Fo Pee Re iew
Impo an
ce o he di ec ionali y o he glycosaminoglycan
chain on he in e ac ion wi h FGF-1
Jou nal:
Glycobiology
Manusc ip ID:
GLYCO-2014-00014.R1
Manusc ip Type:
Communica ions
Da e Submi ed by he Au ho :
n/a
Comple e Lis o Au ho s:
Nie o, Ped o; Ins i u o de In es igaciones Quimicas, Quimica Bio-o ganica
Muñoz-Ga cía, Juan Ca los; CSIC, Ins i u o de In es igaciones Quimicas,
Quimica Bioo gánica, Glycosys ems Lab.
Ca e o, Paula; CSIC, Ins i u o de In es igaciones Químicas, Quimica
Bioo gánica, Glycosys ems Lab.
Canales, Angeles; CSIC, CIB, Dep . o Chemical & Physical Biology
JIMENEZ-BARBERO, JESUS; CSIC, CIB, Dep . o Chemical & Physical
Biology
Ma in-Lomas, Manuel; cicBiomagune, Bio unc ional Nanoma e ials Uni
Imbe y, Anne; CERMAV-CNRS, Molecula Glycobiology
de Paz, Jose Luis; Ins i u o de In es igaciones Quimicas, Quimica Bio-
o ganica
Angulo, Jesus; Ins i u o de In es igaciones Quimicas, Quimica Bio-o ganica
Lo a -Jacob, Hugues; Ins i u de biologie s uc u ale, gagophile;
Ga cia-Jimenez, M. Jose; Ins i u o de In es igaciones Quimicas, Quimica
Bioo ganica, Glycosys ems Lab.
Key Wo ds:
Glycosaminoglican, Hepa in, FGF-1, SPR, p o ein-ca bohyd a e in e ac ion
Glycobiology
Fo Pee Re iew
Impo ance o he pola i y o he glycosaminoglycan chain on he
in e ac ion wi h FGF-1
Juan C. Muñoz-Ga cía,
[a]
M. José Ga cía-Jiménez,
[a]
Paula Ca e o,
[a]
Ángeles
Canales,
[b]
Jesús Jiménez-Ba be o,
[b]
Manuel Ma ín-Lomas,
[c]
Anne Imbe y,
[d]
José L.
de Paz,
[a]
Jesús Angulo,
[a]
Hugues Lo a –Jacob,
[e]
and Ped o M. Nie o*
[a]
a) Ins i u o de In es igaciones Químicas, CSIC, Amé ico Vespucio, 49, 41092 Se illa, Spain
b) Cen o de In es igaciones Biologicas, CSIC, Rami o de Maez u 9, Mad id 28040, Spain
c) CIC biomaGUNE, Bio unc ional Nanoma e ials Uni , San Sebas ian 20009, Spain
d) CERMAV-CNRS, BP 53, 38041 G enoble, cedex 9, F ance
e) Ins i u e de Biologie S uc u al Jean Pie e Ebel, CNRS 41 ue Jules Ho owi z, F-38027 G enoble Cedex 1,
F ance
Phone (+) 34 954 489568, Fax: (+)34 954 460565, e-mail: ped o.nie [email protected]
Keywo ds: Hepa in, Glycosaminoglycan, FGF-1, p o ein-ca bohyd a e in e ac ion, SPR
Wo d coun ing: 4173
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Abs ac :
Hepa in-like saccha ides play an essen ial ole in binding o he FGF-1 and o hei memb ane ecep o s
FGFR o ming a e na y complex ha is esponsible o he in e naliza ion o he signal, ia he dime iza ion o
he in acellula egions o he ecep o . In his s udy we epo he binding a ini ies be ween i e syn he ic
hexasaccha ides wi h human FGF-1 ob ained by Su ace Resonance Plasmon (SPR) expe imen s, and compa e
wi h he induced mi ogenic ac i i y p e iously ob ained. These i e oligosaccha ides di e in he sulpha ion
pa e n and in he sequence. We ha e p e iously demons a ed ha all he i e hexasaccha ides ha e simila 3D
s uc u e o he backbone. Consequen ly, he di e ences in binding a ini y should ha e hei o igin in he
subs i u ion pa e n. Subsequen ly, he di e en capaci y o induc ion o mi ogenic ac i i y can be, a leas
pa ially, explained om hese binding a ini ies. In e es ingly, one o he oligosaccha ides lacking o axially
symme y (3) was biologically inac i e whe eas he o he (2) was he mos ac i e. The di e ence be ween bo h
compounds is he o de o he FGF binding mo i s along he chain ela i e o he ca bohyd a e pola i y. We can
conclude ha he di ec ionali y o he GAG chain is essen ial o he binding and subsequen ac i a ion. The
ela i e biological ac i i y o he compounds wi h egula subs i u ion pa e n can be in e ed om hei alues
o IC
50.
