Severe Low

Autoantibodies to the Low Density Lipoprotein Receptor in a Subject Affected by Severe Hypercholesterolemia

Alberto Corsini, PaolaRoma, DomenicoSommariva,* RemoFumagalli, and Alberico L.Catapano Institute ofPharmacology and Pharmacognosy, and *L. SaccoHospital, Vialba, 20129 Milan, Italy

Abstract

We

studied

a

32-yr-old

man

with

a

benign paraproteinemia (IgA) affected by

severe

nonfamilial hypercholesterolemia.

In

vitro

ex-

periments demonstrated that lipoprotein-deficient

serum

(LPDS)

from the patient

inhibited

the

binding of

low

density

lipoprotein (LDL) to human skin

fibroblasts

cultured in vitro

(up

to70%) whereas LPDS

from

controls had no

effect.

Removal of IgA from the patient's serum by

immunoprecipitation

with mono-

specific antisera abolished

the

inhibition of

LDL

binding.

IgA

isolated

from the serum

of

the

patient

by

affinity

chromatography

inhibited, in

a dose-dependent

manner,

the

binding of

LDL to human

skin fibroblasts

in

vitro, thus showing

an

IgA-mediated effect.

Ligand-blotting

experiments

demonstrated

that the para-

protein

directly interacts with the

LDL

receptor,

thus inhibiting

the

binding of

the

lipoprotein.

Treatment of the receptor

protein with reducing

agents

blocked the interaction of

the

antibody with

the LDL receptor. From

these

data we

speculate

thatthis au-

toantibody

may be

responsible

for the severe

nonfamilial

hy-

percholesterolemia of

the

patient.

Introduction

Familial

type

Iha hypercholesterolemia ^is characterized by high

levels

of plasma cholesterol and

premature coronary

heart disease (1).

Brown,

Goldstein, and their co-workers in

^a

series ofelegant studies have elucidated the molecular basis of

^this

syndrome, i.e., partial or almost complete lack of

low

density lipoprotein

(LDL) receptors

(2). The clinical

counterpart

of this biochemical defect is the

presence

of xanthomata,

with

typical distribution, and early

coronary

and aortic atherosclerosis which eventually leads

to

myocardial infarction.

Type Ila

phenotype, however,

may

also result from

anumber

of secondary

causes.

Hypothyroidism,

^for

instance,

^induces

hy- percholesterolemia

^{dueto a}

down-regulation of

LDL receptors,

which

canbe

reversed by specific

^treatment

(3).

The concept

of autoimmune hyperlipoproteinemia

was

proposed by

Beaumont

and

Beaumont

(4), ^and since then patients

^with

hyperlipemia have been found

who

have autoantibodies

to

circulating lipo- proteins (5-7)

as

well

asto enzymes

related

to

lipoprotein

^ca-

tabolism (8).

Sofarno

patient

has been

reported

^tohaveauto-

antibodies

^tothe LDL receptor. In

this

articlewereport ona

subject affected by

severe

nonfamilial hypercholesterolemia

whose serum

contains

a

paraprotein

that

binds

tothe LDLre- ceptor, thus

inhibiting ^the ligand-receptor

interaction.

Address

reprint

requeststoDr.Catapano, Institute ofPharmacology and Pharmacognosy, ViaAdeSarto 21, 20129 Milan, Italy.

Receivedfor publication 12February1986.

Case

report. F.F., a

31-yr-old

man, with a 10-yr history of hypercholesterolemia, was referred to us in January 1984 because

of hypercholesterolemia (typically

in the range of 600-900 mg/

dl). The patient had xanthoma

at theelbows and thickening of

achilles

tendon was

observed.

Known secondary causes of hypercholesterolemia were ex-

cluded.

The

familial anamnesis

wasnegative and biochemical

analysis indicated that his relatives

were

normocholesterolemic, suggesting

a

nonfamilial type hIa hypercholesterolemia. All

rel- evant

laboratory

testsgave normal

findings

with the

exception of immunoelectrophoresis of the

serum

proteins

which

showed the

presence

of

a

spike in

the

,B-globulin

zone. Thispeak was

identified

as an IgA

(K-chain)

by use

of

commercially available

antibodies. IgA, IgG, and IgM plasma concentrations

were

within the normal

range.

