BioMol Concep s 2016; 7(1): 1–15
*Co esponding au ho : Philipp Rohne, Ins i u e o Pha macy
and Biochemis y, The apeu ical Li e Sciences – Ins i u e o
Biochemis y, Johann-Joachim-Beche Weg 30, D-55128 Mainz,
Ge many, e-mail: philipp[email p o ec ed]
Hans P ochnow: Ins i u e o Pha macy and Biochemis y,
The apeu ical Li e Sciences – Ins i u e o Biochemis y,
Johann-Joachim-Beche Weg 30, D-55128 Mainz, Ge many; and
Depa men o Chemical Biology, Helmhol z Cen e o In ec ion
Resea ch GmbH, D- B aunschweig, Ge many
Claudia Koch-B and : Ins i u e o Pha macy and Biochemis y,
The apeu ical Li e Sciences – Ins i u e o Biochemis y,
Johann-Joachim-Beche Weg 30, D-55128 Mainz, Ge many
Re iew Open Access
Philipp Rohne*, Hans P ochnow and Claudia Koch-B and
The CLU- iles: disen anglemen o a mys e y
DOI 10.1515/bmc-2015-0026
Recei ed Oc obe 1, 2015; accep ed No embe 5, 2015
Abs ac : The mul i ace ed p o ein clus e in (CLU) has
been challenging esea che s o mo e han 35 yea s. The
cha ac e iza ion o CLU as a molecula chape one was
one o he majo b eak h oughs in CLU esea ch. Today,
sec e o y clus e in (sCLU), also known as apolipop o ein
J (apoJ), is conside ed one o he mos impo an ex a-
cellula chape ones e e ound. I is in ol ed in a b oad
ange o physiological and pa hophysiological unc ions,
whe e i exe s a cy op o ec i e ole. Desc ip ions o a i-
ous o ms o in acellula CLU ha e led o u he and e en
con adic o y unc ions. To un angle he cu en s a e o
knowledge o CLU, his e iew will combine old iews
in he ield, wi h new disco e ies o highligh he na u e
and unc ion o his ascina ing p o ein(s). In his e iew,
we u he desc ibe he exp ession and subcellula loca-
ion o a ious CLU o ms. Mo eo e , we discuss ecen
insigh s in o he s uc u e o CLU and assess how s uc-
u al p ope ies as well as he edox en i onmen de e -
mine he chape one ac i i y o CLU. E en ually, he e iew
connec s he biochemis y and molecula cell biology o
CLU wi h medical aspec s, o o mula e a hypo hesis o a
CLU unc ion in heal h and disease.
Keywo ds: Alzheime ’s disease; apolipop o ein J; clus-
e in; LDL- ecep o s; molecula chape ones.
In oduc ion: clus e in (CLU),
i s his o y and he challenge o
esea che s
In he yea 1979, a p o ein was i s disco e ed, whose
complexi y, abundance and in ol emen was ini ially
unknown (1). La e , his sul a ed glycop o ein was iden i-
ied by i ue o i s blood cell agg ega ing abili y in i o,
and was he e o e named clus e in (CLU) (2). Mos s ik-
ingly, i su aced in a b oad spec um o issues and is
o e exp essed in he ace o pa hological p ocesses, such
as a he oscle osis, cance and Alzheime ’s disease (3–6).
Mo eo e , CLU was ound in he bodily luids o almos
all e eb a es om zeb a ish o humans (7, 8). In acco d-
ance wi h hese indings, an ex ensi e epe oi e o names
eme ged anging om complemen lysis inhibi o (CLI)
and es os e one ep essed p os a e message-2 (TRPM-2)
o apolipop o ein J (apoJ). Ne e heless, in 1992 he name
CLU was he gene ally accep ed e m o all disco e ed
p o eins (9).
Thus a , CLU has been demons a ed o be a highly
glycosyla ed glycop o ein o 80 kDa, consis ing o wo
polypep ide chains connec ed by ou o i e disul ide
bonds (10). The p o ein is one o he mos p ominen
ex acellula chape ones. The chape one ac i i y o CLU
has been in ensi ely s udied by Ma k Wilson and his col-
leagues (11–14). In connec ion wi h i s chape one ac i i y,
CLU is desc ibed as a p o ein ha allows o he clea ing
o cellula deb is and mis olded p o eins, as well as he
clea ance o Aβ ia he blood-b ain ba ie (BBB) (15–17).
The conce ed ac ion o chape one ac i i y, sca enging-
and clea ance- unc ion, may be one basis o he cy o-
and issue p o ec i e ole o he p o ein (5, 15, 18, 19).
P e iously, i was u he shown ha he p o ein ac s as
a signaling molecule, inducing cellula p osu i al and
p oli e a o y pa hways, which may con ey ano he mech-
anism o i s cy op o ec i e unc ion (20–22).
This unde s anding o CLU (as p e iously desc ibed)
became complica ed when in acellula CLU o ms we e
desc ibed in damaged cells, in addi ion o he p edomi-
nan sec e ed o m, and a ibu ed o a di e se and e en
opposing ole (23–26). This e iew will summa ize he
©2015, Philipp Rohne e al., published by De G uy e .
This wo k is licensed unde he C ea i e Commons A ibu ion-NonComme cial-NoDe i a i es 3.0 License.
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2      P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y
biosyn hesis o CLU, i s s uc u al ea u es and chap-
e one ac i i y as well as ocus on aspec s o in acellu-
la CLU o ms o assess hei cellula and physiological
unc ion. Finally, he in ol emen o CLU in he modu-
la ion o a ious signal ansduc ion pa hways, i s ole
in he immune sys em and i s in ol emen unde pa ho-
logical condi ions, such as ischemia and nec osis will be
discussed. The goal is o summa ize cu en iews abou
CLU o enable u u e s udies o inally sol e he ‘CLU
mys e y’.