Rema kably, he sulpha e in posi ion 6 o D-Glucosamine was essen ial o he mi ogenic ac i i y bu no
o he in e ac ion wi h FGF-1.
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In oduc ion
FGF-1 is a membe o he Fib oblas G ow h Fac o amily ha in e ac s wi h hepa in/hepa an sulpha e
(Hep/HS) polysaccha ides and he memb ane ecep o s FGFRs. The o ma ion o a e na y complex be ween
FGF, FGFR and Hep/HS is he key s ep o he ac i a ion o he FGF signalling pa hway. This is a he o igin o
di e en cellula essen ial unc ions as egula ion o emb yonic de elopmen , homeos asis and egene a i e
p ocesses (Be n ield M e al. 1999, Eswa akuma VP e al. 2005, K euge J e al. 2006). Dime iza ion o he
ecep o s and subsequen in acellula au ophospho yla ion ac i a es a mi ogenic esponse h ough an enzyma ic
cascade (Mohammadi M e al. 2005, Pelleg ini L e al. 2000, Schlessinge J e al. 2000).
The helical s uc u e o hepa in, a highly sulpha ed o m o HS, di ec s he sulpha e g oups owa ds
opposi e sides o i s longi udinal molecula axis (Mulloy B e al. 1993). Acco ding o ha , he i s
c ys allog aphic s uc u es o he hepa in and human FGF-1 complexes (pdb code: 1amx and 2amx)
co esponded o dime ic s uc u es linked by a egula hepa in chain in i s na i e helical s uc u e (DiGab iele
AD e al. 1998). Howe e , NMR da a in solu ion co esponded o a 1:1 complex (pdb code: 2e m) (Canales A e
al. 2006). Rema kably, he dime s ha e wo al e na i e symme y ela ionships: while o 1amx he FGF-1
p o eins a e ela ed by a cen e o symme y, in he case o 2amx, he symme y elemen is a plane along he
binding si e (DiGab iele AD e al. 1998). In addi ion, he e na y complexes o hepa in and FGF wi h he
ex acellula domains o he memb ane ecep o FGFR a e assembled in o wo di e en o ms (pdb codes 1 q9
and 1e0o espec i ely) (Schlessinge J e al. 2000)
,
(Pelleg ini L 2001, Pelleg ini L e al. 2000)
The analysis o hese s uc u es indica es ha he Hep/HS binding si e co esponds o a swallow dep ession
on he su ace o he g ow h ac o ha could be conside ed di ided in o wo sub-binding si es (Digab iele AD e
al. 1998). Consequen ly, as hepa in does no much change i s helical 3D s uc u e upon binding, in he
monome ic case as is he NMR complex (pdb code: 2e m)(Canales A e al. 2006) some o i s sul ama e g oups
will be di ec ed owa ds he sol en and will no in e ac wi h he FGF-1, as he s uc u al s udies ha e shown.
