
`
`This is an Open Access article under the CC BY license.
`
`Bausch Health Ireland Exhibit 2037, Page 1 of 5
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`
`

`

`22398
`
`Characterization n( Human Prnguanylm Expressed in E. coli
`
`pC'rnatanl wos nridified with 0.1',, Ltifluomcctic acid. brought to 15';
`aceL0111trilt•. and alluwed llJ sil ovl'rnighl at 4 "C. The turbid solution
`was ctintrifoged for 30 min at 5000 >< g, the supernatant filtered throngh
`:i 0.2-1101 filt<:r, and pumped onto a 105 Amicon CS 200 column 10.9 x 50
`cm> :1.l a flow rate uf 9 mVmi11. The column was w.ished with 15'}
`arctonitrile. f!.1 '1 t.rHluor:icct.ic acid 11ntil the ahsorhance base line
`lm e3surcd at 2 14 11w 1 reacbeu zcrv. 'l'be mtJbile phuse w>1s increasud LO
`a:)•;; :icetonitrile, and n grnd1en1 was nm from 33 to 48',, ocetronitrile
`OV('r 60 min. Fractions won• colll'cled at 1-min intervals. and s mall
`nllquoLS from each fraction w1,rt• analyzed hy r .. v .. rs~•phase !RP)-HP!.C
`column f 105 Amicon CS 1.6 x 250 mm) using an isocratic mobile phust•
`of 40'" acctonitrill', 0. 1 •·1 tnfluor11cctic acid_ Fracti(ms ,vere c,,11eeted
`nnd Jyophilized, a nd tht• composition was vi·nfit.'d by mass spcct1•ome1.ry
`arHI/Or puptide se(tUl.!ncing, Fnictions from the RP-HPLC t hat contained
`lhc correct peptide wi•rc pooled and further purified by hydrophobic
`,n1en1etion chromatography using a phenyl-Sepharose 1()/10 Pharma(cid:173)
`ciu FPLC column Buffer A wns50 m,1Na, Po,_ 2 M 1NH ,1,so •. pH"' 7.B:
`hufTer B wes 50 mM Na, PO, . pH : 7.8- A gradient of75'.! A to 25% Bat
`a flow rate oJ 1 ml/min el uted proguanylin al approx.imatdy 5or4 A. Thi!
`pure proguanylin peak was collected, d!!sahecl loy RP-HPl,C, and ly(cid:173)
`ophili:i:ed.
`1'o purify the proguonylin from the indnction media, the exact same
`procedure was followed as for the purilicaLion of the pcriplasmJc fra~•
`tion, except that no osmotic shock step wos neccss:iry and the media
`were acidified with 0.1 ~i trill uoracctic acid and hrought to )5r,; aceto•
`nitrile as the first step. Snhscqucnt s1.eps were the same us desc1ihed in
`the preceding parugmph f'or the periplasmic fracuon.
`1)ypsin Digest11,n rmd P1mficnt1nn- 2.5 mg of proguanylin m 0.1 M
`n,s-HC'I (pH 8.01 was incubated overnight at 37 C with 2<:: trypsin
`(w/w1 /Sigma l in a total reaction voluml' of 0.5 ml. 1'he total digei;t WRS
`injucted on a Sychron C<I (2 x 160 mm) column. Peptides were $eparated
`using a linear gradient of 0. 1 'ii trinuoracctic acid to 70% acetonitrile in
`30 min at a flow rate of0.2 ml/min. Peaks were detected by monitoring
`ahsorbancc al \!14 and 280 nm.
`l.,y.~111e-C D11Jrsti(111 am/ Pvri/it•c,/1011- For the complete digest, pro(cid:173)
`guanylin 12 nmolJ was digt:stcd with 5o/, lw/wl of Lys-C !Worthi ngton) in
`100 µl of O. L ~• 'Tns-llCI. 1p.H RO). al 37 C fur 17 h. For the partial
`digest. proguanyli11 (2 nmoll was digested with 5f{ (w/w l ofLys-C in 100
`r,I of(). I M l'l-is-HCl, (pH r:l,0/, at 23 •c for5 mir1. The Lys-C digests was
`injected on a Sychron <.:4 12 x 160 mm I column. Peptides were separated
`u!ring a linear gradient of0, 1''1- trinuoracetic acid lO 70% acetonilrile in
`30 min al a flow rate of 0.2 mVmin. Peaks were d<'lectcd by monitoring
`ollsorhance at 214 a nd 280 nm.
