Purification
`Secretagogue
`
`a New Pancreatic
`of Exendin-3,
`and Structure
`from Heloderma horridurn Venom*
`Isolated
`
`(Received for publication, April 10, 1990)
`
`Vol. 265, No. 33, Issue of November 25, pp. 20259-20262,199O
`Printed
`in U.S.A.
`
`John Eng@Y,
`From
`the @olomon
`of Medicine,
`Mount
`University
`of Michigan
`State
`University
`of New
`
`A. Kleinman$,
`Wayne
`P. C. Andrewsll,
`Laboratory, Veterans Affairs
`A. Berson
`Research
`Sinai
`School
`of Medicine,
`New
`York,
`New
`York
`Medical
`School,
`Ann
`Arbor,
`Michigan
`48109,
`York-Health
`Science
`Center,
`Brooklyn,
`New
`
`Latika
`Medical
`10029,
`and
`York
`
`and Jean-Pierre
`Singh**,
`Bronx,
`New
`York
`Center,
`the 11 Department
`of Biological
`the **Department
`of Medicine,
`11203
`
`Raufman**
`the SDepartment
`10468,
`Chemistry,
`
`is char-
`(Hisl)
`structure
`histidyl
`An amino-terminal
`superfamily.
`the glucagon
`acteristic
`of most peptides
`in
`by amino-ter-
`performed
`An assay
`for His’
`peptides
`to screen
`venom
`was used
`minal
`amino
`acid sequencing
`Heloderma
`horridum.
`from
`the Gila monster
`lizard,
`a
`Two His’
`peptides
`were
`identified:
`helospectin
`and
`to
`new His’
`peptide
`that
`has been
`named
`exendin-3
`in an
`is
`indicate
`that
`it
`the
`third
`peptide
`to be
`found
`has
`exocrine
`secretion
`of Heloderma
`lizards
`which
`(ex-
`endocrine
`activity,
`the
`first
`two being
`helospectin
`endin-1)
`and helodermin
`(exendin-2).
`In
`the
`lot of H.
`horridum
`venom
`tested,
`exendin-3
`was
`5-lo-fold
`more
`abundant
`in molar
`concentration
`than
`helospec-
`tin. The structure
`of exendin-3
`was analyzed
`by amino
`is
`acid
`sequencing
`and mass spectrometry.
`Exendin-3
`a 39-amino
`acid
`peptide
`with
`a mass of 4200.
`It con-
`tains
`a carboxyl-terminal
`amide
`and has a strong
`ho-
`mology
`with
`secretin
`its amino-terminal
`12 amino
`at
`acids. The complete
`structure
`of exendin-3
`is His-Ser-
`Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-
`Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-
`Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-
`he-
`with
`Pro-Ser-amide.
`It
`is 32 and 26%
`homologous
`lospectin
`and helodermin,
`respectively.
`It has greatest
`homology
`with
`glucagon
`(48%)
`and human
`glucagon-
`like
`peptide-l
`(50%).
`Exendin-3
`(3 PM)
`stimulated
`in-
`creases
`in
`cellular
`CAMP
`and
`amylase
`release
`from
`dispersed
`guinea
`pig pancreatic
`acini.
`
`group of
`of a diverse
`consists
`superfamily
`The glucagon
`related
`by
`that are structurally
`biologically
`active peptides
`having an amino-terminal
`histidine
`residue
`(His’) and a phen-
`ylalanine
`residue
`at position
`6 (Phe’)
`(1) or one of several
`variant
`structures
`such as Tyr’-Phe’
`(2, 3), His’-Tyr’
`(4), or
`His’-Leu’
`(5). An assay
`for His’
`can potentially
`be used
`to
`identify
`peptides
`belonging
`to
`this
`family.
`Although
`special
`chemical methods
`have been
`reported
`for
`the detection
`of
`His’ peptides
`(6, 7), a more general method
`of assaying
`for
`His’
`is amino-terminal
`amino
`acid sequencing.
`This
`assay
`was used recently
`to monitor
`the purification
`of glucagon
`from
`chinchilla
`pancreas
`(8). To
`test the hypothesis
`that His’
`is a
`chemical marker
`for biologically
`active peptides,
`amino-ter-
`
`in part
`supported
`was
`work
`* This
`of
`of publication
`costs
`The
`Affairs.
