Functional Characterization of p.(Arg160Gln) PCSK9 Variant Accidentally Found in a Hypercholesterolemic Subject

Ci a ion: La ea-Sebal, A.; T en i, C.;
Jeba i-Benslaiman, S.; Be olini, S.;
Caland a, S.; Neg i, E.A.; Bonelli, E.;
Beni o-Vicen e, A.;
U aga-G acian epa aluce a, L.;
Ma ín, C.; e al. Func ional
Cha ac e iza ion o p.(A g160Gln)
PCSK9 Va ian Acciden ally Found in
a Hype choles e olemic Subjec . In . J.
Mol. Sci. 2023,24, 3330. h ps://
doi.o g/10.3390/ijms24043330
Academic Edi o : Tzong-Shyuan Lee
Recei ed: 27 Decembe 2022
Re ised: 31 Janua y 2023
Accep ed: 3 Feb ua y 2023
Published: 7 Feb ua y 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
In e na ional Jou nal o
Molecula Sciences
A icle
Func ional Cha ac e iza ion o p.(A g160Gln) PCSK9 Va ian
Acciden ally Found in a Hype choles e olemic Subjec
Asie La ea-Sebal 1,2,3 , Chia a T en i 4, Shi a Jeba i-Benslaiman 1, S e ano Be olini 5, Sebas iano Caland a 6,
Emanuele A. Neg i 4, E em Bonelli 4, Asie Beni o-Vicen e 1, Lei e U aga-G acian epa aluce a 1,
Césa Ma ín1,2,* and Tommaso Fasano 7,*
1Depa men o Biochemis y and Molecula Biology, Uni e sidad del País Vasco UPV/EHU,
48080 Bilbao, Spain
2
Depa men o Molecula Biophysics, Bio isika Ins i u e, Uni e si y o Basque Coun y and Consejo Supe io
de In es igaciones Cien í icas (UPV/EHU, CSIC), 48940 Leioa, Spain
3Fundación Bio isika Bizkaia, 48940 Leioa, Spain
4Depa men o In e nal Medicine Uni , Azienda USL—IRCCS di Reggio Emilia, 42122 Reggio Emilia, I aly
5Depa men o In e nal Medicine, Uni e si y o Geno a, 16132 Geno a, I aly
6Depa men o Biomedical, Me abolic and Neu al Sciences, Uni e si y o Modena and Reggio Emilia,
41125 Modena, I aly
7Clinical Pa hology Uni , AUSL Romagna, 47521 Cesena, I aly
*Co espondence: cesa [email p o ec ed] (C.M.); [email p o ec ed]e.i (T.F.)
Abs ac :
Familial hype choles e olaemia (FH) is an au osomal dominan dyslipidaemia, cha ac-
e ised by ele a ed LDL choles e ol (LDL-C) le els in he blood. Th ee main genes a e in ol ed in FH
diagnosis: LDL ecep o (LDL ), Apolipop o ein B (APOB) and P o ein con e ase sub ilisin/kexin
ype 9 (PCSK9) wi h gene ic mu a ions ha led o educed plasma LDL-C clea ance. To da e, se e al
PCSK9 gain-o - unc ion (GOF) a ian s causing FH ha e been desc ibed based on hei inc eased
abili y o deg ade LDL . On he o he hand, mu a ions ha educe he ac i i y o PCSK9 on LDL
deg ada ion ha e been desc ibed as loss-o - unc ion (LOF) a ian s. I is he e o e impo an o
unc ionally cha ac e ise PCSK9 a ian s in o de o suppo he gene ic diagnosis o FH. The aim o
his wo k is o unc ionally cha ac e ise he p.(A g160Gln) PCSK9 a ian ound in a subjec suspec ed
o ha e FH. Di e en echniques ha e been combined o de e mine e iciency o he au oca aly ic
clea age, p o ein exp ession, e ec o he a ian on LDL ac i i y and a ini y o he PCSK9 a ian
o he LDL . Exp ession and p ocessing o he p.(A g160Gln) a ian had a esul simila o ha
o WT PCSK9. The e ec o p.(A g160Gln) PCSK9 on LDL ac i i y is lowe han WT PCSK9, wi h
highe alues o LDL in e nalisa ion (13%) and p.(A g160Gln) PCSK9 a ini y o he LDL is lowe
han WT, EC50 8.6
±
0.8 and 25.9
±
0.7, espec i ely. The p.(A g160Gln) PCSK9 a ian is a LOF
PCSK9 whose loss o ac i i y is caused by a displacemen o he PCSK9 P’ helix, which educes he
s abili y o he LDL -PCSK9 complex.
Keywo ds: amilial hype choles e olemia; PCSK9; GOF; LOF; cha ac e isa ion; ac i i y
1. In oduc ion
Familial hype choles e olaemia (FH) is an au osomal dominan dyslipidaemia, cha -
ac e ised by ele a ed LDL choles e ol (LDL-C) le els in he blood. He e ozygous amilial
hype choles e olaemia is one o he mo e common gene ic diseases wi h a p e alence o
1:250–300 in he gene al popula ion [
1
]. FH pa ien s ha e a high p obabili y o de eloping
ca dio ascula disease, due o a he oscle o ic lesions in he a e ial endo helium caused
by high plasma LDL-C concen a ions. Rega ding he ae iology o he disease, mu a ions
in h ee genes in ol ed in choles e ol me abolism a e mainly esponsible o he de elop-
men o he disease: LDL ecep o (LDL ), Apolipop o ein B (APOB) and P o ein con e ase
sub ilisin/kexin ype 9 (PCSK9) [
2
]. Gene ic al e a ions ha occu in hese genes educe
In . J. Mol. Sci. 2023,24, 3330. h ps://doi.o g/10.3390/ijms24043330 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2023,24, 3330 2 o 12
plasma LDL-C clea ance by di e en mechanisms. Mu a ions in he LDLR, esponsible o
LDL-C clea ance om plasma, ha e been de ec ed in he majo i y o FH pa ien s (85%).
