Molecular basis of complement C3 deficiency in guinea pigs

Molecular basis of complement C3 deficiency in

guinea pigs.

H S Auerbach, … , A Dodds, H R Colten

J Clin Invest.

1990;

86(1)

:96-106.

https://doi.org/10.1172/JCI114721

.

In experiments to ascertain the biochemical basis of a genetically determined deficiency of

the third component of complement (C3) in guinea pigs, we found that C3-deficient liver and

peritoneal macrophages contain C3 messenger RNA of normal size (approximately 5 kb)

and amounts, that this mRNA programs synthesis of pro-C3 in oocytes primed with liver

RNA and in primary macrophage cultures. In each instance, heterodimeric native C3 protein

was secreted with normal kinetics but the C3 protein product of the deficient cells failed to

undergo autolytic cleavage and was unusually susceptible to proteolysis. These data and a

selective failure of C3 in plasma of deficient animals to incorporate [14C]methylamine

suggested either a mutation in primary structure of the C3 protein or a selective defect in

co-or postsynthetic processing affecting the thiolester bridge, a structure impco-ortant fco-or C3

function. A mutation in the primary structure of C3 was ruled out by comparison of direct

sequence analysis of C3 cDNA generated from two C3 deficient and two C3 sufficient

guinea pig liver libraries. Three base pair differences, none resulting in derived amino acid

sequence differences were identified. Finally, restriction fragment length polymorphisms

were identified in the C3 gene that are independent of the deficiency phenotype. This

marker of the C3 gene permits testing of these hypotheses using molecular biological and

classical genetic […]

Research Article

Find the latest version:

Molecular Basis of Complement C3 Deficiency in Guinea Pigs

Harvey S.Auerbach, Reinhard Burger,*Alister

Dodds,*

and HarveyR.Colten

EdwardMallinckrodtDepartment ofPediatrics, and tThe Department ofGenetics, Washington UniversitySchool ofMedicine, St. Louis, Missouri63110;*TheDepartment ofImmunology, Robert Koch Institut, Berlin, Federal Republic of Germany

Abstract

In

experiments

to

ascertain

the

biochemical

basis of a

geneti-cally

determined

deficiency of the third component of comple-ment

(C3)

in

guinea pigs,

we

found

that

C3-deficient

liver and

peritoneal macrophages

contain

C3

messenger RNA of normal

size

(-

5

kb)

and amounts, that

this

mRNAprograms

synthe-sis

of

pro-C3

in oocytes

primed

with

liver

RNAand in

primary

macrophage cultures. In each

instance,

heterodimeric

native

C3

protein

was

secreted with normal kinetics

but

the

C3

pro-tein

product

of

the

deficient

cells

failed

to

undergo autolytic

cleavage and was

unusually susceptible

to

proteolysis.

These data

and

a

selective failure of C3

in

plasma

of deficient

animals

to incorporate

1"4Cjmethylamine

suggested either

a

mutation

in primary structure

of

the

C3

protein

or a selective

defect

in co-or

postsynthetic processing affecting

thethiolester

bridge,

a structure

important for C3 function.

A

mutation

in the

primary

structure

of

C3

was ruled outby

comparison

of direct sequence

analysis of C3

cDNA

generated from

two

C3

deficient and

two

C3

sufficient

guinea pig

liver

libraries.

Three base

pair

differ-ences, none

resulting

in

derived amino acid

sequence

differ-ences were

identified. Finally, restriction fragment length

polymorphisms

were

identified

in the

C3

gene that are

inde-pendent of

the

deficiency phenotype. This

marker

of

the

C3

gene

permits testing of

these

hypotheses using molecular

bio-logical and

classical genetic

methods. (J.

Clin.

Invest. 1990.

86:96-106.) Key

words:

immunodeficiency

*

complement

com-ponent C3

Introduction

The

importance of

the complement

(C)'

system

in

host

de-fenses and

immunopathology has

been

fully

appreciated in the

study of congenital

and

acquired complement deficiencies.

The

third

component

of complement (C3) in particular

plays a

pivotal

role

in

many

facets of

the

inflammatory

response.

Alper et al. (1, 2)

first described

homozygous

deficiency of

C3 in a

child

with

a

history of

recurrent

infections.

The principal

Addressreprintrequests to Dr.Auerbach, Departmentof Pediatrics, Washington University School of Medicine, 400 S. Kingshighway Blvd., St. Louis, MO 63110.

Receivedfor publication 16June 1989 and in revisedform 13 March 1990.

1. Abbreviations used in thispaper: C, complement; C3, third

compo-nentof complement; C3D, C3 deficient; DOC, sodium deoxycholate; PCR, polymerase chain reaction.

pathogens

affecting

this child,

as

in

most

C3-deficient

individ-uals

(3-6),

were

encapsulated

bacteria,

thus

helping

to

estab-lish in vivo the importance of C3 in

opsonization,

phagocyto-sis,

and

intracellular

killing of these

organisms.

However, sub-sequent reports

of

C3-deficient individuals have presented

conflicting data

on

defects

in other C3-dependent

functions

such

as

immune adherence, development of

leukocytosis

in

response to

infection, vasopermeability,

and

antibody

re-sponse

(1-1

1). One possible

explanation

for these

conflicting

data is that, in C3 deficient individuals, C3 is

synthesized

and

secreted, albeit

at

markedly reduced

rates.

