The nucleotide sequence of the gene for human protein C

Proc. Natl.Acad. Sci. USA Vol.82,pp.4673-4677,July 1985 Biochemistry

The

nucleotide

sequence of the gene for human protein C

(DNAsequenceanalysis/vitamin K-dependentproteins/bloodcoagulation) DONALD C. FOSTER, SHINJI YOSHITAKE, AND EARLW. DAVIE

DepartmentofBiochemistry, University of Washington, Seattle, WA 98195

ContributedbyEarl W. Davie,April 9, 1985

ABSTRACT Ahuman genomic DNA library was screened for the gene forprotein C byusingacDNA probecoding for the human protein.Three different overlapping A Charon 4A phagewere isolated that contain inserts for the gene for protein C.Thecomplete sequence of the gene was determined by the

dideoxymethod and shown to span about 11 kilobasesof DNA. The coding and3' noncoding portion of the gene consists of

eight exons and seven introns. The eight exons code for a preproleader sequence of 42 amino acids, a light chain of 155 aminoacids, a connecting dipeptide of Lys-Arg, and a heavy chain of 262 aminoacids. The preproleader sequence and the

connecting dipeptideareremoved duringprocessing, resulting in thematureproteincomposedofa heavy and alight chain

heldtogether by a disulfide bond. The heavy chain also contains thecatalyticregionforthe serine protease. Two Alu sequences and two homologous repeats of about 160 nucleotides were found in intronE.The sevenintrons in thegene for protein C arelocated inessentiallythe samepositionsintheamino acid sequence as the sevenintronsinthe gene for human factorIX,

while thefirstthree introns inprotein Carelocated in the same

positionsasthefirstthree in thegene for humanprothrombin.

Protein C isaprecursor to aserine proteasepresentin plasma thatplaysanimportant physiologicalrole in theregulationof blood coagulation (1, 2). Human protein C is a vitamin K-dependent glycoprotein containing nine residues of

-carboxyglutamic acid and one equivalent of

p-hydroxy-aspartic acid. Protein C shows considerable structural ho-mology with the other vitamin K-dependentplasma proteins

involvedinbloodcoagulation, includingprothrombin, factor VII,factor IX, and factor X. Protein C is synthesizedas a single-chain polypeptide that undergoes considerable pro-cessingtogiveriseto atwo-chain molecule heldtogetherby a disulfide bond. The two-chain form is converted to acti-vated protein Cby thrombinby the cleavage ofa12-residue

peptidefrom the amino terminus of theheavy chain (2). This reaction is greatly accelerated by the presence of

thrombomodulin (3). Activatedprotein Cregulates the co-agulationprocess bytheinactivation of factorVa (4, 5)and

factor VIIIa (4, 6) by minor proteolysis. Consequently,

individuals lacking protein C often have a history of thrombotic disease (7, 8).

Studies fromourlaboratory(9) andthatof others(10)have ledtotheisolation and characterization of thecDNAcoding

forhumanand bovineproteinC.Inthepresent

investigation,

thecDNA for humanproteinC has been used for theisolation ofoverlappinggenomic clones from a X Charon 4A phage

library. The nucleotide sequence of the gene was then

determinedandcomparedwith thegenesforhumanfactorLX

(11, 12)andprothrombin (13).

MATERIALS ANDMETHODS

Screening of the Genomic Library. A human genomic

library in X Charon 4A phage (14) wasscreened for genomic clones of human protein C by the plaque hybridization procedure ofBenton andDavisasmodifiedbyWoo (15) using acDNA for human protein C (9) as thehybridization probe. ThecDNAstartedatamino acid64ofhumanprotein Cand extendedtothe second polyadenylylation signal (9). It was

radiolabeled bynick-translationto aspecificactivity of 8 X 108

cpm/,ug

with all four radioactive ([a-32P]dNTP) deoxynucleotides. Theprobe wasdenaturedandhybridized

to the filters at a concentration of 1 x 106 cpm/ml in a

hybridization solution containing 6xNaCl/P,(lx NaCl/P,=

0.15MNaCl/0.015Msodiumcitrate,pH 7.0), 5xDenhardt's solution (1x = 0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02%bovine serumalbumin), 0.1% sodiumdodecyl sulfate,100 ,ugofyeast tRNA perml, and50o formamideat 420C for 60 hr. The filters were washed in lx NaCl/P1 containing 0.1% sodium dodecyl sulfateat680C for1 hrand exposed to x-ray film for 16 hr. Positive cloneswere then isolated and plaque-purified.

DNA Sequence Analysis. Phage DNA was prepared from

positiveclonesby the liquid culture lysis methodasdescribed by Silhavy et al. (16). The genomic DNA inserts in the purified phage wereremoved by digestion withEcoRI and

thensubclonedinto pUC9 for subsequent restriction mapping and sequencing. In order toobtain overlappingDNA

frag-ments,theDNA insertsweredigested also withBglII, and thefragments corresponding to thegeneforprotein Cwere

subclonedinto the BamHI site of pUC9.