Biogenesis o clus e in (CLU)
The biosyn hesis o human sec e o y CLU (sCLU) ollows
he canonical pa hway o sec e o y p o eins (Figu e 1).
T ansla ion o CLU s a s om a s a codon loca ed on
exon 2 o he CLU-mRNA, esul ing in a p e-p op o ein
composed o 449 amino acids. The i s 22 amino acids
ep esen a signal sequence ensu ing co- ansla ional
ansloca ion in o he endoplasmic e iculum (ER). Subse-
quen ly, he signal sequence is clea ed o and ou o i e
disul ide bonds a e o med (10). He ea e , N-glycosyla ion
a six Asn- esidues (Asn86, 103, 145, 291, 354, 374) akes place
con e ing he p op o ein o a high-mannose ER-p ecu so
(p e-sec e o y CLU, psCLU) o 60kDa (27, 28). A e ans-
loca ion o he golgi-appa a us, complex ca bohyd a e
moie ies a e a ached o he ma u a ing psCLU, composed
o galac ose, ucose, mannose, N-ace ylglucosamine and
N-ace ylneu aminic acid (29–31). The esul ing 80 kDa
p o ein is u he clea ed by a u in-like p op o ein con-
e ase (FC) (amino acid ecogni ion mo i : RIVR) be ween
A g227 and Se 228 o p oduce an N- e minal α-chain and a
C- e minal β-chain which a e in e linked by disul ide
bonds (29–31). Finally, ma u e sCLU is sec e ed as a he -
e odime ic complex o wo 40-45kDa subuni s (28, 29).
Unde cellula s ess addi ional CLU o ms eme ge by
di e sion om he canonical sec e o y pa hway (Figu e1).
They encompass co e-glycosyla ed o ms, p esumably
de i ed om e o ansloca ed CLU ou o he ER (32, 33)
o in acellula o ms which ailed o be seg ega ed in o
he ER and hus a e no ca ying any suga esidues o
disul ide bonds, espec i ely (34). In addi ion, in acel-
lula o ms may a ise om al e na i e splicing e en s o
om non-canonical/al e na i e ansla ion-ini ia ion s a
si es downs eam o he ER-leade pep ide (Figu e 1) (34).
All men ioned in acellula CLU o ms a e single-chain
p o eins, since hey a e no p o eoly ically p ocessed.
FC clea age si e
Al e na i e Exon 1
(P ochnow e al. 34)
Exon 2–9
ER-Leade
Diso de ed/mol en
globule-like egion
O de ed egion
Disul ide bonds
Amphipa hic helices
O he in amolecula
in e ac ions
Ca bohyd a es
Sec e ion
Co e-glycosyla ed
CLU o m
Co ansla ional
ansloca ion
Disul ide
bonds
Complex
glycosyla ion
FC-
clea age
sCLU
High mannose
co e-glycosyla ion
Endoplasmic
Re iculum
psCLU
Cy osolic
CLU o ms
R
e
o
a
n
s
l
o
c
a
i
o
n
Figu e 1: Illus a ion o CLU biosyn hesis.
The canonical pa hway o sec e o y p o eins leads o he syn hesis o sec e o y CLU (sCLU). I unde goes p o eoly ic ma u a ion and ca ies
complex ca bohyd a e moie ies as well as disul ide bonds. Upon cellula s ess, non-canonic CLU o ms eme ge, mainly de i ed om ailed
ansloca ion, al e na i e splicing o ansla ion-ini ia ion e en s on exon 3, as well as om e o ansloca ion. All non-sec e ed CLU o ms
a e incomple ely ma u a ed. Fo de ails please see ex .
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P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y      3
S uc u e and unc ion o sec e o y
clus e in (sCLU)
S uc u e
So a , one o he mos challenging ques ions pe ains o
he s uc u e o sCLU. O e he pas h ee decades nume -
ous s udies ha e been conduc ed, bu he s uc u al p op-
e ies o sCLU a e s ill no ully unde s ood. One eason
o his lack o knowledge lies in he agg ega ing na u e
o sCLU. Besides in e ac ing wi h a ple ho a o ligands, i
agg ega es wi h i sel , o ming di-, e a- and e en highe
oligome s, depending on he pH- alue (2, 12–14, 35). In
combina ion wi h dis inc ligands, such as ib inogen,
sCLU can o m high molecula weigh complexes wi h a
pu a i e molecula mass up o 40 000kDa and a diam-
e e anging om 50 o 100nm (14). These cha ac e is-
ics oge he wi h a he e ogeneous glycosyla ion pa e n,
ende s i di icul o ob ain X- ay s uc u es o eliable
NMR spec a om pu i ied sCLU-samples (13, 36). Addi-
ionally, i is ha d o ob ain sui able p o ein samples in
su icien quan i ies (30, 36). Ne e heless, a numbe o
s udies ha e been conduc ed, which ocus on dis inc
p ope ies o sCLU and shed ligh on i s s uc u al ele-
men s (10, 13, 37–39). Beyond doub , he p ima y s uc u e
o sCLU is highly conse ed be ween di e en species,
wi h he highes homologies ound in he egions o
disul ide bonding cys eins and he FC clea age si e poin -
ing o hei signi icance ega ding sCLU- unc ion (8, 40).
