Ac i i y-associa ed e ec o LDL ecep o missense a ian s loca ed
in he cys eine- ich epea s
A. E xeba ia1#, A. Beni o-Vicen e1#, M. S e 2, H. Os olaza1, L. Palacios2, C. Ma in1*
1Unidad de Bio ísica (CSIC, UPV/EHU) and Depa amen o de Bioquímica,
Uni e sidad del País Vasco, Apdo. 644, 48080 Bilbao, Spain.
2 P ogenika Biopha ma, a G i ols Company, De io, Spain.
* Co esponding Au ho E-mail: cesa .ma [email protected]. Tel. +34-94-601.80.53; Fax +34-
94-601.33.60
# A.E. and A.B-V. ha e equally con ibu ed o his wo k
Numbe o Tables: 2
Numbe o Figu es: 7
Numbe o supplemen a y Figu es: 6
Numbe o supplemen a y Tables: 1
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in A he oscle osis 238(2) : 304-312 (2015),
which hasbeenpublished in inal o m a h ps://doi.o g/10.1016/j.a he oscle osis.2014.12.026. © 2014 Else ie unde
CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/)
Abs ac
Backg ound: The LDL ecep o (LDLR) is a Class I ansmemb ane p o ein c i ical o
he clea ance o choles e ol-con aining lipop o ein pa icles. The N- e minal domain o
he LDLR ha bou s he ligand-binding domain consis ing o se en cys eine- ich epea s
o app oxima ely 40 amino acids each. Mu a ions in he LDLR binding domain may
esul in loss o ecep o ac i i y leading o amilial hype choles e olemia (FH). In his
s udy he ac i i y o six mu a ions loca ed in he cys eine- ich epea s o he LDLR has
been in es iga ed.
Me hods: CHO-ldlA7 ans ec ed cells wi h six di e en LDLR mu a ions ha e been
used o analyse in i o LDLR exp ession, lipop o ein binding and up ake.
Immunoblo ing o cell ex ac s, low cy ome y and con ocal mic oscopy ha e been
pe o med o de e mine he e ec s o hese mu a ions. In silico analysis was also
pe o med o p edic he mu a ion e ec .
Resul s and conclusion: F om he six mu a ions, p.A g257T p u ned ou o be a non-
pa hogenic LDLR a ian whe eas p.Cys116A g, p.Asp168Asn, p.Asp172Asn,
p.A g300Gly and p.Asp301Gly we e classi ied as binding-de ec i e LDLR a ian s
whose e ec is no as se e e as null allele mu a ions.
Keywo ds: LDLR, mu a ions, Familial hype choles e olemia, ligand binding domain,
mu a ion Class de ec
1. In oduc ion
Familial Hype choles e olemia (FH; MIM#143890) is an au osomal dominan diso de
causing p ema u e co ona y hea disease (CHD) [1] ha is cha ac e ized by inc eased
plasma LDL choles e ol, endon xan homas, deposi s o choles e ol in pe iphe al issues
and accele a ed a he oscle osis. FH has a homozygous equency o 1:1,000,000, and i s
he e ozygous equency has ecen ly been es ima ed o be as high as 1/200 in he gene al
popula ion [2,3], sugges ing ha he disease is hea ily unde diagnosed and unde ea ed.
FH is mainly due o mu a ions in he LDL ecep o (LDLR; MIM# 606945) gene, which
is esponsible o he up ake o LDL pa icles in o cells [1].
The LDLR is a modula p o ein ha combines i e di e en domains: he ligand binding
domain, he EGF-like module ha con ain a 280 amino acid β-p opelle , he O-
glycosyla ed domain and he ansmemb ane and cy oplasmic domains. The binding
domain o LDLR comp ises ~40-amino acid long cys eine- ich epea s in andem,
s uc u ed in se en disc e e ex acellula modules (R1-R7), which a e esponsible o he
binding and elease o i s lipop o ein ligands [4]. A e binding, he LDLR-lipop o ein
complex is in e nalized h ough cla h in-coa ed pi s and a ics o endosomes, whe e
lipop o ein ca go is eleased [5,6]. Lipop o eins a e subsequen ly deg aded in lysosomes,
while he LDLR ecycles back o he cell su ace o u he ounds o lipop o ein up ake.
Nowadays mo e han 1300 di e en a ian s ha e been desc ibed in he LDLR gene [7],
no all o hem pa hogenic. Acco ding o he na u e and loca ion o he mu a ions wi hin
he LDLR and o he pheno ypic e ec s on he p o ein, mu a ions ha e been di ided in o
i e di e en classes [8]: Class 1: no de ec able LDLR syn hesis; Class 2: de ec i e LDLR
anspo ; Class 3: impai ed LDL o LDLR binding; Class 4: no LDLR/LDL
in e naliza ion due o de ec i e clus e ing in cla h in-coa ed pi s; and Class 5: no LDLR
ecycling.