`This
`of page charges.
`by
`the payment
`marked
`“aduertisement”
`in accordance
`fact.
`to
`solely
`indicate
`this
`B To whom
`correspondence
`Research
`Laboratory,
`Veterans
`York
`10468.
`Tel.:
`212-584-9000
`
`should
`Affairs
`(ext.
`
`of Veterans
`the Department
`by
`defrayed
`in part
`article
`were
`this
`article
`must
`therefore
`be hereby
`with
`18 U.S.C.
`Section
`1734
`
`be sent:
`Medical
`1710).
`
`Solomon
`Center,
`
`A. Berson
`Bronx,
`New
`
`abbreviations
`’ The
`high
`pressure
`liquid
`ment-mass
`spectrometry;
`
`phenylthiohydantoin;
`are: PTH,
`used
`FAB-MS,
`fast atom
`chromatography;
`VIP,
`vasoactive
`intestinal
`peptide.
`
`HPLC,
`bombard-
`
`20259
`
`for new His’ peptides
`to screen
`was used
`sequencing
`minal
`the glucagon
`superfamily.
`Gila
`that might
`be members
`of
`for screening
`because
`venoms
`monster
`venom was chosen
`from Heloderma
`suspectum and Heloderma
`horridurn
`had been
`shown previously
`to contain
`two biologically
`active His’-Phe’
`peptides,
`helospectin
`(9) and helodermin
`(10). When H. hor-
`ridum
`venom was examined with
`the His’ assay, a previously
`unrecognized
`His’-Phe’
`peptide was detected.
`In
`this study,
`we report
`the purification
`and structural
`characterization
`of
`this new peptide.
`It is named exendin-3
`to
`identify
`it as the
`third
`peptide
`found
`in an exocrine
`secretion
`of Heloderma
`lizards which has endocrine
`activity.
`Like
`the earlier exendins,
`it is a pancreatic
`secretagogue.
`
`chloride-
`P were
`was
`
`AND METHODS
`MATERIALS
`74F-0240),
`diphenylcarbamyl
`venom
`(lot
`horridum
`H.
`Y, and
`carboxypeptidase
`carboxypeptidase
`trypsin,
`treated
`Sigma.
`Endoproteinase
`Glu-C
`(V8
`protease)
`from
`purchased
`Boehringer
`Mannheim.
`from
`purchased
`amino
`and Amino
`Acid
`Sequencing-Amino-terminal
`Assay
`His’
`degradation
`was performed
`by a single
`cycle
`of Edman
`acid
`analysis
`combination
`using
`an automated
`gas-phase
`protein
`sequenator
`in
`with
`an on-line
`PTH’-derivative
`analyzer
`(Applied
`Biosystems).
`The
`weak
`molarity
`of
`the
`analyzer’s
`solvent
`was
`adjusted
`to position
`PTH-Ala
`PTH-His
`to elute
`between
`and PTH-dehydroser.
`Purified
`peptides and peptide
`fragments were sequenced with
`the gas-phase
`sequenator.
`(25 mg)
`Venom-Venom
`from Heloderma
`Peptides
`of His’
`Isolation
`and passed
`of 10 mg/ml
`in water
`to a concentration
`was
`dissolved
`Associates,
`Milford,
`MA).
`through
`a C,” Sep-Pak
`cartridge
`(Waters
`2
`The C,,
`cartridge
`was washed
`with
`5 ml of water
`eluted
`with
`and
`the
`ml
`of 0.1%
`trifluoroacetic
`acid,
`60%
`acetonitrile.
`Peptides
`in
`eluate were
`separated
`by HPLC
`on an 8-mm
`x
`lo-cm
`MB CIR Radial-
`Pak
`column
`(Waters
`Associates).
`The
`column
`was eluted with
`a linear
`gradient
`from 20 to 60% acetonitrile
`in 0.13% heptafluorobutyric
`acid
`at a
`flow
`rate
`of 2 ml/min.
`One-minute
`fractions
`were
`collected.
`Aliquots
`from
`each
`fraction
`were
`assayed
`for His’
`content.
`dried
`Enzyme
`Cleauages-Purified
`exendin-3
`(10 nmol)
`was
`digested
`separately
`with
`fig of
`trypsin
`or 2 Kg of V8 protease
`0.1 ml of 0.1 M ammonium
`bicarbonate
`overnight
`at
`room
`tempera-
`ture.