Al eady a e he i s success ul isola ion o sCLU, i s
amphipa hic cha ac e was appa en (2). La e on, sec-
onda y s uc u al elemen s we e in es iga ed by means
o ci cula dich oism (CD)- and in a ed-spec oscopy (14,
30, 35, 38, 41, 42). In all o hese a p edominan α-helical
con en o up o 60% was calcula ed. In silico analyses
u he p edic ed i e amphipa hic α-helices (37). On he
e ia y s uc u e le el, sCLU is belie ed o belong o he
amily o in insically diso de ed p o eins, meaning ha
i pa ially lacks a de ined e ia y s uc u e, hus expos-
ing hyd ophobic egions, so-called mol en globule-like
domains, owa ds he ex e nal space (43). This in u n
allows o binding o o he molecules ia hyd ophobic
in e ac ions (Figu e 2A). Rema kably, sCLU sha es his
FC clea age si e
Disul ide bonds
Clien p o ein O he in amolecula
in e ac ions
Amphipa hic helices
O de ed egions
100
Clea ed sCLU
AC
B
Unclea ed sCLU
80
60
40
20
Rela i e chape oneac i i y (%)
Non-
ed.
C l Time a e
DTT emo al C l Time a e
DTT emo al
ed. 0 h 48 h Non-
ed. ed. 0 h 48 h
Di hio h ei ol
Ca bohyd a es
Diso de ed/mol en
globule-like egions
Figu e 2: Schema ic depic ion o sCLU chape one ac i i y and hypo he ical in luence o s uc u al elemen s.
Fully ma u a ed sCLU is s abilized by disul ide bonds and in amolecula in e ac ions, such as hyd ophobic in e ac ions in he egion o
amphipa hic helices (A). In con as , a i icial unclea ed sCLU elies on i s disul ide bonds and he e o e displays a high sensi i i y owa ds
educing condi ions (B). P elimina y da a show ha unclea ed sCLU pa ly egains i s chape one ac i i y a e emo al o DTT wi hin 48h
(C). The p ocedu e o sCLU pu i ica ion, DTT ea men and chape one ac i i y assay a e in acco dance wi h Rohne e al. (30). A e 12h o
incuba ion wi h o wi hou 40 mm DTT a 37°C upon gen le agi a ion, sCLU was subsequen ly used o chape one ac i i y assays (C l educ-
ing and non- educing) o DTT was subs i u ed by PBS using a Vi aspin 4 5000 MWCO PES (Sa o ius) a 4°C and used o chape one ac i i y
assays subsequen ly o a e 48h a 4°C. All chape one ac i i y assays we e pe o med in he p esence o 5 mm DTT wi h Ca alase being
he clien p o ein. Addi ionally, BSA was mixed wi h Ca alase and se ed as a nega i e con ol o calcula e he ela i e chape one ac i i y
o sCLU as desc ibed by Rohne e al. (30). Fo e alua ion, he da a o wo o h ee independen expe imen s, each wi h he mean o wo o
h ee measu emen s we e used (unpai ed - es *p < 0.05). The e o ba s co espond o he mean±s anda d e o s.
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4      P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y
ea u e wi h o he in insically diso de ed p o eins, mos
p ominen ly hea shock p o ein (Hsp) 27 o α-c ys alline
(11, 44). As poin ed ou in he ollowing, sCLU indeed pos-
sesses a chape one ac i i y simila o ha o Hsp27 and
o he hea shock p o eins.
Chape one and sca enging unc ion
Chape ones a e pa o he basic molecula de ense mech-
anism o a cell o e coming s ess si ua ions induced by
UV ligh , ionic i adia ion, hea , oxidan s, hea y me als,
hype oxia o ce ain d ugs. They can be di ided in o di e -
en classes: chape onins, Hsp100, Hsp90, Hsp70, Hsp60
and small Hsps (sHsps) (45). These in acellula chap-
e ones egula e he co ec olding o ma u ing p o eins
o p e en he agg ega ion o dena u ed p o eins hus
adop ing a cy op o ec i e unc ion. Some e en ini ia e
he e olding o mis olded p o eins in an ATP-dependen
manne (Hsp70, Hsp90, chape onins). Fo chape ones,
na i ely diso de ed egions a e equi ed o binding wi h
a ple ho a o s uc u ally di e se clien p o eins (46).
Simila o he abo e men ioned Hsps, CLU-mRNA
is up- egula ed upon cellula s ess due o a hea shock
elemen -like mo i p esen in he CLU p omo o (19, 34,
47, 48). The chape one unc ion o sCLU was e ealed
20yea s a e he p o ein’s disco e y (11). Thus i became
he i s molecula chape one known o ac ou side
o he li ing cell. Simila o sHsps inside he cell, i is
assumed ha sCLU binds o clien p o eins h ough i s
mol en globule-like egions ia hyd ophobic in e ac-
ions (13, 14, 37, 49). In pa icula , p o eins ha a e on
he o - olding pa hway, e.g. as induced by hea o educ-
ing condi ions, se e as clien s o sCLU (12, 30). The
binding occu s in an ATP-independen manne , leading
o o ma ion o soluble high molecula weigh complexes
wi h di e en mola chape one-clien a io be ween 1:5
and 1:0.33 (14, 50, 51). As a consequence, sCLU p e en s
he uncon olled agg ega ion o he dena u ing p o eins.
In e es ingly, binding o sCLU does no e ain po en-
ial enzyma ic ac i i ies o dena u ing clien p o eins.
Howe e , in coope a ion wi h ATP-dependen Hsps, such
as Hsc70, he enzyma ic ac i i y o some p o eins can be
es o ed (11, 50).
