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Dr(a-) polymorphism of decay accelerating

factor. Biochemical, functional, and molecular

characterization and production of

allele-specific transfectants.

D M Lublin, … , C Levene, M J Telen

J Clin Invest.

1991;

87(6)

:1945-1952.

https://doi.org/10.1172/JCI115220

.

The Dra antigen belongs to the Cromer-related blood group system, a series of antigens on

decay accelerating factor (DAF), a glycosyl-phosphatidylinositol-anchored membrane

protein that protects host cells from complement-mediated damage. We studied the rare

inherited Dr(a-) phenotype to ascertain the associated biochemical and functional changes

in DAF and to characterize the basis for this polymorphism. Radioimmunoassay assay and

flow cytometric analysis of Dr(a-) erythrocytes demonstrated 40% of normal surface

expression of DAF but normal levels of several other

glycosyl-phosphatidylinositol-anchored proteins, distinguishing this phenotype from that of paroxysmal nocturnal

hemoglobinuria. Western blots confirmed this reduced DAF expression and indicated a

slightly faster mobility of the molecule on SDS-PAGE. Despite the reduced DAF expression,

Dr(a-) erythrocytes functioned normally in the complement lysis sensitivity assay. Utilization

of the polymerase chain reaction to amplify mononuclear cell genomic DNA from three

unrelated Dr(a-) individuals demonstrated that a point mutation underlies the Dr(a-)

phenotype: a C to T change in nucleotide 649 resulting in a serine165 to leucine change.

This defines the Drb allele of DAF, which can be distinguished from Dra by a Taq I

restriction fragment length polymorphism. We created transfected Chinese hamster ovary

cell lines expressing either the Dra or the Drb allelic form of DAF. These allele-specific

transfectants were tested by inhibition of hemagglutination or flow cytometry and confirmed

the specificity of […]

Research Article

(2)

Dr(a-) Polymorphism of Decay Accelerating Factor

Biochemical, Functional, and Molecular Characterization and Production of Allele-specific Transfectants

DouglasM.Lublin,*E.ScottThompson,*Annette M.Green,* Cyril

Levene,O

andMarilynJ.

Telen*

*DivisionofLaboratory Medicine,DepartmentsofPathologyand Medicine, Washington UniversitySchoolofMedicine, St. Louis,Missouri63110;

*Division

ofHematology/Oncology, Department ofMedicine,DukeUniversity

MedicalCenter,Durham,NorthCarolina27710;andReference Laboratoryfor Immunohematology and Blood Groups, GovernmentCentralLaboratories,Jerusalem, Israel

Abstract

The Dr'antigen belongs to the Cromer-related blood group system, a series ofantigensondecay acceleratingfactor(DAF), aglycosyl-phosphatidylinositol-anchored membrane protein

that protects host cells from complement-mediated damage.

Westudiedthe rare inherited Dr(a-)phenotype toascertain

theassociated biochemical and functional changesin DAF and tocharacterizethebasis for thispolymorphism. Radioimmuno-assay andflow cytometric analysis of

Dr(a-)

erythrocytes

dem-onstrated40%of normal surface expression ofDAF but normal levelsof several other glycosyl-phosphatidylinositol-anchored proteins, distinguishingthis phenotype fromthatof

parox-ysmal nocturnalhemoglobinuria. Westernblotsconfirmed this

reduced DAFexpressionandindicatedaslightly faster mobil-ityof themolecule onSDS-PAGE. Despitethe reduced DAF

expression, Dr(a-) erythrocytes functioned normally in the complementlysis sensitivityassay.Utilization ofthe polymer-asechain reactiontoamplifymononuclear cellgenomicDNA

fromthree unrelated Dr(a-) individuals demonstrated that a

point mutation underlies the Dr(a-) phenotype: a C to T

changein nucleotide 649 resultingin a

serine'65

toleucine

change.Thisdefinesthe Drballele ofDAF, whichcanbe distin-guished fromDr'byaTaqIrestrictionfragment length

poly-morphism.We createdtransfected Chinesehamster ovary cell

lines expressing eitherthe Dr' or the

Dr'

allelicform of DAF. Theseallele-specific transfectantsweretestedbyinhibition of

hemagglutination

orflowcytometry andconfirmedthe specific-ity of anti-Dr' alloantisera. Theallele-specifictransfectants couldformthebasisofa newserologicalapproach to immuno-hematology. (J.Clin.Invest. 1991.87:1945-1952.) Key words:

complement-erythrocytes *Cromer-relatedblood group *

gly-cosyl-phosphatidylinositol membrane anchor *flowcytometry

Introduction

Decay acceleratingfactor(DAF,ICD55) is a70,000Mr

mem-brane

protein

that protects host cells from damage by

autolo-Address reprint requests to Dr. Douglas M. Lublin, Department of Pathology, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8118, St. Louis, MO 63110, or Dr. Marilyn J. Telen, Department of Medicine, Box 3387, Duke University Medical Center, Durham,NC 27710.

Receivedfor publication4January1991.