The ac ha se e al missense a ian s o he LDLR ound in FH pa ien s ha e been
shown no o be he ac ual cause o he disease [9,10] indica es ha e e y de ec ed
a ian needs o be unc ionally cha ac e ized in o de o de e mine i s se e i y, i any.
The aim o his s udy was o analyse he impac on he LDLR ac i i y o six missense
a ian s loca ed in he ligand binding domain o he p o ein, and p e iously ound in FH
pa ien s. The sequence a ia ions s udied p edic he ollowing amino acid changes in he
LDLR: p.Cys116A g, p.Asp168Asn, p.Asp172Asn, p.A g257T p, p.A g300Gly and
p.Asp301Gly. The e ec s on LDLR exp ession, binding capaci y and up ake we e s udied
by Wes e n blo , low cy ome y and con ocal mic oscopy in a ans ec ed LDLR-
de ec i e Chinese hams e o a y (CHO) cell line.
2. Ma e ials and Me hods
2.1. Selec ion o Va ian s
The selec ion o he six missense LDLR a ian s was based on wo c i e ia: o ha e been
p e iously ound in FH pa ien s and o be possibly associa ed o a binding de ec . To
co e he i s c i e ia we selec ed a ian s p e iously desc ibed by o he au ho s in FH
pa ien s ha ha e also been ound in a leas one FH index case by LIPOchip® pla o m
[11] o by SEQPRO LIPO RS® pla o m in P ogenika Biopha ma (De io, Spain), bo h
pla o ms wi h he CE ma k. Jus wo o he a ian s ha e been ound in la ge popula ion
s udies, p.Asp168Asn and p.A g257T p, ha we e coded as s200727689 and
s200990725, espec i ely in he NCBI SNP da abase
(h p://www.ncbi.nlm.nih.go /p ojec s/SNP/snp_ e .cgi?geneId=3949). Bo h o hem
ha e a e y low equency, p.Asp168Asn has been ound in he EVS da abase
(h p://e s.gs.washing on.edu/EVS/) wi h a MAF(%)= 0.0154 and p.A g257T p in he
1000 genomes da abase (h p://b owse .1000genomes.o g.) wi h a MAF(%)=0.001. The
cha ac e is ics o he selec ed a ian s a e compiled in Table 1 and hei loca ion wi hin
he p o ein is shown in Figu e 1.
2.2. Si e-di ec ed mu agenesis
Plasmids ca ying he LDLR a ian s we e cons uc ed by Innop o (De io, Spain) as
desc ibed in Online Supp. Da a.
2.3. Cell cul u e and ans ec ion
LDLR-de icien CHO cell line ldlA7 (CHO-ldlA7) (kindly p o ided by D . Mon y
K iege , Massachuse s Ins i u e o Technology, Camb idge, MA) was cul u ed in
Ham’s F-12 medium supplemen ed wi h 5% FBS, 2 mM L-glu amine, 100 uni s/mL
penicillin, and 100 μg/mL s ep omycin. CHO-ldlA7 cells we e pla ed in o 6- o 24-well
cul u e pla es, and ans ec ed wi h plasmids ca ying he LDLR a ian s using
Lipo ec amine® LTX and PlusTM Reagen (In i ogen) acco ding o he manu ac u e ’s
ins uc ions. T ans ec ed cells we e main ained in cul u e du ing 48 h o achie e
maximal LDLR exp ession.
2.4. Wes e n blo analysis
Cell lysa es we e p epa ed, p o ein concen a ion de e mined, and ac iona ed by
elec opho esis as desc ibed in Online Supp. Da a.
2.5. Lipop o ein isola ion
LDL and VLDL we e isola ed om blood samples o heal hy indi iduals in a wo s ep
cen i uga ion as desc ibed in Online Supp. Da a.
2.6. Lipop o ein labelling
LDL and VLDL we e labelled wi h FITC as p e iously desc ibed [12]. B ie ly,
lipop o eins (1mg/mL) in 0.1 M NaHCO3 (pH 9.0) was mixed wi h 10 µl/mL FITC (2
mg/mL in dime hyl sul oxide). The mix u e was gen ly mixed by slow ocking a oom
empe a u e o 2 h. The un eac ed dye was emo ed by gel il a ion on a Sephadex G-
25 column equilib a ed wi h PBS EDTA- ee bu e . All ac ions we e assayed o
p o ein con en wi h bo ine se um albumin as s anda d (Pie ce BCA p o ein assay,
Pie ce).
