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
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,DukeUniversityMedicalCenter,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-)
erythrocytesdem-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
toleucinechange.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 ofhemagglutination
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 byautolo-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, withpartoftheincreasedsensitivity
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 geneby
restrictionfragment
lengthpolymorphisms
(RFLP)analysis (3, 27). Recently
theCromer blood group antigens
(reviewed
in28)
have beenshowntoresideontheDAFmolecule(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 functionalinvestigation
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). Erythrocytesof the
Dr(a-)
phenotype
lackexpression
of the Dr3antigen
and have lowexpression ofallofthe other Cromerallelesincluding
Cr3,
Tcl,Esa, WESb,
andIFC.Nohematological
abnormalitieshave been reportedto beassociated with the Dr(a-)
pheno-type.
Inthis studywe
investigated
thebiochemicalexpression
ofDAFintheDr(a-) phenotypeandcharacterizedthefunctional properties of thisDAFvariant. Furthermore, we have identi-fied themolecular
(nucleotide) change
thatdefinestheDr(a-)
phenotype, foundanallele-specificRFLPthat can be usedtophenotype individuals, andhavecreated
allele-specific
trans-fectants thatcanbe usedto
identify
alloantibodies.J.Clin.Invest.
©TheAmericanSocietyfor ClinicalInvestigation,Inc.
0021-9738/91/06/1945/08 $2.00
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 a50-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,SV40splicing
andpolyadenylation signals,and theneomycin-resistancegene. Chinese hamster ovary(CHO)cellsweretransfected
by
lipid-mediatedDNA transfectionusing10 Aig DNA and100gtg
Lipofectin (46).
Positive cellswereselectedbyresistancetotheneomycin 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 cellswerewashed,incubatedincysteine-freeHam's F-12 medium with 5%
dialyzed
FCS for 1h,and then 100MCi/ml [35S]cysteinewasadded. For kineticexperiments,the cellswerepulsedfor the indicated timeperiodand 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 celllysatewasimmunoprecipitatedwith rabbit polyclonal DAF
anti-serum asdescribed(48)andanalyzedon9%SDS-polyacrylamide
gels
under
reducing
conditions followedbyfluorography.
Inhibition
of hemagglutination.
Agglutinationof humanerythro-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 notremovesurface 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
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, asjudged bySDS-PAGE, was approximately thesame as on
Dr(a+)erythrocytes; however, comparison of thetwospecies
ongels of
differing polyacrylamide concentrations
suggestedapossible
small decrease (< 2,000) inapparentM,
of
theDr(a-)DAF(notshown).
Expression
of GPI-anchored
proteins on Dr(a-)erythro-cytes.
Having
seenthat Dr(a-) erythrocytesexpress areducedamountofa DAF
species
possessing
nearnormalsize,
we nextstudied
expression
of other GPI-anchored membraneproteins
toascertain whether the defectwaslimitedtoDAF.
Radioim-munoassay wasusedto measurereactivity with antibodiesto
three otherGPI-anchored
proteins: acetylcholinesterase,
mem-brane
inhibitor
ofreactivelysis
(MIRLorCD59), andlympho-cyte
function-associated antigen
3 (LFA-3 orCD58). The Dr(a-) erythrocytes showeda markedlyreducedreactivity withantibodies toDAF,but they showedessentiallynormalreactivity
with antibodies against the other three GPI-anchoredproteins
(TableII). Bycontrast,when cellsfromapatient withNONNMMUNE
Figure
1.Western blotRABBITPOLYCLONALAN-I-OAF RABBI' SERUM
r
>~~,~--
for DAFin
humanerythrocytes. Varying
-~ ; amountsoferythrocyte
membranes
(protein/
lane) preparedfrom
DAf~ db -
Dr(a+)
orDr(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 alsovisualized;
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) to1.8, 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.
Thenextstepin the molecular characterization of the Dr(a-)
phe-notype wasto
identify
theamino acid changeorchangesthatFigure2.CLS assay of
Dr(a-)erythrocytes. Erythrocytes froma
Dr(a-)
individualwere.-
H/
incubated withanti-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
cellswasthencal-0.oI1
10 100 culated and plotted onEquivalnt Milliliters Serum (1/270) the y axis versus
equiv-alentmillilitersserumonthexaxis. The value of CLS
H50
wasfoundasdescribedinMethods;in severalexperimentsthis valueranged
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 regionwasamplifiedandsequenced 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 tothe
high-frequency
DAFallele Dra.Taq I RFLP. The Drb change alters the DAF nucleotide
sequence648-651from TCGAtoTTGA, thus
removing
aTaqIrestriction 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. RFLPphenotyping
ofDr(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 theDr(a+)
andDr(a-) phenotypes603
migrated identically on agarose gels(notshown).The PCR DNAwas
di-(b
p)
gestedwithTaqIandanalyzed byagarosegel electrophoresis, and a 1 2 3 4 photograph of the ethidium bromidestained
gel
isshown. The solidarrowmarks 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
of1,150
and 100(the
latternotvisualizedonthis 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 allelespecificity
(51), AST willpermit
the analysis of antibody specificity (see data below andDiscus-sion).
Wehadpreviously cloned thefull-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, andonehigh-expressing
cell line ofeach allelictypewasselected.
Biosynthesis and decay kinetics ofDAF in AST.The biosyn-thesis ofDAFintheDra and Drb ASTwasstudied in standard
pulse-chase kineticexperiments.Ashort
[35S]cysteine
pulsefol-lowed by a chaseperioddemonstratesessentially identical ki-neticsfor the pro-DAF
species chasing
into thehigher
M,
ma-ture DAF
species (Fig.