1.Abbreviations used in this paper: AST, allele-specific transfectant; CHO,Chinese hamster ovary cellline;CLS, complementlysis

sensitiv-ity;DAF, decay acceleratingfactor,GPI, glycosyl-phosphatidylinosi-tol; LFA,lymphocytefunction-associated; PCR, polymerasechain

re-action; PNH, paroxysmal nocturnal hemoglobinuria; RFLP, restric-tionfragmentlengthpolymorphism; SCR, short consensus repeat.

gouscomplement (reviewedin 1). DAFis one member of the

regulators of complement activationgene family, agroup of

linkedgenes onthe long armofhumanchromosome 1 (2, 3)

thatarecomprised of multiple copiesof a 60-amino acid short consensusrepeat(SCR). The productsof these genes all serve todownregulate the activity ofthecomplementsystem at the leveloftheC3convertase. DAFis widely distributedin periph-eral blood cells andinepithelialand endothelial tissues(4-7).It acts topreventthe assemblyofC3convertases and to dissociate

preformed C3convertases (8-1 1).

Anovelstructural feature ofDAFis itsattachment tothe plasmamembrane throughaglycosyl-phosphatidylinositol (GPI) anchor (12, 13).Theclassof GPI-anchored membrane proteins (reviewed in 14, 15)includesthe

complement-regula-toryfactors DAF, membrane inhibitor ofreactive lysis (16-18), and

C8-binding protein/homologous

restriction factor(19, 20).

Thecomplement-regulatory function ofDAFand theGPI

an-choringboth come intoplay inthe disease paroxysmal noctur-nalhemoglobinuria(PNH;reviewedin21),anacquired

hemo-lytic anemia.Theunderlying molecular basis ofPNHisa

fail-ure toexpress anyGPI-anchoredproteinsonthe cellsurface,

presumably due to adefect inoneofthe enzymesresponsible forsynthesisorattachmentof the GPIanchor. Therefore the affected blood cells inPNH

patients

failtoexpressthe GPI-an-choredcomplement-regulatory molecules andareabnormally sensitivetocomplement, withpartoftheincreased

sensitivity

duetothe absenceofDAFin these cells (9, 22, 23).

The cDNA(24, 25)and gene (26) forhuman DAF have

been cloned. Severalpolymorphisms have been identified in

the

noncoding

regions

ofthe DAF gene

by

restriction

fragment

length

polymorphisms

(RFLP)

analysis (3, 27). Recently

the

Cromer blood group antigens

(reviewed

in

28)

have been

showntoresideontheDAFmolecule(29),so that the

serologi-cally identified Cromerblood groupalleles mark

polymor-phisms in the encodedDAFprotein. These Cromerblood group phenotypes thus provide a basis for biochemical and functional

investigation

of alternate forms ofDAF.

OnerareCromer phenotype is Dr(a-), which has been

foundas a recessivetraitinindividualsinfour unrelated Israeli familiesthat alloriginally emigrated fromtheBukharan

region

of Russia (30, 31;Levene,C.,unpublished data). Erythrocytes

of the

Dr(a-)

phenotype

lack

expression

of the Dr3

antigen

and have lowexpression ofallofthe other Cromeralleles

including

Cr3,

Tcl,

Esa, WESb,

andIFC.No

hematological

abnormalities

have been reportedto beassociated with the Dr(a-)

pheno-type.

Inthis studywe

investigated

thebiochemical

expression

of

DAFintheDr(a-) phenotypeandcharacterizedthefunctional properties of thisDAFvariant. Furthermore, we have identi-fied themolecular

(nucleotide) change

thatdefinesthe

Dr(a-)

phenotype, foundanallele-specificRFLPthat can be usedto

phenotype individuals, andhavecreated

allele-specific

trans-fectants thatcanbe usedto

identify

alloantibodies.

J.Clin.Invest.

©TheAmericanSocietyfor ClinicalInvestigation,Inc.

0021-9738/91/06/1945/08 $2.00

(3)

Methods

Antibodies. Two rabbit polyclonal anti-DAF (29, 32) were used for radioimmunoassays,immunofluorescence, immunoprecipitation,and Western blots. Monoclonal antibodies 3.3.136 and K98 (33) (gift ofDr. Robert Knowles, Memorial Sloan Kettering, New York) directed against DAF were also used inradioimmunoassaysand immunofluo-rescence. Monoclonal antibodies againstacetylcholinesterase (34) (American Type Culture Collection, Rockville, MD) and CD58 (35) have been previously described. (Anti-CD58 was a gift from Dr. Timo-thy Springer, Dana Farber Cancer Institute, Boston.) Monoclonal anti-CD59 (2/24D4) was used as part of investigations performed for the Second International Workshop on Monoclonal Antibodies Against Red Cells and Related Antigens (Lund, Sweden, 1990). Polyclonal anti-CD59 has also been previously described (16) and was provided by Dr. Charles Parker, University of Utah, Salt Lake City.

Radioimmunoassayoferythrocyte expression ofGPI-anchored pro-teins.Radioimmunoassaysto measure antibody reactivity with erythro-cytesfrom Dr(a+) or Dr(a-) individuals were performed as previously described (36) except that radiolabeled Staphylococcal protein A was usedtodetectbinding of rabbit antisera to DAF and CD59 (29).

Western blots. Western blotting of erythrocyte proteins was per-formed as previously described (37), except that antibody binding was detected using goat anti-rabbit IgG conjugated with alkaline phospha-tase (Promega Biotec, Madison, WI) and a chromogenic substrate (a mixture of5-bromo-4-chloro-3-indolyl-phosphateand nitro blue tetra-zolium) prepared according to manufacturer'sinstructions.

Complementlysissensitivity assays. The complement lysis sensitiv-ity (CLS) assay was carried out as described (38). Briefly, erythrocytes were incubated with saturating amounts of human anti-I at0Cand thenincubated with varyingdilutionsoffresh human serum contain-ing complement at 370C. Lysis of erythrocytes was measured by spec-trophotometric analysis of hemoglobin released into the supernatant fluid. The CLS

Hm

isdefined as the reciprocal of the dilution of serum useddivided by the milliliterequivalentsof serum required to obtain lysis of 50% of the cells.