On he o he hand, mu a ions in APOB, which a e esponsible o 5–10% o FH cases,
impai LDL–LDL binding, hus leading o LDL-C accumula ion in he plasma [
2
]. Finally,
gain-o - unc ion (GOF) mu a ions in PCSK9, a p o ein in ol ed in he ine- uning o LDL
exp ession a he plasma memb ane, accoun o 1% o FH cases by an inc eased lysosomal
deg ada ion o he LDL [3].
The human PCSK9 gene is loca ed in he 1p32.3 egion o he sho a m o he i s ch o-
mosome. The PCSK9 gene, a 22 kb sequence consis ing o 12 exons and 11 in ons, encodes a
p o ein o 692 amino acids [
4
]. PCSK9 exp ession is ansc ip ionally egula ed by he s e ol-
egula o y elemen -binding p o ein (SREBP2), which egula es choles e ol me abolism. In
ac , when he in acellula concen a ion o LDL-C is low, SREBP2 induces LDLR ansc ip-
ion oge he wi h PCSK9 [
5
]. The p o ein is dis ibu ed in i e domains: N- e minal signal
pep ide (SP; 1–30 aa), p opep ide o inhibi o y p odomain (
31–152 aa
), sub ilisin/se in
p o ease-like ca aly ic domain (SCD; 153–451 aa) and he
C- e minal
cys eine- and his idine-
ich domain (CHRD; 452–692 aa) [
6
]. PCSK9 is syn hesised as a 74 kDa p ecu so , which
becomes ma u e a e an au oca aly ic clea age be ween esidues 152–153 ende ing a
14 kDa p odomain, which binds non-co alen ly o he 60 kDa ca aly ic domain [
6
]. The
au oca aly ic clea age is essen ial o p o ein sec e ion and also esul s in ca aly ic ac i i y
inhibi ion by hiding he ca aly ic iad (D186, H226 and S386) [7].
Once sec e ed, PCSK9 binds o he EGF-A domain o he LDL , and he complex
is endocy osed ia cla h in-coa ed esicles. Acidi ica ion o he endosome inc eases he
a ini y o he p o ein o he ecep o ha a ge s he PCSK9-LDL complex o he lysosome
leading o LDL deg ada ion [8].
Basal PCSK9 ac i i y is necessa y o main ain a balance in memb ane LDL le els, hus
allowing an adequa e deg ada ion/ ecycling a io. To da e, se e al PCSK9 GOF a ian s
ha cause au osomal dominan hype choles e olaemia (ADH) by educing LDL le els
ha e been desc ibed [
9
]. On he o he hand, LOF mu a ions ha educe he ac i i y o
PCSK9, showing an impai ed abili y o deg ade LDL ha e also been desc ibed [
9
–
11
]. I
is he e o e impo an o unc ionally cha ac e ise PCSK9 a ian s o alida e he gene ic
diagnosis o ADH.
The aim o his wo k is o unc ionally cha ac e ise he ac i i y o he p.(A g160Gln)
PCSK9 a ian , a a ian acciden ally ound in a hype choles e olemic pa ien . In o de o
ca y ou he unc ional cha ac e isa ion, biophysics and molecula biology echniques ha e
been combined o de e mine he e iciency o he au oca aly ic clea age, p o ein exp ession,
he e ec o p.(A g160Gln) PCSK9 on LDL ac i i y and he a ini y o p.(A g160Gln) PCSK9
a ian o he LDL . To unde s and he mechanism by which p.(A g160Gln) p esen s a
educed a ini y o he LDL , a compu a ional s udy using AlphaFold has been used.
2. Resul s
Nex -gene a ion sequencing (NGS) analysis was employed o sequence genes asso-
cia ed wi h Familial Hype choles e olemia in he p oband’s sample. A missense PCSK9
mu a ion was iden i ied: c.479 G > A in exon 3, p.(A g160Gln). Subsequen cascade sc een-
ing led o he iden i ica ion o he same PCSK9 mu a ion in wo o he i e amily membe s
ha we e subjec ed o gene ic analysis. (Figu e 1). Lipid alues o amily membe s and
PCSK9 geno ypes a e epo ed in Table 1. The in o ma ion abou lipid p o iles ob ained
wi h he amily pedig ee demons a ed no clea co ela ion be ween he PCSK9 mu a ion
and he high LDL-C pheno ype as one o he ca ie s o he mu a ion had no mal–low
LDL-C alues. A high LDL-C pheno ype wi h he p.(A g160Gln) a ian was p esen only
in one o he h ee mu a ion ca ie s., which is no expec ed o a GOF PCSK9 a ian . The
ClinVa da abase desc ibes he equency o he iden i ied PCSK9 mu a ion (
c.479 G > A
,
p.A g160Gln) o 0.00013 as i was ound in 19/282782 ch omosomes in he gene al popula-
ion (da a om he Genome Agg ega ion Da abase, gnomAD). The mu a ion was epo ed
in h ee subjec s wi h suspec ed au osomal dominan hype choles e olemia and in one
In . J. Mol. Sci. 2023,24, 3330 3 o 12
subjec wi h hypobe alipop o einemia (ClinVa da abase). The a ian has been also e-
po ed in he li e a u e in a GWAS s udy obse ed in one indi idual wi h low LDL-C [
12
].
The a ailable e idence was no su icien o de e mine he ole o his a ian in disease
conclusi ely. Fo ha eason, we decided o unc ionally cha ac e ise he e ec on he
PCSK9 p o ein.