Bonemarrow plasma cell count was normal (<5%) as well as

the radionuclide bone

scan

and the complete

skeletal x-ray

analysis.

Bence-Jones

proteins

were absent

from

the

urine.

These

findings

suggested the diagnosis of a monoclonal

gammopathy of undetermined significance (9).

The

patient

hadangina and

had had

a

myocardial infarction;

he wasalso hypertensive

(190/

100 mmHg) for which

he wastreated

with j3-biockers.

At age

of 31

he

suddenly died

at

home during

the

night.

Anautopsy was not

performed.

Methods

Materials.Eagle's minimum essential medium (F- 1), fetalcalf serum, trypsin-EDTA (X 1), penicillin (10,000 U/ml), streptomycin (10 mg/

ml), tricinebuffer (1M, pH 7.4), Hepes(1M, pH 7.4) and nonessential aminoacids (X 100),werepurchased fromGibco (GrandIsland,NY);

disposable culture flasks andpetridisheswerefromCorningGlass Works (Corning, NY),filtersfromMillipore Corp. (Bedford, MA).Sodium1251 iodide, carrier free in 0.1 N NaOH was purchased fromAmersham (Amersham, UnitedKingdom).

SephadexG-25 andSepharose4Bactivatedwith cyanogen bromide (CNBr)^wereobtainedfrom Pharmacia(Uppsala, Sweden).Kitsforen- zymatic determination^ofplasma lipidswerefromBoehringer(Mann- heim,FederalRepublicof Germany).MonospecificantiseratoIgA and immunodiffusion platesfor IgA andIgGweregifts fromDr. Porta(Behr- ing, Scoppito, Italy).Nitrocellulose membranes and allelectrophoresis material and equipmentwere purchased from Bio-Rad Laboratories (Richmond, CA). All the reagentswereanalytical grade.

Plasmalipids and lipoproteins.Plasma wasobtained inEDTA(0.1%), cholesterolandtriglyceridesweredetermined byenzymatic procedures, apolipoproteins (apo)'BandA-I weredetermined by radial immuno- diffusionusingantibodies raised inourlaboratoryinrabbitsagainstpu- rified apo^Band apoA-I.Lipoproteinswerefractionated by ultracentri- fugation (10)using thefollowingdensitycuts:very lowdensitylipoprotein (VLDL)-d< 1.006g/mI,LDL-d< 1.019-1.063 g/mI,high density lipoprotein subfraction3(HDL3)-d<1.125-1.21g/mI.Fractions were spun for16, 24,and48hrespectivelyat40,000 rpmin a Beckman 50.2 1.Abbreviations usedinthis

paper:

apo,apolipoprotein; LPDS,

lipopro-

tein-deficientserum.

J. Clin. Invest.

© The AmericanSocietyforClinicalInvestigation,Inc.

0021-9738/86/10/0940/07

$1.00

Volume78, October 1986, 940-946

Ti Rotor (Beckman Instruments, Inc., Palo Alto, CA) at 12'C. Lipo- protein triglyceride and cholesterol content was determined by enzymatic procedures. Lipoprotein-deficientserum (LPDS) was obtained as the serumfraction at d<1.25 g/ml, by ultracentrifugation for 72 h at 40,000 rpm at 12'C in a Beckman 60 Ti Rotor. Densities were adjusted by addition of solid KBr to the solutions.

Alllipoproteins and the LPDS were dialyzed extensively against NaCl 0.15 M, EDTA0.3 mM,pH 7.4, and storedat4VCafter sterilization throughaMillipore 0.45-Mm filter. The LPDSwasstoredat-20'Cand thawedjustbefore use. HDL3werefurtherpurifiedby heparin-Sepharose affinity chromatography toremoveapo B- and apoE-containinglipo- proteins (1 1).