Amplification

of genomic DNA by PCR. Mononuclear cells were prepared from peripheral blood samples by Ficoll-Hypaque density gradient centrifugation (39). Genomic DNA was prepared from these cells by direct lysis in polymerase chain reaction (PCR) buffer with nonionic detergents and proteinaseKasdescribed (40). PCR amplifi-cation of the genomic DNA with Taq polymerasewasperformed in a

50-Mlvolcontaining0.2-1.0 ,gDNA, 1 x PCR buffer(50mMKC1, 10 mM TrispH 8.3, 1.5 mMMgCl2,0.01% gelatin),200MAM deoxynucleo-sidetriphosphate mixture, 0.5MMof eachprimer,and 1.5 U TaqDNA

polymerase (41). The primers of 17-26 nucleotidesweresynthesized on a synthesizer (Pharmacia Fine Chemicals,Piscataway,NJ) andwere

designed based on the sequence of the humanDAFgene(26); in gen-eral,oligonucleotidepairs were synthesized to match sequences in the 5'- and 3'-introns surrounding eitherone orseveralexons. ThePCR was done with an initial denaturation of 94°C for 2 min, 35 cycles consistingof denaturation at 94°C for 1min/annealingat45-55°C for 1 min/elongation at 72°C for 1-3 min, andafinal elongation stepat

72°Cfor 10 min.

DNA sequencing. PCR products were treated withT4DNA poly-merase to repair any possible ragged ends, extracted with phenol-chlo-roform, precipitated with ethanol, kinased withT4polynucleotide ki-nase, and then purified on agarose gels andsubclonedinto the EcoRV

siteof the plasmid pBluescript KS+(Stratagene,LaJolla, CA) by

stan-dardtechniques (42). The double-stranded plasmidDNA(43)was

se-quencedwith Sequenase DNApolymerase (44). Any differences from wild-type sequence were confirmed on several independent isolatesto

rule out a PCR or subcloning artifact.

RFLPanalysis.PCR productsweredigested with TaqIrestriction endonuclease in the PCR bufferat65°C for 1.5 hundermineraloil,

and then analyzed on an agarose gel with ethidium bromidestaining. Transfections. DAF cDNA wassubclonedinto the EcoRIsite of theexpression vector pSFFV.neo (45;agift of S. Fine andD. Loh,

Washington University)

containingthespleen focus-formingvirus5'

long

terminalrepeat,SV40

splicing

andpolyadenylation signals,and theneomycin-resistancegene. Chinese hamster ovary(CHO)cellswere

transfected

by

lipid-mediatedDNA transfectionusing10 Aig DNA and

100gtg

Lipofectin (46).

Positive cellswereselectedbyresistancetothe

neomycin analogueG418(0.25 mg/mlactivedrug)and individual subcloneswereproduced by limitingdilution.

Biosynthetic

labeling of

cell linesand immunoprecipitations.CHO cell transfectantswereplated24 h beforelabelinginmultiple100-mm dishesso astobeat70% confluence atthe time oflabeling.The cells

werewashed,incubatedincysteine-freeHam's F-12 medium with 5%

dialyzed

FCS for 1h,and then 100MCi/ml [35S]cysteinewasadded. For kineticexperiments,the cellswerepulsedfor the indicated timeperiod

and then the chasewasbegun by addinga10,000-foldexcessof unla-beledcysteine.Atgiventimepointsaplatewasprocessedbywashing

with cold PBS andsolubilizingthe cells in 1% Triton X-I14/Tris buff-ered-salinecontainingthe protease inhibitors 2 mMPMSF,1MM

pep-statin,and 100

U/ml

aprotinin. A detergentextract (47)of the cell

lysatewasimmunoprecipitatedwith rabbit polyclonal DAF

anti-serum asdescribed(48)andanalyzedon9%SDS-polyacrylamide

gels

under

reducing

conditions followedby

fluorography.

Inhibition

of hemagglutination.

Agglutinationof human

erythro-cytes with human alloantiserumplusantiglobulin (Coombs')reagent

(Gamma Biologicals, Houston, TX)wascarriedoutbystandard

sero-logicaltechniques.Inhibition ofhemagglutinationwasdoneby prein-cubating plasmasamplesortissue culture supernates with the

alloanti-serumfor 15minat370Candthenproceedingwith the

hemagglutina-tion reachemagglutina-tionasabove.

Immunofluorescence.

CHO transfectantswereremovedfrom tissue culture flasksby incubatingfor 2min withtrypsin-EDTA;preliminary experimentsdemonstrated that this brieftreatmentdid notremove

surface DAF, consistent with thepreviously described resistance of DAFtodegradation by trypsin (49).Cellswerestained withspecific antibodyfollowedbyFITC-labeled secondantibodyand analyzedby

flow cytometryto assesssurfaceexpressionofDAF(50).Because

hu-manalloantisera gavehigh backgroundsonthisanalysis,the human alloantibodieswerefirst partially purifiedfromserumbyabsorption

and elution from humanerythrocytes usingElu-Kit II accordingtothe manufacturer's directions(GammaBiologicals).