`M ass Spectrometr_v ond Peptid1• Sl!qtic11d1,g- Automated Edman
`degradation was performed on a model 477 Applied Biosystems Se(cid:173)
`quencer equipped with a 120A phenylthiohydantoin-derivalive ana•
`lyzer. Elertrospray spoctra were ohtAined on a Sciex APil tnple quad(cid:173)
`rapole mass spectrometer. Spectra were oblaincd by direct infusion al a
`now rate of 1.5 ml/min.
`1,;,'f-STu bindiog assays and guany(yl(cid:173)
`Bi11d111g and cGMP As$a_v-
`cyclase stimulation assays were performed using 29::1,STaR cells as
`described (de Sauvage rl (I/ .. 1991).
`C1rculor Dichroism- Circular clichroism ICD) spectra were obtained
`for proguru,ylin al 0.1 mg/ml in 10 m" NaC~CO~ tpH 6.0), Spectra
`were m1:asured from 250 to 190 nm tfar UV! in an Aviv/Cary 60-0S
`spcctropolarimeuir with the use of a 0.01-cm cell. Five scans were made
`uver the wavelength range al a time constant of 0.3 s. and the values
`were averaired. Spectra were corrected by subtraction of CD spectra
`obtained with buffer alone in the cell. Data arc reported as mean resi(cid:173)
`due ellipticity hy taking Ill g/mol as the mean residue molecular
`weight.
`
`RESULTS ANO DISCUSSION
`In order to genera te large quantities ofbioactive proguanylin
`for functional and s tructura l studies . we have expressed in E.
`roli a fusion protein consisting of the bacterial STH signal
`pe ptide and huma n proguanylin. It was hoped that the STll
`signal pe ptide would lea d to the translocation of the fusion
`protein thro ugh the inner bacterial membrane to the periplas(cid:173)
`m ic space. accompanied by cleavage of the STII signal pe ptide.
`Ther e a r e many examples where such membrane translocation
`t.o t he net oxidizing environment of the periplasm results in
`controlled and correct folding of proteins cont.aining mulLiple
`disulfide bonds tCarter et al., 1992; Stader and Silhavy. 1990).
`The S1'IJ-proguanylin fus ion protein is expressed unde r th e
`
`control of the E. coli alJtaline phos phatase prom oter, which is
`induced under conditions of' phosphate starvation . The media
`ar e designed so that phosphate is de pleted as the cells achieve
`a high de nsity, lea ding to incluction of this promoter. T he a d(cid:173)
`va ntage of this system is that ind uction occurs at a hig h cell
`density, and the expression of the protein is g radual enough
`that there is time for the STII fusion protein to be correctly
`trans!ocated to the periplas m (Carter el aL 1992). SOS-PAGE
`analyses of cell paste and supernata nt during a 10-liter fer(cid:173)
`m ent a tion are s hown in Fig. 1. The bacteria secreted progua(cid:173)
`nylin from about 12 t.o 32 h, a t which point, the induction was
`te rminated . S ubs lantially m ore proguanylin
`leaked out.
`through the cell membra ne into the supe rnatant (Fig. lA l than
`remained in the cells (Fig. 1B ). From a 10-liter fermentation,
`a pproximately 3,2 g of proguanylin were found in the 8 liter s of
`supernatant, and Q_5 g of proguanylin in lhe 1 kg of cell paste.
`The proguanylin was purified from both the induction media
`supernatant and the periplasmic fraction of the cell paste_ The
`periplasmic fraction is obtained by an osmotic (hypotonicl lysis
`of the E. coli. F or both the induct.ion media supernatant a nd the
`periplasmic fraction of the cell paste, a s ubstantial first-step
`purification was achieved by an acid precipitation followed by a
`151H acetonitrile precipitation. The proguanylin remains in the
`s upernatant. and is approximately 50% pure. as judged b.v
`HPLC. LC-MS analysis gave a molecular mass of 10,707 Da for
`the progua nylin. 1'his is the mass expected if the STIJ signal is
`propel'ly removed. The s upernatant of the acid-precipitated os(cid:173)
`motic s hock fraction was analyzed by analytical RP-HPLC (Fig.
`
`A) Supernatant
`
`Hours
`
`MW
`n<g O 3 6 9 12 IS 18 21 24 26 28 JO 32 std.