In cell cul u e expe imen s i was obse ed, ha
sCLU is able o acili a e he up ake o he bound clien
complexes in o su ounding issue cells o allow hei
emo al ia lysosomal diges ion (Figu e 3) (15, 52). Recep-
o s esponsible o he binding o sCLU and/o sCLU-p o-
ein complexes, such as megalin (15, 42, 53), LRP1 (15) and
ApoER2, VLDLR (22, 54) a e membe s o he LDL- ecep o
gene amily. In a ecen s udy, sCLU-clien complexes
we e in a enously injec ed in o mice, con i ming he
sCLU-chape one/sca enging ac i i y (17). sCLU-ligand
complexes we e ound o be en iched in he mice li e
and kidneys, which a e in ol ed in deg ada ion and sub-
sequen exc e ion o cellula oxins. The inding ha he
en ichmen in he li e can be blocked by adminis a-
ion o ucoidan (a po en inhibi o o sca enge ecep o
class A, E and F) indica es ha sCLU is able o in e ac
wi h sca enge ecep o s beside hose o he LDL- ecep-
o gene amily. In addi ion, a dec ease in he emo al o
glome ula p o ein deposi s was ound in CLU-K.O. mice
(55). In humans, educed sCLU sec e ion is accompanied
by a highe isk o Alzheime ’s disease (56). These ind-
ings a e in line wi h obse a ions ha show ha sCLU
p e en s he agg ega ion and oligome iza ion o Aβ and
ans hy e in (57, 58). Conclusi ely, hese da a a gue o
he p onounced ole o sCLU in p o ein homeos asis in
he body.
Disul ide bonds & p o eoly ic ma u a ion:
C ucial componen s o dispensable ea u es?
One o sCLU’s p ominen ea u es is i s ma u a ion in o an
α- and a β-chain occu ing wi hin he golgi appa a us o
e eb a e cells. The wo subuni s a e connec ed by 4–5
highly symme ical disul ide bonds. As sugges ed by Bon-
Hong Min’s g oup, he o ma ion o disul ide bonds is a
p e equisi e o sCLU ma u a ion/syn hesis (59).
In e es ingly, a educ ion o hese disul ide bonds
in ma u e sCLU does no inhibi i s ac i i y in chape -
one ac i i y assays (11, 12, 50). This ole ance o sCLU o
educing condi ions aises he ques ion whe he he α-
and β-chain o sCLU ac independen ly as chape ones o
whe he he disul ide bonds a e ex aneous o hei in a-
molecula associa ion. I mus be no ed ha he p ope
ma u a ion o sCLU is dependen on app op ia e disul ide
bond o ma ion. T ea men o cells wi h di hio h ei ol
(DTT), which p e en s disul ide bond o ma ion in he ER,
abolishes he sec e ion o sCLU (60). Mo e ecen ly i was
demons a ed ha mu a ions in he cys- ich egion o he
CLU p o ein a e leading o educed sec e ion o sCLU in
pa ien s wi h Alzheime ’s disease (56).
Addi ionally, i was shown ha he inhibi ion o
he p o eoly ic ma u a ion by in i o mu agenesis o
he u in-like p op o ein con e ase (FC) clea age si e,
which gene a es unclea ed sCLU, does no in e e e wi h
sCLU-ma u a ion and i s chape one ac i i y bu a he
ende s i highly sensi i e o educing condi ions (30).
The lack o p o eoly ic ma u a ion may he e o e cause
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P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y      5
an impai ed ‘ lexibili y’o sCLU (Figu e 2B). S udies o
Bailey e al. e ealed ha he N- and he C- e minus o
sCLU a e egions wi h diso de ed/mol en globule-like
domains which a e likely in ol ed in clien binding (37).
The e o e, he only egions le o s abilizing he sCLU
p o ein a e p obably he disul ide bonds and he pu a i e
amphipa hic helices nex o he FC clea age si e. As men-
ioned abo e, ma u e sCLU is s ill su icien ly ac i e e en
a e long- e m educ ion (11, 12, 30, 50). Howe e , he
ac i i y o unclea ed sCLU a e long- e m educ ion is
emendously impai ed (Figu e 2) (30). Thus, he amphi-
pa hic domains o o he neighbo ing egions may be
esponsible o sCLU p o ein s abiliza ion. In iguingly,
p elimina y da a om ou lab indica e ha unclea ed
educed sCLU pa ly egains i s chape one ac i i y when
DTT is emo ed (Figu e 2C). In conclusion, we p opose
Spe m
p o eins
Cellula p o eins
dsRNA
P dSe
P dSe
ecep o
Endocy osis
Endosome
Ac i a ion o
anc ip ion ac o s
Dab1/AKT/Co ilin
VLDLR/
ApoER2
ECM emodelling/mig a ion
Pseudomonas
ae uginosa
Su i al o
ca diomyocy es
P oli e a ion o
neu onal
p ecu so s
Modula ion o
mi ochond ial
in eg i y
Rep esen a i e cell
Lysosome
Endoplasmic
Re iculum
Au ophagosome
o ma ion
AKT/GSK3β
CLU-media ed
complexa ion
LDL- ecep o s
Nec o ic cell
Spe m cell
Complemen inhibi ion
MAC
Immune
modula ion
B ain
Blood
Figu e 3: Role o CLU in cell biology, pa hology, immunobiology and beyond.
Fo desc ip ion please see ex .
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6      P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y
ha he disul ide bonds a e on one hand impo an o
co ec olding and ma u a ion bu on he o he hand a e
no essen ial o he chape one unc ion o ma u e sCLU.
Howe e , we canno exclude he possibili y, ha hey
a e in ol ed in o he p ocesses. The la e is suppo ed
by he no ion ha sCLU is able o es o e he unc ion
o glu amine syn he ase by ac ing as a hiol speci ic-
an ioxidan . Howe e , his ac i i y is impai ed when he
disul ide bonds o sCLU a e alkyla ed o when he elec-
on dono is no hiol-based (59).
Ca bohyd a es: negligible o essen ial?