Results

DAFexpression onDr(a-)erythrocytes. Dr(a-)erythrocytes

showed reducedhemagglutinationwith antibodiestonon-Dra

Cromer-related bloodgroupantigens (30, 31),suggestinga

re-duced level of all Cromer-related antigens and hence of DAF. To confirm this directly, aradioimmunoassay wasusedto

measurethe levels of anti-DAF reactivity withDr(a+) and

Dr(a-) erythrocytes.As shown in TableI,erythrocytes froma

Dr(a-)individual demonstrated reduced reactivity with botha

rabbitpolyclonalandonemonoclonal anti-DAF (3.3.136),

av-eraging - 40% of normal in eachcase. Interestingly,

asecond monoclonal anti-DAF(K98)showed almostnoreactivity with

Dr(a-) erythrocytesbut reacted strongly with Dr(a+)

erythro-cytes, suggesting that this monoclonal antibody might

recog-nizeanepitopeatornearthe polymorphic amino acids

corre-spondingtotheDr(a-) phenotype.

Measurementsof surface DAFexpression were alsomade

by immunofluorescence. Erythrocytes fromtwoadditional

Dr(a-)andtwoDr(a+) individualsweretreated witha

differ-entrabbitpolyclonalanti-DAF and FITC-labeledsecond

anti-bodyand assessed by flowcytometry.ThetwoDr(a-)

erythro-cytesyieldedmeanchannel fluorescence valuesthat were32% and 48% oftheaveragevalues for the Dr(a+) erythrocytes

(data

(4)

TableI.ReactivityofNormal and Dr(a-) Erythrocytes withAntibodiesto DAF

Specific cpm bound Monoclonal Polyclonal

Cell type Rabbit anti-DAF 3.3.136 K98

Normal 1 2945 5246 3181 Normal 2 3149 5566 4797 Normal3 3406 4980 4009 Dr(a-) 1488 2034 258 Erythrocytesfrom normal,i.e., Dr(a+),donors or aDr(a-) individual

weretreatedwith anti-DAFantibodiesfollowed by appropriate ra-diolabeled secondantibodyorStaphylococcal proteinA.Specific countsboundwascalculatedasthedifferencein boundradiolabel betweentestantibodyandanegativecontrolantibody(normalrabbit serum or anonspecificmonoclonalantibody).

Tofurther characterize the DAFmembraneprotein from

Dr(a-)erythrocytes,Westernblotswere donewith rabbit

poly-clonal anti-DAF. Western blots consistently demonstrated the markedly reducedexpression ofDAFby Dr(a-)erythrocytes (Fig. 1). Theapparent

M,

ofDAF on Dr(a-)erythrocytes, as

judged bySDS-PAGE, was approximately thesame as on

Dr(a+)erythrocytes; however, comparison of thetwospecies

ongels of

differing polyacrylamide concentrations

suggesteda

possible

small decrease (< 2,000) inapparent

M,

of

theDr(a-)

DAF(notshown).

Expression

of GPI-anchored

proteins on Dr(a-)

erythro-cytes.

Having

seenthat Dr(a-) erythrocytesexpress areduced

amountofa DAF

species

possessing

nearnormal

size,

we next

studied

expression

of other GPI-anchored membrane

proteins

toascertain whether the defectwaslimitedtoDAF.

Radioim-munoassay wasusedto measurereactivity with antibodiesto

three otherGPI-anchored

proteins: acetylcholinesterase,

mem-brane

inhibitor

ofreactive

lysis

(MIRLorCD59), and

lympho-cyte

function-associated antigen

3 (LFA-3 orCD58). The Dr(a-) erythrocytes showeda markedlyreducedreactivity withantibodies toDAF,but they showedessentiallynormal

reactivity

with antibodies against the other three GPI-anchored

proteins

(TableII). Bycontrast,when cellsfromapatient with

NONNMMUNE

Figure

1.Western blot

RABBITPOLYCLONALAN-I-OAF RABBI' SERUM

r

>~~,~--

for DAF

in

human

erythrocytes. Varying

-~ ; amountsoferythrocyte

membranes

(protein/

lane) preparedfrom

DAf~ db -

Dr(a+)

or

Dr(a-)

indi-vidualsweresubjected toWesternblotanalysis

withrabbitpolyclonal ;anti-human DAFor

controlnonimmune Drphenotype: + + + - - rabbit serum. The arrow

Protein/lone 10. 20D3p2OAg.g30 4 K 30,g marks theband

corre-spondingtoDAF.A minorbandof approximatelytwice the

M,

ofDAFis also

visualized;

this DAF-2specieshas beenreportedpreviously(56, 57).

TableI. Comparison ofReactivity of Dr(a-) and PNH III

ErythrocyteswithAntibodiestoGPI-linked MembraneProteins

Percentageof normal binding Antibody type and specificity Drfa-) PNH III

Polyclonal anti-DAF 45%* 15%*

Monoclonal3.3-136anti-DAF 39%* 24%* MonoclonalK98 anti-DAF 6%* 10%*

Polyclonalanti-AChE 98% ND Monoclonalanti-AChE 95% 5%* Polyclonalanti-CD59 99% 23%* Monoclonalanti-CD59 100% ND Monoclonalanti-CD58(LFA-3) 88% 39%*

Thereactivity of various antibodiesagainstGPI-linked membrane proteins with erythrocytes from normal donors,aDr(a-)individual, or aPNH IIIindividual wasmeasuredasspecificcountsboundas

described in the legendtoTable I. The resultsarelistedas a percent-ageofthecountsfor normals.

*Indicates thatdifferenceisstatisticallysignificant,comparedto nor-mal Dr(a+) erythrocytes.

