Pathways of processing of the gastrin
precursor in rat antral mucosa.
A Varro, … , S Voronina, G J Dockray
J Clin Invest.
1995;
95(4)
:1642-1649.
https://doi.org/10.1172/JCI117839
.
The precursor of the acid-stimulating hormone gastrin gives rise to multiple peptides
differing markedly in biological activity, but the relevant biosynthetic pathways are poorly
understood. We have used antibodies to amidated gastrins, gastrins with COOH-terminal
glycine (Gly) gastrins with COOH-terminal hydroxyglycine (GlyOH) and to the COOH
terminus of progastrin, to immunoprecipitate peptides labeled with [35S]sulfate or
[3H]tyrosine during incubation of rat antral mucosa in vitro. Labeled progastrin was
detectable after 30 min of continuous incubation with isotopic precursors, G34 and G34-Gly
after 60 min, and G17 and G17-Gly after 120 min. Pulse chase experiments indicated that
progastrin is converted to G34-Gly which then follows one of two pathways: (a)
hydroxylation of COOH-terminal Gly and conversion to G34 followed by cleavage yielding
G17, or (b) cleavage to G17-Gly. The kinetics of G17-Gly and G17 labeling were similar,
suggesting that G17-Gly is a product in its own right, and not simply an intermediate in G17
synthesis. Since the two peptides are reported to have distinct biological activities, they
appear to be alternative mature products of progastrin processing.
Research Article
Find the latest version:
Pathways of Processing of the Gastrin Precursor in Rat Antral Mucosa
A. Varro, S.Voronina,and G. J. Dockray
Physiological Laboratory, Universityof Liverpool, Liverpool, L693BXUnited Kingdom
Abstract
The precursor of the acid-stimulating hormone gastrin gives rise to multiple peptides differing markedly in biological
activity,
but therelevant biosynthetic pathways are poorly understood. We have usedantibodies to amidated gastrins,gastrins with COOH-terminal glycine (Gly) gastrins with COOH-terminal hydroxyglycine (GlyOH) and to the COOH terminus of progastrin, to immunoprecipitate pep-tideslabeled with[35S] sulfate or
[3H]tyrosine
during incu-bation of rat antral mucosa in vitro. Labeled progastrin was detectable after 30 min of continuous incubation with isotopic precursors, G34 and G34-Gly after 60min,
and G17 and G17-Gly after 120 min. Pulse chase experiments indicated that progastrin is converted to G34-Gly which then follows one of two pathways: (a) hydroxylation of COOH-terminal Gly and conversion to G34 followed by cleavageyielding G17, or (b) cleavage to G17-Gly. Theki-netics of G17-GlyandG17 labeling were similar, suggesting thatG17-Gly isaproduct in its own right, and not simply
anintermediate in G17synthesis. Since thetwopeptidesare
reported to have distinct biological activities, they appear
tobealternative mature products ofprogastrin processing. (J. Clin. Invest. 1995.95:1642-1649.) Keywords:
biosynthe-sis *posttranslational processing * amidation *gastrin
Introduction
Theproduction of biologically active hormonal and
neurotrans-mitter peptides generally requires'multiple posttranslational modifications ofalarge, usually inactive, precursor. Common processing steps include cleavage of thepeptidechain atpairs of basicresidues,tyrosine sulfation,serinephosphorylation,and
COOH-terminalamidation ( 1, 2).Inthe precursor of the
acid-stimulatinghormone gastrin,all of these
processing
events oc-curinadecapeptidesequence that includes the shortestfragmentabletostimulate acid secretion(3). Previous studies have iden-tifiedawidevariety of differentpeptides derivedfrom progas-trin, the best characterized of whicharetheCOOH-terminally
amidatedpeptides, G17and G34(4-7).
It has been recognized for many years that G17 and G34 have similar potencies for stimulation of acid secretion, but differmarkedlyin metabolic clearancerates(8-10). Theyare
Address correspondence to G. J. Dockray, Physiological Laboratory, UniversityofLiverpool,CrownStreet,P. 0.Box 147, Liverpool, L69
3BX, UnitedKingdom.Phone:51-794-5324;FAX: 51-794-5315. Receivedforpublication 8 August 1994 andin revisedform 15 November 1994.
bothfound in the human circulation after a meal, although it is thought that G17 is mainly of antral origin and G34 of duodenal origin ( 11). These peptides are generally considered to be the primary active products of the gastrin precursor. However, re-cent reports indicate that gastrins extended at the COOH termi-nus by glycine also have biological actions, notably growth.
promoting effects on the AR4-2J cell line and stimulation of H+/K+ ATPase a-subunit gene expression, that are distinct from those of the amidated gastrins and appear to be mediated by novel receptors (12-14). Peptides terminating in gastrins with COOH-terminal glycine(-Gly)' are converted to COOH-terminally amidatedpeptides by theenzyme,peptidyl a-amidat-ing mono-oxygenase (PAM) (15). Several groups have identi-fied naturally occurring Gly-extended gastrins (16-19), and havesuggested that these peptides are intermediates in the syn-thesis of the amidated product (Fig. 1). However, there have been no direct studies of thebiosynthetic relationships between progastrin-derived peptides, and consequently it is not clear whether there are separate and distinctive biosynthetic routes leadingtoproduction of -Gly extended, and amidated gastrins.