We analysed he po en ial consequences o he p.(A g160Gln) PCSK9 a ian wi h
he in silico p edic ion p og ams Mu a ion Tas e , Polyphen-2 PROVEAN; SIFT, Mu a ion
Assesso , FATHMM and PANTHER. The a ian was classi ied as a p obably damaging
o disease causing PCSK9 a ian (Table 2). To ou knowledge, no expe imen al e idence
s udying he impac o he mu a ion on p o ein unc ion has been epo ed.
In .J.Mol.Sci.2023,24,xFORPEERREVIEW3o 12


p.A g160Gln)o 0.00013asi was oundin19/282782ch omosomesin hegene alpopu‐
la ion(da a om heGenomeAgg ega ionDa abase,gnomAD).Themu a ionwas e‐
po edin h eesubjec swi hsuspec edau osomaldominan hype choles e olemiaandin
onesubjec wi hhypobe alipop o einemia(ClinVa da abase).The a ian hasbeenalso
epo edin heli e a u einaGWASs udyobse edinoneindi idualwi hlowLDL‐C
[12].Thea ailablee idencewasno su icien  ode e mine he oleo  his a ian indis‐
easeconclusi ely.Fo  ha  eason,wedecided o unc ionallycha ac e ise hee ec on
hePCSK9p o ein.
Weanalysed hepo en ialconsequenceso  hep.(A g160Gln)PCSK9 a ian wi h
heinsilicop edic ionp og amsMu a ionTas e ,Polyphen‐2PROVEAN;SIFT,Mu a ion
Assesso ,FATHMMandPANTHER.The a ian wasclassi iedasap obablydamaging
odiseasecausingPCSK9 a ian (Table2).Toou knowledge,noexpe imen ale idence
s udying heimpac o  hemu a iononp o ein unc ionhasbeen epo ed.

Figu e1.Familypedig eeshowing heca ie so  hep.(A g160Gln)PCSK9 a ian .Hal ‐blackened
indica eshe e ozygousca ie so  hep.(A g160Gln) a ian .Thea ow ep esen s hep oband.
Age(inyea s)andLDL‐C(mg/dL)a egi en.“?”unknown.
Table1.Sex,age,lipidp o ilesandmu a ionals a uso p obandand amilymembe scha ac e ised
du ing hes udy.TC,LDL‐C,HDL‐CandTGa eexp essedinmg/dL.
Subjec SexAgeTCLDL‐CHDL‐CTGp.(A g160
Gln)
P obandF5034127559139YES
SonM2728217576199NO
B o he M542001543998YES
Nephew1M28169995388YES
Nephew2M24136785151NO
Nephew3M2918713444132NO
Table2.Insilicop edic ionso  heimpac o  heA g160Glnmu a iononPCSK9p o einusingdi ‐
e en online ools.
Mu a ion
Tas e 
PolyPhen‐
2
PROVEA
NSIFTMu a ion
Assesso 
FATHM
MPANTHER
p.(A g160G
ln)
Disease
Causing
P obably
damaging
Dele e i‐
ous
Damag‐
ing
HighIm‐
pac 
Damag‐
ing
P obablyDam‐
aging
?
?
??
PCSK9Ex.3c.479G>A
A g160Gln
50y/o
275mg/dl
27y/o
175mg/dl
54y/o
154mg/dl
28y/o
99mg/dl
24y/o
78mg/dl
29y/o
134mg/dl
Figu e 1.
Family pedig ee showing he ca ie s o he p.(A g160Gln) PCSK9 a ian . Hal -blackened
indica es he e ozygous ca ie s o he p.(A g160Gln) a ian . The a ow ep esen s he p oband. Age
(in yea s) and LDL-C (mg/dL) a e gi en. “?” unknown.
Table 1.
Sex, age, lipid p o iles and mu a ional s a us o p oband and amily membe s cha ac e ised
du ing he s udy. TC, LDL-C, HDL-C and TG a e exp essed in mg/dL.
Subjec Sex Age TC LDL-C HDL-C TG p.(A g160Gln)
P oband F 50 341 275 59 139 YES
Son M 27 282 175 76 199 NO
B o he M 54 200 154 39 98 YES
Nephew 1 M 28 169 99 53 88 YES
Nephew 2 M 24 136 78 51 51 NO
Nephew 3 M 29 187 134 44 132 NO
Table 2.
In silico p edic ions o he impac o he A g160Gln mu a ion on PCSK9 p o ein using
di e en online ools.
Mu a ion
Tas e PolyPhen-2 PROVEAN SIFT Mu a ion
Assesso FATHMM PANTHER
p.(A g160Gln) Disease
Causing P obably
damaging Dele e ious Damaging High
Impac Damaging P obably
Damaging
In . J. Mol. Sci. 2023,24, 3330 4 o 12
2.1. PCSK9 Exp ession, Ma u a ion and Sec e ion
Exp ession, ma u a ion and sec e ion o he WT, D374Y and p.(A g160Gln) PCSK9
a ian s we e s udied in ansien ly ans ec ed HEK293 cells, a well-es ablished cell line,
o pe o m PCSK9 unc ional s udies [
9
,
13
–
15
]. As shown in Figu e 2, he p.(A g160Gln)
PCSK9 a ian showed simila exp ession, ma u a ion and sec e ion pa e ns han WT
PCSK9 as de ec ed by Wes e n blo . The p.(A g160Gln) a ian showed a ain ex a-band
o sligh ly highe molecula weigh in he cul u e media. In e ms o o al PCSK9 exp ession
and sec e ion a ios, no signi ican changes we e obse ed (Figu e 2B–D).