Some C3 functions

might therefore

be

retained, especially in extravascular sites.

This suggestion is supported by observations that

monocytes

isolated from individuals with

<

0.0 1% normal

serum

levels of

C3, produced C3

at a rate - 25%

of

normal

(12). Thus,

al-though the hepatocyte is the

primary

source

of plasma C3,

studies of C3

biosynthesis

in

monocytes,

fibroblasts and

syno-vial tissue from patients with rheumatoid arthritis (13)

sug-gested that

regulation

of C3

synthesis

is

independent

in

hepa-tocytes

and

other

cells

in

extrahepatic

sites. Ethical

consider-ations and other technical

constraints,

however, did

not

permit further

exploration

of these

questions

in

C3-deficient

humans.

An

isolated genetically determined deficiency of

C3 in

the

guinea pig

has

recently

been

described

(14).

These

animals

were

derived from

an

inbred strain

2 colony.

Sera

from

C3

deficient

(C3D)

guinea pigs

contain

5%

of normal C3

as

assessed

functionally and antigenically, i.e.,

the

C3

appeared

to have

approximately normal specific hemolytic activity.

Pre-liminary studies

(14)

suggested

that

peritoneal macrophages

and

hepatocytes from the

C3D

animals produced normal

amounts

of C3

and

studies

of the clearance of

1251I-radiolabeled

normal

guinea pig C3 in

C3D

guinea pigs demonstrated

a

normal

catabolic

rate. It

is

interesting

that

C3-deficient

guinea

pigs mounted only

a

minimal primary antibody

response to X

X 174, a T

cell-dependent antigen,

and

failed

to undergo

iso-type

switching

from IgM

to

IgG

with

booster

immunizations

(15).

This inadequate antibody

response has

also

been

found

in

deficiencies of complement components of

the

classical

pathway

(16,

17); all

are

believed

to be

due

to

deficiency

of

activated C3.

The availability of

a

small

animal model deficient in C3

prompted further studies of

the molecular

basis for C3

defi-ciency.

In

this

report we show

that cells from

C3D

animals

express

C3

mRNA

similar in size

and amounttothat

found in

normal

cells. The

C3D

mRNA programs

synthesis of

a

C3

protein that fails

to

undergo autolytic

cleavage

after

heating

in

SDS,

a property

associated

with the

presence

of

a

thiolester

bridge

in

the

a

chain.

In

addition,

a

restriction

length

poly-morphism of

the

C3

gene

has

been

identified in C3D

and

some, butnot

all,

C3-sufficient

guinea pig

strains,

providing

a marker

for the C3

gene

independent

of

the

deficiency

pheno-type.

J.Clin.Invest.

©TheAmerican SocietyforClinicalInvestigation,Inc. 0021-9738/90/07/096/11

$2.00

200-

92-68-

_{_.}

_b

46-

30-NN

C3DC4D1

N

03G

C4D

IC

XC

Figure 1. Biosynthesis of C3byadherent peri-toneal macrophages fromnormal(N), C3 deficient (C3D), and C4 deficient (C4D) guinea pigs.Autoradiograph of reduced SDS-PAGE analysis of intracellular (IC) pro-C3 and extra-cellular (XC) native C3 precipitated from cul-ture media.

68-

-

-

46-

30-kD

cIiv-,6,-',

200

-11

-a

I

>aS

",

,"

IC XC

ICXC

I

Figure3.Biosynthesis

ofC4 in normal and C3D guinea pig perito-nealmacrophages.

Analysis of intracellular (IC) pro-C4 and

extra-cellular (XC) native C4

onSDS-PAGE.

Methods

[IC

X

C

XC

N

C3D

Figure2.Biosynthesis

ofC3 inXenopus

oo-cytesprimedwithliver

mRNAfromnormal

(N)orC3Dguinea pigs.

Pro-C3 (intracellular)

and C3(extracellular)

areanalyzed by

SDS-PAGE under reducing

conditions.

Cell culture.Peritoneal cellswereharvested in HBSS without calcium and magnesium from normal Hartley, C3D, and C4D guinea pigs (C4D guinea pig plasma contains C3atnormalconcentrations). The cellswereisolated bycentrifugationandresuspendedinmedium 199 (GibcoLaboratories,GrandIsland,NY)with50 Upenicillinand 50Ag

streptomycin (Gibco Laboratories) per ml supplemented with 10% heatinactivated (56°C for 2h)fetal calfserum(IrvineScientificCo.,

Santa Ana,CA)to 4X106/ml.0.5mlofthe cellsuspensionwasplaced

in 16-mm flat-bottom tissue culture wells(ComingGlass Works,

Coming, NY) and the cellswereallowedtoadhere for2h. The

mono-layerswereextensivelywashedwith HBSS(viabilitywasassessedby

trypan blueexclusion, differentialcountsbyWright'sstain and stained for esterase) and incubated forspecifiedtimeperiodsat37°Cin Dul-becco's modified essential medium(DMEM; Gibco) lacking methio-nine and containing

[35S]methionine

(- 1,000

Ci/mmol;

ICN

Bio-medicals,Irvine, CA).Attimed intervals mediawerecollected,cleared of cell debris by centrifugation at600 g for 15 min, then stored at

-70°C.Macrophagecultureswerethen washed

extensively

with HBSS andlysedbyfreeze-thawinginasolution of 50mM

Tris-HCI (pH

=7)

containing 100 mMKC1,0.5% Triton X-100 (SigmaChemicalCo.,St. Louis, MO) 0.5% sodiumdeoxycholate(DOC; Sigma),2mMPMSF

(Sigma),and 10 mM EDTA.