PNH weretested, they showedsignificantly reduced expression

of all fourGPI-anchoredproteins, consistentwiththe known

failure of surfaceexpression ofallGPI-anchored membrane proteins in PNHerythrocytes.Thesedata clearlydemonstrated the differentphenotypes ofDr(a-) andPNHerythrocytes.

CLSassayofDr(a-) erythrocytes.NexttheDr(a-) erythro-cytes weretested forfunctional activity ofDAF. DAF serves to protectcellsfrom lysisby complement byinhibiting formation

andaccelerating the decay of the C3andC5convertases

(8-11), thuspreventing the complement cascade from proceeding

tofinal hemolysis. Thiswastested bythe CLSassayofRosse and Dacie(38). Dr(a-)erythrocytes gave a normal result in

thisassay,with values for CLS

Hm

ranging from1.35(Fig.2) to

1.8, values well within the normalrange. Even with reduced

expression of DAF, the Dr(a-) erythrocytes hadnofunctional

defectmeasuredby thisassay.

Single nucleotide change underlies

Dr(a-)

phenotype.

The

nextstepin the molecular characterization of the Dr(a-)

phe-notype wasto

identify

theamino acid changeorchangesthat

Figure2.CLS assay of

Dr(a-)erythrocytes. Erythrocytes froma

Dr(a-)

individualwere

.-

H/

incubated with

anti-CLH501.35 / bodyandvarying dilu-a f tions of fresh human

serum complement.

0l-~: 1 The percenthemolyzed

.0.1 fcellswasmeasuredby

spectrophotometer,and theratio of lysedto

un-O .

...,.

..lysed

cellswasthen

cal-0.oI1

10 100 culated and plotted on

Equivalnt Milliliters Serum (1/270) the y axis versus

equiv-alentmillilitersserumonthexaxis. The value of CLS

H50

wasfound

asdescribedinMethods;in severalexperimentsthis valueranged

(5)

determine the phenotype, including the antigenicity ofthe pro-tein. This was accomplished by using PCR amplification to

cloneand sequence the coding region of membraneDAF. We hadpreviously cloned the human DAF gene, which spans

- 40 kb andconsists of11 exons(26). The membraneform of

DAFisencoded on nineexons (the two additional exons are the first exon encoding the 5'-untranslated region and signal

peptide and analternatively splicedexon thatisabsentfrom

themRNAencoding membraneDAF; 25, 26).Allofthe exons encoding membrane DAF were amplified by PCR from

geno-micDNAoftheoriginalDr(a-)propositus M.D. (30), and the PCR DNA products were subcloned into the vector

pBlue-scriptKS+ and sequenced. There was only a single nucleotide

change intheentire coding region,andthis is shown in Fig. 3:

nucleotide 649 (numberingperreference1) changesfrom C to T in Dr(a-) correspondingto a serine to leucine change at

amino acid residue 165which is inthethird SCR.

Since the entire DNA sequenceencoding the membrane DAFproteinhad been sequenced, the single nucleotide change

found should correspondtothe Dr(a-) phenotype. Toconfirm this

finding,

DNAfrom the identified regionwasamplifiedand

sequenced in two unrelated Dr(a-) individuals, and both showed the same nucleotide change as the propositus. This

nucleotide change thus identifies theseDr(a-) phenotype indi-vidualsas homozygousforaspecific, identifiablealleleofDAF

whichwedesignate

Dr',

alow-frequencyantithetical allele to

the

high-frequency

DAFallele Dra.

Taq I RFLP. The Drb change alters the DAF nucleotide

sequence648-651from TCGAtoTTGA, thus

removing

aTaq

Irestriction site (see bottom ofFig. 3). This allele-specific

RFLPcanbe usedto typeindividualsat the Drlocus, andthis is demonstratedinFig.4. APCR-amplifiedDNAfragmentof

- 1,250 bpwasproduced fromgenomicDNAof eitherDra or

Dr" individuals. ThisDNA wasdigested withTaq Iand

ana-Dr(o+)

C G T A

Intron

-Exon

-_--a

5

i-_

vo

C-A_

_OMA+

D~r(a-) C G T A

.;m

*--_ _

".... WI.

-M . ...

-h

-'

TaqI

Dr(a+): 5 ...TTTGGCTCGACTTCT...

3-PheG ySerThrSer

Dr(a-): 5 ...TTTGGCTTGACTTCT...3'

P heG I yL e uTh rSe r

Figure 3.Single nucleo-tidedifference between

Dr(a+)andDr(a-) in

exonencodingSCR 3B. PCR was used to am-plify genomicDNAof Dr(a+) and Dr(a-) in-dividuals. The5'-primer was a20-merfromexon 4

(5'-AGATTGATG-TACCAGGTGGC-3')

andthe3'-primerwas a24-merfrom intron 5

(5'-GAGTACT-

CAGCCTCACAATCT-GAG-3),thus

amplify-ing the entire exon 5 which encodes SCR 3B. This autoradiograph of thesequencinggel shows thesingle nucleo-tidechangefrom CtoT inthisexon.The

se-quence of thetwoallelesofDAFfrom theregionsurroundingthe

polymorphicnucleotide is shownbelow,with that base marked with

anasterisk. The Taq Irestrictionendonucleasesite that is present in theDr(a+)sequence but absent in theDr(a-)allele is marked with

anoverline.