Lipoproteins (LDL and HDL) were labeled with 125I accordingto Bilheimer et al. (12).Specific activities were 150-230 cpm/ng protein for LDL and 150 cpm/ng protein for HDL3. Free1251Iwasremoved by columnchromatography onaSephadex G-25 column eluted with 0.15 MNaCi,0.3 mM EDTA, pH 7.4. Fractionscontaininglipoproteins were collected anddialyzed against0.15 MNaCl,0.3 mMEDTA, pH 7.4, overnight at4°C. Free iodine accounted for <1% of total radioactivity.

Lipid-associated radioactivity was always <5% for LDL and 4% for HDL.

Immunoglobulin purification. Thed < 1.25 g/ml plasma fraction (LPDS) wasused to studytheeffect ofthepatient'sserum onlipoprotein metabolism. In someexperimentsIgA wasimmunoprecipitated using monospecific antisera at 4°Cfor24 h and the supernatantwascollected aftercentrifugation for 20 minatroomtemperature. The effectiveness of theprecipitationwasevaluated by radial immunodiffusion.

IgAwaspurified from control and thepatient'sLPDSbyaffinity chromatographyusingarabbitanti-human IgAimmunoglobulincoupled to aSepharose 4Bactivated with CNBr as recommendedbythe producers.

The LPDS was loaded onto the column(2.6 X4cm)and elutedwith phosphatebuffer, pH 7.4(10 vol of the column) followed by 10 vol of glycinebuffer (1 M, pH2.5). Fractions of 0.5 mlwerecollected and immediately adjusted to pH 7-7.4 with 1 M NaOH. Protein content was measured asdescribed by Lowry et al. (13). IgA was >95% pure as de- termined byradial immunodiffusionusingcommerciallyavailable plates.

Theinteraction of LPDS (from control and patient F.F.'s serum) with LDL was alsodetermined byaffinity chromatography usingaLDL- Sepharosecolumn preparedusing Sepharose4Bactivated with CNBras describedby the producers. The LPDSwasloadedontothecolumn(2.6 X3 cm) and elutedfirstwithphosphate-buffered saline (PBS) (10 vol of thecolumn) and then withglycine buffer (I M, pH 2.5). Fractions of 0.25 ml werecollected andimmediately adjusted to pH 7-7.4 with 0.1 MNaOHandtheir protein content was determined according to Lowry etal.(13). The columnwasusedwithin 2 d after itspreparation.

Cellculture. Humanskinfibroblasts weregrown fromexplants of skinbiopsies obtained fromthepatient andfrom normolipidemicclin- icallyhealthyindividuals.Cells were grown in monolayers and maintained in75-cm2plastic flasks at 37°C in a humidified atmosphere of 95% air, 5%CO2 in F- 1 medium supplemented with 10% fetal calf serum, non- essentialaminoacidsolution (1%, vol/vol), penicillin (100 U/ml), strep- tomycin (100

gg/ml),

tricine buffer (20 mM, pH 7.4), and NaCO3 (24 mM). For allexperiments,cellsfrom the stock flask were dissociated with 0.05% trypsin-0.02% EDTA at confluency (5-15 passages), seeded in 60-mm plastic petri dishes (2.5 X

10'

cells), and usedjustbefore reachingconfluency, usually 6 d after plating. The medium was changed every 2-3 d. Cell viability, assessed by Trypan blue exclusion, was always >95%.

The cell surface binding of

1251I-LDL

wasdetermined as heparin- releasable radioactivity (14). Monolayers were then digested in 0.1 N NaOH at room temperature overnight; one aliquot was counted for re- sidual cell radioactivity asa measureof LDL internalization and another aliquotusedfor cell protein assay (13). For total uptake (binding and internalization)of LDL, cell monolayers were directly digested in0.1 N NaOHafter standard washing procedures. The specific LDL binding, internalization, and total uptake were computed by subtracting values observed in the presenceof 50-fold excess of unlabeled LDL from those obtained in their absence. LDL

degradation

^wasmeasured from theac- cumulation ofnoniodide trichloroaceticacid-soluble '25Iin theincubation

medium inexcessofthatoccurringin the absence of cells(14). Each experimental point represents the average value oftriplicate incubations.

Inallexperimentscellswerepreincubatedfor 24 hat370Cinamedium containing5%human LPDStoinduceLDLreceptors.