2.7. Quan i ica ion o LDLR ac i i y by low cy ome y
T ans ec ed CHO-ldlA7 cells we e g own in 24-well cul u e pla es. 48 h a e
ans ec ion, cells we e incuba ed o 4 h, a 37ºC o a 4ºC wi h 20 µg/mL FITC-LDL
o de e mine LDLR ac i i y o LDL-LDLR binding, espec i ely. A e incuba ion wi h
FITC-LDL, CHO-ldlA7 cells we e washed wice in PBS-1%BSA, ixed on 4%
o maldehyde o 10 min and washed again wice wi h PBS-1%BSA. To de e mine he
amoun o in e nalized LDL, T ypan blue solu ion (Sigma-Ald ich, S einheim,
Ge many) was added di ec ly o he samples o a inal concen a ion o 0.2%,
elimina ing he ex acellula signal due o he non-in e nalized LDL-LDLR complexes.
Measu emen o VLDL was pe o med by incuba ion o cells wi h 20 µg/mL FITC-
VLDL o 4 h, a 37ºC as desc ibed o LDL. Fluo escence in ensi ies we e measu ed
by FACS, in a Facscalibu Flow cy ome e acco ding o he manu ac u e ins uc ions
as p e iously desc ibed [9]. Fo each sample, luo escence o 10,000 e en s was
acqui ed o da a analysis. All measu emen s we e pe o med a leas in iplica e.
2.8. Quan i ica ion o LDLR exp ession by low cy ome y
To de e mine LDLR cell su ace exp ession by FACS, ans ec ed CHO-ldlA7 cells
g own du ing 48 h we e incuba ed wi h a mouse p ima y an ibody an i-LDLR (1:100;
2.5 mg/L; P ogen Bio echnik GmbH) o 1 h, a oom empe a u e, hen washed wice
wi h PBS-1%BSA and incuba ed wi h seconda y an ibody Alexa Fluo 488-conjuga ed
goa an i-mouse IgG (1:100; Molecula P obes). Fo each sample, luo escence o
10,000 e en s was acqui ed o da a analysis. All measu emen s we e pe o med a leas
in iplica e.
2.9. Con ocal Lase Scanning Mic oscopy (CLSM)
CLSM was used o analyse LDL-LDLR binding and LDL up ake in LDLR ans ec ed
CHO-ldlA7 cells. B ie ly, cells we e pla ed in co e slips and hen ans ec ed wi h he
LDLR con aining plasmids and cul u ed o 48 h, a 37ºC in 5% CO2. Then he medium
was emo ed and co e slips washed wice wi h PBS-1%BSA. To de e mine LDL-LDLR
binding and LDL up ake, non-labelled lipop o eins (20 µg/mL LDL) we e added and cells
we e incuba ed o addi ional 4 h a 4ºC o 37ºC, espec i ely. Cells we e ixed wi h 4%
pa a o maldehyde du ing 10 min, washed h ee imes wi h PBS-1%BSA and
pe meabilised wi h 1% T i on X-100 o 30 min a oom empe a u e. Samples we e hen
washed and blocked in PBS-10%FBS o 1 h and washed in PBS-1%BSA h ee imes.
Then samples we e incuba ed wi h he app op ia e p ima y an ibodies o 16 h a 4ºC,
ollowed by incuba ion wi h he app op ia e luo escen seconda y an ibodies. Co e slips
we e moun ed on a glass slide and samples we e isualised using a con ocal mic oscope
(Olympus IX 81) wi h sequen ial exci a ion and cap u e image acquisi ion wi h a digi al
came a (Axiocam NRc5, Zeiss). Images we e p ocessed wi h Fluo iew .50 so wa e.
Image analysis o quan i y he luo escence in ensi ies was accomplished using he public
domain so wa e ImageJ (a ailable a h p:// sb.in o.nih.go /ij) unning on a s anda d PC.
2.10. In silico p edic ed e ec o molecula e en on LDLR
The possible impac o amino acid subs i u ions on he s uc u e and unc ion o
missense a ian s was p edic ed by using ou di e en so wa es as desc ibed in Online
Supp. Da a.
2.11. Conse a ion analysis
Conse a ion analysis among species o nucleo ide and amino acid was ca ied ou as
desc ibed in Online Supp. Da a.