`Peptide
`fragments
`were
`purified
`by HPLC
`on a Nova
`CIK Radial-
`Pak
`column
`(Waters
`Associates).
`The
`elutions
`were
`performed
`with
`a linear
`gradient
`from
`0
`to 40%
`acetonitrile
`in 0.1%
`trifluoroacetic
`acid
`at a flow
`rate
`of 1 ml/min.
`Y or
`carboxypeptidase
`with
`incubated
`Exendin-3
`(1 nmol)
`was
`acetate,
`carboxypeptidase
`P
`(1 pg each)
`in 0.05 ml of 0.05 M sodium
`pH
`5.5, at
`room
`temperature
`for
`timed
`intervals
`up
`to 6 h. Aliquots
`from
`the mixtures
`were
`analyzed
`for
`release
`of
`free amino
`acids.
`Amino
`Acid
`Analysis-Peptides
`were
`dried
`and
`hydrolyzed
`gas-phase
`6 M HCl
`at 150
`“C
`for 60 min. Amino
`acids were
`with
`an automated
`amino
`acid derivatizer
`(Applied
`Biosystems)
`nected
`to an on-line
`phenylthiocarbamyl-derivative
`analyzer.
`
`0.2
`
`and
`in
`
`with
`analyzed
`con-
`
`SANOFI-AVENTIS Exhibit 1024 - Page 20259
`
`IPR for Patent No. 8,951,962
`
`

`
`20260
`
`Purification
`
`and Structure of Exendin-3
`
`A
`
`ELUTKN
`
`VOLUME
`
`-ml
`
`0
`
`20
`ELUTDN
`
`IO
`
`8
`
`30
`40
`VOLUME-ml
`El
`1
`
`E3
`
`50
`
`60
`
`horridurn
`in H.
`peptides
`1. His’
`FIG.
`on a C,, Sep-Pak
`cartridge
`was
`concentrated
`acid,
`60%
`acetonitrile
`0.1%
`trifluoroacetic
`by
`reverse-phase
`HPLC.
`eluate were
`separated
`assayed
`for His’
`by amino-terminal
`sequencing.
`His’
`peptide
`is a combination
`of helospectins
`peak
`is exendin-3.
`
`venom.
`that
`(AC&‘).
`Elution
`The
`1 and
`
`(25 mg)
`Venom
`was
`eluted
`with
`Peptides
`the
`in
`fractions
`were
`smaller
`peak
`of
`(9). The
`larger
`
`II
`
`its
`and
`of exendin-3
`masses
`Spectrometty-The
`Mass
`flight mass
`desorption/time
`of
`were
`determined
`by plasma
`spectrometer.
`Mass
`accuracy
`etry
`on a BioIon
`Nordiq
`mass
`spectrometry
`is 20.1%
`of
`desorption/time
`of flight mass
`Mass
`determinations
`for
`proteolytic
`fragments
`were
`using
`greater
`accuracy
`fast
`atom
`bombardment-mass
`(FAB-MS).
`The masses
`of
`the
`carboxyl-terminal
`determined
`by FAB-MS
`with
`a mass
`accuracy
`unit
`by peak matching
`to appropriate
`cesium
`FAB-MS
`was performed
`on a Kratos
`MS-50
`a high
`field magnet
`as described
`previously
`Dispersed
`Pancreatic
`Acini-Dispersed
`were
`prepared
`by methods
`described
`by
`the pancreatic
`acini was measured
`Cyclic
`AMP
`production
`was measured
`
`fragments
`spectrom-
`by plasma
`ionized mass.
`obtained
`with
`spectrometry
`fragments
`of greater
`than
`chloride
`cluster
`mass
`spectrometer
`(11).
`pig pancreatic
`guinea
`(12). Amylase
`previously
`the Phadebas
`using
`by
`radioimmunoassay
`
`were
`fO.l
`ions.
`with
`
`acini
`release
`test
`(13).
`(14).
`
`RESULTS
`on a Cl8
`venom were concentrated
`in H. horridum
`Peptides
`prior
`to separation
`by reverse-phase
`HPLC.