5A). Previous work has shown that this largeM,
shift ismainly
the resultofextensive 0-linked glycosyl-ation(48).
Next,tolookatthestability
ofthetwoallelic formsof DAF,the AST were
biosynthetically
radiolabeled byalong pulse, chased in nonradioactivemedium,
toallow alloftheintracellular DAF to get to the
surface,
and thenchased for various timeperiods. Thetwoallelic formsofDAFshow the samedecay kinetics(Fig. 5B). Thisexperiment
wasrepeated withtheaddition of20% human serum to the chasemedium(for
thepossible
addition ofany protease orother moleculein human serum thatmight
beresponsible
for the differentialstability
of thepolymorphic
forms ofDAFonhumanerythro-cytes
circulating
in blood),butagain
therewas nosignificant
A Time- Dr(a+) Dr(a-)
(mninI'~
r673bU60
90' 306090' FigureS.Kineticanaly-MrNx10-) sis of DAF biosynthesis
anddecayin AST. (A)
TheDr(a+)and Dr(a-) 92.5
-(Di'an
Drb,
respec-66-
WW_
, tively)ASTwerebio-syntheticallylabeled with [35S]cysteinefor 15
45dI.
.min and then chasedfor 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 andtrans-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
ofthetwoDrallelic forms ofDAFin human erythrocytes.
Inhibition of anti-Dr'
hemagglutination by
AST. Since therewasonlyasingle nucleotide change in thecoding region
oftheDr'
allele of DAF, itwasclearly
implicated
in theDrpolymorphism.
Inordertoconfirm thisfinding,
wetestedthe DAFAST forreactivity
with anti-Draalloantibodies,
there-agents usedtodefine theactual
polymorphism.
Sincetheal-loantibodies are normally identified in standard blood bank hemagglutination
reactions,
weusedaninhibition ofhemagglu-tination test. Material from the ASTwereusedtoinhibital-loantibody
toDra, whichwasthenassayed
by
hemagglutina-tion.Asseenin TableIII,the DraASTinhibited anti-Drabutneither theDrbASTnorthe control cell
transfected
withvectoralonecaused anyinhibition.As
further controls,
material from the HeLa cell line orplasma
from a Dra individual caused inhibitionbutplasma fromaDrbindividualdidnot cause inhi-bition ofhemagglutination,
asexpected.
Flow
cytometrictestingofAST.
Wealso tested theASTinadirect flow
cytometric
analysis.
Specifically,
thealloantibodies wereusedtosensitizetheAST, whichwerethenstained with FITC-labeled secondantibody
andanalyzed
by
flow cytome-try. The results inFig.
6 demonstrate that the AST show the expectedDrspecificity, i.e.,
thealloanti-Drarecognizes
theDra butnottheDrbAST,
whereas therabbitpolyclonal
anti-DAFrecognizes
both AST. Thus, the ASTtesting
allowsustocon-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 (-)
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
showrelativenumberofcells versus relative
logfluorescenceona four decade scale.(Top) DraAST(thick line)
andDib AST(thin line)
both showstrongDAF
expressionasassessed
Log
Fluorescence
with rabbit anti-humanDAF 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 theappropriate specificity.
This method ofantigen 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 reducedex-pression
doesnotmaptothesamevariation causingtheallelic difference(e.g.,
there isasecondchange, perhaps
intheregula-tory
region
of the gene,that leadstoreduced transcription), orthe CHOtransfectant systemdoes not reproduce the
condi-tions
causing
the reducedexpression
in humanerythrocytes.
Sincetwoseparate,
widely spaced
mutationsseemlesslikely
apriori,
onehastoconsider themanydifferences that ariseincomparing
the CHO cells andtheerythrocytes.
Some of themajor
considerationsare:(a) the altered amino acid mightre-sult in a
change
in posttranslational processing (since itis a serinetoleucinechange,
loss of0-linked glycosylation ispossi-ble)
inhumancellsthat isnotreproduced faithfullyin theCHOcells; (b)
thetissue culturesystem ingeneraldoesnotmatch thein vivo bloodstreamconditions;
anid
(c) the CHOcell type(and
perhaps
allnucleatedcells) isnot agoodmodelfor the erythro-cyte asfarasDAFexpression
andstability.
Inregardtothis latterpoint,
it should be noted that the reducedexpressionof DrbDAFhas only been documentedonhuman erythrocytes,soinvestigationofhuman leukocytes shouldalso be anavenue
for future
study
ofthisquestion.
Thefinal impactofthe studiesreportedhere isin the areaof
serological
analysisthatformspartofthe foundationsofimmu-nohematology. Through
theuseofallele-specificRFLPanaly-siswe canphenotypeindividuals at the DAF Drpolymorphic locus (Fig. 4). Thiswasmadepossiblein the presentsituation becausethe
polymorphism
involvedthe loss ofaTaqI restric-tionendonucleasesite,
but if thiswere notavailablethe DNA sequencedifferencecould beascertained by hybridizationwithallele-specific
oligonucleotides. That approach has been usedextensivelyfortyping 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 ofthema-jor advantagesof this approachusingAST is that only a
single
allele ofonebloodgroup system isexpressed byeachAST ina
foreign
cellbackgroundthatlacksall of the otherbloodgroupantigens of thatorother systems. Therefore one can test an
alloantiserumthatmightcontain multipleerythrocyte alloan-tibodies
(and
possibly alsoan autoantibody) againstan ASTand 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
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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