In .J.Mol.Sci.2023,24,xFORPEERREVIEW4o 12


2.1.PCSK9Exp ession,Ma u a ionandSec e ion
Exp ession,ma u a ionandsec e iono  heWT,D374Yandp.(A g160Gln)PCSK9
a ian swe es udiedin ansien ly ans ec edHEK293cells,awell‐es ablishedcellline,
ope o mPCSK9 unc ionals udies[9,13–15].AsshowninFigu e2, hep.(A g160Gln)
PCSK9 a ian showedsimila exp ession,ma u a ionandsec e ionpa e ns hanWT
PCSK9asde ec edbyWes e nblo .Thep.(A g160Gln) a ian showeda ain ex a‐band
o sligh lyhighe molecula weigh in hecul u emedia.In e mso  o alPCSK9exp es‐
sionandsec e ion a ios,nosigni ican changeswe eobse ed(Figu e2B–D).

Figu e2.Exp ession,ma u a ionandsec e iono  hep.(A g160Gln)PCSK9 a ian issimila  oWT
PCSK9.(A)Rep esen a i eimmunoblo so exp essionandsec e iono PCSK9onHEK293cells
ansien ly ans ec edwi hmock(emp yplasmid),WT,D374Yandp.(A g160Gln).(B)Ra iobe‐
weenp ocessed/non‐p ocessedPCSK9quan i iedbydensi ome y.(C)Amoun o exp essed
PCSK9de e minedas he a iobe weenin acellula PCSK9/GAPDHquan i iedbydensi ome y.
(D)Amoun o sec e edPCSK9de e minedas he a iobe weenmedia/GAPDHquan i iedbyden‐
si ome y.His og ams ep esen  hemean±SDo  h eeindependen expe imen s.*p<0.05com‐
pa ed oWTPCSK9.
2.2.E ec o  hep.(A g160Gln)PCSK9Va ian onLDLUp ake
Analysiso LDL ac i i ywasassessedin ansien ly ans ec edHEK293cellswi h
hePCSK9 a ian ,andLDLup akewasde e minedby lowcy ome yasdesc ibedin
Me hods.AsshowninFigu e3,cellsexp essing hep.(A g160Gln) a ian showasigni ‐
ican highe LDLup akecompa ed oWTPCSK9.
Figu e 2.
Exp ession, ma u a ion and sec e ion o he p.(A g160Gln) PCSK9 a ian is simila o
WT PCSK9. (
A
) Rep esen a i e immunoblo s o exp ession and sec e ion o PCSK9 on HEK293
cells ansien ly ans ec ed wi h mock (emp y plasmid), WT, D374Y and p.(A g160Gln). (
B
) Ra io
be ween p ocessed/non-p ocessed PCSK9 quan i ied by densi ome y. (
C
) Amoun o exp essed
PCSK9 de e mined as he a io be ween in acellula PCSK9/GAPDH quan i ied by densi ome y.
(
D
) Amoun o sec e ed PCSK9 de e mined as he a io be ween media/GAPDH quan i ied by
densi ome y. His og ams ep esen he mean
±
SD o h ee independen expe imen s. * p< 0.05
compa ed o WT PCSK9.
2.2. E ec o he p.(A g160Gln) PCSK9 Va ian on LDL Up ake
Analysis o LDL ac i i y was assessed in ansien ly ans ec ed HEK293 cells wi h
he PCSK9 a ian , and LDL up ake was de e mined by low cy ome y as desc ibed
in Me hods. As shown in Figu e 3, cells exp essing he p.(A g160Gln) a ian show a
signi ican highe LDL up ake compa ed o WT PCSK9.
In . J. Mol. Sci. 2023,24, 3330 5 o 12
In .J.Mol.Sci.2023,24,xFORPEERREVIEW5o 12



Figu e3.Exp essiono  hep.(A g160Gln)PCSK9 a ian inc easesLDLup akecompa ed oWT.
T ansien ly ans ec edHEK293cellswi h hedi e en PCSK9 a ian swe eincuba edwi hFITC‐
labelledLDLandlipop o einup akewasmeasu edby lowcy ome y.His og ams ep esen  he
mean±SDo  h eeindependen expe imen s.*p<0.01compa ed oWT.Mockco esponds oan
emp yplasmid.LDLup akewasno malised o he o alamoun o PKSC9sec e ed o hecul u e
medium48hpos ‐ ans ec ion.
2.3.p.(A g160Gln)PCSK9A ini y(EC50) o  heLDL 
Wenex assessed hea ini yo  hep.(A g160Gln)PCSK9 a ian  o  heLDL .Bind‐
inga ini ieswe ede e minedbysolid‐phaseimmunoassayasdesc ibedinMe hods.As
showninTable3andFigu e4, hea ini yo p.(A g160Gln)PCSK9 o  heLDL was
signi ican ly educedcompa ed o ha o WT.
Table3.EC50o  heWT,D374YGOFPCSK9 a ian and hep.(A g160Gln)PCSK9 a ian .
WTD374Yp.(A g160Gln)
EC508.57±0.761.54±0.38*25.96±0.72*
*p<0.01compa ed oWT.

Figu e4.Thep.(A g160Gln)PCSK9 a ian showslowe a ini y o heLDL compa ed oWT.A ‐
ini ycu es ep esen ing hebindinga ini yo PCSK9 a ian s o  heLDL de e minedbysolid‐
phaseimmunoassaya pH7.4.Da a ep esen s hemeanso  h eeindependen expe imen s.EC50
aluesa eshowninTable3.
% LDL up ake
*
*
Figu e 3.
Exp ession o he p.(A g160Gln) PCSK9 a ian inc eases LDL up ake compa ed o WT.
T ansien ly ans ec ed HEK293 cells wi h he di e en PCSK9 a ian s we e incuba ed wi h FITC-
labelled LDL and lipop o ein up ake was measu ed by low cy ome y. His og ams ep esen he
mean
±
SD o h ee independen expe imen s. * p< 0.01 compa ed o WT. Mock co esponds o an
emp y plasmid. LDL up ake was no malised o he o al amoun o PKSC9 sec e ed o he cul u e
medium 48 h pos - ans ec ion.
