Cell surface expression of the 70 kD component of Ku, a DNA binding nuclear autoantigen

(1)

Cell surface expression of the 70-kD component

of Ku, a DNA-binding nuclear autoantigen.

B S Prabhakar, … , J Srinivasappa, A L Notkins

J Clin Invest.

1990;

86(4)

:1301-1305.

https://doi.org/10.1172/JCI114838

.

The Ku complex, a heterodimer of 86- and 70-kD proteins, is a nuclear DNA-binding

autoantigen. However, hydrophobicity analysis of the deduced amino acid sequence of the

70-kD protein had strongly suggested that this might also be a membrane protein. In the

present study, using antibodies to synthetic peptides and a polyclonal antiserum to the

purified protein, we show that the 70-kD protein of the Ku complex is present in isolated

plasma membranes of human cells. By indirect immunofluorescence microscopy and

fluorescein-activated cell sorting, we demonstrate that this autoantigen is exposed on the

cell surface. In addition, we have identified several domains of the protein that are exposed.

Our study provides one of the first demonstrations of a eukaryotic, nuclear DNA-binding

protein that is also on the cell membrane. Moreover, our results might help explain how

autoantibodies to the Ku autoantigen could target cells for an autoimmune attack.

Research Article

(2)

Cell Surface Expression

of the 70-kD

Component

of

Ku,

a

DNA-binding Nuclear Autoantigen

BellurS. Prabhakar,Graham P. Allaway, Javaraiah Srinivasappa, and Abner Louis Notkins

Laboratory of Oral Medicine, NationalInstituteofDentalResearch, National Institutes ofHealth, Bethesda, Maryland 20892

Abstract

The Kucomplex,a

heterodimer

of 86- and 70-kD

proteins,

isa

nuclear

DNA-binding autoantigen. However, hydrophobicity

analysis of

the deduced amino acid sequence of the 70-kD protein hadstrongly suggested that this

might

also bea

mem-brane

protein.

In the present

study,

using

antibodies to

syn-thetic

peptides

anda

polyclonal antiserum

tothe

purified

pro-tein, we show that the

70-kD protein of

the Ku

complex

is present inisolated plasma membranes

of

human cells. By indi-rect

immunofluorescence microscopy

and

fluorescein-activated

cell

sorting,

wedemonstrate that

this

autoantigen

is exposed

onthe cell

surface.

In

addition,

wehave

identified

several

do-mains

of the

protein

thatareexposed.

Our study provides

one

of the

first demonstrations of

a

eukaryotic,

nuclear

DNA-bind-ing protein that is alsoonthe cell membrane.

Moreover,

our

results might helpexplain how

autoantibodies

tothe Ku

au-toantigen

could target cells foranautoimmune attack.

(J.

Clin. Invest.

1990.

86:1301-1305.) Key words: nuclear autoantigen-cell

surface

expression * DNA binding protein-Ku protein

Introduction

Many

autoimmune diseases

are

characterized by

the presence

of autoantibodies

tonuclear

proteins.

However, it is

notclear how these

"hidden" antigens

could become targets

for

au-toimmune

attack.

Recently,

wecloned and

expressed

ahuman cDNA

encoding

a

70-kD

nuclear

protein

and showed that

patients

with

autoimmune Graves' disease

have

autoantibod-ies

to

this

protein

(1,

2).

Independently,

Reevesand

Sthoeger

(3)

clonedacDNA

encoding

thesame

protein and

showed that

it is

acomponent

of

the Ku

complex.

The Ku complex

consists

of

the

70-

andan

86-kD

protein (4-8). Earlier studies

showed that some

patients with SLE

and scleroderma

polymyositis

have

antibodies

to

the

Ku

complex (4,

9). Furthermore, it

had been demonstrated that the Ku

complex binds DNA,

and

this

binding is mediated by

the 70-kD component

(5).

These

ob-servations

were

confirmed

and extended

using

the

recombi-nant 70-kD

protein expressed

in the

baculovirus

system

(2).

However,

hydrophobicity

analysis of

the

amino acid

sequence deduced

from the

cDNA sequence had revealed several

poten-tial membrane spanning

domains,

strongly

suggesting

that

this

protein

might

be membrane

associated

(1). The presentstudies

Addressreprintrequests to Dr.Bellur S. Prabhakar, Laboratory of Oral Medicine, NationalInstituteofDental Research, National Institutes of Health, Bethesda,MD20892.

Receivedforpublication 12 December 1989 and in revisedform 18

April1990.

