Autoantibodies to the low density lipoprotein receptor in a subject affected by severe hypercholesterolemia

Autoantibodies to the low density lipoprotein

receptor in a subject affected by severe

hypercholesterolemia.

A Corsini, … , R Fumagalli, A L Catapano

J Clin Invest.

1986;78(4):940-946. https://doi.org/10.1172/JCI112684.

We studied a 32-yr-old man with a benign paraproteinemia (IgA) affected by severe

nonfamilial hypercholesterolemia. In vitro experiments 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 to 70%) 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 paraprotein 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 that this autoantibody may be responsible for the severe nonfamilial

hypercholesterolemia of the patient.

Research Article

Find the latest version:

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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

tohave

auto-antibodies

tothe LDL receptor. In

this

articlewereport ona

subject

affected by

severe

nonfamilial

hypercholesterolemia

whose serum

contains

a

paraprotein

that

binds

tothe LDL

re-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).Kitsfor

en-zymatic determinationofplasma 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 inourlaboratoryinrabbitsagainst pu-rified apoBand 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.

940 Corsinietal.

J. Clin. Invest.

© The AmericanSocietyforClinicalInvestigation,Inc.

0021-9738/86/10/0940/07

$1.00

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-containing lipo-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 usinga LDL-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 normolipidemic

clin-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 the

ac-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 determinedas de-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 withafixed

con-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 cholesterolwas640

mg/dl.

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, anddegradationwere de-....<, termined as described in Methods. Each

pointis themeanoftriplicate determi-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_-0

Q

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

(.)

LPDSonthe deg-radationof

'25I-LDL

by cultured skin fibroblasts. Cellswere

preincu-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.LDLdegradationwas

determinedasdescribed in Methods. Eachpointis themeanof

tripli-catedeterminations that didnotdiffer by>10%.

942 Corsinietal.

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.

Cellswere

preincu-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 IgAas

de-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.Eachpointis the

meanof triplicate determinationsthatdidnotdiffer by

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 not

as-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 witha

mem-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 withamedium

con-tainingalbuminfor 24hand thebindingdeterminedat

370C

as

de-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 M

gly-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).

o

I0

- 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.Forthe ligand-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

Figure8.Binding of

"25I-labeled

IgAisolated frompatientF.F.'s LPDS to the LDL receptor. IgA were labeledwith 1251 asdescribedin Meth-ods. 5, 10, 15, and 10

Ag

of membraneprotein(lanesA-D)were ap-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 Mgof

mem-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

otherpatients.

Acknowledgments

The

authors wish

to thank Mrs. Silvana

_Magnani

for typing

the

manu-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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