2.3. p.(A g160Gln) PCSK9 A ini y (EC50) o he LDL
We nex assessed he a ini y o he p.(A g160Gln) PCSK9 a ian o he LDL . Binding
a ini ies we e de e mined by solid-phase immunoassay as desc ibed in Me hods. As shown
in Table 3and Figu e 4, he a ini y o p.(A g160Gln) PCSK9 o he LDL was signi ican ly
educed compa ed o ha o WT.
Table 3. EC50 o he WT, D374Y GOF PCSK9 a ian and he p.(A g160Gln) PCSK9 a ian .
WT D374Y p.(A g160Gln)
EC50 8.57 ±0.76 1.54 ±0.38 * 25.96 ±0.72 *
*p< 0.01 compa ed o WT.
In .J.Mol.Sci.2023,24,xFORPEERREVIEW5o 12



Figu e3.Exp essiono  hep.(A g160Gln)PCSK9 a ian inc easesLDLup akecompa ed oWT.
T ansien ly ans ec edHEK293cellswi h hedi e en PCSK9 a ian swe eincuba edwi hFITC‐
labelledLDLandlipop o einup akewasmeasu edby lowcy ome y.His og ams ep esen  he
mean±SDo  h eeindependen expe imen s.*p<0.01compa ed oWT.Mockco esponds oan
emp yplasmid.LDLup akewasno malised o he o alamoun o PKSC9sec e ed o hecul u e
medium48hpos ‐ ans ec ion.
2.3.p.(A g160Gln)PCSK9A ini y(EC50) o  heLDL 
Wenex assessed hea ini yo  hep.(A g160Gln)PCSK9 a ian  o  heLDL .Bind‐
inga ini ieswe ede e minedbysolid‐phaseimmunoassayasdesc ibedinMe hods.As
showninTable3andFigu e4, hea ini yo p.(A g160Gln)PCSK9 o  heLDL was
signi ican ly educedcompa ed o ha o WT.
Table3.EC50o  heWT,D374YGOFPCSK9 a ian and hep.(A g160Gln)PCSK9 a ian .
WTD374Yp.(A g160Gln)
EC508.57±0.761.54±0.38*25.96±0.72*
*p<0.01compa ed oWT.

Figu e4.Thep.(A g160Gln)PCSK9 a ian showslowe a ini y o heLDL compa ed oWT.A ‐
ini ycu es ep esen ing hebindinga ini yo PCSK9 a ian s o  heLDL de e minedbysolid‐
phaseimmunoassaya pH7.4.Da a ep esen s hemeanso  h eeindependen expe imen s.EC50
aluesa eshowninTable3.
% LDL up ake
*
*
Figu e 4.
The p.(A g160Gln) PCSK9 a ian shows lowe a ini y o he LDL compa ed o WT.
A ini y cu es ep esen ing he binding a ini y o PCSK9 a ian s o he LDL de e mined by
solid-phase immunoassay a pH 7.4. Da a ep esen s he means o h ee independen expe imen s.
EC50 alues a e shown in Table 3.

In . J. Mol. Sci. 2023,24, 3330 6 o 12
2.4. Bioin o ma ic Analysis o p.(A g160Gln) PCSK9 3D S uc u e
Using AlphaFold-2, he 3D s uc u es o WT and p.(A g160Gln) PCSK9 we e compa ed.
Upon s uc u e alignmen , he amino acids p eceding he mu a ion si e (153–160) we e
displaced in he a ian (Figu e 5). Acco ding o he modelling, he eplacemen o an A g
by a Gln causes a displacemen o he amino acids a posi ions 153–156 so ha hey mo e
away om he ca aly ic domain o PCSK9, a key ac o in he in e ac ion wi h he LDL ’s
EGF-A domain (Figu e 5).
In .J.Mol.Sci.2023,24,xFORPEERREVIEW6o 12


2.4.Bioin o ma icAnalysiso p.(A g160Gln)PCSK93DS uc u e
UsingAlphaFold‐2, he3Ds uc u eso WTandp.(A g160Gln)PCSK9we ecom‐
pa ed.Upons uc u ealignmen , heaminoacidsp eceding hemu a ionsi e(153–160)
we edisplacedin he a ian (Figu e5).Acco ding o hemodelling, he eplacemen o 
anA gbyaGlncausesadisplacemen o  heaminoacidsa posi ions153–156so ha  hey
mo eaway om heca aly icdomaino PCSK9,akey ac o in hein e ac ionwi h he
LDL ’sEGF‐Adomain(Figu e5).

Figu e5.AlphaFold‐2s uc u ep edic iono  hep.(A g160Gln)PCSK9 a ian compa ed oWT.
S uc u eso  hea eao in e es (aminoacids153–160)inbo hWTandp.(A g160Gln)we ecom‐
pa ed ounde s and hena u eo  he a ian .(A)F on  iew,(B) op iewand(C)side iew.In
g een,p odomain(31–152);inpu ple,ca aly icdomain(154–451);inpink,C‐ e minaldomain(453–
692).Inside heca aly icdomain,A g160 esidueiscolou edinblue, heca aly ic iadinyellow
(186,226and386)and heLDL bindingsi eing ey(367–381).
3.Discussion
He e ozygousFamilialHype choles e olemiais hemos commongene icdisease,
which equi esea lyde ec ionandapp op ia e ea men  o educeca dio ascula e en s
[1].In ecen yea s,and hanks omassi esequencingo DNA,many a ian so genes
in ol edin hede elopmen o FHha ebeeniden i ied;howe e ,no allo  hema e
pa hogenic[1,9,16,17].Al houghgene icanalysescons i u eanimpo an s ep o diagno‐
sis,es ablishing hepa hogenici yo  hese a ian s h ough unc ionals udiesisessen ial
omakeanaccu a ediagnosis[18].
In hep esen wo kweha e unc ionallycha ac e ised hee ec o  he
p.(A g160Gln)PCSK9 a ian  o indou whe he i s unc ionisal e edo no .This
Figu e 5.
