Isolation of a complementary DNA clone for the human complement protein C2 and its use in the identification of a restriction fragment length polymorphism

Isolation of a complementary DNA clone for the

human complement protein C2 and its use in

the identification of a restriction fragment

length polymorphism.

D E Woods, … , M D Edge, H R Colten

J Clin Invest.

1984;

74(2)

:634-638.

https://doi.org/10.1172/JCI111461

.

Complementary DNA (cDNA) clones corresponding to the major histocompatibility (MHC)

class III antigen, complement protein C2, have been isolated from human liver cDNA

libraries with the use of a complex mixture of synthetic oligonucleotides (17 mer) that

contains 576 different oligonucleotide sequences. The C2 cDNA were used to identify a

DNA restriction enzyme fragment length polymorphism that provides a genetic marker

within the MHC that was not detectable at the protein level. An extensive search for

genomic polymorphisms using a cDNA clone for another MHC class III gene, factor B, failed

to reveal any DNA variants. The genomic variants detected with the C2 cDNA probe

provide an additional genetic marker for analysis of MHC-linked diseases.

Research Article

Isolation of

a

Complementary

DNA

Clone for the Human Complement

Protein C2

and Its

Use

in the Identification of

a

Restriction

Fragment

Length Polymorphism

Derek E.Woods, Michael D. Edge,and Harvey R.Colten Divisionof Cell Biology, DepartmentofMedicine, Children's

Hospital, Ina SuePerlmutterCysticFibrosis Research Center, DepartmentofPediatrics, Harvard Medical School, Boston, Massachusetts 02115; Imperial ChemicalIndustries, PharmaceuticalsDivision,Mereside, AlderlyPark,

Macclesfield, Chesire,England

Abstract.

Complementary

DNA

(cDNA) clones

corresponding to the major histocompatibility (MHC)

class III antigen, complement protein C2, have been

iso-lated from human liver cDNA libraries with the use of

a

complex

mixture of synthetic oligonucleotides (17 mer)

that contains 576 different oligonucleotide sequences. The

C2

cDNA were

used to

identify

a DNA

restriction enzyme

fragment length polymorphism that provides

a

genetic

marker

within the MHC that was not detectable at

the

protein level. An

extensive search

for genomic

polymor-phisms using a cDNA clone

for

another MHC class

III

gene, factor

B, failed to

reveal any

DNA

variants. The

genomic variants detected with the C2 cDNA probe

pro-vide an additional genetic marker for analysis of

MHC-linked diseases.

Introduction

The genes for three of the serum complement proteins, the second (C2) and fourth (C4) components and factor B, have beenlocalizedtothemajorhistocompatibility complex(MHC) in humans,mice,andguinea pigs(1) and have beendesignated classIIIMHCantigens.HumanC4isathree-chainglycoprotein

- 200,000Mrcodedfor bytwoloci,C4A and C4Bonthe short

arm of chromosome 6 (2). The products of these loci differ

structurally (3) and functionally (4) and severalprotein poly-morphic variantshave beenidentified foreach locus (4). Factors

BandC2 are single chain glycoproteins of95,000 (5) and 102,000

Receivedfor publication 20January 1984 andinrevisedformSApril

1984.

Mr (6),respectively, which function in the alternative and

clas-sical pathways ofcomplement activation. Several geneticvariants of factor B protein have been identified by differences in mobility on agaroseelectrophoresis. These have been useful in analysis ofpopulations and families with MHC-linked disorders such asinsulin dependentjuvenileonsetdiabetes mellitus (7).

C2 protein variants have been recognized by isoelectric fo-cusing of serum samples (8) in polyacrylamide gels and detected functionally by overlaying with an agarose gel containing an-tibody-sensitized sheep erythrocytes and either C2-deficient hu-man serum or dilute normal human serum. These reveal a common variant, C2C, a basic variant in 4-5% of unrelated individuals designed C2B, and rarely anacidicvariant (1). Two such acidic C2 variants, designated C2 Al and C2 A2 have been recognized in <1% of the population. Individually, the classIIIgeneproductsdisplay lesspolymorphismthan the class

Iand classIIproteins.