Dr

(a):

+

+

_

-

Figure

4. RFLP

phenotyping

of

Dr(a+) versus Dr(a-)individuals. PCR-amplified genomic DNA includ-ing the Dr(a+)/Dr(a-) polymorphic locus inexon5wasproducedas

de-1353 - 4_ scribed inFig.3. Products of the PCR 1078- - reaction from two individuals each

872

- of the

Dr(a+)

andDr(a-) phenotypes

603

migrated identically on agarose gels

(notshown).The PCR DNAwas

di-(b

p)

gestedwithTaqIandanalyzed by

agarosegel electrophoresis, and a 1 2 3 4 photograph of the ethidium bromidestained

gel

isshown. The solidarrow

marks the bandfrom Dr(a-) individ-uals(lanes 3 and 4) with no TaqIsites, whereas the open arrow marks thebandfrom Dr(a+) individuals (lanes I and 2) that is produced by TaqIdigestion(which also produces a smaller band of 1 16 bp that is not visualized on this gel). Molecular size markers are shown on theleft.

lyzedby agarose gelelectrophoresisand ethidium bromide staining, demonstratingasingle TaqIsite inDra DNA

produc-ing fragments

of

1,150

and 100

(the

latternotvisualizedon

this gel) andtheDrbDNAwithnoTaq Isite.

Allele-specific transfectants. For further analysis of this

DAF polymorphism, we createdallele-specific transfectants (AST).Just asallele-specific oligonucleotides permitthe

analy-sis of

antigen

or allele

specificity

(51), AST will

permit

the analysis of antibody specificity (see data below and

Discus-sion).

Wehadpreviously cloned the

full-length

(wild-type)

DAFcDNA(24)that corresponds to theDraallele. The cDNA

for theDrballelewascreatedbyusing oligonucleotide-directed invitro mutagenesis (52)tointroducethesingle changeat

nu-cleotide 649 (CtoT) into theDracDNA. Both theDraand the Drb allelic cDNA were subcloned into the expression vector

pSFFV.neo (45) and transfected into CHO cells. Permanent

transfectants were selected in the

neomycin

analogue G418. Individual subclones wereproduced bylimiting dilution fol-lowed byscreening for surfaceDAFexpression by flow cytome-try, andone

high-expressing

cell line ofeach allelictypewas

selected.

Biosynthesis and decay kinetics ofDAF in AST.The biosyn-thesis ofDAFintheDra and Drb ASTwasstudied in standard

pulse-chase kineticexperiments.Ashort

[35S]cysteine

pulse

fol-lowed by a chaseperioddemonstratesessentially identical ki-neticsfor the pro-DAF

species chasing

into the

higher

M,

ma-ture DAF

species (Fig.

5A). Previous work has shown that this large

M,

shift is

mainly

the resultofextensive 0-linked glycosyl-ation

(48).

Next,tolookatthe

stability

ofthetwoallelic forms

of DAF,the AST were

biosynthetically

radiolabeled byalong pulse, chased in nonradioactive

medium,

toallow allofthe

intracellular DAF to get to the

surface,

and thenchased for various timeperiods. Thetwoallelic formsofDAFshow the samedecay kinetics(Fig. 5B). This

experiment

wasrepeated withtheaddition of20% human serum to the chasemedium

(for

the

possible

addition ofany protease orother moleculein human serum that

might

be

responsible

for the differential

stability

of the

polymorphic

forms ofDAFonhuman

erythro-cytes

circulating

in blood),but

again

therewas no

significant

(6)

A Time- Dr(a+) Dr(a-)

(mninI'~

r673bU60

90' 306090' FigureS.Kinetic

analy-MrNx10-) sis of DAF biosynthesis

anddecayin AST. (A)

TheDr(a+)and Dr(a-) 92.5

-(Di'an

Drb,

respec-66-

WW_

, tively)ASTwere

bio-syntheticallylabeled with [35S]cysteinefor 15

45dI.

.min and then chased

for the indicated time periods. Celllysates 31- were prepared and

im-munoprecipitated with

2 3 4 5 6 7 8 9 10 anti-DAF andanalyzed

bySDS-PAGE. The

B 100 - fluorograph

demon-stratesthe samekinetics

o\ \ ineachallele for

pro-\ \, cessingof the

intracellu-F50 lar pro-DAFspecies of 45 kD tothe mature : DAFspeciesof74kD.

0 (B)Thekinetics of DAF

U. \\ decaywereassessedby

o \,\ a 2-h biosynthetic label followed bya2-hchase topermitall DAFtobe processed to themature

0-

1 5 10 15 210 DAF species and

trans-Time(h) ported to the cell sur-face. Cell lysateswere

preparedatthis t=0

pointandsubsequenttimepointsandanalyzedasdescribed above. Laserscanningdensitometryof thefluorographwasusedfor the

quantitationthatisdepictedin thegraph (o,Dr'; ,Drb).Linear re-gressionanalysis yieldeda

t11/2

valueof 7.0 hforDra and 6.6 hfor Dr".

ASTtoexplain the difference inthe levelof

expression

ofthe

twoDrallelic forms ofDAFin human erythrocytes.

Inhibition of anti-Dr'

hemagglutination by

AST. Since therewasonlyasingle nucleotide change in the

coding region

ofthe

Dr'

allele of DAF, itwas

clearly

implicated

in theDr

polymorphism.

Inordertoconfirm this

finding,

wetestedthe DAFAST for

reactivity

with anti-Dra

alloantibodies,

the

re-agents usedtodefine theactual

polymorphism.