In o de o analyse he hepa in-FGF-1 binding mode, 2, a hexasaccha ide wi h axially non-symme ic
sulpha e g oups dis ibu ion and unable o o m FGF dime s was p epa ed (Ojeda R e al. 2002). The FGF-1
induced mi ogenic ac i i y o hexasaccha ide 2 was highe han 1 (de Paz JL e al. 2001), which co esponds o
he egula sulpha ion pa e n o hepa in (Angulo J e al. 2004) simila o he ecen ly isola ed om na u al
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sou ces as hexame (Smi s N e al. 2010). This esul pe mi ed o disca d he dime iza ion o FGF-1 h ough a
chain o bound hepa in as a equi emen o he FGF-1 media ed bioac i i y. In e es ingly, 3, which p esen s
simila symme y on he sulpha e g oups dis ibu ion han 2, wi h espec o he longi udinal axis, was inac i e
(de Paz JL e al. 2005). The subs i u ion pa e n o 3 was designed o i wi h he equisi es p oposed by
Pelleg ini o maximise he in e ac ions and symme y in he e na y complex as i was deduced om he analysis
o di e en c ys allog aphic s uc u es (Pelleg ini L 2001). O he syn he ic oligosaccha ides wi h di e se
sul a ion pa e ns p epa ed in ou g oup lacking o sulpha e g oups in all he posi ions 6-O o glucosamines (4)
o in all he 2-O o idu ona es (4) we e also inac i e (Lucas R e al. 2003). Du ing he e ision o his manusc ip
a pape desc ibing he syn hesis o h ee hexasaccha ides and examining hei bioa ini ies p o iles o was
published (Roy S e al. 2014).
Recen ly pe o med was an in dep h analysis o he h ee-dimensional s uc u e o 3 using NMR and MD in
o de o sea ch o any s uc u al di e ences ha migh jus i y he loss o ac i i y (Munoz-Ga cia JC e al. 2013).
F om his analysis i was concluded ha 3 exhibi s he same main s uc u al ea u es cha ac e is ics o hepa in
han he analogues 1 and 2, which a e known o p omo e he in e ac ion wi h FGF-1; a) a well-de ined igid
helical backbone wi h ou esidues pe u n, b) a cha ac e is ic chai
1
C
4
- skew boa
2
S
O
con o ma ional
equilib ium o he idu ona e esidues, and c) a igid beha iou o he glycosidic linkages. This s uc u al
analysis allows o disca d any po en ial di e ence in he h ee-dimensional s uc u e ha could jus i y he
di e ences in he obse ed biological ac i i y be ween 2 and 3 (e.g. modi ica ion o he glycosidic linkages
geome y owa ds an an i disposi ion) (Munoz-Ga cia JC e al. 2013). Consequen ly, he main di e ences in
a ini y o FGF-1 among he hexasaccha ides 1-5 would be due o he capaci y o each sul ona e pa e n o
in e ac wi h FGF-1 as a unc ion o i s spa ial dis ibu ion along he chain.
Resul s
To in es iga e he abili y o he i e syn he ic oligosaccha ides, 1 – 5, o in e ac wi h human FGF-1, an
inhibi ion assay was se up. The g ow h ac o , ei he alone o coincuba ed wi h each o he i e molecules o be
analysed, was injec ed o e bo h a hepa in- unc ionalized senso chip and a s ep a idin senso chip, he la e
being used as a con ol su ace, and he in e ac ion was ollowed by Su ace Plasmon Resonance (SPR)
spec oscopy (Figu e 2). Injec ion o 8.8 nM o FGF-1 o e he hepa in su ace p oduced a binding esponse o
350 esponse uni s (RU) a equilib ium whe eas a esponse o 5 RU was obse ed o e he s ep a idin su ace
(da a no shown). Analysis o he esul s showed ha hese oligosaccha ides s ongly di e in hei abili y o
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p e en FGF-1-Hep binding (Figu e 2). The inhibi o y ac i i y o 1 was cha ac e ized by an IC
50
o 8.3 10
-8
M
whe eas 5 did no display binding ac i i y in he ange o concen a ions es ed indica ing ha , 2-O sulpha e
g oups we e essen ial o he in e ac ion (Angulo J e al. 2004). In con as , 6-O sulpha e g oups, ha a e
in ol ed in he biological ac i i y (Angulo J e al. 2004), seem o be dispensable o he in e ac ion wi h FGF-1,
wi h an IC
50
= 4.7 10
-7
M o 4.