Whenappropriate the preincubation periodwasfollowed by incu- bation for 2 hat370C inamedium containing increasing amounts of LPDSorIgA fractionobtained from control and from the patientserum.

Cellswere then washed three times with PBS, 125I-LDL were added ina fresh medium containing 5% of normal LPDS, and the incubationwas carriedouteitherat370Cor4VCfor 5 hor2h, respectively.Binding, internalization, and degradation of labeled LDL were determinedasde- scribed above. For experiments performedat4VC,cellswereplaced for 30minat40C; the mediumwas then removed and replaced witha chilled F-l 1 medium without bicarbonate and tricine supplemented with 10 mM Hepes (pH 7.4), 5% LPDS, penicillin, streptomycin, 1% non- essential aminoacids, and the indicated concentration of '25I-LDL.

Forcompetition experiments, cellswereincubated withafixedcon- centration of1251I-LDL,(5

gg

of LDL protein) in the presence ofincreasing concentrations ofunlabeled LDL from control and patientserum.

To show specificity of the effect on the binding of LDL to their cellular receptor,westudied thebinding of '25I-HDL3tohuman skin fibroblasts. The experimentswereperformedasdescribed by Biesborek etal.(15) after incubation of the cells inanalbumin containing medium for 24 h. The binding of HDL wasthen evaluatedascell-associated radioactivity because it has been shown that under these conditionsno appreciable internalization and degradation of HDL occur by human skinfibroblasts(15).

Ligand-blotting experiments.Ligand-blottinganalysis wasperformed asdescribed by Daniel et al. (16) using bovine adrenals as a source of membranes rich in the LDL receptor (17). Membranes were prepared asdescribed (17) at 100,000 g and stored at -80'C as a pellet untiluse.

On the day of the experiment the pellet was thawed and solubilized in 1%Triton X-100 (final concentration). The soluble fractionwasseparated from theparticulate material by ultracentrifugation at 100,000 g for 1 hat4°C and applied to a 5% acrylamide gel containing 0.1% sodium dodecyl sulphate.Electrophoresis was run at 15mAperplate for 6 h.

The separatedproteins were then blotted to a nitrocellulose membrane asdescribedby Burnette (18) at 4°C at 250mA for 6 h.Efficiency of blotting was determined by staining of the gels; >85% of the protein was blotted to the nitrocellulose. The cellulose was then incubated with buffer Acontaining 3% albumin to quench the nonspecificbindingsites. '25I- LDLat afinal concentration of 10

Ag

protein/ml were then added to bufferAand incubatedat27°C for 2-3 h. The incubation medium was removed andthenitrocellulose sheet was washed at 4°C with the same buffercontainingnoLDL(threewashes, 30 min each).

Insomeexperiments, after the incubation with albumin, the nitro-

Table I. Plasma Lipids andCholesterolDistribution among Lipoproteinsinthe Patient

Patient Controls§

mg/dl mg/dl

Plasmacholesterol* 600-900 190±18

Plasmatriglyceride* 153-212 94±29

VLDLcholesterolt 40 17±11

LDL

cholesterolt

^{538 123±14}

HDL

cholesterolt

⁴⁰ 48±6.6

Apo

Bt

276 87±12

ApoA-It 121 139±17

* Range in four separate determinations.

t Plasma cholesterol^was640

mg/dl.

§

^{Valuesarethemean±SD of}20normalsubjectsmatchedforsexand age.

ca 0

ai,

10

Z 0

° C

" _

'.3 01.

510 25 50 75 120

LDL /ug ^protein/ml

510 25 50 75

LDL /ug protein/

Figure 1.Specific binding (o), internal- ization(A),anddegradation (o) of con- trol'25l-LDLinvitro by cultured fibro- blastfromanormolipemicsubject(left) andpatient F.F.(right). Increasing^con- centrations of 251I-LDLwereincubated with the cells for 5 h. The binding, in- ternalization, anddegradationwerede- ....<, termined as described in Methods. Each

pointis themeanoftriplicatedetermi- nations that did notdifferby>10%.

