Expression of complement proteins C2 and factor B in transfected L cells

Expression of complement proteins C2 and

factor B in transfected L cells.

D H Perlmutter, … , J G Seidman, D D Chaplin

J Clin Invest.

1985;

76(4)

:1449-1454.

https://doi.org/10.1172/JCI112123

.

Factor B and C2 are structurally and functionally similar complement proteins encoded by

genes that are closely linked within the class III region of the major histocompatibility

complex (MHC). In this study, restriction endonuclease digestion of cosmid DNAs isolated

from an H-2d murine genomic library indicated that the chromosomal organization of these

two genes was similar in mouse to that in man. To further characterize their expression,

cosmid DNAs encoding human and murine factor B and C2 were introduced into mouse L

cells by DNA-mediated gene transfer. Factor B expression was demonstrated in cells

transfected with either the human or the murine gene, but not in cells transfected with a

control plasmid. Synthesis and secretion of factor B by L cells transfected with the human

and murine cosmids was similar to that of human and murine cells in primary culture. An

interspecies variation between human and murine factor B was identified and reproduced

with extraordinary fidelity by the mouse fibroblast. In contrast, C2 RNA and protein were

expressed by L cells alone and by L cells transfected with a control plasmid, as well as by L

cells transfected with cosmids encoding human and murine complement genes. Expression

of the transferred human C2 gene was demonstrated by the presence of a new distinct C2

RNA transcript and secretion of biologically active […]

Research Article

Find the latest version:

Expression of

Complement Proteins C2

and

Factor

B in Transfected L Cells

David H.Perlmutter, HarveyR.Colten, DarioGrossberger,JackStrominger,J. G.Seidman, and David D.Chaplin

DivisionsofGastroenterology, Nutritionand CellBiology, Children'sHospital,InaSue PerlmutterCysticFibrosis Research Center,

DepartmentofBiochemistry and MolecularBiology,Harvard University; DepartmentsofPediatrics andGenetics, Harvard Medical

School, Boston,Massachusetts;and Howard Hughes Medical Institute and Department ofInternal Medicine, Washington University SchoolofMedicine, St. Louis, Missouri 63110

Abstract

Factor Band C2 arestructurallyandfunctionallysimilar com-plementproteins encoded bygenes that arecloselylinked within the class III region of the major histocompatibility complex

(MHC).Inthis study, restrictionendonucleasedigestionof

cos-midDNAsisolated fromanH-2d murinegenomic library indi-cated thatthe chromosomal organizationofthese two genes was similar inmouse tothat in man. Tofurther characterize their

expression,cosmid DNAs encoding human and murine factor B and C2 were introduced into mouseL cellsbyDNA-mediated genetransfer. Factor B expression was demonstrated in cells

transfected with either thehumanorthe murine gene, butnot

in cellstransfected witha controlplasmid. Synthesisand

secre-tionof factorBbyLcellstransfected with the human andmurine cosmidswassimilartothatof humanandmurinecells in

primary

culture. Aninterspecies variation between human and murine factorB wasidentified and reproducedwith

extraordinary

fidelity bythe mousefibroblast. In contrast, C2 RNA andproteinwere

expressed by L cells alone and

by

L cells transfected with a

controlplasmid,aswell asbyLcells transfected with cosmids

encodinghumanand murinecomplementgenes. Expressionof thetransferredhumanC2gene wasdemonstratedbythe presence

ofa newdistinct C2RNAtranscriptandsecretion ofbiologically

active human C2. Theseresults demonstrate the

similarity

of

organization ofthemurineand human class IIIMHCregions. Expression ofthe twoclosely linked gene products,C2andfactor B,after DNA-mediated gene transferprovidesa systemfor fur-theranalysisofregulationinbothnormalanddeficientstates.

Introduction

FactorB, ofthealternativecomplement-activatingpathway,and

C2, of the classical complement-activatingpathway, havemany structural andfunctional similarities(reviewed inreference 1).

Synthesis

offactorBandC2occurs predominantly in theliver,

but also in extrahepaticmononuclear phagocytes (2).Factor B

is synthesized as a single-chain

'90-95,000-D

glycoprotein.