Nex , o in es iga e he impo ance o he p esen a ion o he sul ona e g oups along he chain, he same
assay was used wi h 2 and 3 since bo h ha e an asymme ic sulpha e dis ibu ion. In e es ingly, i was obse ed
ha , 2 ea u es an IC
50
= 4.6 10
-7
M, hus simila o 4, al hough 2 has i e sulpha e g oups compa ed o six o
he egula oligosaccha ide. Finally, 3, which also displays six sulpha e g oups wi h an axially asymme ic
dis ibu ion, has a much lowe binding ac i i y, wi h an IC
50
= 1.6 10
-6
M. Thus, while he mi ogenic ac i i y
p e iously ob ained was 2 > 1 >> 3, 4, 5 (Angulo J e al. 2004), in he case o he binding a ini y he o de was 1
> 2, 4 >> 3 >> 5.
Discussion
Assuming a undamen al ole o he elec os a ic in e ac ions, and conside ing he s uc u al di e ences
be ween he hexasaccha ides, he sulpha ion pa e n should be a he o igin o he di e ences in he s eng h o
he in e ac ion and he e o e in he ac i i y. Appa en inconsis encies be ween he la ge IC
50
alues o 1
compa ed wi h 2, measu ed by SPR expe imen s, and he induc ion o mi ogenic ac i i y, which is la ge o 2,
can be explained conside ing he assembly o he e na y complex. This is essen ial o he biological ac i i y,
and addi ional hidden equi emen s may play addi ional oles (Pelleg ini L e al. 2000, Schlessinge J e al. 2000).
Compounds 2 and 3 , as hey ha e hei sul ona e g oups di ec ed owa ds one side o he molecula axis,
only can in e ac wi h FGF using one o hei hal . On he con a y, as he 3D-s uc u es o 1, 4 and 5 ha e an
axially symme ic dis ibu ion o sul ona e g oups, hey ha e he possibili y o in e ac wi h wo molecules o
FGF using wo opposi e sides o he oligosaccha ide in a sandwich like ashion, wi h wo simul aneous binding
e en s (Angulo J e al. 2004, de Paz JL e al. 2001, Lucas R e al. 2003). This obse a ion migh also explain he
obse ed di e ences be ween he mi ogenic ac i i y measu ed by p oli e a ion s udies (Angulo J e al. 2004) and
he ela i e binding s eng h o FGF-1, epo ed in his s udy.
The hepa in binding si e o FGF-1 could be di ided in o wo spa ially con iguous sub-si es (DiGab iele AD
e al. 1998). The i s one binds a isaccha ide GlcNS – IdoA2S – GlcN6S, in e ac ing ia h ee nega i ely
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cha ged sulpha e moie ies (Saxena K e al. 2010). Such a angemen o cha ged g oups displays he p ope
numbe and o ien a ion o sul ona e g oups o es ablish a igh in e ac ion wi h FGF-1. Recen s udies ha e
e ealed key di e ences be ween FGF-1 and FGF-2 binding o GAG in his subsi e. Fo he case o FGF-2 he
isaccha ide ha in e ac s in subsi e a is he complemen a y one, Ido2S – GlcNS – Ido2S (Saxena K e al. 2010).
The second sub-si e in e ac s wi h a disaccha ide, GlcN6S – IdoA2S. A cen al idu ona e esidue wi h a non-
pa icipa ing sul ona e g oup links bo h mo i s. Rema kably, hexasaccha ides 2 and 3, display simul aneously
hese wo deco a ions o he in e ac ion wi h FGF-1 bu , in e e se o de i he pola i y o he chain is
conside ed.
In an a emp o ind a sa is ac o y explana ion o he lack o ac i i y o 3 wi h espec o 2, a molecula
modelling docking p o ocol was employed o analyse he molecula in e ac ions om a s uc u al pe spec i e.