`
`43K
`29K
`
`18.4K
`14K
`- -Proguanylin
`6K
`3K
`
`8) Cell Pellet
`
`Hours
`MW
`neg O 3 6 9 12 15 18 21 24 26 28 30 32Std.
`
`43K
`29K
`18.4K
`14K
`;:- Proguanylin
`
`3K
`
`Pm. L Time course of induction and secretio n or proguanylin
`by E.coli. E. coli were transformed with plasmid pHPG2 and grown in
`a 10-lit.er fennentor as described under · Materials and Methods." Ali(cid:173)
`quots of media supernatant (.A) or cells \B > were removed and aualyzed
`by SOS-PAGE (181,i.1 at indicated intervals. Gel was stained wi.th Coo(cid:173)
`massie Blue. Negative control in far left. lane is f'or induced E. coli
`contai ning the expression vector Jacking the proguanyli.n cONA insert.
`
`Bausch Health Ireland Exhibit 2037, Page 2 of 5
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Characterization of Human Proguanylin Expressed in E.coli
`
`22399
`
`2 ~ - - - - -n - - - - - - - - - - ,
`
`Proguanylin
`
`"'
`
`0
`~
`ci
`0
`
`2A) and the peak eluting at 40% acetonitrile was confirmed by
`LC-MS to be proguanylin. For preparative purification, 500 ml
`of the acid-precipitated osmotic shock supernatant (Fig. 2B) or
`!-liter batches of the acid-precipitated induction media (not
`shown) were loaded onto a preparative RP-HPLC and eluted
`with a gradient of 30---50% acetonitrile, 0.1 % trifluoracetic acid
`over 80 min. The proguanylin-containing fractions were deter(cid:173)
`mined by analytical RP-HPLC of fractions from the preparative
`column. An analytical RP-HPLC profile of the final proguanylin
`pool is shown in Fig. 2C.
`Although the proguanylin appeared to be >99% pure by RP(cid:173)
`HPLC, isocratic elution analysis of the material indicated a
`slight nongaussian shape to the peak. The only chromato(cid:173)
`graphic technique that was successful in separating contami(cid:173)
`nants from the proguanylin peak was hydrophobic interaction
`chromatography (HIC). The major eluting peak from the HIC
`column was proguanylin, but a number of contaminants were
`separated (Fig. 3).
`Proguanylin is inactive with respect to binding or activating
`
`Proguanylin +
`
`20
`
`40
`
`60
`
`80
`
`------
`
`--------
`
`---·-··J
`
`10
`
`20
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`30
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`60
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`70
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`80
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`
`E 350
`C ..,.
`£ 250
`>
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`150
`
`50
`
`0
`
`B
`
`1.6
`
`C
`
`~
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`E 1.2
`..,.
`~ 0.8
`::J
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`0.4
`
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`£ 60
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`E
`
`40
`
`20
`
`0
`
`20
`
`I
`80
`
`I
`I
`40
`60
`Time (minutes)
`FIG. 2. Reverse-phase high performance liquid chromatogra(cid:173)
`phy analysis and purification of proguanylin. The samples were
`chromatographed using an acetonitrile, 0.1% trifluoracetic acid gradi(cid:173)
`ent (33-48%) and a CS column as described under "Materials and Meth(cid:173)
`ods." A, analytical RP-HPLC of osmotic shock supernatant after acid
`precipitation. Column is a 105 Amicon CS 4.6 x 250 mm at 1 ml/min. B,
`preparative RP-HPLC of 500 ml of osmotic shock supernatant after acid
`precipitation. Proguanylin peak is highlighted. Mobile-phase gradient
`is indicated by the dashed line and scale at right. Column is a 105
`Amicon CS 200 column (0.9 x 50 cm) at a 9 ml/min flow rate. C, ana(cid:173)
`lytical RP-HPLC of purified proguanylin using a 105 Amicon CS column
`(4.6 x 250 mm). Absorbance for the indicated chromatograms was moni(cid:173)
`tored at 214 nm.
`
`. .,
`
`60
`
`40
`20
`Time (minutes)
`FIG. 3. Hydrophobic interaction chromatography of RP(cid:173)
`HPLC-purified proguanylin. Proguanylin peak is indicated by the
`arrow. Buffer A was 50 mM Na2PO4 , 2 M (NH4 )sSO4 (pH 7.8); buffer B
`was 50 mM Na2PO4 (pH 7.8). A gradient of75%Ato 25% Bat a flow rate
`of 1 ml/min eluted proguanylin at approximately 50% A. Absorbance
`was monitored at 214 nm.