Cleavage intotwofragments, Ba(Mr -30,000 D), andBb(Mr -60,000 D), by factor Dactivates the zymogen. Thecarboxy

terminal fragment(Bb) combines with C3bto form the

alter-This work was supported in part by an American Liver Foundation Postdoctoral Fellowship Award, the Jane Coffin Childs Memorial Fund for Medical Research, and by the Mallinkrodt Foundation. Address Cor-respondence to Dr. Chaplin, Howard Hughes Medical Institute, Wash-ington University School of Medicine.

Receivedforpublication19 December 1984.

nativepathway C3 converting enzyme. C2 is synthesized asa

single-chain -92,000-Dglycoprotein, which is also cleaved into alarge carboxy terminal fragment (C2a) and a small, C2b

frag-ment.The C2a fragment then combines with a cleavage product ofthe fourth component,C4b, to generate the classicalpathway

C3 convertase. Although C3 serves as substrate for both acti-vation pathways, differences in local concentrations of the C2 and factor B proteins may effect the relativeimportance of the classical and alternativepathways in initiation ofthecomplement

effector system.

By genetic linkage analysis, the genes encoding these com-plementproteinshave been mapped to the major

histocompat-ibility complex(MHC)' on the short arm of chromosome 6 in humans and onchromosome17 in mouse(reviewed in reference 3). Recently, cosmid cloning has demonstrated extremely close

linkage of the humangenes with the 3'-end of the C2 gene lying within -2,000 base pairs of the 5'-end of the factor B gene, and

withtheC4geneslyinganadditional 10,000basepairsfurther downstream (4, 5). The gene order in the human class III region

is, thus, C2, factor

B,

and two C4 genes.Althoughsimilar linkage offactorBand C4geneshas beenshown inmouse(6),the order

of the murine C2 and factorB genes on chromosome 17 has notbeenpreviously determined.

Despite

this extensive structural and functional similarity,

and closelinkage within the classIIIregionof theMHC,there aremajor differencesin theexpressionof factor B and C2

(7-11). Infact, both

species-specific

and

tissue-specific

differences

in the expressionof thesetwocomplementproteins havebeen

demonstrated. The present study uses DNA-mediated gene

transfertointroduce thegenesencoding both humanand mouse C2 andfactorBintoLcells. Withthe genes from both man and mouseintroduced intothe samegeneticbackground,it should be possible to investigate which features of the differential

expression of thesegenes arecharacteristic ofthe genes

them-selves andwhicharethe resultof

differential processing

bythe cells in which they aretranscribed. In addition, transfected L cellsmight provide asystem inwhichtoexamine, both at the

transcriptional and translational level, the modulation of expression of thesetwo early acting complement proteinsby

mediators of inflammation.

Methods

Materials.Dulbecco's modified essential medium(DME),DME lacking methionine, Hanks' balanced salt solution, fetal bovine serum, and tryp-sin-EDTA solution were obtained from Gibco Laboratories, Grand Island, NY.

L-[35Sjmethionine

(specific radioactivity 1,000 Ci/mmol),

[32Pldeoxycytidine

triphosphate (specific radioactivity 3,000Ci/mmol), 1.Abbreviationsused in thispaper:DME,Dulbecco'smodified essential medium; HAT, 1.36 mg/ml hypoxanthine, 17.4

_Ag/ml

aminopterin,and 388 ,ug/ml thymidine; kb, kilobase; MHC, major histocompatibility complex; PAGE, polyacrylamide gel electrophoresis; PBS, Dulbecco's phosphate-buffered saline.

J. Clin.Invest.

©The AmericanSociety for Clinical Investigation, Inc. 0021-9738/85/10/1449/06 $1.00

andEn3Hance were purchased from New England Nuclear, Boston, MA, and"C-methylatedprotein standards from Amersham Corp., Arlington Heights, IL. Other reagents included sodium deoxycholic acid, ethidium

bromide,2-mercaptoethanol from Sigma Chemical Co., St. Louis, MO, TritonX-l00from Mallinckrodt Inc., St.Louis,MO,SDS and acrylamide from Bio-Rad Laboratories, Richmond, CA, IgG-Sorb from Enzyme

Center, Cambridge, MA, leupeptin from Peptide Research Institutes, Osaka, Japan, pepstatin A from Calbiochem-Behring Corp., La Jolla,

CA,agarosefromFMCCorp., Rockland, ME, and 37% formaldehyde solution fromFisher Scientific Co., Pittsburgh, PA. Antisera included sheepanti-humanC2from Seward Laboratories, London, England, sheep

anti-humanC2fromMiles Laboratories Inc., Elkhart, IN, and goat anti-humanfactorBfrom Atlantic Antibodies, Scarborough, ME. Restriction enzymeswereobtained from New England Biolabs, Beverly, MA, or Bethesda Research Laboratories, Gaithersburg, MD, and used according

tomanufacturersinstructions. Plasmid pBR-TK (herpes simplex virus

thymidine kinasein pBR327) was a gift from Dr. Lynn Enquist, Dupont,

Wilmington,DE.