TheJournalof ClinicalInvestigation,Inc. Volume86, October 1990, 1301-1305

were initiated to investigate whether the protein is also ex-pressed on theplasma membrane.

Methods

Peptides andantipeptide antibodies. In general, peptides were selected for high solubility, based onthepresence ofseveral charged amino acids. Peptidesweresynthesizedaccordingtothe deducedamino acid sequence ofthe 70-kD protein usinganautomated peptide synthesizer (model430A;Applied Biosystems, Inc.,FosterCity, CA). Deprotec-tion and release of thepeptides from the resinweredoneusing anhy-drous hydrogenfluoride with 10% anisoleat-50Cto0C for 1-2 h. After ethylacetateextraction, the peptidesweredissolved in 10% acetic acid,filteredtoseparate thedissolvedpeptide fromtheresin, andthe filtratewasIyophilizedtogive the crude driedpeptide.Thepurity of thepeptidewasevaluatedusing HPLC withanaqueousphase of 0.1% TFAand amobile phaseof 70% acetonitrile in theaqueousphase.For mostanalyses done on HPLC,alinear gradient of 0-100% mobile phase was usedover30min at25'C withaC8reverse-phase HPLC column(TheNest,Southboro, MA). Amino acidanalysiswasdoneto verify the correctcomposition ofeachpeptide(WatersPicotagSystem; WatersAssociates,Milford, MA). Substitutions ofAfor R, G forQ,S forL, Afor R, andAforR werenoted atresidues 165, 278, 374, 444, and470, respectively. Antipeptide hyperimmunesera wereprepared byimmunizing rabbits with individualpeptides coupledto BSA. Pep-tide-specific antibodies were obtained by affinity chromatography using protein A-Sepharose (Pharmacia FineChemicals, Piscataway, NJ) andpeptide-specific columns.Theindividual antipeptide antibod-iesreactedspecifically with the corresponding peptide inanELISA. Theseantibodiesalso reactedwellwith the 70-kD protein, expressedin insect cells usingarecombinantbaculovirus,bothinWestern blots and ELISA.

Western blotting. HeLa cell plasma membrane ghosts were pre-pared bythe method ofBrunette and Till(10). The production of recombinantAutographa californica nuclear polyhedrosis virus ex-pressing the 70-kDprotein is describedelsewhere (2). Sampleswere run on 10%SDS-polyacrylamidegels andelectroblottedonto nitrocel-lulosefilters.Thefilterswereblockedwithbuffercontaining5%nonfat dry milk and 0.1%Tween-20, then incubated with polyclonalserumto thepurified70-kD componentof theKuautoantigen (purifiedfrom MLA 144 monkey leukemia cells by Dr. J.Quinn[National Cancer Institute, National Institutes ofHealth], manuscript in preparation) diluted 1:500 inthe samebuffer. After being washed,thebound anti-bodywasdetectedby incubation with peroxidase-labeledgoat anti-rabbit IgG antibodies (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD)at2.5 ug/ml, washed andincubated with chloro-naphthol substrate. Alternatively, MAb to proliferatingcell nuclear antigen (Boehringer Mannheim Biochemicals, Indianapolis, IN)at50

gg/ml

wasused astheprimary antibody,andits bindingwasdetected using peroxidase-labeled goatanti-mouse IgG (KirkegaardandPerry Laboratories, Inc.)at10

lsg/ml.

FACSanalysis. All cells were split one day before the analysis and were in log-phase growth. IM9lymphocyteswere growninsuspension culture usingRPMI 1640 medium with 10% FCS. HeLa cells were

(3)

resuspended in FACS buffer (PBS containing 0.1% BSA and 0.1% sodiumazide[Sigma Chemical Co., St. Louis, MO]). Cell viabilityas

determined by trypan blue staining was >95% (IM9) or >90% (HeLa). Allincubationswerecarriedoutat40C.Cellswereincubated withrabbitsera orpurified IgG for 1handwashed in FACSbuffer. The cellpelletwasthenincubated with FITC-labeledgoatanti-rabbit im-munoglobulins(Kirkegaard and Perry Laboratories, Inc.)at adilution of 1:10 for 1 h. The cellswerewashed with FACSbuffer and resus-pended in thesame buffer containing0.2% paraformaldehyde. The cells were analyzed usingaFACSCAN (Becton-Dickinson & Co., MountainView, CA) analyzer. Mostofthedead cells and celldebris were excludedusingforwardand900 scatteranalysis.