Thus, he backbone o he mos ep esen a i e con o ma ion o 3, aken om 500 ns o un es ained molecula
dynamics ajec o y (Munoz-Ga cia JC e al. 2013), was manually supe imposed o he mos ep esen a i e
s uc u e o he NMR complex be ween FGF-1 and 2 (pdb 2e m). As he dis ances be ween he h ee sulpha e
g oups di ec ed owa ds he same side o he molecule we e simila , wo pola i ies o supe imposi ion we e used,
om he educing o non- educing end and i s e e sed al e na i e. We ha e employed as i s c i e ia, he
alignmen o he longi udinal axis o bo h ca bohyd a es. Howe e , he posi ions o he sul ona e and sul ama e
g oups o 3 we e no adequa e o he comple e in e ac ion wi h he complemen a y esidues o he p o ein. A e
ha , he non- educing end isaccha ide o 3 was manually docked in o he main sub-si e in he “ e e se”
o ien a ion. In his case, he es o he oligosaccha ide did no i in he comple e binding pocke and poin ed
owa ds ou side he complex. In addi ion, a s e ic clash was obse ed be ween he p o ein side chains and he
GlcN – IdoA – GlcN isulpha ed isaccha ide o 3 (see Suppl. In o. o desc ip ion o addi ional modes). The
impossibili y o assemble a complex wi h he comple e se o cha ged in e ac ions be ween he FGF-1 and he
hexasaccha ide 3, lead us o conclude ha he co ec pola i y o he GAG chain is essen ial o he in e ac ion
wi h he g ow h ac o (FGF-1).
We decided o pe o m docking calcula ions in o de o ge a deepe insigh in o he possible binding o 3 and
FGF-1. We ha e used Glide, i s using he Induced Fi Docking p o ocol wi h he s anda d condi ions and hen,
he esul s we e subjec ed o a un o Single P ecision Docking. In his case, he ocus was pu in o he h ee
saccha ides o he iad, leading o a displaced sequence. A ema kable supe imposi ion o he poses o his
egion was ound in he solu ions (see supplemen a y ma e ial).
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In e es ingly, 2 (Figu e 3a) has bo h sulpha e clus e s in he igh disposi ion o in e ac simul aneously wi h
bo h binding sub-si es while 3 is only capable o in e ac ing wi h he sub-si e a (Figu e 3) (Canales A e al. 2006).
This can explain he alue o IC
50
o 3, 3.5 old la ge han 2. This di e ence can be explained conside ing ha
he pola i y o he glycosaminoglican chain is essen ial o he maximum numbe o in e ac ions ake place.
While he sub-si e a is in e ac ing wi h 2 h ough he isaccha ide GlcNS – IdoS – Glc6S s a ing a glucosamine
in posi ion i, he equi alen isaccha ide ha binds he main subsi e in he case o 3 is shi ed o he GlcN a
posi ion i+4. The seconda y binding sub-si e does no es ablish any in e ac ion wi h 3, hus explaining he lowe
a ini y measu ed by SPR and he absence o mi ogenic ac i i y due o he ailu e o assemble o highe o de
complexes needed. The e o e, he in e ac ion be ween FGF-1 and 3 should be weake han 2. This should be he
cause why 2 and 3 showed such d ama ic di e ences in hei binding a ini y and bioac i i y in spi e o bea ing
he same wo binding mo i s, bu in opposi e o de .
Addi ional in o ma ion can be ex ac ed om he compa ison be ween he a ini y expe imen s and biological
ac i i y ones. Fo ins ance, he sulpha ion in posi ion 6 o glucosamine ha , acco ding o ou p e ious biological
esul s, is essen ial o he FGF-1 mi ogenic ac i i y (Angulo J e al. 2004), i is no o he in e ac ion wi h FGF-
1. Tha obse a ion migh be exploi ed in he design o po en ial inhibi o s o he FGF-1 media ed mi ogenic
ac i i y ha being able o in e ac wi h he FGF-1, he absence o his key g oup p e en he assembly o he
e na y ac i e complex, and he subsequen biological ac i i y. Ano he impo an conclusion ha can be
ex ac ed om ou wo k is he e idence o he s ong in luence o he pola i y o he GAG chain on he binding.