2.12. S a is ical analysis
All measu emen s we e pe o med a leas 3 imes, wi h n=3 unless o he wise s a ed,
and esul s a e p esen ed as mean ± s.d. Le els o signi icance we e de e mined by a
wo- ailed S uden 's - es , and a con idence le el o g ea e han 95% (p<0.05) was used
o es ablish s a is ical signi icance.
3. Resul s
3.1. In silico analysis
The esul s ob ained by di e en so wa e packages a e p esen ed in Table 2. Depending
on he p og am, especially Align GVGD compa ed o he o he ones, he p edic ion o
he e ec caused by he a ian s is di e en . All he a ian s excep p.A g257T p and
p.A g300Gly we e classi ied as pa hogenic by he majo i y o he p edic ion p og ams.
These 2 a ian s a e he less conse ed ones and, as he p edic ion algo i hms a e mainly
based on conse a ion analysis, he esul s a e he expec ed ones. No splicing de ec s
we e p edic ed o any o he s udied a ian s (da a no shown).
3.2. Exp ession o LDLR a ian s in CHO-ldlA7 cells
CHO-ldlA7 cells we e ans ec ed wi h plasmids ca ying he di e en a ian s and
LDLR exp ession was assayed by immunoblo ing as desc ibed in Ma e ials and
Me hods. As shown in Figu e 2A (uppe panel), all he mu a ed LDLR a e exp essed a
simila le els as w 48 h a e ans ec ion. Equal loading o p o ein was con i med in
each blo by memb ane s ipping and u he incuba ion wi h an ibodies o isualise
cy osolic GAPDH p o ein (Figu e 2A, lowe panel). The ex en o p o ein exp ession
was de e mined by quan i a i e densi ome ic analysis (Figu e 2B). The da a ob ained
by low cy ome y con i med hese esul s, he LDLR exp ession o all he a ian s
being simila o he one in he w (Figu e 3A).
3.3. Analysis o LDLR ac i i y by FACS
CHO-ldlA7 cells exp essing w LDL ecep o o LDLR p.Cys116A g, p. Asp168Ans,
p.Asp172Asn, p.A g257T p, p.A g300Gly and p.Asp301Gly a ian s we e assayed o
LDL binding and up ake by low cy ome y. Fo in e nal me hod alida ion wo con ols
we e used because hei e ec s on exp ession, binding and up ake a e op imal o
compa ison wi h he expe imen al esul s ob ained by FACS. One o he con ol is
p.T p87*, a null allele mu an , ha does no p oduce LDLR. The o he in e nal con ol is
Ex3_4del mu an ha p oduces a de ec i e binding p o ein because he mRNA con ains
an in ame dele ion o exons 3 and 4, essen ial o LDL binding [13]. As shown in Figu e
3B, LDL-LDLR binding ac i i ies we e simila in w and p.A g257T p. Howe e ,
binding ac i i ies o he o he 5 a ian s we e diminished as compa ed o w (Figu e 3B).
As shown in Figu e 3C and in ag eemen wi h LDLR exp ession and binding esul s, LDL
in e nalisa ion in p.A g257T p was simila o w , and LDL up ake de e mined in he o he
5 a ian s was diminished when compa ed o w . Values o LDLR exp ession, LDL
binding and LDL up ake a e shown in Supplemen a y Table 1.
3.4. Analysis o LDLR ac i i y by con ocal mic oscopy
Con ocal mic oscopy was used o con i m he ac i i ies and pheno ypes o he analysed
LDLR a ian s. CHO-ldlA7 cells exp essing ei he w o he LDLR a ian s we e
incuba ed wi h LDL o 4 h and hen immunos ained wi h he app op ia e an ibodies o
de e mine LDLR and LDL localisa ion wi hin he cell. LDL incuba ion was pe o med a
4ºC o de e mine LDL binding, o a 37ºC o de e mine LDL up ake. FITC o Texas Red®
-conjuga ed seconda y an ibodies we e used o isualise LDLR and LDL espec i ely.
Figu es S1 and S2 show LDL binding o CHO-ldlA7 ans ec ed cells a low
magni ica ion in o de o show a wide ield wi h mul iple cells. As shown in Figu e 4A,
LDLR exp ession and LDL binding, de e mined a 4ºC, we e simila in w and in
p.A g257T p. Quan i ica ion o luo escence in ensi ies o he images ob ained by
con ocal mic oscopy showed no s a is ically signi ican di e ences be ween w and
p.A g257T p (Figu e 4C uppe : LDL exp ession, and Figu e 4C middle: LDL binding).
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FIGURE LEGENDS
Figu e 1: Th ee-dimensional model o he s uc u e o he LDLR ec odomain om
he i s cys eine- ich epea o he EGF domain (black). Loca ion o he a ian s
analysed in his s udy is shown in each co esponding R module by a ows. This igu e
was p epa ed wi h PyMOL (DeLano scien i ics).