AlphaFold-2 s uc u e p edic ion o he p.(A g160Gln) PCSK9 a ian compa ed o WT.
S uc u es o he a ea o in e es (amino acids 153–160) in bo h WT and p.(A g160Gln) we e compa ed
o unde s and he na u e o he a ian . (
A
) F on iew, (
B
) op iew and (
C
) side iew. In g een,
p odomain (31–152); in pu ple, ca aly ic domain (154–451); in pink, C- e minal domain (453–692).
Inside he ca aly ic domain, A g160 esidue is colou ed in blue, he ca aly ic iad in yellow (186, 226
and 386) and he LDL binding si e in g ey (367–381).
3. Discussion
He e ozygous Familial Hype choles e olemia is he mos common gene ic disease,
which equi es ea ly de ec ion and app op ia e ea men o educe ca dio ascula e en s [
1
].
In ecen yea s, and hanks o massi e sequencing o DNA, many a ian s o genes in-
ol ed in he de elopmen o FH ha e been iden i ied; howe e , no all o hem a e
pa hogenic [
1
,
9
,
16
,
17
]. Al hough gene ic analyses cons i u e an impo an s ep o diagno-
In . J. Mol. Sci. 2023,24, 3330 7 o 12
sis, es ablishing he pa hogenici y o hese a ian s h ough unc ional s udies is essen ial
o make an accu a e diagnosis [18].
In he p esen wo k we ha e unc ionally cha ac e ised he e ec o he p.(A g160Gln)
PCSK9 a ian o ind ou whe he i s unc ion is al e ed o no . This a ian was iden i ied
in a 50 y/o emale in pos menopausal s a e wi h high LDL-C le els. Cascade sc eening in
i e amily membe s was no conclusi e o es ablish he supposed “gain-o - unc ion” o
he mu a ed PCSK9 p o ein. In pa icula he mu a ion was iden i ied in a 28 y/o male
wi h no mal/low LDL-C. In silico p edic ion o he e ec o he mu a ion suppo ed a
dele e ious impac o he mu a ion on he p o ein unc ion, bu , o ob ious eason, wi hou
gi ing ad ice on gain o loss o p o ein unc ion. Fo he p.(A g160Gln) mu a ion, a e y
low equency was desc ibed in genomic da abases and i was iden i ied in bo h hype -
and hypo-choles e olemic condi ions. The a ailable e idence was conside ed insu icien
o de e mine he ole o his a ian in disease conclusi ely. The e o e, his a ian was
classi ied as a a ian o unce ain signi icance, claiming ha i s associa ion wi h disease
equi e u he in es iga ion.
The p.(A g160Gln) mu a ion is loca ed a he beginning o he PCSK9-ca aly ic do-
main, as shown in he p esen wo k; exp ession/p ocessing and sec e ion o he a ian
is no a ec ed. In e es ingly, he highe alues o LDL in e nalisa ion in cells exp essing
p.(A g160Gln) indica es ha he ac i i y o p.(A g160Gln) on LDL ac i i y is lowe com-
pa ed o WT PCSK9. The e o e, he unc ional cha ac e isa ion pe o med in his wo k
indica es ha p.(A g160Gln) PCSK9 is a LOF a ian , hus, is no esponsible o he pa ien ’s
pheno ype. In addi ion, he LOF ac i i y o he p.(A g160Gln) a ian was co obo a ed by
de e mining i s a ini y o he LDL , which showed a educed EC
50
o he LDL compa ed
o ha o WT PCSK9.
We nex sough o unde s and he mechanism by which he p.(A g160Gln) a ian
shows a educed a ini y o he LDL . I has been desc ibed ha au oca alysis is c i ical
o PCSK9 sec e ion and endows LDL -binding capaci y by exposing he EGF(A)-binding
si e [
19
]. The au oca aly ic clea age occu s be ween he las p odomain esidue, Gln152 (P1
esidue; nomencla u e o Schech e and Be ge ) and he i s esidue o he ca aly ic domain
Se 153 (P1
0
esidue). Once clea ed, a sp ing-loaded mechanism is igge ed and P
0
amino
acids ansloca e in o a g oo e loca ed nex o he LDL -binding si e (Figu e 6). The EGF(A)-
in e ac ing si e o PCSK9 is a sol en -exposed a ea o ~530 Å2 ha is la gely la , ea u eless
and de oid o binding pocke s. The e o e, i elec os a ic o ces a e no s ong enough,
binding and main aining he PCSK9-LDL complex could be comp omised. In e es ingly,
he s e ch o P
0
esidues (Se 153–Th 162) adop s an
α
-helical con o ma ion, e med he
P
0
helix, in which P1
0
Se 153 and P3
0
P o155 con ibu e o he s abilisa ion o he bound
LDL -EGF(A) domain h ough pola and an de Waals in e ac ions [
20
,
21
]. In addi ion,
he A g160 esidue con ibu es speci ically o binding a ini y and speci ici y by o ming a
sal b idge wi h he LDL Asp343 esidue [
19
]. As Gln has no cha ge, eplacemen o he
posi i ely cha ged A g160 by Gln ab oga es he in e ac ion wi h he nega i ely cha ged
Asp343 o he LDL . Hence, he loss o his elec os a ic in e ac ion can con ibu e o he
educed a ini y de e mined he e o he p.(A g160Gln) PCSK9 a ian o he LDL . By
using AlphaFold, we ha e also de e mined ha eplacemen o A g160 by Gln a ec s he
con o ma ion o he 153–168 P’ esidues. P’ helix modelling shows ha he subs i u ion
causes a change in helix o a ion, which esul s in a comple e displacemen o he amino
acids in ol ed in he s abilisa ion o PCSK9-EGF-A binding (Figu es 5and 6). I seems
plausible ha his p edic ed displacemen o he PCSK9 P’ helix in p.(A g160Gln) a ian
ep esen s he main eason o he lowe a ini y o he a ian o he LDL .