Immunofluorescence microscopy. HeLa cells were grown as

mono-layersonglass chamberslides (LabTek;Miles Scientific,Naperville, IL). Thecells wererinsed in PBSand thenfixed incold acetone/metha-nol(1:1) for 10 min, air-dried, and stored at-20°C untilused. For viable cellstaining, HeLa cell monolayers were washed in cold PBS with5%FCS to remove anydead cellsandstained immediately. The fixed and viable cells were stained at room temperature and 4°C, respectively. Staining followed the procedure described above for FACSanalysisexceptthat the reagents werediluted inPBSwith 5% FCS. Theslides wereviewed usinganOlympusAHBT Microscope withfluorescenceattachments.

Results and Discussion

To study the

cellular

distribution of

the 70-kD

protein,

we prepared a panel of 12 synthetic peptides and

raised

polyclonal

hyperimmune

sera

against

each

peptide.

The sequences

for

the

peptides

areshown in Table I. Each

antipeptide antibody

prep-arations

was

affinity-purified

and was shownto react

specifi-cally with the corresponding peptide in anELISA. The

anti-peptide

antibodies were tested for their

reactivity with

the 70-kD

protein

thatwas

expressed using

abaculovirus vector

(2).

Allthe

antipeptide antibodies

reacted

with

the

protein by

Western

blot

analysis (Table

I).

Initially,

we looked

for

the 70-kD

protein

in the plasma membrane

of

HeLa

cells

using

a

mixture of antipeptide

anti-bodies. In addition, apolyclonal hyperimmuneserum to the

purified

70-kD

protein

was used asan

independent

reagent.

HeLa cell plasma membrane ghosts were obtained by the method of Brunette and Till (10), which has been used

suc-cessfully

in a number of studies (11, 12). Western

blotting

showed that the antipeptide antibodies reacted

specifically

witha

single

protein of 70-kD in total HeLa cellextractsand in HeLa cell plasma membranes (not shown). Aneven stronger

reactivitywasfound when the antiserumtothe purified 70-kD proteinwasusedtostain the blots (Fig. 1). The plasma

mem-brane sample (lane a)wasderived from - 50 timesmorecells

thanwereusedtopreparethe total cellextract(lane b) andyet

contained similar quantities of the 70-kD protein. Two methods were used to help exclude the possibility that the membrane preparations had adsorbed nuclear proteins that might be present in the medium. First, the membrane

prepara-tionswerewashedwith 0.5 M NaCltoremoveadsorbed

pro-teins. This treatment did not release the 70-kD protein (results

notshown).Secondly, the membranesweretested for the pres-enceof another nuclear protein, proliferatingcellnuclear

an-tigen. Using an MAb to PCNA, this antigen was found in Western blots oftotal HeLa extracts but not in HeLa cell membranes (not shown). The plasma membrane-associated 70-kD protein had thesamemobility by SDS-polyacrylamide gel electrophoresis asthat of the protein derived from total HeLacell extractsand the recombinant protein (lane c).These

results suggest that the 70-kD protein in the membrane, like

therecombinant protein (2), is not glycosylated.

To seewhether the proteinwasexposedonthe cell surface, we first stained

viable

HeLacells with a

mixture of the

anti-peptideantibodiesusingindirect immunofluorescence micros-copy. In contrast to HeLa cells fixed with acetone/methanol

that exhibited primarily nuclear staining (Fig. 2 A), greater than 90%viable cells showed surface fluorescence (Fig. 2B). Nostaining was detected using normal rabbit immunoglobu-lins asa control (not

shown).

To

quantitate

this

binding

we

TableL.Sequences

of

Synthetic

Peptides

and Reactivities

ofAntipeptide

Antibodies

Reactivityof anti-peptide antibodies with*

HeLa cells (byimmunofluorescence)

Peptide Aminoacid 70-kDprotein

number numbers Aminoacid sequence (byWesternblot) Nuclei Surface

1 164-175 KAIMLFTNEDNP + + +++

2 185-202 RARTKAGDLRDTGIFLDL +++

+++-3 212-228 DISLFYRDIISIAEDED-NH2 + -

-4 244-258 RKVRAKETRKRALSR +++ ++ +

5 270-287 SVGIYNLVGKALKPPPIK ++

+++-6 314-325 SDTKRSQIYGSR ++ +++ ++

7 344-358 GLMLMGFKPLVLLKK ++ +++ +++

8 365-379 SLFVYPEESSVIGSS + ++ +++

9 391-407 EKEVAALCRYTPRRNIP ++-

-10 409-425 YFVALVPQEEELDDQKI +++ ++ +++

11 435-452 VFLPFADDKAKMPFTEKI ++-

-12 463-480 KAIVEKLAFTYRSDSFEN ++ -

-*-_No_reaction;₊_weak_reaction;₊₊_strong_reaction;₊₊₊_very_strong_reaction. _{The sequence}_of_each_synthetic_{peptide is}_shown_together

withthereactivity ofthecorresponding antipeptide antibody. Viableorfixed (acetone/methanol, 1:1)HeLacells werestained withantipeptide