`Two
`cartridge
`His’ peptides were detected. The smaller,
`earlier eluting His’
`peptide
`peak
`in Fig. 1 was determined
`to be helospectin
`by
`sequence analysis. The helospectin
`was heterogeneous.
`It was
`composed
`of
`two closely
`eluting
`UV peaks
`in
`the HPLC
`separation
`shown
`in Fig. 1. These peaks correspond
`to helos-
`pectin-I
`and helospectin-II,
`which differ
`from each other by 1
`serine at the carboxyl
`terminus
`(9). The helospectins
`together
`were only
`IO-20% as great
`in molar concentration
`as the
`later
`eluting His’ peptide.
`to be a new,
`in Fig. 1 proved
`peak
`The major His’ peptide
`peptide.
`It was pure after
`the
`previously
`undetected His’-Phe’
`contaminating
`peptide
`se-
`first purification
`step. No other
`quences were
`found on amino acid sequencing.
`Further
`ana-
`lytical
`separations
`of the peptide
`on a strong cation-exchange
`HPLC
`column
`and on a reverse-phase
`column
`using a differ-
`ent counter-ion
`(trifluoroacetic
`acid)
`revealed
`a single homo-
`geneous UV peak at 214 nm (data not shown).
`exendin-3,
`The amino
`acid sequence
`of the new peptide,
`was determined
`by direct sequencing
`of the intact peptide and
`by sequence analysis
`of overlapping
`peptide
`fragments
`gen-
`erated by separate
`digestions
`with
`trypsin
`and V8 protease
`(Fig. 2, A and B).
`Intact
`exendin-3
`and
`its proteolytic
`frag-
`ments were analyzed
`separately
`by mass spectrometry.
`The
`
`I
`0
`
`IO
`
`50
`
`,
`60
`
`40
`30
`20
`VOLUME-ml
`ELUTION
`reverse-phase
`HPLC
`separation
`2. A,
`FIG.
`exendin-3
`by digestion
`with
`from
`generated
`and monoisotopic
`masses
`obtained
`sequences
`given
`below.
`The mass
`the
`carboxyl-terminal
`of
`residue
`determined
`by FAB-MS.
`The
`serine
`amide
`deduced
`from
`the amino
`acid
`content
`and mass
`was
`obtained
`for T5.
`The
`amino
`acid
`content
`consistent
`the
`formation
`of T5
`from
`T2
`amino-terminal
`glutamine
`in T2
`to a pyroglutamyl
`
`fragments
`of peptide
`amino
`acid
`trypsin.
`The
`Tl-T5
`are
`for peptides
`T4 was
`fragment
`in parentheses
`is
`of T4. No
`sequence
`and mass
`of T5
`are
`by
`cyclization
`of
`the
`residue.
`
`with
`
`Peptide
`
`Residue
`no.
`
`Sequence
`
`obtained
`
`Tl
`T2
`T3
`T4
`
`T5
`
`1-12
`13-20
`21-21
`28-39
`
`13-20
`
`HSDGTFTSDLSK
`QMEEEAVR
`LFIEWLK
`NGGPSSGAPPP-
`(Samide)
`( <QMEEEEAVR
`
`Monoisotopic
`Experimental
`1293.8
`990.2
`947.1
`1022.46
`-
`
`)
`
`973.1
`
`masses
`Theoretical
`1293.6
`990.4
`947.6
`1022.46
`-
`
`972.4
`
`generated
`fragments
`of peptide
`separation
`HPLC
`reverse-phase
`i?,
`acid
`se-
`The
`amino
`V8 protease.
`by digestion
`with
`from
`exendin-3
`obtained
`for peptides
`El-E5
`are
`quences
`and monoisotopic
`masses
`given
`below.
`The mass
`the
`carboxyl-terminal
`fragment
`E3 was
`determined
`by FAB-MS.
`
`of
`
`Peptide Residue
`no.