Thesequenceofgenomicfragmentscontainingthe genefor protein Cwasdeterminedbothby direct cloning of specific restriction fragments into the M13 phage cloning vectors

mplO, mphl, mpl8, and mpl9, as well as by the BAL-31 exonuclease method described by Guo et al. (17) and Yoshitakeetal. (12).

Dideoxy chain termination sequencing reactions were

carried out with 3"S-substituted deoxyadenosine

5'-[a-thioltriphosphate

(dATP[a-35S];

Amersham) essentiallyas

described in thesequencingmanualprovided by Amersham andrunonbuffergradient gelsasdescribedbyBigginetal. (18).More than90% of thesequencewasdeterminedtwo or

more times, and =50% was determined on both strands. DNA sequenceswerestoredandanalyzed bythe computer programs ofLarson and Messing(19).

M13 vectors mplO,

mphl,

mpl8, andmpl9,

deoxynucle-otide triphosphates, and

dideoxynucleotide

triphosphates were purchased from P-L Biochemicals. Restriction

en-zymes, T4 DNAligase, bacterialalkaline phosphatase, and

the Escherichia coliDNApolymerase I(Klenowfragment) werepurchased fromNewEnglandBiolabsorfrom Bethesda ResearchLaboratories.

Abbreviation: kb, kilobase(s).

4673

Thepublicationcostsof this articleweredefrayedinpartbypagecharge

payment. This articlemustthereforebeherebymarked"advertisement"

4674

Biochemistry:

al. Acad. Sci. USA 82(1985)

-EM

3~ ~ ~ ~ ~~~~~ E 3 C-- : 3 ~ E

0- V)Qa. U)U)dIU) ' (/)tfCD ~W) Y( d a U U )c XU) U) (n) LJw Qa.a- coQ. U)

1-ti1

I

~

(

I

I(

C1m

II

I

16

-

1

Ij

o 2 3 4 5 6 7 8 9 10 I

ExonlI Exon1l Exons1l, IV,andV ExonVI ExonVI] ExonV111

FIG. 1. Detailedrestrictionmapandsequencingstrategy for thegenefor humanprotein C.Thelocations of each of theeightexonsareshown withsolid bars. Thelengthanddirection of eachsequencingreactionare shownbythinarrows.

RESULTS ANDDISCUSSION another (PCX8) to the 3'

region,

and the third (PCX6) was AhumngeomicNAlbrar Q X 01page) nkCaron positiveto bothsets of

probes.

4A

phuangenomicrDNA

libhararyi(2ax106

phage in

Charo

obe The

genomic

DNAinserts in PCX6 and PCX8were

mapped

4Aupageroei

wa

Creen

redwithfaradiolabeled

clNpobes fore

by

single-

and

double-restriction-enzyme digestion

followed humaproein. Thee dffernt psitie clnes ere

by

agarose

gel

electrophoresis,

Southern

blotting,

and

hy-isolated, and each was

plaque-purified.

These three clones bridizationtoradiolabeled 5' and 3'

probes

derived from the exhibited

unique

patterns ofEcoRI

fragments

upon electro- cDNAfor human

protein

C. This

analysis suggested

that the

phoresis

in 0.7% agarose but also contained

fragments

in genefor

protein

Cwas presentin threeEcoRI

fragments

of common with each other. Southern blot

hybridization

of 4.4,6.2,and 6.9kilobases(kb)oriented 5'to3'in the

genome.-digests

of these clones with

probes

made from the 5' and3' The 4.4-kb

fragment

wasisolatedfrom

phage

PCX6, and the ends of the cDNA established thatoneoftheclones(PCX1) 6.2-kb and6.9-kb

fragments

wereisolatedfrom

phage

PCX8;

corresponded

to the 5'

region

ofthe gene for

protein

C, eachwassubcloned into the EcoRI site of

pUC9.

To

provide

AGTGAATCTG GGCGAGTAAC ACAAAACTTGAGTGTCCTTA CCTGAAAAAT AGAGGTTAGA GGGATGCTAT GTGCCATTGT GTGTGTGTGT TGGGGGTGGGGATTGGGGGT GATTTGTGAGCAATTGGAGG -2001

TGAGGGTGGAGCCCAGTGCC CAGCACCTAT GCACTGGGGA CCCAAAAAGG AGCATCTTCT CATGATTTTA TGTATCAGAA ATTGGGATGG CATGTCATTG GGACAGCGTC TTTTTTCTTGTATGGTGGCA -1871 CATAAATACA TGTGTCTTAT AATTAATGGTATTTTAGATT TGACGAAATA TGGAATATTA CCTGTTGTGC TGATCTTGGGCAAACTATAATATCTCTGGG CAAAAATGTC CCCATCTGAAAAACAGGGAC -1741