(4)

a b c

200-

97.4-Figure 1. Western blotanalysis of HeLa cell 43- extracts:HeLacellplasma membrane ghosts

from2x 106cells (a),HeLa cell extract from4 X 104cells(b),and 0.5

Ag

ofthe 70-kD proteinexpressedusinga recombi-nantbaculovirusvector(c). The blot was probedwith antibodies to the 70-kD protein. Molecular masses arein kilodaltons.

stained viable cells with antipeptide antibodies and analyzed them using FACS. The antipeptide antibodies bound to the surface of both HeLa cells (Fig. 2C)and IM9 cells (a human B

lymphoblastoid

cell line) (Fig. 2 D). Further support for these

observations

came

from staining viable

HeLa cellswith

anti-serumto the

purified

70-kDprotein. Fig. 2 E shows that

this

700

w

m

z

0

i 600

LC-700

0-600

0

RELATIVE FLUORESCENCE

antibody reacted strongly withthe HeLa cell surface. The

spec-ificity of this reaction was confirmed by the reductionin bind-ing of the serum when preincubated with recombinant70-kD

protein (2) (Fig. 2 F).

To identify regions of the protein expressed on the cell surface, we examined the binding of individual antipeptide antibodiestoHeLa cells(Fig. 3). Using FACS, maximal bind-ing was seen with antipeptides 1, 7, 8, and 10. Antipeptides 5 and 6 bound toalesser degree. The other antipeptide antibod-ies reacted minimally or not at all with the cell surface. Similar findings were obtained with individual antipeptide antibodies by fluorescence microscopy (Table I). These results demon-stratethat several different regions of the proteinareexposed. Sequence analysis had shown a number ofpotential

trans-membrane domains. One

of

these

domains (amino acid

resi-dues374-390) was

of sufficient

hydrophobicitytopredict that theprotein has>90%probability of spanning the membrane (1). That exposed domains are found on either side of this

hydrophobic

region suggests that the protein may traverse the membrane more than once. The lack

of reactivity

of

other antipeptide antibodies suggests that the regions of the protein

Figure2.(Top) Indirect immunofluorescence micros-copyofHeLacellswith rabbitantipeptide antibodies andfluorescein isothiocyanate-labeledgoatanti-rabbit Ig.(A) Nuclearstaining ofacetone-methanol (1:1)fixed HeLacells.(B) ViableHeLacells showspecific mem-branefluorescence (green),whereasnuclei of these cells shownonspecificautofluorescence(orange/brown).

(Bottom)(CandD)showFACSanalysis of the binding ofamixture ofanti-peptideantibodiestoviableHeLa andIM9 cells,respectively.(... )Cellsonly;(---) normalrabbit Ig; ( )antipeptide antibodies.The re-activity withnormalrabbitIgisverysimilartothe re-activity found withthesecondantibodyalone(not shown).Eshows thebindingtoviableHeLa cellsof thehyperimmune rabbitserum to the70-kDprotein (1:200dilution).(... )Cellsonly;(- -) normal rab-bitserum(1:200 dilution);( ) antiseratothe 70-kDaprotein.Fshows theinhibitionofbinding ofthe antisera by the recombinant 70-kD protein. (...) Cellsonly; (* --- - _{) antiserum preincubated with}

the70-kDprotein;and ( ) untreated antiserum. Thesurfacestaining procedurewascarriedout at4°C

usingPBScontaining0.1%BSA and 0.1%sodium azidetopreventcapping.

D

:i'

. .1It

I*

(5)

600 2

2C

5

8

1 1

A I nn n^

)0 0 100 200

RELATIVE FLUORESCENCE

6

9

12

IL

0 100 20(

Figure 3.Binding of individual anti-peptide antibodiestoHeLacells:(---)normalrabbit

Ig,( )anti-peptide antibodies.Numbers 1 through 12 representantipeptides 1 through

0 12,respectively.Forstaining proceduresee legendtoFig.2.

recognized by these antibodiesarenotexposedonthe plasma membraneor areinaccessibletotheantibodies duetotertiary

conformation of theprotein.