Figu e 2: Exp ession o w LDLR and LDLR mu a ions in CHO-ldlA7 ans ec ed
cells. Cells we e ans ec ed wi h he co esponding plasmids ca ying he mu a ions o
in e es , LDLR was o e exp essed o 48 h and hen cells we e lysed and analysed by
Wes e n blo . Whole cell ex ac s (20 µg) we e ac ioned in non educing 8.5% SDS-
PAGE, ans e ed on o ni ocellulose memb anes o incuba ion wi h a abbi polyclonal
an i-hLDLR an ibody and de ec ed by chemioluminiscence as desc ibed in Ma e ials and
Me hods sec ion. The ela i e band in ensi y o ma u e LDLR p o ein exp ession was
calcula ed as he a io o 160 kDa LDLR band in ensi y o ha o GAPDH. A
ep esen a i e expe imen om h ee independen ly pe o med assays is shown in uppe
panel. Le els o signi icance we e de e mined by a wo- ailed S uden ’s - es , and a
con idence le el o g ea e han 95% (p<0.05) was used o es ablish s a is ical
signi icance. No s a is ically signi ican di e ences we e ound among he LDLR
exp ession.
Figu e 3: Func ional cha ac e iza ion o LDLR a ian s. A: LDLR exp ession a
cellula memb ane; B: LDL-LDLR binding a e 4 h incuba ion a 4 ºC; and C: LDL
in e nalisa ion e iciency a e 4 h incuba ion a 37ºC. 10,000 cells we e acqui ed in a
Facscalibu and alues o LDL up ake, binding and LDLR exp ession we e calcula ed as
desc ibed in Ma e ials and Me hods. The alues ep esen he mean o iplica e
de e mina ions (n = 3); e o ba s ep esen ±SD. *P < 0.001 compa ed o he w using a
S uden ’s - es .
Figu e 4: Analysis o w and p.A g257T p LDLR ac i i y by con ocal mic oscopy.
A: LDLR exp ession and LDL binding de e mined a 4ºC; B: LDL up ake de e mined a
37ºC; C: Quan i ica ion o LDLR exp ession, LDL binding and LDL up ake in w and
p.A g257T p. Fo LDLR exp ession and LDL binding assays, ans ec ed cells we e
incuba ed wi h non labelled LDL o 4 h a 4ºC, and o LDL up ake, cells we e incuba ed
wi h non labelled LDL o 4 h a 37ºC as desc ibed in Ma e ials and Me hods. An i-
hLDLR and an i-ApoB100 p ima y an ibodies ollowed by Alexa Fluo ® 488 and Texas
Red® labelled seconda y an ibodies we e used o isualize LDLR o LDL, espec i ely.
Dapi was used o isualize he nuclei o non ans ec ed cells o p.T p87* ans ec ed
cells. The images show a ep esen a i e indi idual cell o n=30. The his og ams ep esen
he mean ± s anda d de ia ion (n=30 cells), S uden ’s - es was pe o med showing no
s a is ical signi ican di e ences be ween w and p.A g257T p.
Figu e 5: Analysis o LDLR exp ession and LDL binding in w , p.Cys116A g, p.
Asp168Ans, p.Asp172Asn, p.A g300Gly and p.Asp301Gly LDLR a ian s. A: LDLR
exp ession and LDL binding de e mined a 4ºC; B: Quan i ica ion o LDLR exp ession
and LDL binding. Fo LDLR exp ession and LDL binding assays, ans ec ed cells we e
incuba ed wi h non labelled o 4 h a 4ºC. An i-hLDLR and an i-ApoB100 p ima y
an ibodies ollowed by Alexa Fluo ® 488 and Texas Red® labelled seconda y an ibodies
we e used o isualize LDLR o LDL, espec i ely. The images show a ep esen a i e
indi idual cell o n=30. The his og ams ep esen he mean ± s anda d de ia ion (n=30
cells), *p< 0.001 compa ed o he wild- ype (w ) using a S uden ’s - es .
Figu e 6: Analysis o LDL up ake in w , p.Cys116A g, p. Asp168Ans, p.Asp172Asn,
p.A g300Gly and p.Asp301Gly LDLR a ian s. A: LDL up ake de e mined a 37ºC;
B: Quan i ica ion o LDL up ake. Fo LDL up ake, cells we e incuba ed wi h non labelled
LDL o 4 h a 37ºC as desc ibed in Ma e ials and Me hods. An i-hLDLR and an i-