`
`""; ·VJVQDGNFSFSL~_g)LQE~P,BVG!(.,BNFAPIPGEPVVPI-
`
`Guany9l-63 +
`
`Gu.-n,otin-32
`
`Guanylin-22
`
`LCSNPNFPEELJg'LC!EPNAQEILQ&LEEIAEDPGTCEICA Y MCTGC ·co;
`
`FIG. 4. Amino acid sequence of human proguanylin showing 3
`potential proteolytic processing sites. Guanylin-63, -32, and -22
`are the C-terrninal peptides that would result from cleavage at residues
`52, 83, and 93, respectively. Alternative basic amino acid residues that
`are possible processing sites are underlined.
`
`STaR (de Sauvage et al., 1992a; Schulz et al., 1992). Prohor(cid:173)
`mone processing enzymes are known to cleave primarily at
`dibasic residues, such as arginine and lysine (Barr, 1991). In
`order to study the biological activity of fragments of the mate(cid:173)
`rial purified from E. coli, proguanylin was completely or par(cid:173)
`tially digested with the proteases trypsin or lysine-C, respec(cid:173)
`tively. While these are not the physiological processing
`enzymes, they are convenient tools to generate smaller frag(cid:173)
`ments since they cleave at similar residues as most known
`processing enzymes. The cleavage sites from which fragments
`were generated for our studies are indicated in Fig. 4. Complete
`digestion of proguanylin with trypsin is expected to cleave at
`Arg-93 and yield a 22-amino acid C-terminal fragment. Com(cid:173)
`plete digestion with Lys-C is expected to yield a 32-amino acid
`C-terminal fragment resulting from cleavage at Lys-83. Partial
`digestion with lysine-C should also generate a 63-amino acid
`fragment resulting from cleavage at Lys-52 of proguanylin.
`The trypsin digestion was injected onto an RP-HPLC column
`(Fig. 5A), and the peaks were analyzed by mass spectrometry
`and amino acid analysis. The peak corresponding to the 22-
`amino acid fragment was re-injected onto the RP-HPLC and
`found to be homogeneous (Fig. 5B ). Mass spectrometry analysis
`indicates a molecular mass of 2258 Da as predicted for the
`completely oxidized peptide.
`The Lys-C digests were also analyzed by RP-HPLC (Fig. 6).
`After exhaustive digestion (17 h at 37 °C), a 32 amino acid
`fragment corresponding to residue Lys-83 of proguanylin was
`purified (Fig. 6A). After partial digestion (5 min at 23 °C), we
`could isolate a 63-amino acid C-terminal fragment resulting
`from cleavage at residue Lys-52 of proguanylin (Fig. 6B ). Both
`
`Bausch Health Ireland Exhibit 2037, Page 3 of 5
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`22400
`
`Characterization of Human Proguanylin Expressed in E.coli
`
`,.._Guanylin-22
`
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`
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`
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`40 > (") z
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`0
`
`A
`
`100.----.-.~
`
`"0
`C :,
`0
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`;f.
`
`180 'A
`140
`
`100
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`60
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`£!.
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`
`400- ~
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`2000 B
`
`1600
`
`::) 1200
`1
`
`800
`
`400
`
`0
`
`0
`
`.,.._ Guanylin-32
`
`5
`
`10
`
`..J ""
`15
`
`20
`
`25
`
`30
`
`Guanylin-63-
`
`5
`
`10
`
`15
`
`20
`
`Time (minutes)
`FIG. 6. Lysine.C digest of purified proguaoylin. A, RP-HPLC of
`complete digest yielding a 32-amino acid fragment. B, RP-HPLC of
`partial digest yielding a 63-amino acid fragment. Details of the chro(cid:173)
`matography are identical to those described for guanylin-22 and are
`described under "Materials and Method!l."
`
`peptides were found to have the expected molecular mass.