In the present study we have examined thepathways of pro-cessing of progastrin inratantrum.Thedata suggestthat progas-trin is convertedtoG34-Gly, which is then processed eitherto
G34-GlyOH (gastrins with COOH-terminal hydroxyglycine), G34and thenG17,or toG17-Gly. The kinetics ofproduction
of G17 and G17-Gly are closely similar, suggestingthat they should both be consideredasmatureactiveproducts, and
conse-quently that a common sequence of progastrin gives rise to
eitheroftwotypes of distinct activepeptide.
Methods
Labeling experiments. Thebiosynthesisofprogastrin-derived peptides
was studiedbyfollowingincorporationof[35SIsulfate and[3H] tyrosine
inratantralmucosaincubated in vitroaspreviouslydescribed(20,21 ). Briefly, antral mucosal segments were incubatedat37°Cinamodified Krebs-Ringer biocarbonate mediumsupplementedwithamino acids and vitamins, andgassedwith95%02and 5%CO2. Mediumcontained 50
,ICi/ml
[3H]Tyrand 100or 200pCi/ml [355]sulfate
(both obtainedfrom AmershamInternational, Buckinghamshire,UnitedKingdom),and omitted unlabeledsulfateortyrosineexcept in chaseexperiments.Two
experimental protocolswereused.First,the pattern oflabelingof
pro-gastrin-derived peptides was examined during continuous incubation with isotopic precursors for periods of30 minto 4 h. Second, the kinetics ofinterconversionofdifferentprogastrin-derived peptideswere
examined inpulse-chaseexperiments. Pulse-labelingofprogastrinwas
produced by incubationat22'Cfor 2h;previousstudies haveestablished that thisleadstoaccumulation of radiolabeledprogastrinin the terminal
regionsof theGolgicomplex (21 ).Chaseexperimentswerethencarried
outinmediumcontainingunlabeledtyrosineand sulfateat37°C; sam-ples of tissue and mediaweretaken at10-160 minduringthechase.
1.Abbreviations used in thispaper:CFP,COOH-terminaltryptic frag-ment of progastrin; -Gly, gastrins with COOH-terminal glycine; -GlyOH, gastrinswithCOOH-terminalhydroxyglycine, PAM, peptidyl a-amidatingmono-oxygenase.
J.Clin. Invest.
©TheAmericanSocietyforClinicalInvestigation,Inc. 0021-9738/95/04/1642/08 $2.00
Chase20 min: L367
400^ Hcpw"
;
Tyr200 1 0]
500 1 ^[S1 sulphate 250-1
to
0]
j j
MRlA
IChas
80 min:L373| 200-11?-04
o4me-* P3HI Tyr
100
*
KI1
100
OUf
=
4w
Chase 160min:1109 I -- 0174'j
I
..o
GTlw~
50
300 2001
3IS
l mfitate ("S sulfate
1501
-100
* U,
,<)
oI)
Jh
J
o
S
0
E ~RIA
E 2.5
0
20 40 60
Time
(min)
0 20 40 60
Time (min)
0 20 40
Time (min)
Progastrin
0 ..
..
-1L367
CFP
I GO--Gy
G34-Gly
JGIy
L373
G17-Gly
LZ|~Z
* NH2G34 *
FNH2
G17
Figure 1.Schematic representation ofprogastrinand itsmajor products, antibodies used forprecipitation, andtypicalHPLCseparations.Bottom
shows therelationships betweenprogastrin, thetwomainGly-extended products (G34-GlyandGJ7-Gly), and thetwomainamidatedpeptides (G34andG17).Thelabeling sites, i.e.,sulfatedtyrosines,areindicatedbyfilledcircles;note, thesearetheonly tyrosines inratprogastrin. Horizontal bars show the specificity of antibodiesused forimmunoprecipitation: L367 (COOHterminusprogastrin),L373(Gly-extended gastrins), and L109 (amidated gastrins). Not shown hereare-GlyOH peptides producedas anintermediate in the amidation reaction. Top,leftshowsa
representativeHPLC result of fractionation ofasample precipitatedwith L367 after20-minchase.Sampleswereappliedtocolumnsequilibrated
in50-mMammonium bicarbonate and eluted withagradientto28%acetonitrileover28min,whichprogressedto50% from 46to56min.The main labeledpeak correspondstothe CFPand thereisasmallpeakcorrespondingtoprogastrin. Note that radioimmunoassay (RIA)showsamajor peakofCFP and minorpeaks correspondingtoprogastrinandlargerCOOH-terminalfragments. Top, center,HPLCprofileafterseparation ofa
sample precipitated withL373 after80-minchase. The elutionsystemwassimilartothatalreadydescribedexceptthatagradientto16% acetonitrile
was run over16min and thento50% acetonitrile. Therearepeaksoflabel, and ofimmunoreactivity correspondingtoG34-GlyandG17-Gly.
There isonly partial separationofsulfated and unsulfatedG17-Glyandnoseparationof sulfated and unsulfatedG34-Gly. Top, right, HPLCprofile
ofasample precipitatedwithL109,after160-minchase.Gradientwasidenticaltothatused for L373immunoprecipitates. Therearepeaksoflabel,
andimmunoreactivity, correspondingtoG34,and G17. The sulfated and unsulfated forms of thelatter,butnottheformer,areseparated.cts,counts
perupdate period (15 s).