Thenonspecific binding,internalization, 120 anddegradation were the values ob-

tained in the presence of 500

Ag/ml

^of

unlabeledLDL.

cellulosewasincubated for 2 h with LPDS(controlorpatient F.F.);final concentrationwas 120

Ag

protein/ml. Theexperimentwasthen per- formedasoutlined for the LDL.

Tostudythedirectbinding ofpatientIgAtothe LDL receptor, the immunoglobulinfractionwasiodinated by theiodogenprocedure(19) to aspecific activity of 1,500-1,900cpm/ng. Theexperimentsofligand blottingwereperformedasfor the LDL. The dried nitrocellulose sheets werethen processedforautoradiographyat-80'Cusinganintensifier screen.

Results

Table

I

shows the plasma lipid

levels and the cholesterol distri- bution among

lipoproteins

in thepatient. Thephenotype ^was

0 L.

z I-

bo

E-

C?

nr 40

30

20

10

Ha. Family studies

showed, however, that both parents were normocholesterolemic

(father

210mg/dl; mother 190 mg/dl), thus

ruling

out the

possibility

that we were dealing with a subject affected

by

familial

hypercholesterolemia.

In fact, studies on

cultured skin fibroblasts obtained from

patient F.F. showed that

the binding, internalization, and degradation of

LDLwas normal

(Fig.

1). The LDL

from

the patient efficiently interacted with

the

LDLreceptor as shown

in Fig.

2. The patient LDL were as

effective

as

control

LDL

in competing

for

'25I-LDL

uptake, as well as

degradation

(data not shown).

Fig. 3 shows

the

effect of

control and patient F.F.'s LPDS on

the 251I-LDL degradation

by human

skin fibroblasts

at

370C.

2504

4. 200_-

0Q

0 E 150^-

ad 100-

a

50-

10 25

5 1525 50 100 200

LDL /ugprotein/mi

Figure 2. Effect of control(m)andpatientF.F.'s(-)LDLonthe up- takeof

'25I-LDL

byculturedhuman skin fibroblasts.

'251I-LDL

(5 ,ug/ml) were incubated for 5 h at 370C with the cells in presence of the indicatedamountof unlabeledLDLfromanormolipemic subject andfrompatientF.F.ThenonspecificuptakewastheLDLuptakeⁱⁿ the presenceof500

,g/ml

^{of controlLDL.Each}

point

^{is themeanof} triplicatedeterminations that didnotdifferby>10%.

.

0

0~~~~

60 100

LPDS /ul^/ml

Figure 3. Effect of control

(i)

^andpatientF.F.'s

(.)

^{LPDSonthedeg-}

radationof

'25I-LDL

by cultured skin fibroblasts. Cellswerepreincu- batedwith the indicatedamountof control LPDS for2 h at

370C.

Proteinconcentration of control andpatientF.F.'sLPDSwas20 mg/ml. The monolayerwaswashedthrice with PBS and then incu- bated for 6hwith

'25I-LDL (10

yg/ml)at370C.LDLdegradation^was determinedasdescribed in Methods. Eachpointis themeanoftripli- catedeterminations that didnotdiffer by>10%.

_- IT

30

c

LO

Q 06

a.0 I

0 m.2

in

20

10

0

5 10 25 50 75 120

LPDS/ul^/ml

Figure 4.Effectof patientF.F.'sLPDSbefore

(.)

andafter (m) IgA^im- munoprecipitationonthebinding of

'25I-LDL.

Cellswerepreincu- batedwith LPDS for 2 hat37°Cattheconcentrations indicated in thefigure.Protein concentrationoftheLPDSwas20

mg/ml.

^Thecells werethen washedthrice with PBS and thebindingof

'25I-LDL

assayed at4°Casdescribedin Methods. Thenonspecificbindingwasthe

125I-LDL

bound inthe presenceof 500,ug/mlof unlabeled LDL. Each pointis themeanoftriplicate determinationsdiffering<10%.

Similar results

were

obtained for

the

binding

at

4°C (data

not

shown). LPDS from the patient clearly inhibited this

process

while

control

LPDS did

not.

Immunoprecipitation of

IgA

from

the LPDS resulted

in

a

loss of the inhibitory activity (Fig.

4),

suggesting

that the IgA was

responsible for

the LPDS

effect.