Immunoprecipitation andanalytical gel

electrophoresis.

Radiola-beledproteinswereassayedas

previously

described

(18).

Briefly,

ali-quots of celllysateormediumwereincubated

overnight

at

4°C

in 1% Triton X-100/1%SDS/0.5%DOC withexcess

antibody.

Asuspension

of formalinfixedStaphylococcus aureus

bearing protein

A(IgSorb;

The EnzymeCenter, Boston, MA)wasthenaddedtoeachsampleand incubatedfor 1hat4°C.After extensive

washing,

the

antigen-antibody

complexwasreleasedbyboilingin

sample

buffer

(0.05 Tris, pH

=6.8, 1% SDS, 10% glycerol, 5%

2-mercaptoethanol,

and 0.01% bromo-phenolblue), andappliedto9% SDS-PAGE under

reducing

condi-tions.Afterelectrophoresis, gelswerestained in Coomassie brilliant

GeneticDeficiencyofComplementC3 Protein in GuineaPigs 97

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:X:: W=as¢L atac:SX.:

3 k @ ei i?aa 3 jrtg;

i >XAsW S E

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Normal

kD

200-

"

92-. /J

92- t

68-

46-

30-j0 30 60 120

240_1

10

30 (min)

IC

C3D

kD

200

-60 120

_240i

(min)

xc

OM

92-Freund'sadjuvant. Blood was drawn 5 d after immunization, and IgG antibody was purified by ammonium sulfate precipitation followed by ion exchange column chromatography (DE52; Pharmacia Fine Chem-icals,Piscataway, NJ). Antibody specificity was determined by inhibi-tion of complement lysis and ouchterlony analysis.

C3proteolysis. Mediawereharvested from normal HartleyorC3 deficient guinea pig macrophage cultures that had been incubated with

[35S]methionine for4 h. Aliquotswere incubated with appropriate concentrations of TPCK-treated trypsin (Sigma) for 4-5 minatroom temperature. The reactionwasstopped by addition of DFP (final

con-centration 1M)at370C for 20min. Radiolabeled C3 proteinwasthen isolated and applied to SDS-PAGEasdescribed above. For autolytic cleavagestudies, 50-Ml aliquots of media from normal HartleyorC3D macrophagespreviously incubated with

[35S]methionine

wereaddedto

750 ul of 0.1 M Tris acetate, pH = 8.5, and 0.1% SDS. After 20 min incubationat750C, 200

,d

ofastocksolutiontoyield final

concentra-tions of 1% Triton X-100, 1.0% SDS, 0.5% DOC,wasadded. Radiola-beled C3 was then analyzed by immunoprecipitation, SDS-PAGE, and

fluorography, as described above. 10 ug ofRNA were subjectedto

Northern blot analysisaspreviously described and probed with 32P-la-beledmouseC3 cDNA generously donated by M. Takahashi and M. Nonaka (KanazawaUniversity, Kanazawa, Japan) or witha 32P-la-beled fulllength guinea pig C3cDNAprobe.

Xenopus oocyteswereinjected with 50 ng of mRNA per oocyte. The oocyteswereincubated in microtiter wells (Corning Medical In-struments)containing 40 Ml of Barth's medium/4 oocytes per wellat

25°C for 6 h to allow puncture healing and recruitment of mRNA; the oocyteswerethen refed with DMEMcontaining 1 mCi/ml of

[35S]-methionine. After 50 h of incubation at 25°C,the media were

re-moved. The oocyteswererinsed with PBSandhomogenizedwitha

loosefitting Douncehomogenizer(FisherScientific,Pittsburgh, PA)in 0.5 mlof 15mMTris,pH=7.5, 50 mMNaCl,and 2 mMPMSF. The homogenateswerecentrifugedat100,000g for5min andaliquotsthen immunoprecipitated for C3,asdescribedabove.

68-

46-

30-10

30 60 1202401L0 30 60 120

_240j

(min) (min)

IC

xc

Figure4.Kinetics of C3 secretion from normal andC3D

macro-phages.Cells were incubated with[35S]methionine-containingmedia for 30 min, then washed and refed with fresh unlabeled media. C3 wasimmunoprecipitated fromintracellularandextracellular samples

atindicated time points.

kD

92-__

_N

68-

_to

46

-blue,destained, impregnatedwith2.5-diphenyloxazole

(EN3HANCE;

NewEngland Nuclear, Boston, MA), and dried for fluorography on XARfilm (Eastman Kodak Co., Rochester, NY).