Extraction ofprogastrin-derivedpeptides. Following incubation,
tis-sueand mediawereseparated. Tissuessampleswereroutinely washed
inice-cold medium, boiledin1mlwater,homogenized, and centrifuged (10,000g, 1min).Thesupernatantwasrecovered,and thepellet washed
withafurther 1 mlwater,centrifuged,andthesupernatantscombined.
Progastrin and its derivatives in media or tissue extracts were then
concentratedaspreviously described (21). Briefly, samples were con-centrated on C18 cartridges (Sep-pak; Waters Associates, Millipore Corp.,Milford, MA)and eluted with 50% acetonitrile in 0.02 M phos-phatebufferpH 7.2; acetonitrilewassubsequently removed inastream
of N2.Progastrin-derived peptides thatwere notretainedby C18
car-tridges(mainly COOH-terminal flanking peptideofprogastrin, COOH-terminaltryptic fragmentofprogastrin [CFP])werediluted with4 vol
isopropanol and concentratedonSep-pak Silica cartridgesaspreviously described (22);thismaterialwaselutedwith 0.02Mphosphate buffer, lyophilized, re-dissolved in 1.5 ml water,and combined with the C18 eluates for immunoprecipitation.
Immunoprecipitation. Radio-labeled progastrin-derived peptides
werepurified byimmunoprecipitation usingfour differenttypesof anti-body. Routinely, samples wereprecipitatedin turnwith antibodies to (a)theCOOH terminus ofprogastrin (L367)whichrecoversprogastrin
and COOH-terminalfragments, including CFP, (b) Gly-extended
gas-trins(L373),and(c)COOH-terminallyamidatedgastrins (L109) (Fig. 1).Ineachcase,50
ILI
ofantiserumwasaddedtothesamplein 2 ml 0.02 MphosphatebufferpH7.4, containing 15 ul liquidBSA(stock22%vol/vol,Advanced Protein ProductsLtd., Brierly Hill,West
lands, United Kingdom) together with 200
Itl
goat anti-rabbit precipitat-ing antibody (Incstar Corp., Stillwater, MN). Precipitates were allowed to form at 40C overnight and were recovered by centrifuging at 3,000 g for 10min. Precipitates were washed five times by resuspension in 3 ml 0.02 M phosphate buffer pH 7.4, containing 0.75% vol/vol liquid BSA, and after the final wash were solubilized by boiling in 1 ml water. The supernatant from the first precipitation was briefly boiled to denature any remaining immunoglobulin, cooled, and then taken for precipitation with the next antibody. Pilot experiments showed that similar patterns of recovery were obtained regardless of the order in which antibodies were used in serial precipitations. The specificity of the antibodies used for precipitation wasverified by radioimmunoassay of solubilized pre-cipitates. Thus, for example, antibody L109 precipitated immunoreactive amidated gastrins, but not peptides detected in radioimmunoassay(seebelow) for progastrin or -Gly-extended gastrins.
The antibody specific for the COOH terminus of progastrin has
previously been described in detail (20, 21). Rabbit antibody specific for the COOH terminus of Gly-extended gastrins was raised by immuni-zation with Gly-extended COOH-terminal nonapeptide of G17
(G9-Gly) coupled to thyroglobulin with glutaraldehyde as previously de-scribed (23). Antibody specific for the COOH terminus of amidated gastrins was raised to the terminal tetrapeptide amide coupled to thyro-globulin as described (24). In all three cases bleedings taken afterthe
second immunization were found to be the most useful for immunopre-cipitation.
HPLC.Solubilized immunoprecipitates were clarified by centrifuga-tion at 100,000 g and fraccentrifuga-tionated by reversed phase HPLCusing an
Altex system with on-line scintillation counting using a Radiomatic detection system (Canberra-Packard,Pangbourne, United Kingdom)and
UltimaFlo AP scintillation cocktail (Canberra-Packard).Samples were
applied routinely to 4.6 X 150 mm columns of 8,u PLRP-S (Polymer Laboratories, Church Stretton, United Kingdom) equilibratedwith 0.05
M ammonium biocarbonate, and eluted with a gradient of acetonitrile (see figure legends for details); but for separations of -GlyOH labeled gastrins, the aqueous phase was 20 mM Tris, pH 7.4. The flow ratewas 1ml/min, and the update time for counting was 15 s. Incontrol separa-tions, a spillover of 30% of35Scounts into the3Hchannel was found, and all data were corrected for this. In someexperiments,columneluates were split to provide a sample for radioimmunoassay, and in thesecases
the counts obtained were corrected for the split-fraction.
Radioimmunoassay. Progastrin and its COOH-terminal fragments in HPLC eluates was detected by radioimmunoassay using antibody
L363 (21). A mouse monoclonal antibody (mAb 109-21, generously provided by Dr. J. H. Walsh, UniversityofCalifornia at Los Angeles)
was used to assay Gly-extended gastrins (18) and antibody L2was used
to assay amidated gastrins (25).