To

directly

address this

question,

IgA were

purified

to

homogeneity

by

affinity

chro- matography.

This immunoglobulin

was as

effective

as whole

LPDS in inhibiting 125I-LDL

uptake and

degradation

whereas

control IgA had

no

effect (Fig. 5).

It was

therefore demonstrated that in

our

patient the

IgA

fraction

was

responsible for

the

effects of LPDS

upon

the receptor-mediated catabolism of

LDL.

0 a. 60

C 40

c20

to cJ

cL

uz

.

2

0

L.CL

14 u4 30

0

~* -E 1

aoo

a, a.

75

c

I-

OZ ²⁰

_

2

1

w an

_3 10

75 vz

a S

10 25 10 25

IgA /ug/ml

Although

the

tissue culture experiments

were

performed

to avoid any

direct interaction

in the medium

between

LDL and

LPDS

or

IgA,

^we

also performed affinity-chromatography

studies toshowthatno

interaction

between LDL and the

patient

LPDS components occurred.

Indeed,

these

experiments

showed that noneof the proteins present in the patient and control LPDS

interacted with

LDL

under the conditions used (Fig. 6) whereas

a

rabbit antiserum

toapo B

contained

a

fraction

that interacted

with

LDL.

To verify the specificity ofthe LPDS from the patient

in

inhibiting the binding of

LDL, we

studied its ability

to

interfere with the binding of

apo

E-free HDL3

to

human

skin

fibroblasts.

Theresults in Table II show that neither control nor

patient

F.F.'sLPDS affected the

'25I-HDL binding.

Using the

ligand-blotting

technique,

wethen addressed the question as to whether the

IgA

from our patient interacted di- rectlywith the LDL receptor. The patient's LPDS

effectively inhibited

the

binding of

LDL to the receptor inthis assayas shown in

Fig.

7

(lanes

E and

F),

thus

demonstrating

adirect effect onthe receptor protein.

Fig.

8 showsthat

1251-labeled IgA

purifiedfrom patient F.F.'s serum directly interacted with a pro- tein of approximately 160,000 mol wt (lanes A-C) with the same mobility of the LDL receptor (lanes E and

F).

Treatmentof the membranes with reducing agents abolished the interaction

of patient F.F.'s

IgA with

the

LDL receptor

(lane

D).

Discussion

Hypercholesterolemia (type Ha) is a risk factor for coronary heart disease (1). Genetic

forms

of

this disease

relate to the

deficiency

(partial or total) of LDL receptors;

hypercholesterolemic

subjects exist, however, that are nonfamilial and therefore no loss of receptoractivity occurs

(1).

Autoantibodies

to

lipoprotein

receptorshave not been pre- viously recognized as "secondary" causes of

hypercholestero-

lemia.

However, autoantibodies to

other

receptors (insulin,

ace-

tylcholine, and thyrotropin) have been described (20-22),

which lead to severe

forms ofdiabetes, myasthenia gravis,

andGraves'

syndrome.

Autoantibodies to

lipoprotein

components as well as to

lipolytic

enzymes

have also been reported (4-8), and

these conditions are related to

hyperlipemia

with

increased

plasma

concentrations

of

triglycerides

and cholesterol.

Wereport here on a

patient

whose LPDS

contains

an

anti- body-like protein

that

inhibits the receptor-mediated binding

Figure 5. Effect of control (n)andpatientF.F.'s

(.)

IgA onthe uptake and degradation of'25I-LDL by cultured

= ~* skin fibroblasts. Cellswerepreincubated with IgAasde- scribed in the legendstoFigs. 3 and 4 and then incu- batedfor5 h at37°Cin presence of 10

;g/mI

^{of 25I-}

LDL. Thenonspecific uptakewastheuptakein the 75 presenceof 500

jg/ml

^{unlabeledLDL.Each}pointis the

meanof triplicate determinationsthatdidnotdiffer by

IgA /ug/ml ^{> 10%.}

20 25 30 35 20 25 30 35 20 25 30 35 FRACTION NUMBER

and catabolism of

LDL

by human skin fibroblasts in vitro. The experiments performed after immunoprecipitation of IgA strongly

suggest

that this factor is

an

antibody (see Fig. 4),

and

the data obtained using purified IgA indeed

show that the

activity is completely attributable

to

this globulin fraction (Fig. 5).