Methods

Preparation and screening of cosmid libraries. The preparation of the murinecosmid genomic library(H-2d)andinitial isolation of clones has beendescribed previously(6). The murine cosmid cluster was extended

usingcosmid walking procedures as described (6). The human cosmid

genomiclibrarywasconstructedfrom DNA isolated from the cell line JY,using methods similar to those used for the murine library except that theinsertDNAs weresizefractionatedby sucrosegradient centrif-ugationbefore ligationtothecosmidvectorpJB8. The humancosmid librarywasscreenedaccordingtothe methodofHanahan and Meselson (12)usingas aprobethe human cDNAinsertofplasmid pBfA28 (13), 32P-labeled bynick-translation (14).Cosmid DNAwasisolatedaccording

tothe methodofIsh-HorowiczandBurke(15).

Cellculture and biosynthetic labeling. Mouse L cells (TK-) and

HepG2 cells were maintained as monolayers in 75-cm2tissue culture

flasks withDMEcontaining109%heat-inactivated fetal bovineserum as

previouslydescribed(16, 17). Mouseperitoneal macrophages were har-vestedand adhered toplastic tissue culture wellsaspreviously described (7). Forbiosynthetic labeling,cells weresubculturedinto 35-mmdishes, incubatedinserum-freemediumfor 12 h, andrinsedthreetimes with DMElacking methionine.

L-[33S]methionine,

500MCi/mlof DMElacking methionine,wasadded and cellsincubated at37°C, 5%C02/95%air

for time periods rangingfrom 30 minto24 h(pulseperiod).Atthe end

ofthepulseperiod,unlabeledmethionine-containingmediumwasthen added in200-1,000-foldexcessfor timeperiodsupto6 h(chaseperiod).

Atspecified intervals,culturemediumwasharvested andmonolayers rinsedsixtimes, solubilizedin 1% TritonX-100,0.5%deoxycholic acid,

10 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, leupeptin, and

pepstatinA, 100

_Ag/ml

each, in Dulbecco'sphosphate-buffered saline (PBS), pH7.6, and subjectedto two freeze-thaw cycles. Supernatants

andlysateswereclarifiedbycentrifugationat15,000g for 5 and 30 min,

respectively,andstoredat-70°C.Trichloroaceticacid-precipitable

pro-teinwasdeterminedaspreviouslydescribed(18).

Immunoprecipitation andSDS-polyacrylamide

gel electrophoresis

(PAGE).ForstaphylococcalproteinAimmunoprecipitation,

100-300-Ml aliquotsof celllysateormediumwereincubated overnight at 4°C in

anequalvolumeof PBScontaining 1%TritonX-100,0.5%SDS,and 5 mg/ml bovineserum albumin, withaliquots of antiserum

previously

determinedtoprecipitateall labeledantigen.Characteristics of the

an-tiserum have been described (16).Excess formalin-fixedstaphylococci

bearingproteinAwasadded for 1 hat4°C.

Immunoprecipitates

were

washed, releasedbyboilinginsamplebuffer,and

applied

to7.5% SDS-PAGE underreducing conditionsasdescribedbyLaemmli(19).

Mo-lecularmassmarkers(200,000,92,500, 68,000, 46,000,and30,000D)

wereincludedonallgels. Afterelectrophoresis, gelswerestained with Coomassie Brilliant Blue,destained, impregnatedwith

2,5-diphenylox-azole anddried forfluorographyonKodakXARX-rayfilm(Eastman

KodakCo.).

DNA-mediatedgene transfer. Gene transfer followed a previously describedmodification ofthe technique of Wigler et al. (17, 20). Cosmid DNA (5 Mg), pBR-TK DNA (0.5-1.0 Mg), and C3H/HeJ liver DNA (20

Mg)

were

incorporated

intoacalcium

phosphate

precipitate

and

applied

to100-mm culture dishes containing 1-2X 106 cells. Within 48 h, me-dium was replaced by a selective meme-dium (DME containing 10% fetal bovine serum, 1.36 mg/ml hypoxanthine, 17.4,g/mlaminopterin, and 388jug/mlthymidine [HAT]). HAT-resistant colonies were isolated and propagated in selective medium.