Recently, cDNA clones correspondingtohuman and mouse classIandIIhistocompatibility antigens(HLA andH2)(9-13) and the classIIIproteins factorB(14, 15) and C4(16-18)have beenisolated and characterized. Theavailabilityof cDNA probes for genes within the MHC havepermitted an analysis of the

finestructureof this region (13, 19). Additionalstructural data

derivedfrom nucleotidesequencingwillleadtoanunderstanding

of the molecular basis ofpolymorphic variants important in regulation of the immune response.

DNApolymorphismsof the C4 genes have been identified by two groups (20, 21). These C4 DNA polymorphisms were

detected among individuals who werehomozygousasdefined by C4protein variants. This permitted subdivision ofthe C4 variants already identified and therefore generated additional markers within the MHC. These results prompted efforts to

isolate human C2 cDNA clones that could be usedtogether withfactor B cDNA (14) probestogenerate additional markers for analysis of MHC-linked diseases. Genomicpolymorphisms

at agivenlocus aredetectedbydigestionofDNAsampleswith restriction endonucleases thatrecognizespecificnucleotide

se-J. Clin. Invest.

©TheAmerican Societyfor ClinicalInvestigation,Inc.

0021-9738/84/08/0634/05 $1.00

quences. TheresultingDNAfragmentsaresize-separated

elec-trophoretically andtransferred to nitrocellulose filters. Radio-labeled DNA probes are then used to detect fragments that contain complementary sequences (22).

Inthis report, we describe the isolation of cDNA clones for human C2. TheC2 clone and afactorB clone(14) have been used to investigate polymorphisms of these genesatthe DNA

level.

Methods

IsolationofC2cDNAclones.A17-nucleotide longoligonucleotide

mix-ture(Fig. 1) wassynthesized byasolid-phase phosphotriester method

(14). Inpositionof nucleotide7and13for the glutamine (Gln) codons, 25%cytosine (C) wasaddedaswell as theguanosine (G)in order to

obtain representation of the codon for glutamicacid (Glu). The oli-gonucleotide mixture was kinase labeled with [32P]dATP (23) and

used to screen a humanlivercDNAlibrary, asdescribed by Woodset

al.(14).

Approximately 40,000 clones were screened. The final posthy-bridization washeswere at45°C in 6 XSSC(0.15 MNaCl, 0.015 M Nacitrate, and 0.05% sodium

pyrophosphate).'

Positive cloneswere

colony purified and rescreened asdescribed above. The five positive

clonesisolatedwereused asprobesinNorthern blotanalysis(24)against

human liver mRNA todetermine if they hybridized to a mRNA of approximately thecorrectsize. One of theclones, pC2-10 (-500base pairs(bp)), waspartially sequenced byusing theMaxamandGilbert sequencing procedure(23).pC2-10wasusedto rescreen200,000 clones of the human adult livercDNAlibrary( 14) and15additional C2 clones

wereidentified.Thelargestof thesewaspC2-6(900 bp). This insertwas used to screen ahuman fetal livercDNAlibrary(25) andaC2 clone (pC2-F2) of 1.6 kilobases(kb)wasisolated.

Restriction enzymeanalysis ofhuman DNA. Restriction enzyme digestionswere carried out and DNA samples wereanalyzed by the

Southernblottechnique (22).Theinsertfromthe C2clone(pC2-6)and

thefactorBclone(pBfA28)(14) werenicktranslated(26) and used as probes.

Complement protein typing.Bloodfor genetic typing of BF, C2, and C4wascollectedinto 1 mg/ml of sodium ethylenediaminetetra-acetate

(Na2 EDTA). Plasma wasobtainedbycentrifugationand immediately

frozenand stored at-80°C. Thespecimenswerethawed immediately

before analysis.

Plasmasamplesweresubjectedtoagarosegelelectrophoresis.Factor Bpatternswerevisualizedbyimmunofixation withgoatanti-humanB

(Atlantic Antibodies, Scarborough, ME)as described previously (27). ForC2 typing, plasma samples were subjected to isoelectric focusing in

polyacrylamide gels (8) and C2patternsweredevelopedwithanagarose

gel overlaycontainingantibody-sensitized sheeperythrocytes andfresh

normalhumanserumdiluted 1:90instead of C2-deficienthumanserum

asoriginally described.