It

is known that monoclonal antibodies

to

Apo

B may

inhibit

LDL

catabolism (23, 24). The lipoprotein profile of

our

patient,

however,

differs from that of subjects whose

serum

contains

au-

toantibodies against

LDL. These

patients have,

in

fact, also

in- creased

plasma triglyceride

levels andmostof the

antibody

can

be found

at

the d

<

1.21 g/ml

top

(5, 8) associated with lipo- proteins. Our patient

was

normotriglyceridemic, his

LDL were

normal both in chemical composition and ability

to

interact with cellular

receptors,

and

the

inhibiting activity

was notas-

sociated with lipoproteins. Along this line

we

have also shown that this antibody does

not

bind

toLDL

(see Fig. 6);

^these

data, however,

must

be regarded with caution, for under the experi- mental conditions used,

an

antibody

may

fail

to

interact with

LDL or may not

be released from the column. Nevertheless, these data further

support

the tissue culture experiments in vitro which

were

designed

to

avoid

any

direct interaction between LPDS

or

IgA and

LDL

in the medium.

The antibody might therefore

act

by directly binding

the receptor or

interacting with the cell membrane

near

the

LDL receptor

producing steric hindrance of the LDL-receptor ^inter- action. Alternatively, the antibody might

interact withamem- Table II.

Effect of

LPDSonthe

Binding

of

Apo

E-free

HDL3toFibroblasts*

'25I-HDL3bound

LPDS F.F. Control

jtz/mi ng ng

- 69.9 69.9

20 63.2 70.1

50 69.0 60.2

150 61.4 72.9

* Dataareexpressed asnanogramsof

'25I-HDL

protein bound per milligramof cellprotein.Cellswerepreincubated withamediumcon- tainingalbuminfor 24hand thebindingdeterminedat

370C

asde-

scribed

in Methods. The dataare meansoftriplicate determinations thatdidnotdiffer by^>10%.

.0.600

0.400

.0.200

Figure 6.Elution profile of a LDL-Sepharose column.

Patient F.F. LPDS (A), control LPDS (B), and a rabbit polyclonalanti-human apo B serum (C) were applied tothe column. Only thefractionselutedwithI Mgly- cine, pH 2.5,arepresented.

brane component distant from the receptor, modulating the LDL-receptor interaction. To study the specificity of this anti-

body,

we

investigated whether it could also affect

the

binding

of HDL3 to human

skin fibroblasts

(seeTable II). Binding sites for HDL are present on a number

of tissues

and are distinct in terms of

specificity

and

regulation from

the LDLreceptor. (15,

25, 26).

No

interference with

the

binding of

HDL was found suggesting that the effect ofthe antibody is specific, at least among the

lipoproteins, for the

LDL receptor.

Finally, the ligand-blotting

experimentsdemonstrate that IgA from the patient interact in vitro with the LDL receptor and that treatment ofthe membrane

proteins with reducing

agents destroys the

epitope

recognized

by

the

antibody, suggesting that the

area

ofthe receptor involved

in

the

LDL

binding contains the epitope(s) recognized

by the

antibody.

A

loss of the

receptor

secondary

structure

abolishes

the

binding

of LDL as

well

as

ofthe antibody. Interestingly,

the

epitope for

a mouse

monoclonal antibody

wasalso not

retained

after exposure

of

the LDL receptor to

reducing

agents (16).