`The ability of the purified 22-amino acid tryptic peptide (gua(cid:173)
`nylin-22) to compete with 1251-STa for binding to the STaR was
`measured using 293-STaR cells (de Sauvage et al., 1992b). As
`was found for guanylin-22 isolated from mammalian cells (de
`Sauvage et al., 1992a), 100 nM guanylin-22 was needed to dis(cid:173)
`place 50% of the bound 1251-STa (Fig. 7A). This result is also
`
`80
`
`0
`
`20
`
`40
`60
`Time (minutes)
`Fie. 5. Trypsin digest of purified proguaoylin. A, RP-HPLC of
`total trypsin digest. B, RP-HPLC of purified C-terminal 22 residue
`fragment. Peptides were separated using a linear gradient of 0.1%
`trifluoracetic acid to 70% acetonitrile in 30 min at a flow rate of 0.2
`mVmin on a Synchron C4 column. Other details of the chromatography
`are described under "Materials and Methods."
`
`o+-.~.,-~....,..~....,..~"'""C~~f-.--.........,
`.0001
`.001
`.01
`.1
`1
`10
`100
`[Guanylin](µM)
`
`B
`50
`
`40
`
`30
`
`20
`
`10
`
`0.,c;c=;c=~=::;:;:..,~~~....-~~-l
`.01
`.1
`10
`100
`
`fGuanvlinlhlMl
`Fm. 7. Functional analysis of guanylin-22. A, displacement of
`1251-STa by guanylin-22 from 293-STaR cells expressing STaR. Various
`concentrations of guanylin-22 were incubated with 25 pM125J-STa and 2
`x 105 293-STaR cells. Nonspecific binding was determined in the pres(cid:173)
`ence of a saturating concentration of guanylin-22 (5 µM). The percent
`specific binding is plotted versus the concentration of STa. Each point
`represents the mean of duplicate determinations. B, cGMP production
`stimulated by guanylin-22. 293-STaR cells were incubated with various
`concentrations of guanylin-22 for 30 min. Intracellular cGMP accumu(cid:173)
`lation was then determined as described (de Sauvage el al., 1991). Each
`point represents the mean of duplicate samples assayed in duplicate .
`
`1 2 0 r - - - - - - - - - - - - ,
`
`80
`
`-c
`C
`::,
`0
`tO
`~ 0
`40
`
`60
`
`20
`
`o Guanylin-22
`• Guanylin,32
`D Guanylin-ti3
`■ Proguanylin
`
`0
`,1
`
`100 1000 10000
`10
`[Peptide] (nM)
`F10. 8. Displacement of •••I-STa from 293-STaR cells by puri•
`fied guanylin-22, -32, and -68. Experiments were performed as in Fig.
`7. Individual curves are identified by the key in the inset.
`
`similar to the affinity measured for the 15-amino acid peptide
`generated by acid cleavage ofproguanylin (Currie et al., 1992).
`The ability of guanylin-22 to stimulate guanylyl cyclase activity
`was studied by incubating 293-STaR cells for 10 min in the
`presence of increasing concentrations of guanylin-22 and de(cid:173)
`termining the intracellular concentration of cGMP (Fig. 7B).
`An 80-fold stimulation was observed at 30 µM guanylin-22,
`where 49 pmol of cGMP/106 cells were measured. This level of
`stimulation is similar to the 150-fold stimulation measured
`with STa at 100 m.i (92 pmol of cGMP/106 cells) (de Sauvage et
`al., 1991).
`The discrepancy between the concentration of guanylin re(cid:173)
`quired for half-maximal cGMP elevation and the binding Kn is
`observed for all of the identified guanylyl cyclase receptors and
`is not yet understood (Schulz et al., 1989; de Sauvage et al.,
`
`Bausch Health Ireland Exhibit 2037, Page 4 of 5
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Characterization of Human Proguanylin Expressed in E. coli
`
`22401
`
`ture (Fig. 9). The single minimum at 208 nm indicates strong
`(3-sheet and (3-turn structure, with negligible a-helix content.
`The minimum of the spectrum (208 nm) is shifted somewhat
`from classical {3-sheet (Yoshimura et al., 1985) CD minimum
`(218 nm), which is a reflection of short sheets connected by
`loops of random structure. Proguanylin is extremely proline(cid:173)
`rich, with 5-7 amino acid residues separating the prolines, so a
`general topological model might be one in which there are
`numerous short tJ-sheets connected by reverse turns compris(cid:173)
`ing prolines. Complete three-dimensional structure determina(cid:173)
`tion of the prohormone by NMR is under way, and should yield
`novel structural information on the role of the unprocessed,
`larger precursors of small peptide hormones.