Sincethe

al-loantibodies are normally identified in standard blood bank hemagglutination

reactions,

weusedaninhibition

ofhemagglu-tination test. Material from the ASTwereusedtoinhibit

al-loantibody

toDra, whichwasthen

assayed

by

hemagglutina-tion.Asseenin TableIII,the DraASTinhibited anti-Drabut

neither theDrbASTnorthe control cell

transfected

withvector

alonecaused anyinhibition.As

further controls,

material from the HeLa cell line or

plasma

from a Dra individual caused inhibitionbutplasma fromaDrbindividualdidnot cause inhi-bition of

hemagglutination,

as

expected.

Flow

cytometrictesting

ofAST.

Wealso tested theASTina

direct flow

cytometric

analysis.

Specifically,

thealloantibodies wereusedtosensitizetheAST, whichwerethenstained with FITC-labeled second

antibody

and

analyzed

by

flow cytome-try. The results in

Fig.

6 demonstrate that the AST show the expectedDr

specificity, i.e.,

thealloanti-Dra

recognizes

theDra butnottheDrb

AST,

whereas therabbit

polyclonal

anti-DAF

recognizes

both AST. Thus, the AST

testing

allowsusto

con-firm thesingleamino acidchangefrom Drato

Dr'

asthebasis ofthe Drpolymorphism.

Discussion

This studyhas analyzed at the biochemical, functional, and molecular level thepropertiesofaDAFpolymorphic variant.

Thishas produced several conclusions concerning DAF, and it has also led to a new approach to serological analysis in immu-nohematology through the use of allele-specific transfectants. At the protein level, the Dr(a-) phenotype was found to be due toreduced surface expression of a slightly altered form of DAF (basedonits mobility on SDS-PAGE), with normal expression ofother GPI-anchored membrane proteins. This form of DAF possessesnormalcomplement regulatory activityasmeasured inthe CLS assay. The actual molecular basis for the phenotype isa variant allele of DAF, named Drb, that encodes a single nucleotide (and amino acid) change from the common Dra allele. Finally, these two alleles of DAF were differentiated by

anallele-specific Taq I RFLP, and were expressed inforeign

cells to create AST that could be used in analyzing the

specific-ityofhuman alloantiserum.

The Dr(a-) phenotype of DAF is one of eight identified genetic variants in the Cromer-related blood group system that resides on the DAF molecule (29). With the exception of the Inabphenotype, which lacks all surface expression of DAF, the Dr(a-) phenotype is the only variant that demonstrates re-duced DAF expression by hemagglutination. Because the PNH phenotype results in reducedexpression ofallGPI-anchored membrane proteins, it was important to compare these two phenotypes. Radioimmunoassays measuring antibody reactiv-ity againsterythrocytes clearly demonstratedthat the Dr(a-) erythrocytes had an isolated defect in expression of DAF with normalexpressionofthree otherGPI-anchored proteins.This is inmarked contrast to the PNH III phenotype with reduced expression of all of these GPI-anchored proteins (Table II). Western blotanalysis confirmedthe reduced expression of DAF protein, and suggested aslightalteration ofSDS-PAGE mobility.

Previousinvestigationsof the Inab phenotype had found a mild defect in complement regulatoryactivityasjudgedby the complement lysis sensitivity assay, comparable to PNH II cells

(53, 54).Itwastherefore of interestto testDr(a-) erythrocytes,

Table III. Inhibition ofAnti-Dra Hemagglutination by AST

Sample Inhibition

CHO/Dra +

CHO/Drb

CHO/SFFV.neo

HeLa +

Dra plasma +

Drb plasma

Tissue culture supernatants from CHO cells transfected with Draor

Drb alleleDAFcDNAortheexpression vectorSFFV.neoorfrom the HeLa cellline,orplasmaof Dr'orDrbhomozygousindividuals wereincubated with alloantibodytoDra,thentestedfor hemaggluti-nation of normal erythrocytes. Inhibition(+)orfailuretoinhibit (-)

(7)

Figure6. Assay using

ASTtoassess

alloanti-serumspecificity by

flow cytometry.TheDr

andDri ASTwere

treated withhuman

anti-Dra

orwith rabbit

: anti-human DAF,

fol-O) .- r : I' \ lowedbyappropriate

O

0 ~ h *@. , I K FITC-labeledsecond

_ ______________________ antibody. Theywere

analyzed byflow

cy-E tometry, and the panels

Z3

showrelativenumber

ofcells versus relative

logfluorescenceona four decade scale.(Top) DraAST(thick line)

andDib AST(thin line)

both showstrongDAF

expressionasassessed

Log

Fluorescence

with rabbit anti-human

DAF compared to the negative control CHO cellstransfectedwith vector alone(dottedline). (Bottom) Testing

with human anti-Dr' demonstrates reactivity with the Dr' AST(thick line)but not theDr'AST(thinline).

which mighthavealtered complement regulatory function ei-therbecauseof thereduced expression (- 40% ofnormal)or because of the underlying minor structural alteration in the DAFmolecule.Infact,theDr(a-)cells had anormalCLS titer (Fig. 2). Thus, neither the structural change nor the greater thantwofold reduction in expression impairDAFfunction(at

least asjudged by this assay). This suggests that there might

normallybe an excess ofrequiredDAF on thesurfaceof eryth-rocytes.

The remaining part of these studies focusedon the actual

molecular variationthatunderliesthe Drpolymorphism:a

sin-glenucleotide changefromC to Tresultinginasingleamino acidchange from serinetoleucine. Thiswas the only

nucleo-tidechange presentinthecoding region ofthe DAFgeneofthe

originalDr(a-)propositus,andthe same changewasfoundin two other unrelated Dr(a-) individuals. Thepresence of this nucleotide sequencecan then be used toidentifythe DAF allele

presentin theseindividuals, whichwedesignate

Dr".