For C4 typing, plasma samples were desialated and detected by

electrophoresis ofdesialatedplasma in agarose gels and thenimmunofixed withgoat anti-human C4 antiserum (Atlantic Antibodies) (4).

Complementgenetic nomenclature. The nomenclature for genetic polymorphism ofthecomplementproteins conformstothe International

1.Abbreviations usedin this paper:SSC,0.15 M NaCl,0.015 MNa

citrate,and 0.05% sodiumpyrophosphate.

System for Human Gene Nomenclature (1979) (28) as revised at the

4thInternational Workshopfor the Genetics of Complement, Boston, July 1982. Alleles of the complotypesarewritten in the order BF, C2, C4A, andC4Balthough thetrueorderof the genesis C2, BF,C4A,

andC4B (29).

Results

The C2 oligonucleotide mixture shown in Fig. 1 was kinase labeled and used as a probe to screen 40,000 clones of the human adult liver cDNA library. The nitrocellulose filters were washedin 6 XSSCat45°C and autoradiographed. Under these conditions, five weakly hybridizing cloneswereidentified. Anal-ysis of these clones by hybridizationto aNorthernblot ofhuman livermRNAshowed that onlytwoclones hybridized tomRNA

largeenough to codeforC2 i.e., -2.7 kb. One of these clones, pC2-10 (-500 bp),was partially sequenced by using Maxam andGilbertDNAsequencing; the resultsare shownin Fig. 2. This sequence represents the 5'-terminus of the clone, which corresponds tothe amino-terminus of the C2a fragment (30)

(the 70,000-D carboxyterminal fragment of C2generated by the Cl enzymeduring C2 activation) plus the nucleotidesfor five amino acids of thecarboxyterminus of the C2b fragment. Fig. 2 also showsacomparison of the protein sequence generated from the cDNA sequence (a), the published sequence for the amino-terminus ofhuman C2a(30) (b),and thepublished

se-quence of guinea pig C2a (30) (c). The amino acid sequence derived from the cDNA sequence is homologoustothe published amino acid sequencesfor both human andguinea pig C2, thus identifying pC2-10 as a C2 cDNA clone. The amino acid

se-quencederived from the cDNA clone differs from the published human C2aprotein sequence atresidue 6 (Arg for Ser) and 10 (Leu for Lys). Differences between thederivedhumanC2a

se-quences and the published sequence forguinea pig C2a (30)

were evident atpositions 18, 20, 24,25, 27 and 28 (Fig. 2).

DNA isolatedfrom leukocytes of six unrelatedindividuals

(Glu) (Glu)

Lys Ile Gln Ile Gln Ser Protein Sequence (30)

1 2 3 4 5 6

AAG AUA CAA AUA CAA UC

A C(G)G C(G)G AG

U U TTC TAT GTT TAT GTT AG

T G(C)C G(C)C TC

A A

I

mRNA Sequence

Oligonucleotide Sequence

Figure 1.Amino acidsequenceofhumanC2ausedtoconstructthe syntheticoligonucleotidemixture,whichwasthen usedtoisolateC2

cDNA clones. ThederivedpotentialmRNA sequences and the

C2b C2a

4-w

GGC AAG CCA GGC CGU AAA AUC CAA AUC CAG CGC UCU GGU CAU CUG AAC CUC UAC CUG C

(a) Gly Lys Pro Gly Arg Lys Ile Gln Ile Gln Arg Ser Gly His Leu Asn Leu Tyr Leu I 1

-uc

Leu

10

(b) Lys Ile Gln Ile Gln Ser X Gly X Lys Asn Leu Tyr

(c) Lys Ile Gln Ile Gln Arg Ser Gly His Leu Asn Leu Tyr Leu Leu

CUA GAC UGU UCG CAG AGU GUG UCG GAA AAU GAC UUU CUC AUC UUC (a) Leu Asp Cys Ser Gln Ser Val Ser Glu Asn Asp Phe Leu Ile Phe