Southern and Northern blotting. High molecular weightDNA and total cellular RNA were isolated using previously describedtechniques

(21,22). DNAwasdigestedtocompletion with fourfold excess of re-striction enzyme and fractionated by agarose gel electrophoresis. RNA wasdenatured by heating in formaldehyde and fractionated in agarose-formaldehyde electrophoresis before transfer to nitrocellulose,

hybrid-izationtothe appropriateradiolabeled cDNA probe, and autoradiog-raphy (23).

Assay ofC2hemolytic activity.Reagents werepreparedand hemolytic titrationspecifically for human C2 activity were conducted according to the method ofRappandBorsos(24).

Results

Cloning and chromosomal localization of the murine second

componentofcomplementgene.Restriction endonuclease

diges-tion of cosmid DNAs isolated from an H-2d murine genomic library (6) followed by Southern blot analysis using as a probe

acDNA encodingaportion of human C2 (pC2-6, reference25)

as well as a probe encoding a portion of murine C2 (kindly providedby Drs. P. Lapre and A. S. Whitehead, Children's

Hos-pital),indicated that the gene encoding murine C2 was located 5' tothegeneencoding murine factor B (Fig. 1). Cosmid D-9 appearedtospan both the C2and factor B genes. Using 5 'and

3 'subfragments of the human C2 cDNA as probes, it was pos-sible to show that the murine C2 gene shares the same 5'-3' orientation as the murine factor B gene. Southern blot analysis

of H-2d genomic DNAshowed co-migrating Bam HI (6.9 kilo-base (kb)), Hind III (4.7 kb), Kpn I (20 kb), and Sac I (14 kb) fragmentsusingthe murine C2 and human factor B cDNAsas

probes(Fig. 2). These probes hybridized to different XbaI

frag-ments(C2, 5.1kb; factorB,7.0kb). An identical hybridization patternwasobservedinSouthern blots of cosmid D-9 using the

sameprobes (datanotshown).The C2 and factorBgeneswere

separated by <4,500basepairs.It was notpossibletodetermine

the exactdistance between thetwogenes becauseacDNA

con-tainingthe 5' end of the murine factorBgeneis notcurrently

available.These data are similar to results obtained fromstudies

of cosmidscontainingthe human C2 and factor Bgenes(4). Isolationofcosmidclonescontaining genesencoding human

C2andfactorB. A human cosmidlibrarywasscreenedusinga

cDNA encodinghuman factorB (pBfA28, reference 13)as a

probe. Two cosmids, designatedcBf1 and cBf2 (Fig. 1), were

isolated,andcontained factorB-hybridizingrestriction

fiagments

thatco-migratedwith humangenomicfactorB restriction

frag-ments on gelelectrophoresis (datanotshown). These cosmids also contained restriction fragments that hybridized with the

human C2 cDNA(datanotshown).Neither cosmid contained

sequenceshomologous toa human C4 cDNA. Insubsequent

experiments, the cosmid DNA encoding the murine C2 and factor B genes, cosmidD-9, will be referred to as the murine

cosmid, and the cosmid DNAsencoding the human C2 and

factorB_genes,cosmids_cBfl and

cBf2,

will be referred toasthe

Mouse M

Chromosome 17

I-15 Cosmid

Clones

C2 Bf

9mt==:t~~~~~~~~~~~~~~~~~~~~i--D-12

D-9

D-24

Restriction BamHI IIIo 1iI l I II I 11lily

Sites Eco RI I II I II I I II I I

Kb F-0 Cosmid

Clones

40 80

cBf

---cBf2 ---Human

Chromosome6

C2 Bf C4A 21-OH A

21-OH Figure1. Molecularorganizationof Sip A the murine and humanC2 and

fac-tor B genes. Four overlapping mu-rinecosmid clones define the relative locationof the murine C2 and factor B genes. Thelocation of thefactorB

andSlp genesaredescribed in

refer-ence6. Thelocation ofthe 2 1-hy-droxylasegene(21-OHA)isas de-scribed in reference 31. The location of thehuman genesistaken from references 3 and 32. All genes are transcribed in thesamedirection, 5'

20 to 3' from the left of the figure to the right. The boundaries of the genes have notbeen determinedprecisely andareshownbywavy lines. The restriction map of the murine cos-midsusingBamH IandEcoRl,as

previously described (6), is indicated. The exact extents of cosmidscBfl andcBf2 have not been determined andareindicatedby dotted lines.