`
`Sequence
`
`obtained
`
`El
`E2
`E3
`
`E4
`
`E5
`
`1-17 HSDGTFTSDLSKQMEEE
`18-24
`AVRLFIE
`25-39 WLKNGGPSSGAPPP-
`(Samide)
`AVRLFIEWLKNGGPSSGAPPP-
`(Samide)
`25-34 WLKNGGPSSG
`
`18-39
`
`Monoisotopic
`Experi-
`mental
`1936.6
`845.9
`1449.81
`-
`
`2277.9
`
`1000.5
`
`masses
`
`Theoreti-
`Cd
`1939.8
`846.5
`1449.72
`-
`
`2278.2
`
`1000.1
`
`SANOFI-AVENTIS Exhibit 1024 - Page 20260
`
`IPR for Patent No. 8,951,962
`
`

`
`Amino
`
`acid
`
`composition
`
`its carboxyl-terminal
`
`I
`
`Purification
`
`and Structure of Exendin-3
`
`TABLE
`and
`of enendin-3
`(T4)
`tryptic
`fragment
`in the sequence data
`All of the amino acids are accounted
`for
`obtained
`for T4
`(Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-
`)
`except an additional
`serine,
`indicating
`that serine
`is the carboxyl-
`terminal amino acid. ND, not determined. Numbers
`in parentheses
`are the expected number of residues.
`Exendin-3
`3.0
`(3)
`4.8
`(5)
`5.7 (6)
`3.9
`(4)
`1.0 (1)
`0.9 (1)
`2.1
`(2)
`2.2
`(2)
`4.2
`(4)
`(0)
`1.0 (1)
`1.0 (1)
`0.9 (1)
`3.1
`(3)
`2.0
`(2)
`2.3
`(2)
`ND
`(1)
`
`were
`databases
`sequence
`and NBRF
`EMBL,
`GenBank,
`with exendin-3
`but did not
`searched
`for sequence homologies
`yield any close matches other
`than with
`those peptides
`known
`to belong
`to the glucagon
`superfamily.
`Exendin-3
`has a strong
`homology with secretin at its amino-terminal
`12 amino acids
`whereas
`the
`remaining
`portions
`of the
`two peptides
`are vir-
`tually unrelated
`(Fig. 3). Glucagon
`and glucagon-like
`peptide-
`1 (human)
`have the greatest degree of homology with exendin-
`3 at 48 and 50%,
`respectively.
`The homologies
`between
`ex-
`endin-
`and
`the glucagon/glucagon-like
`peptides
`appear
`as
`two clusters,
`one at
`the amino
`terminus
`and one
`in
`the
`carboxyl-terminal
`portions
`of the peptides. Helospectin
`and
`helodermin
`have smaller degrees of homology with exendin-3
`at 32 and 26%,
`respectively.
`iso-
`were originally
`Because
`helospectin
`and helodermin
`lated based on
`their
`properties
`as pancreatic
`secretagogues,
`exendin-3
`was
`tested
`for
`its effects on cellular
`CAMP
`and
`amylase
`release
`in a preparation
`of dispersed
`guinea
`pig
`pancreatic
`acini. Fig. 4, A and B, shows
`that exendin-3
`is
`equal
`in efficacy
`to VIP at stimulating
`the release of amylase
`and
`to secretin
`at stimulating
`an
`increase
`in
`intracellular
`CAMP.
`The potentiating
`effects
`of
`the phosphodiesterase
`inhibitor
`theophylline
`on the actions of exendin-3
`(Fig. 4, A
`and B)
`suggest
`that as is the case with VIP and secretin
`(15),
`CAMP mediates
`the exendin-3-induced
`amylase
`release.
`
`DISCUSSION
`from
`isolated
`was detected
`and
`Exendin-3
`horridum
`H.
`rather
`than a
`assay (His’)
`venom on the basis of a chemical
`in
`the bioassays
`used
`in
`biological
`assay.
`Its
`low potencies
`this study would have made
`its identification
`by these assays
`difficult.
`It
`is only 0.01% as potent
`as VIP
`at stimulating
`at
`amylase
`release
`and
`is only
`0.1% as potent
`as secretin
`maximally
`increasing
`cellular CAMP.’ This might explain why
`an earlier
`exploration
`for VIP-like
`peptides
`in H.
`horridum
`venom
`(16) did not detect exendin-3.
`The superior
`sensitivity
`of the chemical
`assay
`for detecting
`exendin-3
`compared with
`either of the two bioassays
`illustrates
`the usefulness
`of chem-
`ical assays as adjuncts
`to bioassays
`for the
`isolation
`of biolog-
`ically active peptides.
