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)