Thescaleis in kbpairs.

DNA-mediated gene transfer. In order to examine the expressionofC2 andfactor B,themurine cosmid and the human

cosmid wereco-transfected with the herpes simplex virus

thy-midinekinase gene into separate L cell monolayers, and

trans-fectantswereselected usingHAT medium. More than 75% of HAT-resistant colonies contained cosmid DNA sequences. By Southern blot analysis, cosmidDNAsequences were present in

HAT-resistantcoloniesin 5-15 copies per cell (data not shown).

Identification offactorBandC2 RNA intransfectedcells. FactorBmRNA was detected inmouse L cells transfected with cosmidCBfl (human) and in cellstransfectedwith cosmid D-9(murine), appearing as a single band of -2.5 kb on Northern blotanalysis (Fig.3). Factor B RNA was not detected in mouse

Lcellstransfected with the control plasmids (Fig. 3). C2 mRNA wasdetected in untransfected L cells and L cells transfected with the control plasmid, appearing at

~-2.6

kb on Northern blot

analysis (Fig. 3) and co-migrating with human liver, human

bronchoalveolar

macrophage (Fig. 4, lanes 1 and 2), mouseliver,

1,2,3,4i5 67,8,910 Figure2. Southern blot

23-jjA t analysisofmousegenomic

DNA. 15 gofbalb/cliver

9.,A-: _ DNA wasdigested to

com-6.6- w pletion withb~i:' theindicated

restrictionenzymesand

an-4.4- _ _{alyzedby electrophoresis}on a0.8%agarosegelfollowed by Southern blot transferas 2.3- previouslydescribed (6).

C2 Bf

_{Duplicate samples}

_were

an-Probe

alyzedonthesame

_gel.

Gel-purifiedprobes (0.25Mg)werelabeledwith 32P by nick-translationto a

specificactivity of2-4x 108cpm/ug(14).Hybridizationwas as indi-catedwith the0.9-kbinsert of pC2MI(lanes1-5) andthe 1.9-kb

in-sertofpBfA28(6-10).Hybridizing restrictionfragmentswere identi-fiedbyautoradiography. 1 and 6,digestionwith BamHI;2 and7,

digestion with HindIII;3 and8,digestionwith KpnI;4and9,

diges-tion with SacI;5and10,digestionwith Xba I.

and mouseperitoneal macrophage C2 mRNA (data not shown). Incellstransfected with the murine cosmid, there appeared to be several fold more RNA hybridizing with the C2 cDNA probe than in L cells transfected with the control plasmid. An additional andpredominant C2 mRNA of 2.0kb was detected only in Lcellstransfected with cosmid DNA containing the human genes (Figs. 3 and 4).

Biosynthesisandsecretion offactor B and C2 by transfected cells.TransfectedLcells wereradiolabeled and products analyzed byimmunoprecipitation, SDS-PAGE, and fluorography. Cells transfected with the murine cosmid synthesized and secreted factor B(Fig. 5, lanes I and2). Twopolypeptides of -87,000

Probe C2 Bf

C r_

a c cs

E E

, , 0 :

o i I t0 _{2 I} kb

-6.75 -4.26 -226 - I .98

Figure 3. Northern blotanalysisofRNAisolated from transfected L cells. Total cellularRNA wasisolated from -1 X10'L cells. 20Mgof theindicated RNAsweresubjectedtoelectrophoresisin agarose-form-aldehyde gels and transferredtonitrocelluloseasdescribed(23). 32p-labeled HindIIIfragments of lambdaDNA wereincludedasrelative size markersoneachgel.Rightandleftpanelsaretaken from replicate gels. Theleft panelwashybridizedwith 0.1 Mg of human factor B

cDNA(pBfA28)andtherightpanel with 0.1MIgof human C2 cDNA (pC2-6) nick-translatedto aspecific activityof 4X 101cpm/Mg. Washing was at500Cin 0.2XSSC(30 mMNaCl, 3mMsodium ci-trate, and 0.1%SDS).Autoradiographywasconducted for 12 hat

-70'CusingKodakXARfilm(EastmanKodakCo.)withan

intensi-fyingscreen.