`pep-
`functional
`new
`assay to purify
`The use of a chemical
`tides originated
`with Tatemoto
`and Mutt
`(17). They used a
`chemical
`assay
`for carboxyl-terminal
`amino
`acid amides
`to
`isolate a series of new, biologically
`active peptides
`from
`intes-
`tinal
`(18-20),
`brain
`(21), and pancreatic
`(22)
`tissues. The
`success of their
`chemical
`assay
`in
`the
`isolation
`of these new
`peptides
`emphasizes
`the growing
`recognition
`that biologically
`active peptides
`tend
`to have one or more unique
`post-trans-
`lational modifications
`that are
`important
`for mediating
`bio-
`logical potency.
`
`‘J.-P. Raufman, L. Singh, and J. Eng, unpublished data.
`
`Asx
`GlX
`Ser
`GUY
`His
`Arg
`Thr
`Ala
`Pro
`‘M
`Val
`Met
`Ile
`Leu
`Phe
`LYS
`Trp
`
`T4
`
`1.0
`
`3.0
`2.9
`
`1.1
`4.0
`
`ND
`
`fragments were in close
`masses of the proteolytic
`experimental
`agreement with
`the
`theoretical
`masses calculated
`from
`their
`amino acid sequences.
`was
`of exendin-3
`terminus
`The structure
`of the carboxyl
`determined
`by a combination
`of amino acid analysis,
`sequenc-
`ing, and mass spectrometry.
`Multiple
`attempts
`at sequencing
`fully
`the carboxyl-terminal
`fragments
`generated
`by
`trypsin
`(T4) and V8 protease
`(E3) ended at the prolyl
`residue at the
`penultimate
`carboxyl-terminal
`position.
`Amino
`acid analysis
`of T4 showed
`that
`it contained
`an additional
`serine
`not
`accounted
`for by
`the sequence
`data
`(Table
`I). Thus,
`the
`carboxyl-terminal
`amino acid of exendin-3
`is serine.
`Y
`Attempts
`at digesting
`exendin-3
`with
`carboxypeptidase
`or carboxypeptidase
`P were unsuccessful
`at releasing
`amino
`acids, which
`suggested
`that
`the carboxyl
`terminus might be
`amidated.
`The presence
`of a carboxyl-terminal
`amide was
`confirmed
`by the masses of T4 and E3 determined
`by FAB-
`MS
`(see legends
`to Fig. 2, A and B). The experimental
`masses
`are 1.0 unit
`less than
`the
`theoretical
`masses calculated
`for a
`free acid at the carboxyl-terminus
`and are the expected masses
`for
`sequences with
`a carboxyl-terminal
`amide.
`The mass
`analysis
`also confirmed
`the
`identity
`of the carboxyl-terminal
`amino
`acid as serine. All sequence,
`composition,
`and mass
`data were consistent
`with each other,
`indicating
`that
`there
`was no remaining
`structural
`ambiguity.
`Thus, exendin-3
`is a
`39-amino
`acid peptide with mass 4200 which has an amidated
`carboxyl
`terminus.
`
`of
`
`exendin-3
`3. Comparison
`FIG.
`of
`other
`peptide
`sequences
`with
`superfam-
`the
`of
`glucagon
`members
`Glucagon-like
`peptides
`1 and 2
`ily.
`and GLP-2)
`(25) and growth
`(GLP-I
`hormone-releasing
`factor (GRF)
`(3) are
`human
`sequences. Pituitary
`adenylate
`cyclase-activating
`polypeptide with 38
`residues (PACAP38)
`(30) is ovine. All of
`the other mammalian peptides are por-
`cine,
`including
`gastric
`inhibitory
`poly-
`peptide
`(2) and peptide having an
`(GIP)
`amino-terminal
`histidine and carboxyl-
`terminal
`isoleucine amide
`(PHI)
`(18).
`The sequence of helodermin
`is the re-
`vised one (16).
`
`SANOFI-AVENTIS Exhibit 1024 - Page 20261
`
`IPR for Patent No. 8,951,962
`
`

`
`Purification
`
`VIP
`
`Ex-3
`
`-
`
`1
`
`venom that was analyzed, helospectin
`In the H. horridum
`and exendin-3 were detected, but
`helodermin
`was
`not.