The present study clearly demonstrates that the 70-kD

protein is present in the plasma membrane of transformed

cells and thatsomeepitopesareexposedonthecell surfaceand areaccessibletoantibodies. Normalcellsexpressconsiderable amountsof Kuproteins.Towhatextentthe70-kDprotein is expressed in the plasma membrane of normal cells and whethertherearequantitative differences in its expression in

variouscelltypesisyet tobedetermined.AfewDNA-binding proteinsofvirusesandbacteriahavebeenshownto be asso-ciatedwith thecell membrane. Examples include the

origin-specific DNA-binding protein

of

Bacillus

subtilis

(13) and the

SV-40largeTantigen(14).Theonlyknownexample of such proteinsineukaryoticcellsmaybe the steroidreceptors,which

bind DNAto exerttheir action,and are thoughttobe asso-ciated withtheplasma membrane (15-17). The function ofthe

70-kDprotein isnot known. Whereasprevious studies have

suggested arole in DNA repair andtransposition (5, 6), the current results suggest that theprotein might be involvedin cellsignaling,as arethose described above.

Earlier, we had found that patientswith Graves' disease have autoantibodies against the 70-kD autoantigen (1, 2).

Other studies have shown that patients with

scleroderma-polymyositis and SLE have autoantibodies against the "Ku

complex,"whichconsists ofthe70-kDproteinandan86-kD

protein (4-9). Together, these studies suggest that the 70-kD

protein might play abroaderrole inautoimmunitythan

pre-viously thought.Inthisconnection it isinterestingto notethat manyother humanautoantigens(e.g., Ro, La,Sm,

proliferat-ingcell nuclear antigen, nuclearribonucleoproteins) are nu-clearproteins (18). However,it has notbeenclear howsuch nuclearantigens, whichareapparentlyinaccessibleto the im-munesystem,could be targets ofanautoimmune attack.The demonstration that the 70-kDproteinisexpressedonthecell surfacemightexplainhowanautoimmuneresponsetoa

"nu-clearprotein"couldadverselyaffectanintactlivingcell. 0

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(6)

Acknowledgments

Theauthorsaregrateful to Dr. Frank Robey (National Institute of Dental Research, National Institutes of Health[NIH])forsynthesizing thepeptides.TheauthorsalsothankDr.JohnQuinn (National Cancer Institute, NIH) for his generosity in supplyingtheantiserum tothe 70-kD protein, Mr. Lloyd Billups for excellent technical help, and Eloise Mangeand Karen Hardmanfor their helpin preparing the manuscript.

References

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2.Allaway, G. P., A. A.Vivino,L. D. Kohn, A. L. Notkins, B. S. Prabhakar. 1989. Characterization ofthe 70 kDacomponentof Ku autoantigen expressed in insect cell nuclei usingabaculovirusvector. Biochem.Biophys.Res.Commun. 168:747-755.

3.Reeves, W.H., andZ. M.Sthoeger. 1989. Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen. J. Biol. Chem. 264:5047-5052.

4.Mimori, T., M. Akizuki,H.Yamagata, S. Inada, S. Yoshida,and M.Homma. 1981.Characterization ofahighmolecularweight acidic nuclear protein recognized by autoantibodies in serafrom patients withpolymyositis-scleroderma overlap. J. Clin.Invest. 68:611-620.

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6.Mimori, T.,J. A.Hardin, andJ. A.Steitz. 1986. Characteriza-tion of the DNA-binding protein antigenKurecognized by autoanti-bodies from patients with rheumatic disorders. J. Biol. Chem. 261:2274-2278.

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13.Laffan,J. L.,andW.Firshein. 1988. Origin-specific DNA-bind-ing membrane-associated protein may be involved in repression of initiation ofDNAreplicationinBacillussubtilis.Proc.Natl. Acad. Sci. USA. 85:7452-7456.

14. Butel, J.S., andD. L.Jarvis. 1986. The plasma-membrane-as-sociated form of SV40 largetumorantigen: biochemical and biological properties. Biochim. Biophys.Acta.865:171-195.

15. Gametchu, B. 1987. Glucocorticoidreceptor-like antigen in

lymphomacell membranes: correlationtocell lysis.Science(Wash. DC). 236:456-461.

16.Peck, E. J., J. Burgner, and J.Clark. 1973. Estrophilic binding sitesof the uterus. Relationto uptakeand retention of estradiol in vitro.Biochemistry. 12:4596-4603.

17. Bression, D., M. Michard, M. L. Dafniet,P. Pagesy, and F. Peillon. 1986. Evidence fora specific estradiol binding site on rat pituitary membranes. Endocrinology. 119:1048-1051.