20 .30

(c) Leu Asp Ala Ser Lys Ser Val Ser Gln Gln Asp Ile Glu

Figure 2. Partialnucleotide se-quenceof human C2cDNA clone pC2-10 and amino acid sequencederived from the nu-cleotide sequence (a); pub-lishedamino acid sequence of theaminoterminal segment of humanC2a(b); and guinea pig C2a (30) (c).Aminoacid resi-dues shown in boldface indi-cateposition of the sequence used toconstruct the oligonu-cleotidemixture.Residues

un-derlined showamino acid dif-ferences between the sequence derivedfrom thecDNAand the published human and guinea pig amino acid

se-quences.

wasdigested witha panel of 36 restriction enzymes (EcoR I, PstI,BamH I,XbaI,Kpn I,MspI,HhaI,SacI,BstNI,Hinf

I, Bgl II, MboI, NciI, RsaI, MluI,Apa I, HindIII, Tthl1 1 I, ScrF I, Mbo II, NdeI, NaeI, ScaI, EcoR V, Taq I, Sin I,

Cla I,NarI,XhoI,Pvu II,StuI,XmnI,Bgl I,BanI, BanII,

andBcl I) specific for different DNAsequences. Thisdigested DNAwasanalyzed by using the Southern blotting technique

andhybridizedwith the factor B and C2(pC2-6)cDNAprobes. No factor B genomic polymorphisms were identified in this

panel although individualswithdiffering factorBproteinvariants

wereincluded.Inasecond experiment, 23oftheenzymes were

usedtoanalyzeDNA from 20 additional unrelated individuals.

No genomic variants of factor B were identified. In contrast,

usingtheC2 probeto screenthe initialpanel ofsix unrelated

individuals, the DNA digestedwith theenzymeBamH I revealed

apolymorphismthatwasrepresentedbytwobandingpatterns, each containing four bands(Fig. 3). Pattern I contains bands

6.9,4.5, 3.35, and 2.3 kb and pattern IIcontains bandsat6.9, 6.6, 3.15, and 2.3 kb. The intensities of the bands in Fig. 3

indicate that the 6.6- and 3.15-kb bandsarederived from the

4.5- and 3.35-kb bands. To date 27 unrelated individuals (54 chromosomes)have been tested for the BamHIpolymorphism;

22 of these individuals werehomozygousfor bandingpattern

I and 5 were heterozygous, i.e., show banding pattern I/IH.

Therefore,the DNAbandingpatternIIisrepresentedon- 10%

ofthe chromosomes.No individualshomozygousfor the pattern II havebeen identifiedyet;thisvariant wouldonlyoccurata

frequency of- 1in100individuals. The 22peoplehomozygous

forthe C2 DNA pattern I include individuals with either the

C2C or the C2B protein variants. In addition, the C2 DNA

bandingpatternsI andI/IHhave been identified in individuals

homozygous for C2protein variant C2C. Thesefindings

dem-onstratethat the C2 DNA variantsrepresentnewgeneticmarkers distinct from the C2proteinvariants. ThepC2-6cDNAprobe

wasdigestedwith BamHI.No internal BamH I sitewasobserved

(data not shown), indicatingthat the restriction sites detected

ingenomicDNAwereinflanking regions, interveningsequences, orincoding regionsnotcoveredbythe -975bpcDNAprobe. The Mendelianinheritance of the C2 DNA variants is shown inFig. 4. Analysisof DNA from membersof threegenerations

in this family are consistent with an autosomal codominant

0)

Kb

E

23

9.4

am

6.6w0

_ a) 03) 0)

oL a. a

zQ zz

a

Kb

.- -6.9

- 6.6

~41

S..

4.4

*W 't--:-l.

3.35

. -.

_3.

_{1 :}

2.2-2.0

A

B

Figure 3. Southern blotanalysisof DNA from five unrelated

individ-ualsprobedwith 32P-labeledpC2-6cDNA clone(A).Banding pat-ternsarerepresenteddiagramaticallyinpanelB.