This also can be exploi ed o he design o inhibi o s ha in e ac ing wi h he FGF-1 hey do wi h he opposi e
pola i y and hey will no be able o o m he ac i e e na y complex.
In summa y, we ha e demons a ed ha he pola i y o he oligosaccha ide chain ela i e o FGF–1 is a
c i ical ac o o he s eng h o he bina y in e ac ion and u he assembly o he e na y complex (B own A e
al. 2013).
Ma e ials and Me hods
Syn heses o compounds 1 - 5 ha e been p e iously desc ibed (de Paz JL e al. 2001, de Paz JL and Ma in-
Lomas M 2005, Lucas R e al. 2003, Ojeda R e al. 2002).
Size de ined hepa in (Hep; 6 kDa) was immobilized on a Biaco e senso chip. Fo ha pu pose, Hep was
bio inyla ed a i s educing end by coincuba ion wi h 10 mM bio in/LC-hyd azine o 24 h a oom empe a u e.
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The mix u e was hen ex ensi ely dialyzed agains H
2
O o emo e un eac ed bio in and eeze-d ied. Two low
cell o a CM4 senso chip we e hen unc ionalized wi h app ox. 2500 esonance uni s (RU) o s ep a idin as
desc ibed (C uble E e al. 2008) and bio inyla ed HP (5 µg/ml), in HBS-EP (10 mM HEPES, 150 mM NaCl, 3
mM EDTA, 0.005% su ac an P20, pH 7.4) was injec ed ac oss one low cells o ob ain an immobiliza ion le el
o 50 RU. The o he low cell was le un ea ed and se ed as nega i e con ol. Fo binding assays, 150 µl o
FGF-1 (8.8 nM), co incuba ed wi h a ange o concen a ion o he di e en oligosaccha ides, we e
simul aneously injec ed, a a low a e o 50 µl/min, o e he con ol and he HP su aces. The o med complexes
we e washed wi h unning bu e o 3 min and he senso chip su aces we e egene a ed wi h a 3 minu e pulse
o 2 M NaCl. Con ol senso g ams we e sub ac ed on line om HP senso g ams.
The p o ein da a bank s uc u es 1amx, 2amx and 2e m we e used o he p elimina y s udies o docking
desc ibed in his pape , pe o med wi h GLIDE (F iesne RA e al. 2004). The monome C om he 1amx
complex was isola ed om he es o he agg ega es and used o p epa e he model o he hexasaccha ide 3 wi h
FGF-1 by supe imposi ion o he isaccha ide o i s educing end wi h he one a he non- educing end o he
1amx and/o 2e m complexes aligning he sul a e g oups. Hyd ogens a oms we e added o he c ys allog aphic
s uc u e when i was necessa y using he Maes o p o ein p epa a ion module. The co esponding hexa- and
pen asaccha ides we e p epa ed and named consis en ly and using pa ial cha ges om GLYCAM (Ki schne
KN e al. 2008), ligand p epa a ion module was un and he s uc u e was minimized. A g id (10 x 10 x 10 Å)
cen e ed in he glycosaminoglycan was cons uc ed. We i s un an Induced Fi Docking wi h he s anda d
condi ions keeping he GLYCAM cha ges. The esul ing s uc u es we e submi ed o a Single P ecision
Docking, wi h a 10 Å g id using GLYCAM pa ial cha ges wi h an elec os a ic cu o o 2.0. The minimiza ion
was pe o med using OPLS-2005 o ce ield wi h a dielec ic cons an o 4
Funding
This wo k was suppo ed by CSIC (JAEP e_09_01999 o J.C.M-G and 2004FR0025), Jun a de
Andalucía (P07-FQM-02969), Spanish Minis y o Science and Inno a ion (CTQ2009-07168,
CTQ2012-32025, and CTQ2012-32605; and RYC-2007-01791 o J.A.), and he Eu opean Union
(FEDER).
Abb e ia ions
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Fo Pee Re iew
293x305mm (300 x 300 DPI)
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