Ano he p ominen ea u e o sCLU is i s high le el o
glycosyla ion esul ing in ca bohyd a es, which com-
p ise abou 30% o he molecula weigh (61). The gly-
cosyla ion pa e n o human se um sCLU was elucida ed
in 1997. Six N-linked ca bohyd a e a achmen si es a e
ound wi hin he molecule, wi h h ee on he α- and h ee
on he β-chain in humans (29). The ca bohyd a e compo-
si ion is highly di e se and depends on he exp essing
issue (4, 31). Se um sCLU mainly con ains ca bohy-
d a e moie ies wi h sialic acid in a mono- and bian en-
na y ashion (29, 31). Simila glycosyla ion pa e ns we e
ound wi h ecombinan human sCLU (30). In con as ,
semen sCLU ca ies mainly ucose- ich glycans, enabling
semen sCLU o bind o he C- ype lec in ecep o DC-SIGN
ound on dend i ic cells. This binding was no obse ed
wi h se um sCLU (31, 62). The e o e, he glycan moie-
ies ei he change he spa ial s uc u e o sCLU so ha
i s ecep o a ini y is al e ed, o he ecep o s and o he
in e ac ing molecules ha e a ying a ini ies o dis inc
glycan moie ies.
In e es ingly, i was obse ed ha he glycosyla ion
o sCLU is c ucial o co ec pola sec e ion in epi he-
lial cells (28), bu no o chape one unc ion (41). sCLU
ea ed wi h he endoglycosidase PNGase F only showed
li le dec ease in chape one ac i i y (41). Indeed, i was
ecen ly shown ha he emo al o he e minal suga s by
exoglycosidases does nei he al e he chape one ac i i y
no he seconda y s uc u e o sCLU. Howe e , i became
ob ious ha plain PNGase F-diges ion esul s in esidual
suga s s ill being a ached o he p o ein-co e. Fully degly-
cosyla ed ecombinan human sCLU was ob ained by
applica ion o a mo e elabo a ed p o ocol and was shown
o possess a emendously dec eased chape one ac i i y.
CD-spec oscopy u he e ealed ha upon ull degly-
cosyla ion he amoun o α-helices lowe ed om 60% o
40%, whe eas he amoun o diso de ed egions inc eased
signi ican ly om abou 20% up o 30%. A e emo al
o all ca bohyd a es he ea angemen o he seconda y
s uc u e indica es ha he olding o sCLU is co ela ed
wi h i s glycosyla ion (30). In summa y, he co e-glyco-
syla ion is a p e equisi e o he olding and chape one
ac i i y o sCLU and e minal suga s migh be ins umen-
al o o he mechanisms, such as ecep o binding and
signal ansduc ion (21, 31, 62).
In acellula clus e in: signi icance
s. occu ence
Nuclea CLU (nCLU)
Since i s disco e y in he ea ly 1980s un il he mid-1990s,
CLU was ega ded solely as a sec e ed p o ein (sCLU). I
was no un il 1995 ha esea che s obse ed an associa-
ion o CLU wi h he nucleus a e induc ion o cell dea h
wi h an i-es ogens (26) o TGF-β (63). They specula ed
ha i ansla ion would s a a a s a codon loca ed on
exon 3 o he CLU-mRNA, an o he wise c yp ic nuclea
localiza ion sequence (NLS) could be ac i e. Thus, Reddy
e al. pos ula ed in e nal ansla ion ini ia ion as one pos-
sible mechanism leading o N- e minally unca ed o ms
o CLU. These unca ed o ms a e lacking he signal
sequence o seg ega ion in o he ER, exposing an NLS
and he e o e localizing CLU in he nuclea compa men
(Figu e 1). This hypo hesis, howe e , is s ill lacking sup-
po ing da a.
A ound he u n o he millennium, he g oup o Da id
Boo hman epo ed he exp ession o a nuclea o m o
CLU (nCLU) in MCF-7 cells a e ea men wi h ionizing
adia ion. They p oposed ha his nCLU o m would ac
as a p o-dea h ac o by in e ac ing wi h he DNA- epai -
associa ed p o ein Ku70. While i was ini ially assumed
ha nCLU de i es om an ER-bo ne psCLU o m (64), he
au ho s s a ed in la e publica ions ha ionizing adia-
ion a o s in e nal ansla ion ini ia ion a a s a codon
on exon 3, esul ing in he exp ession o nCLU (Figu e 1)
(24, 65). Finally, in 2003, he same g oup epo ed ha
an exon-skipping e en , esul ing in a ansc ip lacking
exon 2 wi h i s signal sequence coding egion (he ea e
e med a ian 1 [Δex2]), p ecedes he exp ession o nCLU
(25). Su p isingly, o e exp ession-expe imen s wi h
a ian 1 [Δex2] esul ed in s ong cy osolic localiza ion
o he co esponding p o ein as de e mined by con ocal
mic oscopy. Nuclea associa ions, howe e , could only
be de ec ed by o e exp ession o a i icial cons uc s
lacking dis inc po ions o a ian 1 [Δex2]. Despi e his
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P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y      7
con o e sial obse a ion, he au ho s hypo hesized ha
he p o ein ansla ed om he exon 2-lacking mRNA is
he p e-nuclea o m o nCLU. I would hen be ans-
o med in o ma u e nCLU o unknown pos - ansla ional
modi ica ions induced by ionizing adia ion. Howe e ,
expe imen al p oo o his hypo hesis is s ill missing.