C2 andFactorBGeneExpression 1451

2 3 4 5 6 Figure4.Northernblot analysis of

kb 's. _{RNAisolated from transfected L cells}

compared with RNAisolated from

6.75- cells inprimaryculture. Totalcellular

RNAwasisolated and subjected to

4.26

_{agarose-formaldehyde}

electrophoresis

asdescribed above. RNA from human - liver(lane 1),human bronchoalveolar

226

macrophages

(2),

Lcellstransfected

2298- withthe murinecosmid(3),and L

cellstransfected with the human cos-mid (4) were compared wwith L cells transfected with the control plasmid (5) andLcells alone (6) andhybridizedwith the human C2 cDNA as described in Fig. 3. The -2.6 kb C2 RNA is indicated.

A 2 3 4 5 6 7 8 9 10 I1 12 Mr (x 0-3)

925 -68

-46

B 2 3 4 5 6 7 8 9 10 11 12

and "-89,000Dweredetectedinthe intracellularcontents,and

twopolypeptides of -90,000 and -92,000 D were secreted, co-migrating with factorBpolypeptides synthesized andsecreted

bymurine peritoneal macrophages (Fig.5,lanes 3 and 4). Cells transfected with the human cosmid synthesized factor B asa

singlepolypeptide, -90,000 Dinapparentsize, and secreteda

single -92,000-D factor B polypeptide (Fig. 5, lanes 5 and 6). Single factorBpolypeptides of the sameapparentsizewerealso

synthesized and secreted by human hepatoma-derived

hepato-cytes(Fig.5,lanes7and 8). Therewasnoevidence for synthesis

andsecretion of factorBbymouseLcellstransfected with the controlplasmid (Fig. 5, lanes 9 and 10).

Thekinetics of synthesis and secretion of factorB by L cells

transfected with the human cosmid (Fig. 6 A)werecompared

withthose ofLcells transfected withthemurine cosmid (Fig. 6 B) in pulse-chase experiments. The single, -90,000-D

intra-cellularfactorBpolypeptide synthesized by L cells transfected with thehuman cosmid begantodisappearat30-60min of the

chaseperiod, coincidentwithitsappearance inthe fully

glyco-sylated-92,000Dformintheintra-and extracellularcontents

(Fig. 6A). Factor B is secreted with similar kinetics overthe

remaining 360min of the chase period. Two intracellular factor

Bpolypeptides aresynthesized by L cells transfected with the

murine cosmid(Fig. 6 B). Kinetics of synthesis and secretionof thesetwopolypeptidesweresimilartothose of the single poly-peptide in Lcells transfected with the human cosmid.

Synthesis andsecretionof C2wasdetected inmouseLcells transfected with the control plasmidsaswellasin L cells

trans-2 3 4 5 6 7 8 9 10 _FigureS.Biosynthesis and

se-M, cretion of factorB byLcells

lxlo') transfectedwithhuman and -92.5 murinefactorBgenes.

Intracel-lularlysates (lanes1, 3, 5, 7,

-68 and 9) andextracellularmedia

(2, 4, 6, 8,and10)of

trans-fectedLcells radiolabeled for 4 hwereimniunoprecipitated

-46 withgoatanti-human factorB

for SDS-PAGEand fluorogra-phynext tomolecularmass

markers. L cells transfected withmurine cosmid (1 and 2)arecompared withthose transfected

withhumancosmid(5 and 6),tomouseperitoneal macrophages(3

and4),andtohumanhepatoma-derived hepatocytes (7and8).

Ly-satesand media of L cells transfected with the control plasmidarealso included(9and10).

Y <

*X- *. As' At

**'t'i.t Ax 4:+

!

i%§t.

t..S

..wiR,,''..

bout.,

W -92.5

-68

-46

Figure 6.Kineticsof synthesisandsecretion offactorBbytransfected

Lcells. (A)Lcells transfected with human cosmid.Lcellswere la-beledfor 0.5 h with

L-[35S]methionine

andchased with unlabeled

me-thionine forintervalsupto6 h. Cellswerethen solubilized,

superna-tantsandlysatesimmunoprecipitated and analysed by SDS-PAGE

andfluorography. Lanes1-6containcelllysatesand7-12 contain ex-tracellular media from chase time points 0, 0.5, 1, 2, 4, and 6 h. (B) L

cells transfected withmurine cosmid. Exactlyasin A.Fastermigrating

radiolabeled bandswerenonspecific andnotreproduced in other

ex-periments.