Antibodies. Antiserum to guinea pig C4wasraised inC4-deficient guineapigs (19)bypreviouslydescribed methods(18).Antiserum to

guineapigC3wasraised in albinorabbits by subcutaneous injection of 1.0 mlofamixturecontaining equal volumes of purified guinea pig C3, 1.0 mg/ml (supplied by M. Thomas, Washington University SchoolofMedicine,St. Louis, MO)andcompleteFreund'sadjuvant,

followed bytwoinjectionsat2-wkintervals of0.5 mg C3 in incomplete

30-,N

C3D

N

C3D,

Before

After

Figure 5.Autolytic cleavageof C3synthesizedin normal and C3Dmacrophagecultures. SDS-PAGE of C3

immunopre-cipitatedfrom extracellular mediabefore and after incuba-tion in 0.1%SDS,0.1Mtris acetate,pH=8.5,at75°Cfor 20 min.

Figure 6. Incorporation of

["4C]-methylamine into guinea piga2

macroglobulin,and the achainof

C3and C4. Normal and C3D guinea pigsera werefractionatedon aQ Sepharose column. Fractions

were incubated with

[14C]-methylamineandanalyzedby SDS-PAGE andautoradiography. (A) Normal, (B) C3 deficient guinea pig

serum. No

['4C]methylamine

was

incorporated intothe C3inthe

C3Dserumfractions (B).

DNA analysis. 1 g liverfragments from C3D, C4D, C2D, and normal Hartley guinea pigsweredigestedin0.5% SDS, 0.5 M EDTA, pH=8, and 100Ag/mlproteinase K foratleast6 hat50'C.DNAwas extracted from digested solutions by phenol/chloroform, dialyzed against 1,000 times the volumeof10mMTris, pH 8, 1 mMEDTA, and storedat4VC.Restriction endonuclease digestion ofI0-ugaliquots wasperformed formorethan 18 h, and the samplesweresubjectedto agarose gel electrophoresis, transferredto nitrocellulose paper, and hybridized witha32P-labeled fulllength guinea pig C3 cDNA probe.

Methylamine incorporation inserumC3.A4cmX 1cmQ Sepha-roseFast Flow (Pharmacia Fine Chemicals) columnwasequilibrated with 10mMTris, 5 mM EDTA, 50 mM e-aminocaproic acid, 0.2% Na

azide, 0.2 mM PMSF, 100mMNaCl, pH7.4. 2 mlofnormalorC3D guinea pigserumwasapplied andelutedwitha20-mlgradientto500 mMNaCl in thesamebuffer. -ml fractionswerecollected. For me-thylamine incorporation, 22.5-,ulaliquotsofeachfractionwere incu-bated with 2.5 of 2 M Tris, pH 8.8, containing 50 mM [14C]-methylamine, 48 mCi/mmol (New England Nuclear) for 30 minat

37°C, then were reduced and subjectedto gel electrophoresison a modified Laemmli gel (20).ThegelwasstainedwithCoomassieblue, dried, andexposedtox-rayfilmat-70°C.

a-a'-

_p

go.

ta .

RNA.Total liver RNAwasisolated from three C3-sufficient

(Hart-ley,oneanimal, C4-deficient subline of NIH multipurpose guinea pigs,

twoanimals) andtwoC3-deficient guinea pig livers accordingto pre-viously described techniques (21). The polyadenylated RNA fraction wasisolated by affinity chromatographyonoligo-dT cellulose (22).

Construction of cDNA libraries. ThecDNA synthesissystemwas purchased from Bethesda Research Laboratories (Bethesda, MD); re-strictionenzymes,T4 DNA polymerase, and T4 DNA ligasewere pur-chased from New England Biolabs (Boston, MA); dNTPswere pur-chased fromPharmacia;S-adenosyl methionine and Eco RI methylase were purchased from Promega Co. (Madison, WI). Radionuclides [32P]a-dCTP (800 Ci/mmol) and [35S]a-dATP werepurchased from NewEngland Nuclear; oligo-dT cellulosetype3waspurchased from Collaborative Research (Bedford, MA); nitrocellulose HAW filters werepurchased from Millipore Corp. (Bedford,MA);phosphorylated Eco RIlinkers, lambda ZAP, XL- I Blue, and Gigapakwerepurchased from Stratagene (La Jolla, CA);sequenaseDNA sequencing kitwas purchased from U. S. Biochemicals (Cleveland, OH); SP6/T7 Tran-scription kitwaspurchased fromBoehringer-Mannheim(Mannheim, WestGermany).

TheC4- andC3-deficient polyA'wasusedtodirect cDNA synthe-sis, whichwasthencloned into lambda ZAP byamodification of the methods described by Okayama and Berg (23, 24). Oligo-dT (12-18) orspecific oligonucleotides designed from the guinea pig C3sequence were utilized as primers for first strand synthesis ingenerating the

primer extension libraries fromoneadditional C3-sufficient (C4 defi-cient) and C3-deficient animal.

IsolationofC3 cDNA clones. cDNA librarieswereplatedata den-sity of 8,000 plaque-forming unitsper 100-mmdish. Developed plaques weretransferred to nitrocellulose filters. Filterswere dena-tured, neutralized, baked invacuoat80°C for 2 h, prehybridized for 2 hat42°C in 6x SSC, 1X Denhardt's solution, 0.5% SDS, 1%dextran

A.