Gastrins terminating in hydroxyglycine (i.e., -GlyOH)were sought
using antibody (L394) raised to G9-GlyOH. Peptide inthe form of
the mixed enantiomers of G9-GlyOH (740 nmol) was conjugated to
thyroglobulin (2 mg) in 1.0 ml 0.1 M phosphate buffer pH 7.4 by
addition of glutaraldehyde (50
,1).
The product was dialyzed overnight against 4 liters distilled water and emulsified in Freund's complete adjuvant for immunization of four rabbits (50nmolequivalent); rabbits were boosted at 6-wk intervals and bleedings taken 7-10 d later. Forradioimmunoassays, G9-GlyOH was radiolabeledwith 125I using
iodo-gen and the products purified by HPLC.Radioimmunoassay incubations were made in1 ml 0.02 M phosphate bufferpH 7.4containing0.75%
vol/vol liquid BSA, for 48hat4°C andseparated by addition of 200
dil
of a suspension ofcharcoal (100 g/liter) coated withdextran (10g) and fat-freemilk powder (5 g). The specificity of the assay was
determined by comparison of inhibition of bindingof label by the mixed
enantiomers of G9-GlyOH, G17, G9-Gly,mixedenantiomers of CCK8-GlyOH, the separate enantiomersofG4-GlyOH, G4-Gly, and G4.
Stability of-GlyOHpeptides. The stability of -GlyOH-modified gas-trinswas determined by incubation at40, 220,or 37°C of the mixed
enantiomers of G4-GlyOH (6.7 nmol/ml) in 0.05 Mphosphate buffer
at pH 6.0, 7.4, or 8.0, for up to 8 d. Aliquots were separated by HPLC
on a VydacC18 (protein and peptide) column, using 16% acetonitrile
100 y4FP Pr" 4 4Pr" 100
30mff
1001 300
IE 5 \_;S60As t [
30 [600
,_300- -boo
-150 12 400°
55501
16002751
~~~~~~~240
min:0
Time (min) Time (min)
Figure2.Patternoflabelingof progastrin and itsCOOH-terminal frag-ments during continuousincubationat370C.Inthis andsimilar
subse-quentfigures,left panel, shows incorporation of[3H]Tyrandright panel showsincorporationof ["SIsulfate.SeeFig.1for details ofthegradient used forelution. After30-minincubation there isincorporationinto
progastrin,
and withlongerincubation,thereisaprogressiveincrease in labeled CFP (note thedifferentscalesforordinates).and 84% trifluoroacetic acid(0.1%vol/vol).This system separates the two stereo-enantiomers ofG4-GlyOH,fromG4-Glyand G4. The latter was detected by absorbanceat280nmand determinedby radioimmuno-assay usingantibody toG4(24).
Results
Time course of production ofnew gastrin. Continuous incuba-tion of rat antral mucosal segments at 370C with
[3H]Tyr
and[35S]sulfate
was associated with linearincorporation
of both labels intoprogastrin-derived peptides. Labeledprogastrin
wasidentified within 30 min of
starting
theincubation;
the relative magnitude of the labeledprogastrinpeak
didnotchange
thereaf-ter, but there was aprogressive increase in thelabeling
of its COOH-terminal tryptic fragment(Fig.
2).
At the earliest time point studied (30 min) thelabeling
of the latter was greater with [35S1sulfate than [3H]Tyr, which iscompatible
with the incorporation of sulfate atthetrans-Golgi
network(21),
and of amino acids into newly translatedpeptide
inendoplasmic
reticulum(Fig. 2).
A small peak of
[35S]G34-Gly
was seenwithin 30 min of starting theincubation (Fig. 3).The appearance of[35S]G34-Gly preceded that of
[3H]G34-Gly
which was first identified after 60 min. Moreover,["SS]G34-Gly
alsoappeared
earlier than[35S]G34.LabeledG17-Gly
and G17 firstappeared
at120min, and the relative magnitude of these
peaks
increased with continued incubation (Fig. 3).Pulse-chase experiments. The results described above pro-vide information on the time taken to generate
newly
synthe-sized gastrins, but provide
relatively
little information on the pathways ofposttranslationalprocessing.
To address this issue weusedapulse-chaseprotocolthat made ofuseofthefact that at22°C progastrinis labeled with both[35S]
sulfate and [3H]-Tyr, andaccumulates in the terminal
regions
of theGolgi
com-plex (21 ). Furtherprogressionofprogastrin
along
thesecretory
100 7-v4 044 4+34-GWo h 100 1001 a4034-Gi2h
17-
4-50]~~~~~~~~~oWA
0
50
Jo
0 LoL 0i
lo
100- 200 200 300
"'~~~~smiI 40WA
50 t°oIL 100j t 1o
2000-=w
120. . soWAR0
loo ~~~~~~~~~~20C~ FE oo 100
IL~~~~~~~~~~
200]- 240Nib0 0
0
100] 20J[0
20 40 60 0 2 40 60 0 20 40 60 0 20 40 60
017 01768
soe
1017.1
4034100 100
0l7.1,
4034 a s A0O
203
503,2°-E530°ni
E50
25-
so~0
1.II
so
20
so.