In . J. Mol. Sci. 2023,24, 3330 8 o 12
In .J.Mol.Sci.2023,24,xFORPEERREVIEW8o 12



Figu e6.Sugges edmechanismsleading olosso a ini yo  hep.(A d160Gln)PCSK9 a ian  o 
heLDL .(A)Uponp odomainclea age,P′ helixisloca ednex  o heLDL ‐bindingsi eand
s eng hens heelec os a ic o ces o bindingandmain aining hePCSK9‐LDL complex.Addi‐
ionally, heA g160 esidue o msasal b idgewi h heLDL Asp343 esidue.(B)Replacemen o 
A g160byaGlnmodi ies hechange hedi ec iono  o a iono  heP’helix,whichnolonge  e‐
mainsclose o heEGF‐A esiduesimplica edinPCSK9binding.
Acco ding o heob ained esul s,wecanconclude ha  hep.(A g160Gln)PCSK9
a ian isaLOFPCSK9 a ian whoselosso ac i i yiscausedbyadisplacemen o  he
PCSK9P’helix,which educes hes abili yo  heLDL ‐PCSK9complex, husno causa‐
i eo  amilialhype choles e olemia.
4.Ma e ialsandMe hods
4.1.Pa ien s
A50y/opos menopausal emalewasp esen ed o heLipidClinic o  he indingo 
e yhighLDL‐Ccholes e ol(Table1)anda amilyhis o yo hype choles e olemiaand
ea men wi hlipid‐lowe ingagen s.Thediagnosiso p obableFamilialHype choles e ‐
olemiawasmadebasedonaDu chLipidClinicalNe wo k(DLCN)(Re )sco eo 6.Nex ‐
gene a ionsequencing(NGS)analysiswaspe o medusing heIlluminaMiSeqDXpla ‐
o m(Illumina,SanDiego,CA,USA) osequencegenesassocia edwi h amilialhype ‐
choles e olemia(APOB,APOE,LDLR,LDLRAP1,PCSK9,ABCG5,ABCG8,CYP27A1,
LIPA,MYLIP)[22].Gene icanalysiswasalsope o medon i e amilymembe s(son,
b o he and h eenephews).Lipidp o ileso p obandand amilymembe sa e epo ed
inTable1.
4.2.SampleAnalysis
To alcholes e ol(TC),HDL‐C,LDL‐Cand iglyce ides(TG)we emeasu edusing
ullyau oma iclabo a o yins umen a ionwi henzyma iccolo ime icassays(A ellica
CH,SiemensHeal hinee s,E langen,Ge many).
Fo allsubjec sin ol edin hes udy,samples o lipidp o ileswe eob aineda e 
ano e nigh  as ingpe iodandwi hou adminis a iono lipid‐lowe ingd ugs.As
showninFigu e1andTable1, womu a ionca ie shadhigho mildele a iono plas‐
ma icLDL‐C alues(275mg/dLand154mg/dLin hep obandand heb o he , espec‐
i ely).Thi dmu a ionca ie (Nephew1)hadno mal‐lowLDL‐C alues(99mg/dL).
4.3.Si e‐Di ec edMu agenesisandCloning
Plasmidsca yingPCSK9 a ian swe econs uc edbyInnop o .Mu a ionswe ein‐
oducedin o hehumanPCSK9cDNA(NM_174936.3),in hemammalianexp ession
Figu e 6.
Sugges ed mechanisms leading o loss o a ini y o he p.(A d160Gln) PCSK9 a ian o he
LDL . (
A
) Upon p odomain clea age, P
0
helix is loca ed nex o he LDL -binding si e and s eng hens
he elec os a ic o ces o binding and main aining he PCSK9-LDL complex. Addi ionally, he
A g160 esidue o ms a sal b idge wi h he LDL Asp343 esidue. (
B
) Replacemen o A g160 by a
Gln modi ies he change he di ec ion o o a ion o he P’ helix, which no longe emains close o he
EGF-A esidues implica ed in PCSK9 binding.
Acco ding o he ob ained esul s, we can conclude ha he p.(A g160Gln) PCSK9
a ian is a LOF PCSK9 a ian whose loss o ac i i y is caused by a displacemen o he
PCSK9 P’ helix, which educes he s abili y o he LDL -PCSK9 complex, hus no causa i e
o amilial hype choles e olemia.
4. Ma e ials and Me hods
4.1. Pa ien s
A 50 y/o pos menopausal emale was p esen ed o he Lipid Clinic o he inding o
e y high LDL-C choles e ol (Table 1) and a amily his o y o hype choles e olemia and
ea men wi h lipid-lowe ing agen s. The diagnosis o p obable Familial Hype choles-
e olemia was made based on a Du ch Lipid Clinical Ne wo k (DLCN) (Re ) sco e o 6.
Nex -gene a ion sequencing (NGS) analysis was pe o med using he Illumina MiSeq DX
pla o m (Illumina, San Diego, CA, USA) o sequence genes associa ed wi h amilial hype c-
holes e olemia (APOB, APOE, LDLR, LDLRAP1, PCSK9, ABCG5, ABCG8, CYP27A1, LIPA,
MYLIP) [
22
]. Gene ic analysis was also pe o med on i e amily membe s (son, b o he
and h ee nephews). Lipid p o iles o p oband and amily membe s a e epo ed in Table 1.
4.2. Sample Analysis
To al choles e ol (TC), HDL-C, LDL-C and iglyce ides (TG) we e measu ed using
ully au oma ic labo a o y ins umen a ion wi h enzyma ic colo ime ic assays (A ellica
CH, Siemens Heal hinee s, E langen, Ge many).