18S-Figure7.Trypsincleavageof

C3 fromnormal(N) and C3D guinea pig peritoneal macro-phages. Native C3 immuno-precipitated before and after incubation withtrypsin.

Tryp-sincleavagegeneratesana'

N C3D N C3D fragment

noimmunoprecipitableC3

Before

Af ter

protein in the C3Dsample.

28s-B.

Figure8. Northern blot analysisofequivalent

amounts of RNA

iso-latedfrom normaland C3D liver visualized under ethidiumstain

(A) and autoradiogra-phy (B).Theblotwas probedwithafull N C3D lengthC3 cDNA.

GeneticDeficiency ofComplement C3Protein in GuineaPigs 99

a2M

--

-__.w

C3

04

NORMAL

C3

D

US&46'..

qmbo,...-A.

PST 1

23

-9.4

-'

6.5-4.3

.-4.3

-

2.0-

0.5-A

B

C

D

E

F

B.

ECO RI

23-94:

6.5-

_

4.

2.3

-

2.0-Constructionofpolymerasechainreaction (PCR) fragments. Oligo-nucleotides(29")containing guinea pigC3 sequences with 5'

restric-tion endonuclease recognition sites wereused to prime first strand

cDNAsynthesisfromC3-deficientandC3-sufficient(Hartley) guinea piglivermRNA from additional animals ofeachtype. The RNA-DNA

hybridwasamplified inabuffercontaining 50mMKCI, 35mM Tris

(pH =8.3), 2.5 mM MgCl2, bovineserumalbumin (100,ug/ml), 1.25 mNdNTPs,sense andantisense primers (500ngeach)and 5 U Taq

polymerase ina totalvolumeof 100

til.

Thereactionswerecarriedout

for 40 cycles of denaturation (2 minat950C), primer annealing (3 min

at610C) and primer extension(7 min at 720C), in an automated

thermalcycler (Perkin Elmer-Cetus, Norwalk, CT). The productwas

subjectedto electrophoresison a 1%agarosegel (Seaplaqueagarose;

FMC Bioproducts, Rockland, ME), and the appropriate sized

frag-ment isolated, phenolextracted andethanolprecipitated.The DNA was digested withthe appropriate restriction enzyme, ligated into

bluescriptphagemid, and sequencedasabove.

Results

C3

biosynthesis.

Net

biosynthesis of C3

was

assessed

in normal Hartley, C3D, and C4D guinea pig peritoneal cell cultures pulsed with [35S]methionine for 4 h. Theamountof radiola-beled pro-C3 (intracellular) and native C3 protein (extracellu-lar)produced was quantitatively similar, and the apparent mo-lecular mass ofa chain (1 15 kD) and ,B chain (65 kD) were indistinguishable in cells from the normal and C3D guinea pigs (Fig. 1). In this experiment, pro-C3 produced by the C4D cells was apparently present in reduced amounts but the amountof extracellular native C3 was similar to that produced by the normal and C3D cultures. Thisdecreasedamount of pro-C3 in the C4D cells was not a

reproducible finding

since in four other experiments, the amountofpro-C3 synthesized in C4D cells was similarto thatproduced in normal and C3D cells. In each culture (normal, C4D, andC3D),thenet

incor-0.5

-A

B

C

D

E

F

Figure9.Southern blotanalysisofPst I(A)andEco RI(B)

digests

of

DNAfrom (A) normal outbredHartley, (B)strain2,

(C)

strain

13,

(D)C2-deficient,(E)C3-deficient,and(F)C4-deficient

guinea

pigs.

Additional 2.3 kb(Pst1)and 1.6 kb(Eco

RI)

bandsarevisualized in theC3D and C4D lanes.

sulfate,100

_Ag/ml,

andhybridized inthesamesolutionat42°C usinga

radiolabeled

32P-labeled

mouseC3cDNAprobekindly suppliedby M.

Nonaka andM.Takahashi. Filterswerewashedat55°Cin 1XSSC, 0.1%SDSfor 1 hX2, andsubjectedtoautoradiographyusing Kodak

XAR-5film. Positivecloneswerepurified bytwosubsequentroundsof plating and screening. After purification, candidate C3 cloneswere digestedwithEco RIandsubjectedto 1% agarosegelelectrophoresis.

cDNAsequencing. 10 ggofcDNAwasself-ligated, sheared

ran-domly by sonication (six bursts of40 s), subcloned into

Ml

3mp18

accordingto methods described by Messing (25), andsubjected to dideoxy-sequencing.Inotherexperiments, double-stranded bluescript

phagemidsweredenatured in 0.2MNaOH, 1 mM EDTAfor5min,

ethanolprecipitated,and thensequencedasabove.

A

B

5' 3'

0 1000 2000 3000 4000 5000 base pairs

C3D

..._+ _ +5 C4D(NL)

5.1

E

83

T 1630G1 2789 C3D

i. NL(C4D)] 3 665 1165 1760 2440 3440 4240 5010

C .

C₆₀ 1235 1690 2500 3373 4273

Figure10. (A) Sequence strategyforC3D-and C3-sufficient

(C4-de-ficient, C4D) C3cDNA clones.Solidarrows representindividualgel

readings from randomly sheared(shotgun)fragments.Dashed arrows representadditionalsequenceobtained from oligonucleotide-directed sequencing. (B) Solid barrepresents sequenceofC3D (above)and C3

sufficient (C4D,below)C3 cDNA, with base pair differences identi-fied.Arrowindicates origin of primer extension libraries. (C) PCR-generatedfragmentsderivedfrom C3Dandnormalguineapig liver

mRNAforfurthersequenceanalysis.