100- 60MD 100- 100 - 150
yiC500-__1 _fiE
3w200 100
31~~~~~~~r
0In1o3120MM~ o t I-33
=00 540MWEtUA40 so50
0 20
Tk. (mu
40 6o 0 20 40 6o
in) Tine mmin)
0 20 40
Tmm (Mim)
60 20 40
Time (min)
Figure3.Labeling of -Gly-extended and amidated gastrins during
con-tinuousincubation at370C with[3H]Tyr and[35S]sulfate. Top, HPLC separation of precipitates using antibodyto-Gly-extendedgastrins. Bot-tom, HPLCseparation of precipitates using antibodytoamidated
gas-trins. NoteG34-Gly and G34appearbeforeG17-Gly and G17. The
latterpredominates by 240 min. See Figs. 1 and 2 for further
informa-tion.
labeled progastrin-derived peptide which appeared earliest in
chase experiments was G34-Gly (Fig. 4). The magnitude of
thepeak oflabeled G34-Gly was maximal after 20-40 min,
and thereaftertherewasaprogressivedecline(Fig. 5).Aclearly
identifiable peak oflabeled G34 was alreadyevident after20
min, wasmaximalat40-80min, andthen declined. Thus, the appearanceof G34wassomewhatdelayed compared with G34-Gly, but in both cases, and with both isotopes, there was a
decline in thelabeledmaterial from80to160 min of the chase (Figs.4and5).
Clearly defined peaks of labeled G17-Gly and G17 were
foundafter80 min chase, but unlike the labeling of G34-Gly,
or G34, these peaks then increased furtherup to 160min of chase(Figs. 4and5). By 160min, labeled G17andG17-Gly
werethepredominant material identified in immunoprecipitates
with L109 and L373, respectively. Thus the time course of
labelingofG17-GlyandG17 was similar, andtheappearance
of thesepeptides coincidedwithdecreasesinG34-Gly and G34.
Thepattern oflabeling with [35S] sulfatewasclosely similarto that with [3H]Tyr which labels both sulfated and unsulfated peptides exceptthat sulfated peptides appeared slightly earlier forreasons noted above. These data suggestthat amidation is
not influenced by sulfation at Tyr 7. In support of this, the
Figure 4.Labeling of -Gly-extended and amidatedgastrinsina
pulse-chaseexperiment. Pulse labelingwasconductedat22°( for 120min; chaseswereconductedat370(. Note theearlyappearanceofG34-Gly andG34, followed by G17-Gly and G17. See Figs. 1-3 for further details.
data inFig. 4 showclearly resolved peaksof[3H]Tyr-labeled unsulfatedand sulfated G17, which exhibitsimilar labeling
ki-netics (Fig. 4). Moreover in other experiments using HPLC systemsthatseparatethesulfated andunsulfated forms of G34, G34Gly,andG17-Gly,therewerealso similar labeling kinetics (resultsnotshown).
Secretion of labeled progastrin-derived peptides. A
de-creasein cellularcontentoftotal amidated and-Glyextended
gastrins labeled with both [35S]sulfate and [3H]Tyr occurred
from 80 to 160min inchase experiments (Fig. 5, left). This might be duetosecretion into the media,andtoprovide further
informationonthispoint, secreted labeled gastrinsweredirectly
identifiedbyimmunoprecipitation of mediaatintervals during
the chase period. Antibodies to both amidated and
-Gly-ex-tendedgastrinsfailedtoreveal labeled peptides inthe mediaat chase timesof 10-40min,andat80min, labeled gastrinswere
notreproducibly identified. However, at achase time of 160 mintherewasboth labeled amidatedand -Gly-extended gastrins in themedia,indicatingthat thetwoformsaresecretedin
paral-lel. ThepredominantamidatedpeptidewasG17,and therewas a small amount ofpoorly resolved material corresponding to
G34; wellresolvedpeaks corresponding tobothG34-Gly and
G17-Gly weredetected in themedia(Fig. 6).
Identification of -GlyOH peptides. The data obtained by pulse-chase experiments indicatethatG34-Glyis convertedto
0o
|Labeing of G17/34 and G17/34-GIyI
ITotal
Counts incorporated: -NH2 & -Glypeptidee1000-3000
E
] 500
INS)S04 U)
2000 0
/ / Tvr 0
1000
E
o 500
co
m0
0 160 0 20
-Glypeptides
034-Gly
-~~~~~~~~~
I7-Gly.--;
...w.80
Time
(min)
1000
-E
a.
O 500
-
0-1000
-E a.a 500
-0-,
160 0 20
Figure 5. Kinetics of labeling of different progastrin-derived peptides in pulse-chase experiments. (Left) The totalcountsincorporated into -Gly andamidatedgastrins.Note theslightlyearlierappearanceof sulfatedpeptides. (Center) Labeling with[35S]sulfateofG34-Gly and G17-Gly (top) and G34 and G17(bottom). For reference thepatternoflabeling of G34 is also shownas abroken line in thetoppanel. (Right) Similar data for [3H]Tyr labeling. Note, the decline in G34-Gly and G34 after 40-80min,and increase in G17-Gly and G17upto 160 min. Mean data, from four experiments.