Th oughou he i s decade o he 2nd millennium
many esea ch g oups claimed a nuclea localiza ion o
CLU in s essed cells, including cellula s ess induced by
a ious agen s such as E oposide (66–68), In e leukin-6,
Soma os a in (69), hea (70), Ca2+-deple ion (67, 71),
se um-s a a ion (72), TNF-α- ea men (73), indocyanine-
ea men wi h g een ea ex ac s (74), p o easome
inhibi ion (75), ea men wi h 5′-Fluo ou acil and Fas
ecep o -binding an ibodies (76) o anadium- ea men
(77). Fu he mo e, nuclea localiza ion o CLU has been
obse ed upon o e exp ession o c- os (77), In e leukin-24
(78) o pVHL (79), as well as spon aneously in un ea ed
cells (69, 76, 80–82). Addi ional o he mechanisms dis-
cussed abo e, i was a gued ha inhibi ion o sCLU
sec e ion could lead o in acellula accumula ion and
subsequen ly nuclea localiza ion (83). Howe e , dimini-
shed sec e ion and he e o e in acellula accumula ion
o CLU, as desc ibed by Be ens e al. (56), did no p o ide
any e idence o nuclea localiza ion o CLU.
Ano he poin o in ense deba e is he po en ial
exon 2-skipping o CLU-mRNA (Figu e 1). The esul ing
a ian 1 [Δex2], was commonly conside ed o explain
he occu ence o nCLU in many s udies, al hough igo -
ous mRNA-analyses ha e no been pe o med. This led
o con o e sies, as some g oups could no alida e he
exis ence o a ian 1 [Δex2] (84–86). I was specula ed
ha i s exp ession migh be limi ed o MCF-7 cells o e en
an expe imen al a i ac (75, 84–87). Howe e , P ochnow
e al. con i med he exis ence o a ian 1 [Δex2] in se e al
human cell lines using alida ed, a ian -speci ic p ime
se s o RT-PCR (34). Ye , mRNA-quan i ica ion indica ed
ha a ian 1 [Δex2] accoun s o  < 0.13% o o al CLU-
mRNA, e en in s essed cells. The exon 2-con aining
a ian 1, in con as , ep esen s he p e-dominan CLU-
mRNA ( > 99% o o al CLU-mRNA). In his s udy i was also
shown ha he p o ein encoded by a ian 1 [Δex2] local-
izes solely in he cy oplasm o uns essed and s essed
cells. In ac , e y ecen s udies suppo his obse a-
ion, challenging he heo y o a nuclea localiza ion o
CLU (32, 79, 80, 88–91). Only by inco po a ing an a i i-
cial nuclea localiza ion sequence (NLS) a he 5′-end o
a ian 1 [Δex2], a nuclea localiza ion o he ansla ed
p o ein could be achie ed (92). This in u n ende s he
exis ence o a unc ional NLS hidden in he CLU p e-
p op o ein unlikely.
CLU associa ed wi h mi ochond ia
O e he cou se o he las ew yea s, an associa ion o
CLU wi h mi ochond ia has equen ly been epo ed
and in e ac ions wi h in insic apop ose- ela ed p o eins
Bax and Bcl-xL ha e been discussed (32, 93–96). Thus, i
was specula ed, ha ei he psCLU o sCLU migh ac an i-
apop o ic by seques e ing Bax in i s inac i e s a e in he
cy osol (95, 96). Howe e , in none o hese s udies i was
in es iga ed, how ex acellula sCLU o ER/golgi- esiden
psCLU can each he cy osol in o de o bind Bax (Figu e1).
Li e al. (32) ecen ly showed ha a hypoglycosyla ed o m
o sCLU can escape he sec e o y pa hway wi h he aid o
he chape one GRP78 (BiP) and s abilize he mi ochon-
d ial memb ane o a oid pacli axel-media ed apop osis
(Figu es 1 and 3). An in e ac ion wi h Bax was, howe e ,
no in es iga ed in his s udy. O he epo s sugges ha
CLU can ac p o-apop o ic by p e en ing Bcl-xL om
binding o Bax (88, 97). The au ho s assumed ha non-
sec e ed CLU o ms simila o nCLU, a e esponsible o
his e ec . This was a ibu ed o a po en ial BH3-domain
ound in silico wi hin he nCLU-sequence (97). Howe e ,
upon o e exp ession o dis inc in acellula CLU o ms,
no p o-apop o ic p ope ies could be assigned o any case
(34). Thus, he signi icance o CLU associa ion wi h mi o-
chond ia s ill needs o be challenged.
How signi ican a e dis inc in acellula CLU
o ms?
The di e se esul s and p oposals pu o wa d in he
s udies on in acellula CLU o e he pas 20 yea s a e
inconclusi e and e en con adic o y. One eason o his
may be ha he desc ibed e ec s a e es ic ed o s essed/
damaged cells and ha he abundance o in acellula
CLU o ms is negligible (34). Mos impo an ly, o ou
knowledge, no dis inc in acellula CLU o ms ha e been
desc ibed in o he o ganisms besides humans, ye . Addi-
ionally, CLU o ms lacking ca bohyd a es and/o p o eo-
ly ic ma u a ion a e no capable o main ain a chape one
ac i i y in he cell (30). Fu he mo e, P ochnow e  al.
ound no e idence o an apop osis-modula ing e ec o
unglycosyla ed cy osolic CLU o ms. The e o e, we sugges
ha in acellula CLU o ms may acciden ly e ol e in com-
p omised cells and migh ha e no bene icial unc ion o
he cell. One excep ion may be hypoglycosyla ed psCLU
o ms e o ansloca ed om ER/golgi. These o ms ha e
been desc ibed in he cou se o modula ing mi ochond ia
in eg i y o au ophagy o p omo e cell su i al (Figu e 3)
(32, 91).