100 Auerbach,Burger,Dodds,and Colten

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I

GeneticDeficiency

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ComplementC3 ProteininGuinea Pigs 101

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poration of

[35S]methionine

in C3 of a chain was substantially greaterthan

for

ft

chain. Two additional polypeptides (- 70 kDand48 kD) were detected in the normal and

C4D

butnot

the

C3D

extracellularculture media (see below).

Isolated

liver

poly

A'

selected mRNAfrom

C3D

and

nor-mal

Hartley

guinea pigs

generated comparable amounts of

pro-C3

and secreted

native

C3 when injected into Xenopus

oocytes

pulse labeled for 72

h

with

[35S]methionine

(Fig. 2).

The

size and subunit

composition of the

C3D

and normal

guinea pig liver-derived native

C3 were

similar,

but againthe

70- and 48-kD products

were only

visualized

when theoocytes

were

injected with

normal mRNA (even overexposed gels

failed

todemonstrate

70- and 48-kD

products

in C3D-injected

oocytes

[data

not

stown]).

Immunoprecipitation

of C4, a

complement

protein homologous with C3, from

thenormal

and

C3D cultures (Fig. 3)

revealed

comparable

amounts of

pro-C4

intracellularly

and

native

C4 inthe

extracellular

me-dium.

Secretion

of C3. To ascertain the rates of C3 secretion

in

normal

and

C3-deficient cells, peritoneal cell

cultures

were

pulse labeled for 30 min with

[35S]methionine

(500

MCi/ml),

washed,and

refed

with

medium

containing unlabeled

methio-nine. Cell lysates and

media

werethen

assayed

for radiolabeled

C3

protein

(Fig.

4). These data showed that the

kinetics

of C3

secretion and

net recovery

of C3 protein

were

similar in

the

C3D

and

normal

peritoneal

cells.

Autolytic cleavage and thiolester bridge. Since the 70- and

48-kD

polypeptides precipitable

with

antisera

to

C3 observed

in

Fig.

1 are

similar in size

to

the

previously

reported C3

autolytic

cleavage

products,

we

attempted

togenerate

these

fragments from

35S-labeled native C3

protein

produced in

C3D and normal

peritoneal

cell

cultures

by

incubating

the

media in

dilute alkaline SDS

at

75°C for 20

min

(21,

26).

Again

(Fig. 5), the 70- and 48-kD bands

were

visualized only

in the

normal but

not

in

the

C3D-derived culture media.

Methylamine incorporation

into

plasma

C3, C4,

and

a2

macroglobulin.

Previous

reports

have

shown that

C3D

guinea

pig plasma contains C3 both

functionally

and

antigenically

at

around

5%

of the level

found

in normals.

To

examine the

possibility

that

residual C3 in

plasma

from C3D

guinea

pigs

differed from that

produced

in

tissue

culture

the

following

experiment

was

performed.

Plasma

from

C3D

and normal

guinea

pigs

were

fractionated

on an

ion

exchange

column and

aliquots

of each fraction

examined

for the

ability

to

incorpo-rate

'4C-labeled methylamine,

a

function

of

an

intact thiolester

bridge.

Fig.

6 demonstrates that

methylamine

was

incorpo-rated

into

a2-macroglobulin

and

C4,

two

proteins

that

contain

thiolester bonds, in both normal and C3D

plasma;

methyl-amine

incorporation

into C3

was

detected

only

in normal but

not

C3D

plasma

even

when the C3D

plasma

fractions

were

concentrated

-

20-fold

(data

not

shown)

to account

for

the

difference in

content

of C3 in'the deficient

plasma.

Proteolysis. Trypsin cleavage

of native C3

protein

pro-duced

by

normal

guinea

pig peritoneal

cells

resulted

in

genera-tion of

C3b,

as

expected

(Fig. 7). However,

incubation of

the

C3D-derived native C3

protein

with between 0.01 and 0.1

g

of

trypsin

(10-fold

less than the minimal concentration

re-quired

for C3b

generation

from normal

C3)

resulted in

com-plete

loss

of

immunoprecipitable

C3

protein.

C3

transcription.

As shown in

Fig. 8,

Northern

blot

analy-sis

of total

RNA

isolated

from C3D and C3 sufficient

guinea

pigsrevealed mature C3 mRNA transcripts (- 5 kb)

compara-ble

in size

and amount.That is, C3 mRNA from normal and

C3D

guinea pig liver appeared

qualitatively

and quantitatively

indistinguishable. The ethidium-stained gel indicated that

comparable

amounts

of RNA

were

analyzed.

C3 gene structure.

DNA was isolated

from Hartley,

C2-de-ficient (strain 13), C4-deC2-de-ficient (strain

13), and

C3-deficient

(strain 2) animals, digested with

several

restriction

enzymes (Pst I, Eco RI, Bam HI, Hind III, Bgl I, and Sma I) and

sub-jected

to

Southern

blot

analysis (27) using

a

full

length normal

guinea pig C3

cDNA probe.