fected with thehuman and iutrinecosmids(Fig. 7).Theprinciple intracellular form of C2 was 86,000 mol wt, with additional

fastermigrating C2 polypeptidesat84,000,70,000, 67,000,and

65,000Dsimilartotheadditionallower molecularweight

cell-associated forms of C2 protein observedin human

hepatoma-derivedhepatocytes (Fig.7, lanes 7 and8,reference 16).C2was

secretedbyLcells transfected with the human and murine

cos-midandcontrol plasmidsas a polypeptidewithapparentsize of94,000 D (Fig. 7, lanes 2, 4, and 6). Another -70,000 D

form of C2proteinwasalsoseen inthe extracellular

compart-mentandthoughttobe the C2a fragment.All the C2

polypep-tides, intracellular and extracellular, produced bymouseL cells

2 3 4 5

Figure 7. Biosynthesis and secre-tionof C2by L cells transfected

with human andmurinecosmids. Intracellularlysates (lanes 1, 3, and

M r _{5) and extracellularmedia(lanes 2,}

(x10

3)

_{4, and 6) from transfectedL cells}

radiolabeledfor 4 hwere immuno--92.5 precipitatedwithsheep

anti-hu-manC2 for SDS-PAGE and fluo--68 rography next to molecularmass

markers. L cells transfected with human cosmid(1and2),murine

-46 cosmid(3and4),controlplasmid

(5 and 6)arecompared with

hu-manhepatoma-derived

hepato-6 7 8 cytes(7and8).

1452 Perlmutter, Colten, Grossberger, Strominger, Seidman, and Chaplin

Mr

{xlo-)

towin

0

ww so

wereslightlyslower in migration than thecorresponding poly-peptides identified in the HepG2 cells. Extracellular fluid from Lcells transfected with the human cosmid was hemolytically

active in the human C2 hemolytic assay (Fig. 8). Culture fluid from L cells transfected with themurine cosmid or control plas-mid (Fig.8) orculture fluid frommurineperitonealmacrophages

in primaryculture(data notshown)did notproducehemolysis inthis assay.

Discussion

Structural and functionalsimilarities between factor B and C2 have beenattributedtoprimary sequencehomology and have

suggestedevolutionbygeneduplication. In fact, recent molecular mapshave indicatedthat thehuman factorBand C2genes are less than

-2,000

base

pairs

apartin the genome of the Class III

region ofthe

MHIC

(4, 5). Nevertheless, failuretodemonstrate

cross-hybridization of the two genes, even at very low stringency,

indicatesasubstantialdegreeofnucleotidesequence divergence

(26).Inadditionto thenucleotidesequencedifferencesimplied

by thecross-hybridization experiments, differences in the

reg-ulation of C2 and factor B by hepatocytes and mononuclear phagocytes from several species have also been identified (7-11, 16).

Inthisstudy, we have demonstrated that themurine genes

encoding C2andfactorB are very closelylinked within theclass IIIregion of the murine MHC, with organization similarto that

inman.Expression ofhuman andmurinefactorB and human

C2wasdemonstratedin amurine fibroblast cellline transfected

with cosmid DNA containing these genes. Factor B RNA was

detected incellstransfected with the humanandmurine cosmid

but notincellstransfectedwith the controlplasmid. Biosynthesis

andsecretion of factorB was detected in cellstransfected with the human and murine cosmids by incorporation of a radiola-beled amino acid precursor into newly synthesized protein.

Expression ofthetransferred human C2 gene was demonstrated

byadistinct 2.0-kbC2 RNA species(Figs.3 and 4) and functional

activity

of the transfected cell culture fluid inthe human C2

hemolytic assay (Fig. 8). L cells transfected with the murine cosmid contained highersteady state levels of C2 RNA than

thosetransfectedwith the control plasmid; however, expression

of the transferred murineC2 gene could not be further

distin-guished fromendogenous C2 expressed in the control transfected cellsbecause of the absence of polymorphic differences in C2

between thedonor

H-2d

mice and the recipient

H-2k

L cells.

Figure

8.Secretionof

he-13 _/

molytically

active C2

by

transfected L cells. Cell

cul-0

s_/ turefluid from L cells

trans-fectedwith human cosmid

(closedcircles), L cells

transfected with murine

5- cosmid(X),and L cells

transfectedwith the control

*

/ plasmid

(open

circles)

were

testedin the human C2 he-O 24 48 72 96 molytic assay.Forthis

ex-T/ME(hours) periment, cell culture fluid

was collected at the indi-catedtime

_intervals,

andthe

_{monolayers resuspended}

infresh culture

fluid.