I0

o

- 200 ^X

...~~~~~~~~-

S

_I)

- 116 3

- 94 _i

- 68 i

- 43 ⁰

-A

AB8 C D E F 0

Figure7.Effect ofpatientF.F.'sLPDSonthebindingof

1251-LDL

to theLDLreceptor from bovine adrenalcortexes.Fortheligand-blot- tingassay, membranesweresolubilized with TritonX-100 (1%

vol/

vol) andrun on a5%sodiumdodecylsulfate-acrylamide gel,blotted to anitrocellulose filter.25

jsg

^{(lanesA,C,andE)or50},g (lanes B, D, andF) of membraneproteinwereappliedtothegels.The lanes were cutandincubatedat27°C for 2 h with control LPDS(120

jig/ml,

lanes A andB)orpatientF.F.'s LPDS(120

jtg/ml,

lanes E and F). The sheetswerewashedandincubatedat27°Cfor 2hwith

12511

LDL(10

Mg/ml)

without unlabeled LDL(lanes A,B,E,andF)or with 0.5 mg/ml unlabeledLDL(lanesC andD).Thehighmolecular weight components in laneBmay representaggregatedreceptor pro- tein.

0.600 E

c

C41

0 0

0.400

0.200

A B C

1. -1IJ..., -

I# I I I I I IIa

h

m

I0

V- x

_ 200 -

11I

3:

- 94

- 68

- 43

AB C D E F

w

-i

0

Figure^{8.Binding of}

"25I-labeled

IgAisolated frompatientF.F.'s LPDS to the LDL receptor. IgA were labeledwith 1251 _asdescribed^{in Meth-} ods. 5, 10, 15, and 10

Ag

of membraneprotein(lanesA-D)wereap- plied to the sodium dodecyl sulfateacrylamidegels and were blotted tonitrocellulose. The binding was performed in PBScontaining2%

bovineserum albumin for 2 h at 270C. The sample in lane D was pre- treatedwith 5%

fl-mercaptoethanol.

^Forcomparativepurposes the bindingof

25I-LDL

is shown (lanes E andF,¹⁰ and 15 Mgofmem- braneprotein applied). The area of higher

moleculer

^{weightin lanes}

A,B, andCmay represent aggregated receptor protein.

The degree ^of hypercholesterolemia in our patient

is com- parableto thatofhomozygotesfor familial hypercholesterolemia,

although

at the most theantibodyreduces LDLbindingto the

receptor by 70%. Patients receptor-defective ^also

^{have severe}

hypercholesterolemia

even though 15-20% of thereceptor ac-

tivity

ispresent (1).

The causal relation between antibodies and hypercholester- olemia in our patient, although likely, ^is

^not

demonstrated.

Bei-

siegel ^{et al.} (27) and Kita et al. (28) reported ^that

^antibodiesto

the LDL receptor interfere with LDL catabolism

invitro as well as in vivo. In this respect our patient will be very similar to

patients with severe insulin resistance, Graves' disease,

or myas-

thenia gravis ^{who have} autoantibodies to the insulin,

^acetylcho-

line, ^and thyrotropin receptors, respectively ^(20-22).

^{These an-}

tibodies have a number of physiologic ^effects

^{and some also}

mimic the effect of the physiological ligand (29).

^Thequestion

as to whether binding ^{of the} autoantibody to the

LDLreceptor

in our patient ^{leads to the} receptor internalization

^{and down-}

regulation as it occurs for LDL (1, 2) ^{remains to}

be addressed.

Finally, ^this autoantibody recognized ^{also the LDL}

^receptor

present on bovine adrenal cortexes. A mouse monoclonal

^an-

tibody against ^{the bovine} receptor cross-reacts with

^thehuman,

bovine, and dog ^LDL receptor (27) but does not

recognizethe

rabbit receptor. ^{We are} currently ^{addressing the}

^{questionas to}

whether patient ^F.F.'s IgA has a broad interspecies specificity.

In summary, we have described in the plasma

of a hyper-

cholesterolemic patient ^the presence of autoantibodies

^{to the}

LDL receptor that inhibit the in vitro catabolism

ofLDL. The

clinical history ^ofthe patient is similar to

that

of

the homozygous

and heterozygous ^{forms of} hypercholesterolemia.

^Screening

among ^the nonfamilial forms of moderate

^{and severe hyper-}

cholesterolemia may result in the discovery of

other^patients.

Acknowledgments

The authors wish to thank Mrs. Silvana Magnani ^{for typing}

^themanu-

script.

This work was supported ⁱⁿ part by ^a grant ^{to Dr. Catapano}

^from

P.

F. Malattie Degenerative ^{No. 850049356 and from the}

^Ministero

Pubblica Istruzione.

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