G34. It is believed that this conversionproceedsvia the forma-tion ofa hydroxyglycine intermediate (15). Although the
re-arrangement of-GlyOHto thecorrespondingCOOH-terminal amide isenzymically mediated,thisstepcanalsooccur sponta-neously. To characterize the production of amidated gastrins
wesought -GlyOH intermediates, andasfirst towards this end
we investigated the stability ofG4-GlyOHwithrespect topH
and temperature. At pH 8.0 and 370C there was progressive
conversion ofG4-GlyOHtoG4, invitro (Fig. 7).Conversion
wasinhibitedat220C,andatpH 7.4; itwasvirtuallyabolished
at40C andatpH6.0.
The relative stability of-GlyOH atphysiological pH
sug-gestedthat it would be worthimmunizing rabbitsinanattempt
toobtainspecificantibodies. Fourrabbitswereimmunized with amixture ofthetwostereoenantiomers ofG9-GlyOH;all four
produced antibodies that bound '25M-labeled G9-GlyOH. The antiserum with the highest titer and affinity was selected for use inradioimmunoassay. The results ofcompetitive binding experiments,indicated thatradioimmunoassays usingantibodies to -GlyOH gastrins showed about 10-fold higher affinity for thesepeptides comparedwiththecorresponding -Gly-extended
andamidatedgastrins (Fig. 8).Theimmunoreactivityof
chole-cystokinin (CCK)-GlyOH was 30-fold lower affinity than
that ofG9-GlyOH, and there was an 100-fold difference in
immunoreactivitybetweenG9-GlyOHandG4-GlyOH.Thetwo
Figure 6. Secretion of
amidated and -Gly-ex-tendedgastrins into the media. Afterpulse label-au-Gy ingwith200pCi/ml 100-
(17-Glys4
G17 034[35S]
sulfate for2hat-Gly gastrin
22TC,
sampleswerechasedat370C for
10-160min and media
se-50 - quentially precipitated
E withL109and L373to
pep-0
~~~~~~~~~tides.
-, 150- peptidesweredetected in
Ca -
N
,
astrinsamplesat 160min,but
not at10-80min. Top,
75- HPLC separationof
la-beled-Gly-extended
gas-trins, and bottom, HPLC
separationof amidated
0 gastrins from media after
o 20 40 60 chasingfor160min.See
Time (min) Fig. 1for further details.
Q4-GOl
0.041i
.4-geqi
0.02
0.01
0-i
10 20 30
Time(min)
ISO 104-OyOHM 04-Ni,atpH&o
E WC
f0o
00 a
Time(d)
Figure7. StabilityofG4-GlyOH. Synthetic G4-GlyOHwasincubated
atpH6.0-8.0 and4-370C,forvarying periodsof time and theproducts separated (left) byisocratic HPLC(16%acetonitrile in 0.1% vol/vol trifluoroacetic acid).In controlexperiments, itwaspossibletoresolve
twostereoenantiomers ofG4-GlyOH. Both eluted well beforeG4, or
G4Gly.Conversion ofG4-GlyOHtoG4wasmostrapidatpH8.0 (bottom right)and 370C(topright).
1646 A. Varro, S. Voronina, andG. J. Dockray
E a. u
0.
of/
'"' '1,
1000
-0
01020 80 Time
(min)
80
Time
(min)
160
I
I-Gly poptides
I
W4-G*I...9
. G3%
I
I
I
20
0.001 0.01 0.1 1 10 100
Concentration (nmol/Iiter)
Figure 8. Radioimmunoassay dilution curves of -GlyOH gastrins, and related peptides, with an antibody raised to G9-GlyOH. The antibody shows ~10-foldhigher affinity for G9-GlyOH than G9-Gly or G17, and progressively lower affinity for cholecystokinin (CCK)8-GlyOH andG4-GlyOH. Mixed stereoenantiomers of the -GlyOH peptides were used; reversed phase HPLC separated the two G4-GlyOH peptides, which reacted equally in radioimmunoassay. B/F, ratio of antibody bound to free radiolabeled peptide.
stereoisomers ofG4-GlyOH were relatively readily separated
byreversedphaseHPLC(Fig. 7), and bothwere foundto react equally in radioimmunoassay.
When antisera raised against G9-GlyOH were used in im-munoprecipitation,peptides corresponding to labeled -Gly and
amidated gastrins were recovered, in addition to putative -GlyOH gastrins. To demonstrate unequivocally the synthesis
of-GlyOH gastrins, we sequentially precipitated samples with
antibodies selective for -Gly-extended and amidated gastrins,
before precipitation using the antibody to -GlyOH peptides. When this approach was applied to samples from a 20-min chase, sequential precipitation with the appropriate antibodies recoveredmaterialcorresponding to G34-Glyand asmall peak
ofG34(Fig. 9); theefficiencyof theseprecipitationsteps were
establishedbyreprecipitation with thesameantibodies,whenno
further labeled materialwas recovered. Subsequent precipitation
withantibody to -GlyOH gastrins then recovered a single peak of labeled material eluting slightly later than G34 (Fig. 9). Radioimmunoassay of the column eluates with antibody
re-acting with-GlyGHpeptides,revealedtwopeaks of
immunore-activity one of which coincided with 35S-labeled peptide, and neither ofwhich coincidedwith a clear
peakc
ofimmunoreactiv-ity in assays specific for amidated or -Gly-extended gastrins.