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8      P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y
Cellula challenges: om nec osis
o immunobiology and beyond
sCLU and nec osis
The exp ession o sCLU is highly co ela ed o issue
degene a ion, nec osis and edox imbalances (98,
99). The s ess-associa ed up egula ion in esponse o
UV ligh , hea -shock, oxidan s, hea y me als and in
pa icula p o eo oxic s ess is media ed by he clu p o-
mo o which con ains HSF1, NFκB and AP-1 binding si es
(100). Cy o oxic s ess can also be induced by nec osis
in neighbo ing cells (Figu e 3) (101). As a consequence,
mis olded p o eins, ee adicals, lipids and o he com-
ponen s accumula e in he ex acellula space causing
an ac i a ion o he immune sys em and a ec neighbo -
ing cells (101, 102). Rema kably, i was obse ed ha CLU
up egula ion also occu s in cul u ed cells upon exposu e
o nec o ic cell deb is (103). This up egula ion is d i en
by componen s o he nec o ic cells, such as memb anous
phospha idylse ine (P dSe ) (103) o by RNA eleased
om he cy osol (Figu e 3) (104). P dSe , unde i al con-
di ions, is es ic ed o he cy osolic side o he plasma
memb ane and is exposed o he ex acellula en i on-
men upon damage o apop osis (105). Ex acellula RNA
is endocy osed by su ounding cells and up egula es
CLU ia he TLR3-signaling pa hway (104). Once sCLU
is sec e ed in o he ex acellula space, i media es he
binding and endocy ic up ake o cellula deb is by ecep-
o s o he LDL- ecep o gene amily, such as megalin o
LRP1 hus ees ablishing he ex acellula p o eos asis
(Figu e 3) (15, 30).
sCLU and immunobiology
Qui e ecen ly, da a sugges ha semen sCLU can bind
s ess-damaged p o eins, acili a ing he up ake by den-
d i ic cells ia DC-SIGN hus making a p iming o immune
cells easible (Figu e 3) (31, 62). As a u he consequence,
sCLU has been a ibu ed o media e ole ance o he
emale geni al ac owa ds male an igens (Figu e 3) (62).
This sugges s an immune egula o y ole o sCLU, which
is e iden om mul iple in i o and in i o s udies. Ini-
ially, sCLU was ound o be co-localized wi h complemen
SC5b-9/MAC (memb ane a ack complex) in glome ulo-
neph i i is bu no in heal hy indi iduals (106). Subse-
quen ly, in associa ion wi h he SC5b-9 complex sCLU was
cha ac e ized as an inhibi o o he complemen sys em
(Figu e 3) (38, 49, 107). The complemen sys em causes cell
damage unde enal pa hologies. Thus, sCLU may alle ia e
enal inju ies by p e en ing an uncon olled MAC ac i i y
(108). Howe e , in 1999 Hochg ebe e al. (109) showed ha
complemen inhibi ion by sCLU is no possible a physi-
ological concen a ions. Despi e hese ac s, he mos
ecen da a sugges ha Pseudomonas ae uginosa can
p o ec i sel om complemen a ack by ec ui ing sCLU
(110). Simila sugges ions we e made o spe m cells in he
emale geni al ac (Figu e 3) (49). Thus, an inc ease in
he local sCLU concen a ion may achie e a complemen
inhibi ing e ec .
Addi ionally, o he da a suppo a ole o sCLU in
immune modula ion: I was shown ha sCLU syne gize
wi h IL-2 o enhance he p oli e a ion o na u al kille cells
(111). Mo eo e , sCLU is desc ibed as a ansc ip ional
a ge o he lympho oxin-β ecep o in ge minal cen e s
du ing an immune esponse (112, 113). I was u he dem-
ons a ed ha sCLU imp o ed he iabili y o B lympho-
cy es (113). In addi ion, i is impo an o no e ha CLU
was desc ibed as a necessa y ac o o cu a ion o au oim-
mune myoca di is, which is accompanied by an inc eased
le el o nec osis and in lamma ion (102, 114). These con-
clusions we e u he suppo ed by obse a ions a e
in a enously adminis a ion o sCLU in a s du ing myo-
ca dial in a c ion. A e ea men wi h sCLU, he in a c
size was signi ican ly educed and led o mac ophage
in asion (115). This inding is suppo ed by an ele a ed
chemo ac ic mig a ion o mac ophages due o inc eased
ma ix me allop o einase-9 (MMP9) exp ession induced
by sCLU ia he ERK1/2-, PI3K/AKT- and NFκB-signaling
pa hway (Figu e 3) (21, 116). The abili y o sCLU o modu-
la e mac ophage unc ion was u he con i med by an
addi ional s udy which demons a ed ha he mig a o y
e ec is ela ed o TNF-α sec e ion induced by sCLU (116).
Howe e , an Dijk e al. (20) ha e ound no e idence o
a di e ence in wound healing a e sCLU applica ion.
Ra he , sCLU p o ec s ca diomyocy es om apop osis by
inducing he PI3K/AKT-signaling pa hway (Figu e 3).
I is impo an o no e ha nei he he p o ec i e
e ec o sCLU on ca diomyocy es, he educ ion o in a c
size no he in e ac ion wi h dend i ic cells a e ela ed o
ecep o s o he LDL- ecep o gene amily (20, 62, 115). I
is he e o e impo an o emphasize ha he cha ac e iza-
ion o o he sCLU binding ecep o s is essen ial o he
un a eling o sCLU unc ion.
sCLU and issue epai
The signi icance o sCLU in issue p o ec ion/ emodeling
was ound o be impo an because o i s po en ial o
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P. Rohne e al.: The CLU- iles: disen anglemen o a mys e y      9
p e en o gans om damage unde ischemic condi ions,
e.g. ischemia- epe usion inju y (IRI) a e ansplan-
a ion (117). The ischemia- epe usion damage occu s
when he bloods eam is ees ablished a e pe iods o
limi ed nu ien and oxygen supply. The consequence
o his mechanism leads o ini ial nec osis, ollowed
by massi e apop osis o he su ounding issue (102).