Fig.

9 shows

that

digestion with

either

Pst I or Eco RI resulted

in

restriction fragment

length

polymorphisms (additional

2.3 kb [Pst I]

and

1.6 kb

fEco RI]

bands)

in

the

C3D

and

C4D

samples.

Since

C3

protein

synthe-sis

and

C3

serum

concentrations

are

normal in

C4-deficient

guinea

pigs, this polymorphism is

not a marker

for

the C3

deficiency

phenotype.

Comparison

of C3 cDNA

from C3-sufficient and deficient

liver libraries.

17

C3

clones from the C3-deficient library and

25 clones

from

theC4-deficient library were detected; the larg-est

insert, 3.8

kb

in

the

C3-deficient

and 4.2 kb

in

the

C4-defi-cient libraries.

Inserts

from

the three largest clones of each

library

were

isolated

and

sequenced.

As

shown in

Fig. 10,

70%

of

the cDNAwas

analyzed

using

this

sequencing

strat-egy,

with

most

regions sequenced several times

on

both

strands. To

complete

the sequence,

oligonucleotides

were

constructed and used

as

primers for

dideoxy sequencing

di-rectly

from

phagemids.

Using this initial

strategy, the C3

cDNA

sequence extended from

the

poly(A)

tail through

the

750 and

1150

most5'

nucleotides of

the ,B

chain for

C3-defi-cient and C3-suffiC3-defi-cient clones,

respectively.

To complete the

C3

cDNA sequence,

primer extension

cDNA

libraries

wereconstructed

using

a

C3

specific

oligonu-cleotide (20 mer)

corresponding

to

positions

2469-2488

of

complete

guinea pig C3

cDNA sequence.

Clones isolated from

each

library

contained

sequences

that extended 29 nucleotides

5'

of the C3

signal peptide

and

overlapped

by

1312 and 1712

nucleotides, the largest clones from the deficient and

C3-sufficient libraries. Confirmation of

the normal and

C3

defi-cient

guinea

pig

C3

sequence was

accomplished by

direct

se-quence

analysis

of PCR

fragments

generated from C3

suffi-cient (Hartley) and C3 defisuffi-cient

mRNA

(Fig. 10).

The complete

C3-sufficient

(C4

deficient)

guinea pig C3

cDNA

sequence

and

derived amino

acid

sequenceareshown in

Fig.

1 1.

The

sequence spans

5081

nucleotides,

from

29 base

pairs

upstream

of

the

signal peptide

to

the

beginning

of

the

poly(A) tail.

A

putative

polyadenylation

signal,

AATAAA, is

located 16

nucleotides

upstream

from the

polyadenylation

site,

and

is identical

to

the

polyadenylation

signal

described for

murine C3 and

Slp. The deduced C3

protein

sequence

consists

of

1666

amino

acids

in a

(3-a chain orientation with four

argi-nines interposed

between the

_#

and a

chains. The

coding

re-gion is,

therefore, equal in size

to

murine

C3

and

three amino

acid residues longer

than human

C3. Overall,

77-80%

nucleo-tide

and

amino acid

identity

wasobserved

for human,

mouse,

and

guinea

pig

C3.

Complete nucleotide

identity

between

human and

guinea pig

C3

sequences was

found

at

the

thioles-ter 1

5-base

pair

segment

within the

flanking

45 base

pairs

in

each

direction. Guinea

pig

C3

shares

other structural

and

functional characteristics with

human

and

murine

C3,

includ-ing

two

asparagine carbohydrate attachment sites

at

positions

944

and 1620,

a

trypsin cleavage site

between

lysine

at position 1006 and histidine at 1007, and two Factor I cleavage sites at

positions 1310

and

1327.

C3-sufficient (C4 deficient)

and

C3-deficient

guinea pig C3 cDNA sequences were

compared. Only

three

nucleotide

dif-ferences,

at

positions 83, 1630,

and

2789

were

found (Fig.

10). These

nucleotide differences

are

"silent"

in that they do not

result in

a

difference in the derived amino acid

sequences. The sequence

of PCR fragments from

an

additional C3-deficient

liver mRNA

was

identical

to the

sequence generated from

the

C3-deficient

cDNA

and primary extension libraries.

The

Hartley C3 PCR

fragments

showed

identity

with the other

C3-sufficient (C4 deficient) C3

cDNAsequence except at

po-sitions 83 (T

to

C); i.e.,

a

substitution identical

at

this position

with

the

C3-deficient

sequence.

Discussion

Several

possible explanations

were

considered

toaccount

for

discrepancies

between

serum

concentration of C3 and

rate

of

C3

biosynthesis

in

previous studies of

humans

with genetically

determined C3 deficiency (1-11).

However,

those experiments

necessarily monitored C3 biosynthesis

only at an

extrahepatic

site (blood monocytes) since ethical considerations

precluded adirect test of thehypothesis that C3 synthesis rate in liver was more

closely

related to the

deficiency

than C3

synthesis

in

other

tissues. One possibility that

was not

considered

at that time was

that

a

defective

C3

protein

susceptible to

accelerated

catabolism

was

produced by the deficient

cells even

though

the

newly

synthesized C3 protein appeared

grossly normal by

esti-mates

of size and subunit composition.