The relevant standards (syntheticG34-GlyOH)were not
avail-able to us, but we tentatively identify the two peaks ofL394 immunoreactivity as sulfated and unsulfated G34-GlyOH, on the basis of theirimmunochemicalproperties andsimilar
reten-tion times to G34. The concentration ofG34-GlyOH in antral mucosa was estimatedto be <1% thatof theamidated gastrins, i.e., ofthe orderof 10pmol/g.
Discussion
The present study providesdirectexperimentalevidence on the rates androutes ofgastrin biosynthesis in antral mucosa. The
I
Recovery ofG34-GIyOHI
100
-Ab L373 A
601
E Ab L109
8.40
n 601 co AbL394
0 4]-f
80
-80
Ab L109(re-ppt)
40
0
0 20 40 60 s0 100
Time (min)
I of a-34WO ppt'sI
EbL2 0.2
n o
Mob109-21 02 E '0.1 .
Of~L
..
~ ~0.2:
-Giu o
TimeWM)
Figure 9.Identification of G34-GlyOH inapulse-chase experiment. A sampleobtained after a chase of 20min, following pulse labelingwith
[35S]sulfate(see Figs.4and5)wassequentially precipitated (ppt)with
antibodies L373 (toremove-Gly-extended gastrins), L109 (toremove
amidated gastrin), and then divided intotwoequalparts.Onepart was
precipitated with antibody L394 (raisedtoG9-GlyOH) and the other
wasreprecipitated (re-ppt) with antibody L109 (to verify the efficiency of the first precipitation). Left, shows the elution of labeled peptides.
Intheseexperiments,thestarting conditionsfor column equilibration and sampleapplicationwere10% acetonitrile in 20 mM Tris pH 7.4; gradientelution from 10 to 25%acetonitrilewascarriedout over 80 min.Right, shows the results of radioimmunoassay of the HPLC eluates of L394 precipitates using three different antibodies (L2, specific for amidated gastrins; mAb 109-21, specific for -Gly-extended gastrins, and L394 reacting with -GlyOH peptides); for reference,the elutionof 35S-labeled material in the L394 precipitates has been superimposed (broken line,axis toleft)onthe radioimmunoassay dataonthebottom
right.TheL394precipitates contain materialthatelutesafterG34-Gly
andG34.The same material is identified inradioimmunoassay with antibodyto-GlyOH gastrins,butnotantibodiestoamidatedor-Gly gastrins.A secondpeak detectedinradioimmunoassayshas the
proper-tiespredicted for unsulfated G34-GlyOH. Inother experiments,
repre-cipitation with L373wasusedinplace of the second L109 precipitation:
aswithreprecipitation with L109,nofurther labeled materialwas recov-ered.
results indicate thatthemajor acid-stimulating product, the
hep-tadecapeptide G17, is produced -2 hafter mRNA translation
and isgeneratedfromprogastrinviaG34-Gly,G34-GlyOH, and G34. There are atleast three alternative routes by which the
59-93 sequence of progastrin, i.e., G34-Gly, might be pro-cessed (Fig. 10). Thepresentdatasuggest thatputative path-ways in whichG17-Gly and G17-GlyOH are intermediates in
G17 synthesis, areunlikely to be ofmajorimportance. There are,however, substantial amounts ofG17-Glygenerated inrat antrum; the datasuggestthat this material should be regarded
as anendproductinits own right. This conclusion is interesting
inthe context of recent reports that -Gly-extended gastrins might
havecharacteristic biological activities, notably stimulationof cellgrowth and of proton pump gene expression, independent of those of the amidatedgastrins (12-14).
There is an extensive literature on the identification and
chemical characterization ofprogastrin-derived peptides. All
|
Progastrin|
I
G34-GIy
G34-GIyOH
. G34Storage
GI7-GIy... (GI7-GIyOH)...*G17Relea3
Figure10. Schematic representation of the pathwaysby which
progas-trinisconvertedtoG17. Thepresentobservationssupportthe ideathat
themajorpathway for synthesis of G17 is via G34-Gly, G34-GlyOH, andG34(bold arrows). Although G17-Gly is produced the kinetic evidencedoesnot supportthe idea of conversiontoG17; neither do the datasupportthe ideathatG34-GlyOH is converted via G17-GlyOHto G17.
predicated from thegene sequencehavenowbeenisolatedand
chemically characterized (4, 5, 19, 22, 26); variants differing
in sulfation state atTyr87 and phosphorylation state at
Ser'
havealso been defined together withmanyputative biosyntheticintermediates. However, little has been done to establish the biosynthetic pathways by which these moleculesarelinked. The
ideathatG34 might be aprecursorof G17wasfirst suggested
onthebasisofidentities inprimary amino acidsequence and
receivedsupportfromthefindingthat theNH2 terminal tryptic
fragment ofG34 existedinasinglepopulation of cellsin
stoi-chiometricratioswithG17 (27).But thesubsequent discovery
of-Gly-extendedvariants of G17 suggestedalternative biosyn-thetic routes (16, 17), for example that progastrin was first
converted to G34-Gly, which was then cleaved to G17-Gly,
followedbyformation of G17. Thepresentdatadonotsupport
the latter scheme, but instead suggest that G34 is the main immediateprecursor ofG17.