The su ounding apop osis is belie ed o be due o he
elease o ee adicals, gene a ing lipid adicals in he
cellula memb ane and he e o e causing emendous
cellula damage (Figu e 3) (101). In ac , sCLU is up eg-
ula ed a e oxida i e s ess caused by H2O2 o ciga e e
smoke, p o ec ing he cells and main aining hei iabil-
i y (19, 20, 59, 118). In he case o ansplan medicine, i
was shown ha supplemen sCLU in Uni e si y o Wis-
consin-solu ion (UW-solu ion) is easible o p olong he
handling- ime be o e an o gan can be ansplan ed (119).
Simila ly o his inding, i was obse ed ha a gene ic
inac i a ion o he CLU gene leads o an inc eased o gan
ejec ion as well as impai ed cell iabili y. These p o ec-
i e ac ions a e p obably due o inc eased memb ane lu-
idi y in he p esence o sCLU, minimizing cold-induced
s i ening and shea s ess (117). Fu he mo e, sCLU is a
candida e bioma ke o damage in he p oximal ubule
o he kidney (120). Addi ionally, i was shown ha
o e ime, he absence o sCLU leads o an inc eased
accumula ion o componen s om he immune sys em
wi hin he glome uli (55). Se e al in i o s udies sugges
a cy op o ec i e unc ion o sCLU a e gen amicin- o
H2O2-induced cy o oxici y in kidney cells (121, 122).
When inducing IRI by clamping he enal pedicles o
mice, sCLU is an essen ial ac o o enal egene a ion.
In con as o mac ophages, howe e , sCLU has no p o-
mig a o y e ec on enal ubula epi helial cells (TECs)
bu p omo es p oli e a ion and cell iabili y o TECs. In
con as o wild ype mice, CLU K.O.-animals exhibi a
ema kably dec eased su i al a e o abou 90% o 30%,
espec i ely (123). P e iously i was shown ha a e uni-
la e al u e e al obs uc ion (UUO) he p esence o sCLU
was a p e equisi e o supp ess TGF-β media ed up egu-
la ion o plasminogen ac i a o inhibi o -1 (PAI-1). PAI-1
p e en s ea angemen o he ex acellula ma ix and
hus a enua es plasmin ac i a ion leading o ib osis
(124). Howe e , in con as o Nguan e al. (123) no in lu-
ence o sCLU on p oli e a ion o apop osis on ubula
epi helial cells was obse ed. Finally, i is impo an o
men ion ha al hough Gi on e al. (121) ha e excluded
an in ol emen o megalin, he pa icipa ing ecep o s
media ing he cy op o ec i e e ec o sCLU (di ec ly o
indi ec ly) a e s ill unknown.
sCLU and he ne ous sys em
Al hough i is well es ablished ha sCLU is cy op o ec-
i e in he kidney and in he hea , he ole o sCLU in
he ne ous sys em is s ill challenging. As men ioned ini-
ially, sCLU was also desc ibed as an apolipop o ein (apoJ)
which is p esen in se um ( ee and bound o high densi y
lipop o ein (HDL) pa icles). sCLU(apoJ)-con aining HDLs
accoun o app oxima ely 2% o plasma HDLs (125) wi h
abou 20% o he ci cula ing sCLU being ac ually bound o
HDLs (126). In con as o he b ain, sCLU(apoJ)-con ain-
ing HDL pa icles in plasma a e mo e lipid- ich (38, 127).
In he mammalian b ain sCLU (apoJ) and apoE a e he wo
mos abundan apolipop o eins [ e iewed by Wang and
Eckel (128)].
In iguingly, bo h apolipop o eins a e in ol ed in
simila mechanisms, such as Alzheime ’s disease o cog-
ni i e and memo y p ocesses (18, 128–130). I is impo an
o no e ha e en hough sCLU is a cons i uen o HDLs,
which a e he only lipop o ein subclass c ossing he BBB,
only LRP1 and SR-B1 a e in ol ed in lipid homeos asis o
he b ain (128). The emaining lipop o ein ecep o s a e
in ol ed in a ple ho a o o he unc ions (128, 129).
Inside he b ain, CLU is p ima ily exp essed and
sec e ed by as ocy es bu also by o he ypes o cells, e.g.
neu ons (127, 131, 132). Upon neu onal dea h, ischemia
and wi h ad ancing age, howe e , a signi ican up egula-
ion o CLU is obse ed (7, 131, 133, 134). P e iously, i was
no clea whe he an ele a ed le el o CLU in he b ain was
help ul o ha m ul. In 2001, wo g oups pe o med b ain-
ischemia expe imen s and epo ed con adic o y esul s
(133, 134). Whe eas Han e al. (133) ound ha he absence
o CLU educes cell dea h in neona al hypoxia-ischemia,
Weh li e al. (134) obse ed a neu op o ec i e e ec o CLU
in pe manen ocal ce eb al ischemia. I is belie ed ha
hese di e en indings a e due o di e en expe imen al
se ings; mo e bene icial e ec s o CLU in i o and in i o
we e obse ed elsewhe e. Fo ins ance, b ain ischemia
induced by pe manen middle ce eb al a e y occlusion
shows hea ily impai ed issue emodeling in CLU K.O.-
animals. In wild ype animals, howe e , a signi ican
CLU up egula ion by as ocy es and inc eased mic oglia/
mac ophage in asion was de ec ed (135). In e es ingly,
ano he s udy has shown ha mic oglia ac i a ion by
sCLU leads o ch onic in lamma ion and hus neu o oxic-
i y due o obse ing an inc eased elease o ni i e oxide
and TNF-α (136). Mo eo e , sCLU in as ocy e-condi ioned
media om he hippocampus and midb ain had a con-
ibu ing e ec on cell su i al and di e en ia ion bu no
on p oli e a ion o neu al p ecu so cells (137). Howe e ,
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