`
`REFERENCES
`
`Nanometers
`FIG. 9. CD spectra of 0.1 mg/ml progu.anylin. Spectra were ob(cid:173)
`tained at pH 6.0 and 23 •c in 10 mM NaCH3C02. Recording of spectra
`is described under ~Materials and Methods."
`
`1991; Schulz et al., 1990).
`The two lysine-C-generated peptide fragments of progua(cid:173)
`nylin were compared to proguanylin and guanylin-22 in a 1251-
`STa competition binding assay (Fig. 8). The ability of gua(cid:173)
`nylin-32 to compete with 1251-STa for binding to 293-STaR cells
`is comparable to guanylin-22 (IC50 = 100 nM), whereas gua(cid:173)
`nylin-63 and intact proguanylin are far less effecUve (IC50 > 5
`µM). These results suggest that processing of proguanylin at
`sites located upstream from Lys-52 is not likely to generate an
`active hormone. Therefore, the dibasic Lys-Lys site that is con(cid:173)
`served between human and mouse at position 37-38 probably
`does not represent the physiological processing site of progua(cid:173)
`nylin.
`Very little is known about the structure of prohormones. We
`were interested to see whether the intact prohormone, progua(cid:173)
`nylin, folds into a molecule with stable and definable structure
`in aqueous solution. CD spectroscopy will yield information
`concerning whether the proguanylin is folded into definable
`secondary structural elements, or exists as an unstructured
`random coil. CD spectra on the intact E. coli expressed progua(cid:173)
`nylin indicates a folded molecule, which has secondary struc-
`
`Barr, P. J. (1991) Cell 66, 1-3
`Carter, P, Kelly, R. F., Rodrigues, M. L., Snedecor, B., Covarubias, M., Velligan, M.
`D., Wong, W. L. T., Rowland,A. M., Kotts, C. E., Carver, M. E., Yang, :M., Bourell,
`J. H., Shepard, H. M., and Henner, D. (1992) Bio'Il!chn.owgy 10, 163-167
`Chan, S. K., and Giannella, R. A. 0981) J. Biol. Chem. 200, 77 44-77 46
`Currie, M. G., Fok, K. F., Kato, J., Moore, R. J., Hamra, F. K., Duffin, K. L., and
`Smith, C. E. U992) Proc. Natl. Acad. Sci. U.S. A. 89, 947-951
`de Sauvage, F. J., Camerato, T. R., and Goedde), D. V. (1991) J. Biol. Chem. 266,
`17912-17918
`de Sauvage, F. J., Keshav, S., Kuang, W..J., Gillett, N., Henzel, W., and Goeddel, D.
`V. (1992a) Proc. Natl. Acad. Sci. U. S. A. 89, 9089-9093
`de Sauvage, F. J., Horulr., R., Bennett, G., Quan, C., Burnier, J. P, and Goedde!, D.
`V. (1992bl J. Biol. Chem. 267, 6479--648Z
`Field, M., Graf, L. H., Laird, W. J., and Smith, P. L. (1978) Proc. Natl. Acad. Sci.
`u. s. A. 111, 2800-2804
`Hughes, J. M., Murad, F., Chang, B., and Guerrant, R. L. (1978) Nature 211,
`755--756
`Se.xena, V. P, and Wetlaufer, D. B. (1971) Proc. Natl. Acad. Sci. U.S. A. 66,
`969--972
`Schulz, S., Singh, S., Bellet, R. A., Singh, G., Tubb, D. J., Chin, H., and Garbers, D.
`L. (1989) Cell 118, 1155--1162
`Schulz, S., Green, C. K., Yuen, P. S. T., and Garbers, D. L. 0990) Ce/I 63, 941-948
`Schulz, S., Chrisman, T. D., and Garbers, D. L. (1992) J. Biol. Chem. 261, 16019-
`16021
`Stader, J. A., and Silhavy, T. J. (1990) Methods Enzymol. Ul5, 166-187
`Wiegand, R. C., Kato, J., Currie, M. G., (1992) Biochem. Biophys. Res. Commun.
`185, 812-817
`Yoshimura, S., Ikamura, H., Watanabe, H., Aimoto, S., Shimonishi, Y., Hara, S.,
`Takeda, T., Miwatani, T., and Takeda, Y. /1985) FEBS Lett. 181, 138--142
`
`Bausch Health Ireland Exhibit 2037, Page 5 of 5
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`