Fo all subjec s in ol ed in he s udy, samples o lipid p o iles we e ob ained a e an
o e nigh as ing pe iod and wi hou adminis a ion o lipid-lowe ing d ugs. As shown in
Figu e 1and Table 1, wo mu a ion ca ie s had high o mild ele a ion o plasma ic LDL-C
alues (275 mg/dL and 154 mg/dL in he p oband and he b o he , espec i ely). Thi d
mu a ion ca ie (Nephew 1) had no mal-low LDL-C alues (99 mg/dL).
4.3. Si e-Di ec ed Mu agenesis and Cloning
Plasmids ca ying PCSK9 a ian s we e cons uc ed by Innop o . Mu a ions we e in o-
duced in o he human PCSK9 cDNA (NM_174936.3), in he mammalian exp ession ec o
WT-PCSK9 plasmid (pCMV-PCSK9-FLAG) by oligonucleo ide si e-di ec ed mu agenesis,
using he QuickChange Ligh ning mu agenesis ki (Agilen Technologies Inc., La Jolla, CA,
In . J. Mol. Sci. 2023,24, 3330 9 o 12
USA) acco ding o he manu ac u e ’s ins uc ions. This ec o con ains a 6×His ag o he
pu i ica ion and a FLAG epi ope (DYKDDDDK) as a speci ic a ge o an ibodies. Res ic ion
enzyme diges ion o he app op ia e agmen s and he in eg i y o he emaining PCSK9
cDNA sequences o all cons uc s we e e i ied by di ec sequence analysis.
4.4. PCSK9 Exp ession on HEK293 Cells
A o al o 5
×
105 HEK293 cells we e ans ec ed wi h an emp y plasmid o wi h
1.5
µ
g o a plasmid encoding WT-PCSK9, GOF-PCSK9 p.(Asp374Ty ) o he analysed
a ian p.(A g160Gln) using a Calcium Phospha e T ans ec ion Ki (In i ogen, The mo
Fishe Scien i ic, Pie ce, CA, USA). The nex day cells we e washed and incuba ed wi h
DMEM medium con aining 10% FBS, 2 mM L-Glu amine and an ibio ics (100 uni s/mL
penicillin; 100
µ
g/mL s ep omycin) (comple e medium) o 48 h. Nex , cell supe na an s
we e collec ed and cells we e lysa ed o analyse bo h PCSK9 sec e ion and exp ession.
4.5. PCSK9 Sec e ion and Exp ession Analysis by Wes e n Blo
PCSK9 exp ession and sec e ion analysis in HEK293 cells ans ec ed wi h emp y plas-
mid and he di e en PCSK9 a ian s was pe o med by Wes e n blo ing. Fo ha pu pose,
p o eins om cell lysa es o he supe na an s we e esol ed by 8.5% T is-Glycine SDS-PAGE.
Gels we e nex blo ed on o Ni ocellulose memb anes (P o an BA 83, Wha man
™
, GE
Heal hca e, Munich, Ge many), blocked o 1 h in TBS (50 mM T is-HCl,
pH 7.5
,
150 mM
NaCl, 0.1% Tween 20) con aining 5% BSA and immunoblo ed wi h a a monoclonal
an i-FLAG an ibody (1:1000) (Ca . No:MA1–142; In i ogen, The mo Fishe Scien i ic,
Pie ce, CA, USA) o 16 h a 4
◦
C. Then, hey we e coun e s ained wi h a ho se adish
pe oxidase-conjuga ed goa an i- a an ibody (Ca . No: 7077S; Cell Signalling Technology
®
Inc., Dan e s, MA, USA). The signal was c ea ed by adding Supe Signal Wes Du a Ex-
ended Subs a e (The mo Fishe Scien i ic, Pie ce, CA, USA) and i was measu ed by he
ChemiDoc XRS chemiluminescence sys em (Bio-Rad, He cules, CA, USA) a 425 nm. The
ela i isa ion was pe o med using Glyce aldehide 3-phospha e dehyd ogenase (GAPDH)
as con ol.
4.6. Ac i i y o PCKS9 Va ian s by Flow Cy ome y
4.6.1. LDL Labelling wi h Fluo escein Iso hiocyana e (FITC)
LDL was pu i ied om blood plasma by cen i uga ion a 120,000
×
ga 4
◦
C o 19 h.
LDL (1.019–1.050 g/mL) was isola ed h ough isopycnic ul acen i uga ion by adjus ing
plasma densi y o 1.21 g/mL by he addi ion o KB . LDL pa icles we e labelled wi h FITC
as p e iously desc ibed [
23
]. B ie ly, 10
µ
L o FITC (2 mg/mL) we e added o 1 mL LDL
(1 mg/mL apoB) in 0.1 M NaHCO3, pH 9.0, and mixed o 2 h by slow ocking a oom
empe a u e. 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
using bo ine se um albumin as s anda d (Pie ce BCA p o ein assay; Pie ce, The mo Fishe
Scien i ic, Pie ce, CA, USA).
4.6.2. Flow Cy ome y
HEK293 cells we e ans ec ed wi h he ec o con aining PCSK9 a ian s as explained
p e iously, and hey we e incuba ed wi h 20
µ
g/mL o FITC-labelled LDL o 4 h p io o
he expe imen . A e he incuba ion, he in e nalised LDL was measu ed by luo escence-
ac i a ed cell so e (FACS) as desc ibed be o e [
23
]. Fluo escence was measu ed by
FACSCalibu
™
(BD Bioscience, San Jose, CA, USA). Each sample was iplica ed and
10,000 e en s we e measu ed in each case.
4.6.3. PCSK9 Quan i ica ion
Aliquo s om he cul u e mediums we e collec ed a 48 h and PCSK9 le els we e
de e mined by ELISA ollowing manu ac u e ins uc ions (Quan ikine
®
ELISA; R&D
Sys ems, McKinley Place, MI, USA, Ca . No: DPC900)