In addition to demonstrating that C2 and factor Bcan be

expressed byL cells afterDNA-mediated genetransfer, differ-encesin the waythese twocloselylinkedcomplementgenesare

expressed have also been identified. Untransfected, orcontrol transfected, Lcells synthesize and secreteC2,but not factor B. While we have previously shown that the fourth component of complement(C4) is not expressedbyuntransfected L cells(17),

expression of C2byL cells has not beenpreviouslyaddressed. Nevertheless,C2isnotuniquein this respectsinceothers have shown that the third component of complement (C3) is secreted by anumberof established fibroblast cell lines(27,

28).

After transfection of the human factor B and C2 genes into Lcells on the same cosmid, both genes were expressed. Whereas factor B wassynthesized, processed,and secreted with extraor-dinary fidelity, C2 was transcribed, or the transcript modified,

in an altered form as seen in Northern blot analysis (Figs. 3 and 4). The nature of the differences between the 2.0-kb C2 hybrid-izingsequences recovered from cells transfected with the human cosmid and the usual 2.6-kb C2 mRNA obtained from human and murine hepatocytes and macrophagesis notknown. Dif-ferences in the 2.0-kb RNA could reside in the untranslated

region. Itis also possible that this apparently smaller RNA en-codes C2in analternative polypeptiJe form, which is similar to thepreviously reported cell associated forms of C2(16).

Differences inthe expression of otherclosely linked genes

has also beendemonstratedbefore and after DNA-mediated gene

transfer. Transcription of an alpha globin gene was different from that of a beta globin gene despite transfer into mouse

erythroleukemia cellson the same plasmid (29). In that case,

regulation of expressionwasattributedtosequences within the

individual structuralgenes.Further studies with cosmid DNAs

containingthe C2andfactor B genes individually will be nec-essary to localize sequences regulating the expression of C2 and factor B intransfected Lcells. It is still possible that asingle promoter on the 5' side ofthe C2 gene, individual promoters

onthe 5' sides ofthe C2andfactor B genes, or structural elements

controltheexpression ofC2 and factor B.

Several additional features of C2 and factor B

expression

havebeen identified in these experiments. Although notidentical, multiple intracellular forms ofC2protein in transfectedL cells aresimilartomultiple forms of C2 synthesized by humanliver

(16).Themultiple intracellular forms ofC2identifiedin human

liverhavedifferent fates,oneform being secreted and two forms

remainingcellassociated. These forms are derived from distinct

primary translationproducts andpresumably distinct RNA

spe-cies, asthere isonly asingle C2 gene (16). With these

consid-erations in mind, further studies ofthe relationship between the

additionaldistinctC2 RNA and the multiple intracellular forms

of C2 in L cellstransfectedwith the human cosmid DNA will beimportant.

An

interspecies

variationbetween murine and human factor Bwasalso identified. Factor B was synthesized and secreted as twopolypeptides by L cells transfected with the murine cosmid, but as asinglepolypeptide by L cells transfected with the human

cosmid. This difference was also observed for biosynthesis of

factorBbymurinecells (peritoneal macrophages) (30) in primary culture compared with that of human cells (hepatoma-derived

hepatocytes).Since this variation in the way factor B is translated andprocessed is preserved when murine and human factor B genes are expressed in the same cell, the variation must reside

inthefactorB genes

themselves,

ratherthan indifferential

These experiments, therefore, indicate that C2 and factorB areexpressed bymouseL cellsafterDNA-mediated genetransfer, and that intracellular processing of these complement proteins isreproduced with extraordinary fidelityby the L cells. Further

analysis of C2 and factor Bin thissystem in comparisonwith

human and murine hepatocytes and macrophages will allow a

greaterunderstanding of their differential regulation,both atthe level of RNA metabolism and at the level of posttranslational processing and secretion.

Acknowledgments

The authors are indebted to Claudia S. Leonard for technical assistance, Dr. Shu-hei Takemura for performing hemolytic assays, and Helen Hourihan for secretarial assistance.

This work was supported by U. S. Public Health Service grants AM07114,A118436,AI 19148, HD17461, AI20032,AI21157, HL32361, and AM13230.

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