-5

"'N

t _{2 3 4} markers

Kb

9.4 _

6.6 _

4.4

-~~~~~~~

₂₂

,-

4o

2.20

2.0

7

SC3 /

fr C, 3 tISt

Figure4. Inheritancepatternofprotein complotypes;

variants. Complotypesgiven below eachfamilymemb

C4A, C4B-,/DNApattern.Individual number4w

alignthe twoSouthern blots.

modeof inheritance of the C2genomicpatterns.

of the protein complotypes ofeach family me reveals thatinthis family the C2DNApattern

with the F protein variant offactor B. Howev with the F variant do not always show the typeI

since two other unrelated individuals with this have been identified and were homozygous

pattern I.

4 5 6 7 used forisolation of many low abundant cDNA clones. Only limited publishedamino acid sequence data for human C2were

available (30). This sequence necessitated synthesis of an

ex-tremely complex mixture of oligonucleotidesin order to include

the authentic coding sequence (Fig. 1). In the regions

corre-sponding to amino acids atpositions 3 and 5 (Glu) we

con-structed amixture that contained 75% glutaminecodons and

25%glutamic acid codons in orderto accountforpossible protein sequencing errors. The resulting oligonucleotide mixture

con-tained 576 different sequences.A difference was found between

theamino acid atposition 6 of thepublishedsequence andthat

specified by the cDNA clone(pC2-10). This difference ledto a

nucleotide sequence error in thesyntheticoligonucleotideprobe used toisolate the clone.Despite thiserrorand thecomplexity of theoligonucleotide mixture,aweakhybridization signalwas

obtained in the initialscreeningof the cDNAlibrary. The

dif-ferences betweenthepublishedand derived amino acidsequence

at positions 6 and 10 could be the result of errors in protein sequencing or may represent geneticvariants.

Several interestingdifferences between the derived human

aminoacid sequence and thepublishedsequenceforguinea pig

C2a were noted. Forinstance, a Cys at position 18 in the human

5 sequenceis Ala in theguinea pig, whichmay result inahigher J

31 order structural difference between the guinea pig and human

C 30/it C2proteins. Suchasubstitutionmightaccountfor the previously

recognized functionaldifferences between guinea pig and human

C2 (33).

Overall structural and functional similarities between C2

/ andfactorBledtospeculationthat these werehomologous and . therefore may have arisen as a result ofgeneduplication. Ex-and C2 DNA periments designed to detect cross hybridization even at low 3erBF,C2, stringency failed to demonstrate a high degree of homology vasrepeatedto between these two genes. Thus, it was possible to separately

analyze genomicpolymorphisms for each gene. The fact that nogenomicvariants offactor B were detected plus the obser-vation that mouse and human factor B genes display 83%

nu-Ascertainment

cleotide sequence homology (34) suggests that thisregionof the

ember

(Fig.

4) _{genome has been relatively highlyconserved when compared} II

_{iS associated}

with other genes within the MHC.

er, individuals A new marker for C2 polymorphism has been identified at [DNA pattern the DNA level by using the Southern blotting technique. The factor B type availability of this marker coupled with C4 variants (protein

for the DNA and DNA) and C2 and factor B protein polymorphisms has

increasedthepotential forapplication of complementgenetics

to the studyofhuman diseases andfortissuetyping. Discussion

Thesecond component ofcomplement is synthesized in liver (31) and mononuclearphagocytes(32)atextremely lowlevels,

(0.1%of totalprotein),henceC2-specificmRNAisof relatively

low abundance in these tissues. The availability ofa human

liver cDNA library (14) containing -200,000 recombinants allowed isolation ofa C2 cDNA with the use ofa

synthetic

oligonucleotide mixture. Synthetic oligonucleotides have been

Acknowledgments

WethankDr.ChesterAlper forperformingtheproteincomplotyping,

JaneIpsen andElizabeth Davidsonfor technical assistance,and Ms.

Helen Hourihanforhersecretarial assistance.

ThisworkwassupportedbyU.S. PublicHealthServicegrants HD

17461, Al 20032,HL32361, Al 18612, and byagrantfrom the Charles

H.HoodFoundation.

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