Theincorporation of[35S]sulfate intoamidatedgastrins in ratantralmucosa wasfirstdescribed byBrandetal. (28).After 4h, incorporationof[35S]sulfateand[3H]prolineintoG34was observed, but therewas onlyaminorpeak oflabeled G17. In similarexperimentsinthepresentstudywefound that labeled G17 wasthe major peak after 4 h incubation. Thereasons for
this difference are not known, but it is worth noting that to obtaindetectable incorporationoflabel,Brandetal. increased
gastrin synthesis by fundectomy, which may have influenced
patterns of progastrin processing (28). There have been no
previous studies of the kinetics of progastrin processing. We
have found that thepoolofprogastrinpresent in thetrans-Golgi
network can be converted within minutes into G34-Gly. The timecourseofappearance ofsulfatedG34-Gly inpulse-chase experimentsissimilartothatforthedisappearanceofprogastrin
(21), suggesting relatively rapid cleavageatbothArg57-Arg58
andArg94-Arg95, togetherwithearlyremovalof
COOH-termi-nalarginineresidues. Aproportionof theproduct, G34-Gly, is thenconvertedtoG34, and thereisrelatively slowcleavageat
Lys74-Lys75ofbothG34 andG34-GlytogenerateG17-Glyand G17.ThetimecoursesofappearanceoflabeledG17and
G17-Gly were similar which suggests that thetwopeptides might
be storedtogetheringastrincells.
In keeping with the idea that amidated and-Gly-extended gastrins arecostored ingastrin cells, ithas beenreported that inratantralmucosainvitro,and in humans andsheepinvivo,
there isparallel secretion of amidated and -Gly-extended gas-trins (18, 29, 30). In the present study we examined the secre-tionof newly synthesized-amidated and -Gly-extended gastrins, by detection of labeled peptides in the media in pulse-chase experiments. Amidated and -Gly-extended gastrins were identi-fied after achaseinterval of 160min, which coincided with a decrease in the cellular content of the two groups of labeled peptide. Presumably at earlier periods in chase experiments, the biosynthetically labeled peptides were located in maturing
secretorygranules that are unavailable for exocytosis. It is inter-esting to note that the relative abundance of G17 and G34, and of G17-Gly and G34-Gly, were similar in cells and media after 160 min chase. The observation that labeled -Gly-extended and amidatedgastrinsaresecreted inparallel provides direct support for the idea that both groups of peptides should be regarded as maturesecretoryproducts of the G-cell.
Theenzyme PAM, which converts -Gly-extended peptides to the corresponding COOH-terminal amide, possesses two catalytic domains (15). The hydroxylating activity is responsi-ble for the production of -GlyOH intermediates, and a lyase activity then converts this to the COOH-terminal amide. The latter conversion can occur spontaneously at alkaline pH and is accelerated by warming; however, in the relatively acidic conditions of thesecretorygranule, the spontaneous conversion isunlikely to be physiologically important (31). We sought the putative -GlyOH intermediate usinganantibody thatreactswith this molecular species, although it also reacts with amidated and -Gly-extended gastrins. After taking steps toexclude the latterpeptides, wehaveidentified anewlysynthesized gastrin that hasproperties compatible with those of G34-GlyOH, and
so provides support for the proposed route from G34-Gly to G34. Wehave not, as yet, been abletodetect material
corre-spondingtoG17-GlyOH. Further work will be neededto deter-minewhy PAMingastrin cells appearstoshowpreference for the conversion of G34-Gly to G34, over that of G17-Gly to
G17. There is clear evidence that final production of amidated
peptidesoccursinsecretory granules(21, 32). One possibility is that the progressive maturation ofsecretorygranules leadsto
conditions,e.g., lowintragranularpH(31),thatareunfavorable for the PAM-hydroxylating activity. The present data suggest that such a change might occur 40-80 min after progastrin
leaves the trans-Golginetwork,and thereafter that either G34-Gly orG34 is an acceptable substrate for cleavage at
Lys74-Lys75
generating the corresponding COOH-terminalhepta- oroctadecapeptide.
The processing of progastrin varies considerably between cell types.Inantral gastrin cells of thecommonlystudied
mam-malian species, including humans, G17 is a major product.
However, in other cells, e.g., the human duodenum, there are
higher relative concentrations of G34(33-35),and inpituitary
therearehighrelative concentrations ofGly-extended gastrins (36). In this context it is interesting that some tumors, e.g., colorectal carcinomasarenowrecognizedtoexpress thegastrin
gene, albeitatlow levels(37-40). Thesecells donotcontain
morethantraceamountsofmatureamidated gastrins,but sev-eral laboratories have reported that the occurrence in tumor extracts ofbiosynthetic intermediates including -Gly-extended gastrins (39, 41, 42).Thepotential autocrineorparacrine
tro-phic activityof suchpeptidesisplainlyof interest. Elucidation of the mechanisms that determine the productionof
G17-Gly
and G17 in antral gastrin cells may therefore be relevant in
attemptingto understand howprocessing isdisruptedintumor cells.
Acknowledgments
We aregrateful to Dr. J. H. Walsh for the giftofantibody 109-21and
toProfessor R. Ramage, Fellows of the Royal Society for the synthesis of-Gly and-GlyOH gastrin analogs.
Thiswork was supported by grants from the Wellcome Trustand
the Medical Research Council.
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