Moreover, a near

nor-mal

rate

of catabolism of

normal

purified radiolabeled

C3

protein

was

observed in

a

C3-deficient patient (3). Similar

findings

were

obtained in the initial studies ofC3 catabolism in

C3-deficient

guinea

pigs (14).

Preliminary

data

from those

studies also

suggested that C3

synthesis

is normal in

C3-defi-cient

guinea pig

hepatocytes.

In

the

present report we

established that liver and

perito-neal cells

from

C3-deficient,

normal,

and

C4-deficient guinea

pigs (a) contain C3

specific

mRNA

indistinguishable

in

size

and

quantity

by Northern blot

analysis,

and

(b) synthesize

pro-C3

and secrete

native C3

protein

at

similar

rates.

Although

the

C3

protein

produced by

the

C3D

peritoneal

cells

or

se-creted

by

oocytes

primed with C3D liver

mRNA appears

nor-mal in

size and

subunit

composition

it

differs from normal C3

in

at

least

two respects.

That

is, the C3D-derived C3

protein

fails

to

undergo

autolytic

cleavage and is

unusually susceptible

to

proteolytic

digestion.

C3, C4, and

a2

macroglobulin

are

proteins

that sharean

unusual structural

feature,

a

thiolester

bridge, important

in

their

respective

functions.

The

phenomenon

of

autolytic

cleav-age

in

dilute alkaline SDS is associated with

the presence

of

an

intact thiolester

bridge.

The

C3

protein

produced in

C3-defi-cient

guinea

pigs, by this

criterion,

appears to

display

a

defect

in

the thiolester bridge

or

its

accessibility. Support for this

concept and

for

the

selectivity of

the

defect is

provided by

demonstrating incorporation

of

methylamine

into C4 and

a2

macroglobulin,

but

not

into C3 of C3 deficient

plasma

even when

the

samples are

concentrated

-

_20-fold

_to

_increase

the

C3

contentin the

C3D

sample

to near

normal levels.

An

ab-normality

in

primary

or

higher

order

structure

of the

C3-defi-cient C3 protein is

alsosuggested

by the unusual susceptibility

of this

protein

to

proteolysis.

The rate

of

in

vivo catabolism of

this abnormal C3 protein is

unknown because

previous

esti-mates

of C3 metabolism

in the

deficient animals

as in studies

of C3D humans

were

obtained using normal purified

radiola-beled

C3 protein (14).

These

data

are

consistent with

(a) a

mutation

in the

coding

region of

the

C3

gene

leading

to a

defect in primary

or higher

order

structure

affecting the thiolester bridge

or

its

accessibil-ity,

(b)

a C3

specific

co- or

post-translational processing defect

that affects the thiolester

bridge;

i.e.,

a

genetic

defect unlinked

to the

C3

gene. The

finding

of

a

restriction fragment length

polymorphism

(RFLP) that does

not correlate

with

the

C3

deficiency phenotype

lends support to the latter

hypothesis.

That

is, the

RFLP

provides

a

marker for the C3

structural gene

distinct

from the deficiency phenotype. Sequence analysis of

C3

cDNA

from C3 deficient

guinea pig liver libraries

ruled out a

mutation

in

primary

structure

of

the C3 gene as the basis

for

this

genetic

defect.

Sequencing

of C3

cDNA

clones from

twoseparate

C3

defi-cient liver libraries and from PCR

fragments

generated from

a

third

C3-deficient

liver

mRNA

preparation

established

that

the C3

coding regions in

C3-sufficient

and deficient

guinea

pigs is identical

except

for

positions

83, 1630, and 2789.

None

of these

substitutions result in

an

amino acid difference.

At

position

83 the Hartley (C3

sufficient)

sequence

is identical

to the

C3-deficient

sequence.

These

data thus support

the

hy-pothesis that C3

deficiency

in

guinea

pig is

due to a co- or

postsynthetic processing modification

that renders

the C3

pro-tein

highly

susceptible

to

proteolysis.

The

biochemical mechanism

by which the

thiolester

bridge

is generated in C3 is uncertain.

Kahn

and

Erickson (28)

sug-gested that isomerization of

a

lactam

ring

togenerate

the

thio-lactone

proceeds spontaneously under

physiological

condi-tions, though they could

not rule out an

enzymatically

facili-tated

process.

lijima

et

al.

(29)

on

the other hand

provided

tentative evidence for

participation

of

a

cytoplasmic

factor

(possibly

an

enzyme) in

the

formation of

the

C3

thiolester.

Heretofore,

investigators

have

assumed

but not

tested

the

as-sumption

that

the mechanisms for

generation

of

the

C3,

C4

and

a2

macroglobulin

thiolesters

are

identical. Since the

mu-tation

accounting

for C3

deficiency

is

notin the

coding region

of the C3

gene,

this defect

provides

a

probe

to

examine

co-and

postsynthetic processing

of this

important

group

of

proteins.

Molecular

biological

and classical

genetic

studies

are

under-way to test

this

hypothesis.

Acknowledgments

We thank Barbara Pellerito for excellent secretarial assistance. This workwassupported by National Institutes of Healthgrants AI-24836andHL-37591.

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