`Hel-
`odermin has been reported to be found only in H. suspecturn
`venom (16). Since helospectin was also originally
`isolated
`from H. suspecturn venom, it would appear that helospectin
`is found in the venoms of both H. horridum
`and H. suspecturn
`whereas helodermin
`is restricted to H. suspectum
`venom.
`Whether exendin-3 is present in the venom of both species of
`Gila monster lizard remains to be determined.
`It might be speculated that the helospectin/exendin-3 pair
`of peptides is derived from a common precursor since VIP
`and PHM have a common precursor (29) and glucagon, GLP-
`1, and GLP-2 likewise have a common precursor (25). How-
`ever, the disporportionate molar ratio of the two peptides in
`the venom would argue against this
`unless
`exendin-3
`were
`present in multiple copies in the precursor. Alternative expla-
`nations for the greater abundance of exendin-3 include differ-
`ential synthesis of separate precursors for the peptides, dif-
`ferential secretion of the peptides into the venom or different
`solubilities of the two peptides during reconstitution of the
`venom.
`
`and Structure of Exendin-3 1 shorter His’-Phe’ form is the actual active product (26-28).
`I I I
`
`20262
`
`None
`
`c2
`E
`-Jc
`i-t
`
`c
`a
`
`z
`”
`
`100
`
`80
`
`60
`
`40
`
`20
`
`o-
`
`Ex-3
`5ec
`None
`from
`release
`on amylase
`and VIP
`of exendin-3
`4. A, effect
`FIG.
`10 nM VIP
`or
`were
`incubated
`with
`acini.
`Acini
`pancreatic
`dispersed
`release
`was
`(Ex-3)
`for
`30 min
`at 37 “C. Amylase
`3 pM exendin-3
`at
`acini
`the
`percent
`of amylase
`activity
`in
`the
`the
`calculated
`as
`beginning
`of
`the
`incubation
`which
`was
`released
`into
`the extracellular
`was
`medium
`during
`the
`incubation.
`In each
`experiment,
`each
`value
`from
`determined
`in duplicate,
`and
`results
`given
`are means
`+ SE.
`three
`separate
`experiments.
`Open
`and solid bars
`represent
`the absence
`or presence
`of 5 mM
`theophylline,
`respectively.
`B, effect
`of exendin-
`3 and
`secretin
`on cellular
`CAMP
`in dispersed
`pancreatic
`acini.
`Acini
`were
`incubated
`with
`10 nM secretin
`(Set)
`or 3 pM exendin-3
`(Er-3)
`for 30 min at 37 “C. Results
`for cyclic
`nucleotide
`content
`are expressed
`as a ratio
`of
`the value
`obtained
`with
`a particular
`agent
`(experimental)
`divided
`the value
`obtained
`with
`acini
`incubated
`with
`no additions
`(control).
`In each experiment,
`each
`value was determined
`in duplicate,
`and
`results
`given
`are means
`+ S.E.
`from
`four
`separate
`experiments.
`Open
`and
`solid
`bars
`represent
`the
`absence
`or
`presence
`of 5 mM
`theophylline,
`respectively.
`
`by
`
`The amino-terminal histidine residue in His’ peptides is an
`important determinant of biological potency. Des-His’-glu-
`cagon and des-His’-secretin both have greatly reduced activity
`when compared with the native peptides (23, 24). His’ pep-
`tides are formed as the result of specific cleavages between
`Lys-His
`or Arg-His
`sequences
`during
`the
`processing
`of
`their
`precursor peptides. The presence of Phe’ in the mature pep-
`tide sequence may be a necessary condition
`for the His’-
`specific cleavage to occur. Preproglucagon contains two po-
`tential His’ cleavage sites at the amino terminus of glucagon-
`like peptide-l
`(25). One cleavage site produces a 37-amino
`acid His’-Phe4 peptide, and the other produces a shorter 31-
`amino
`acid
`His’-Phe’
`peptide.
`There
`is
`evidence
`that
`the
`
`C. H., Brown,
`
`A. L.
`
`(1971) Acta Physiol.
`
`-
`
`-
`
`
`
`_ 1 Y
`
`-‘,.
`
`.‘,”
`
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`SANOFI-AVENTIS Exhibit 1024 - Page 20262
`
`IPR for Patent No. 8,951,962