This isthe first case, to our knowledge, whereanerythrocyte antigenwas firstdefinedatthenucleotide leveland not by thediscoveryof analloantiserum of the

appropriate specificity.

This method of

antigen identificationat the DNA levelwillbeespecially useful for low-frequency alleles where it isvery unlikely that an

al-loantiserumwill befound.

Expression of the twoDralleles in CHO cellstocreate AST

hasallowed us toprobetheproperties conferedby theallelic difference,as well as to set up a systemfor alloantibody identi-fication (seebelow). The pulse-chase experiments showed no

difference betweenthe twoallelic forms ofDAF asfaras the

kinetics ofprocessing ofDAFfrom a precursor form to the

mature, glycosylated form orin the

kinetics

of decay.

There-fore, thepolymorphic difference astested in these kinetic

ex-periments inCHOtransfectants doesnotexplain the reduced expression ofDAFinDrb erythrocytes. There are two broad

classesof explanations for this

finding:

either the reduced

ex-pression

doesnotmaptothesamevariation causingtheallelic difference

(e.g.,

there isasecond

change, perhaps

inthe

regula-tory

region

of the gene,that leadstoreduced transcription), or

the CHOtransfectant systemdoes not reproduce the

condi-tions

causing

the reduced

expression

in human

erythrocytes.

Sincetwoseparate,

widely spaced

mutationsseemless

likely

a

priori,

onehastoconsider themanydifferences that arisein

comparing

the CHO cells andthe

erythrocytes.

Some of the

major

considerationsare:(a) the altered amino acid might

re-sult in a

change

in posttranslational processing (since itis a serinetoleucine

change,

loss of0-linked glycosylation is

possi-ble)

inhumancellsthat isnotreproduced faithfullyin theCHO

cells; (b)

thetissue culturesystem ingeneraldoesnotmatch the

in vivo bloodstreamconditions;

anid

(c) the CHOcell type

(and

perhaps

allnucleatedcells) isnot agoodmodelfor the

erythro-cyte asfarasDAF

expression

and

stability.

Inregardtothis latter

point,

it should be noted that the reducedexpressionof DrbDAFhas only been documentedonhuman erythrocytes,

soinvestigationofhuman leukocytes shouldalso be anavenue

for future

study

ofthis

question.

Thefinal impactofthe studiesreportedhere isin the areaof

serological

analysisthatformspartofthe foundationsof

immu-nohematology. Through

theuseofallele-specificRFLP

analy-siswe canphenotypeindividuals at the DAF Drpolymorphic locus (Fig. 4). Thiswasmadepossiblein the presentsituation becausethe

polymorphism

involvedthe loss ofaTaqI restric-tionendonuclease

site,

but if thiswere notavailablethe DNA sequencedifferencecould beascertained by hybridizationwith

allele-specific

oligonucleotides. That approach has been used

extensivelyfortyping polymorphic HLAloci

(55).

Themorecommonprobleminimmunohematologythan theidentificationofalloantigen specificity (phenotyping)is the

identificationofalloantibody. The system that we have

estab-lished withASTallows that alloantibody analysis as

demon-stratedbyeither oftwoassays,inhibitionof

hemagglutination

(Table III)orflowcytometry(Fig. 6). In the present casethey allowedustocomefullcircleand confirm the molecularbasis

of the polymorphism usingthealloantisera toDra that

origi-nally definedthepolymorphism.Inthe general case they would allowserological analysisof any alloantiserum. One ofthe

ma-jor advantagesof this approachusingAST is that only a

single

allele ofonebloodgroup system isexpressed byeachAST ina

foreign

cellbackgroundthatlacksall of the otherbloodgroup

antigens of thatorother systems. Therefore one can test an

alloantiserumthatmightcontain multipleerythrocyte alloan-tibodies

(and

possibly alsoan autoantibody) againstan AST

and get an answer regarding the specific allele of that AST. Similar testing against human erythrocytes isambiguous

be-cause the erythrocytes express multiple alloantigens. As the

major human bloodgroup systems arecloned,one could create

apanelofAST coveringmostorall of theclinically important

systems, allowing identification of all of the specificities in a

complex alloantiserum. Neither of the two assays that were used in this study would be the most practical for routine use in serological analysis in the clinicallaboratory,buttheycould be replaced with other methods oftestingthealloantiserum versus the AST, such as agglutination reactions or some form of en-zyme-linkedimmunoassay. The actualdemonstration in this report that one can useallele-specificRFLP andallele-specific transfectants for the analysis of erythrocyte alloantigens and

(8)

We thank MiguelArceand KatharineCoyneforexperttechnical

assis-tance, Kelli Simburger for oligonucleotidesynthesis, and JulieNegrete

forexpertassistance inpreparationofthe manuscript.

This work was supported bygrantsfromtheNational Institutes of

Health(AI-15322)andfromthe American AssociationofBlood Banks

Foundationto D. Lublin, and bygrants HL-33572 and HL-44042

from the NIH to M. Telen. Dr. Telen is the recipient ofResearch

Career Development Award HI-02233from the NIH.

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(9)

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Med glavne prednosti načrtovanja izdelka s pomočjo metode QFD so med drugim Benefits of QFD, 2000:  majhno število sprememb tekom procesa razvoja,  manj potrebnega časa za