AACGTTCCTC CCTCAGCCAG CCACTATGGG GCTAAAATGA GACCACAICT GTCAAGGGTT TTGCCCTCAC CTCCCTCCCT GCTGGATGGCATCCTTGGTA GGCAGAGGTG GGCTTCGGGC AGAACAAGCC -1611 GTGCTGAGCT AGGACCAGGA GTGCTAGTGC CACTGTTTGT CTATGGAGAG GGAGGCCTCA GTGCTGAGGGCCAAGCAAAT ATTTGTGGTTATGGATTAAC TCGAACTCCA GGCTGTCATG GCGGCAGGAC -1481

GGCGAACTTG CAGTATCTCC ACGACCCGCC CCTGTGAGTC CCCCTCCAGG CAGGTCTATG AGGGGTGTGGAGGGAGGGCT GCCCCCGGGA GAAGAGAGCT AGGTGGTGAT GAGGGCTGAATCCTCCAGCC -1351

AGGGTGCTCA ACAAGCCTGA GCTTGGGGTA AAAGGACACAAGGCCCTCCA CAGGCCAGGC CTGGCAGCCACAGTCTCAGG TCCCTTTGCC ATGCGCCTCC GTCTTTCCAGGCCAAGGGTC CCCAGGCCCA -1221 GGGCCATTCC AACAGACAGT TTGGAGCCCA GGACCCTCCA TTCTCCCCACCCCACTTCCA CCTTTGGGGG TGTCGGATTT GAACAAATCT CAGAAGCGGC CTCAGAGGGA GTCGGCAAGA ATGGAGAGCA -1091 GGGTCCGGTA GGGTGTGCAG AGGCCACGTG GCCTATCCAC TGGGGAGGGT TCCTTGATCTCTGGCCAccA GGGCTATCTCTGTGGCCTTT TGGAGCAACC TGGTGGTTTG GGGCAGGGGTTGAATTTCCA -961

GGCCTAAAAC CACACAGGCC TGGCCTTGAG TCCTGGCTCT GCGAGTAATG CATGGATGTAAACATGGAGA CCCAGGACCT TGCCTCAGTC TTCCGAGTCT GGTGCCTGCA GTGTACTGATGGTGTGAGAC -831

CCTACTCCTG GAGGATGGGG GACAGAATCT GATCGATCCC CTGGGTTGGT GACTTCCCTG TGCMATCAAC GGAGACCAGC AAGGGTTGGATTTTTAATAA ACCACTTAAC TCCTCCGAGT CTCAGTTTCC -701 CCCTCTATGA AATGGGGTTG ACAGCATTAATAACTACCTC TTGGGTGGTT GTGAGCCTIA ACTGAAGTCA TAATATCTCA TGTTTACTGA GCATGAGCTA TGTGCAMAGC CTGTTTTGAG AGCT'TTATGT -571 GGACTAACTC CTTTAATTCT CACAACACCC TTTAAGGCAC AGATACACCA CGTTATTCCA TCCATTTTAC AAATGAGGAAACTGAGGCAT GGAGCAGTTAAGCATCTTGC CCAACATTGC CCTCCAGTAA -441

GTGCTGGAGC TGGAATTTGG ACCGTGGAGT GTGGCTTCAT GGCGTGCGCT GTGAATCCTG TAAAMATTGT TTGAAAGACACCATGAGTGTCCAATCAACG TTAGCTAATA TTCTCAGCCC AGTCATCAGA -311 CCGGCAGAGG CAGC-CACCCC ACTGTCCCCA C-GGACGACACAAACATCCTG GCACCCTCTC CACTGCATTC TGGAGCTGCT TTCTAGGCAG GCAGTGTGAG CTCAGCCCCA CGTAGAGCGG GCAGCCGAGG -181 CCTTCTGAGG CTATGTCTCT AGCGAACAAG GACCCTCAATTCCAGCTTCC GCCTGACGGCCAGCACACAG GGACAGCCCT TTCATTCCGC TTCCACCTGG GGGTGCAGGC AGAGCAGCAGCGGGGGTAGC -51

-42

Met TrpGin Leu Thr Ser Leu Leu Leu Phe Val Ala ThrTrp Gly Ilie Ser GiyThr ProAla

ACTGCCCGGA GCTCAGAAGT CCTCCTCAGA CAGGTGCCAG TGCCTCCAGA ATG TGGCAG CTC ACA AGC CTC CTG CTG TTC GTG GCC ACC TGG GGAATT TCC GGC ACA CCA GCT 63 -20

Pro Leu

CCT CTTGIGTAAGGCCAC CCCACCCCTA CCCCGGGACC CTTGTGGCCT CTACAAGGCC CTGGTGGCAT CTGCCCAGGC CTTCACAGCT TCCACCATCT CTCTGAGCCC TGGGTGAGGTGAGGGGCAGA 190

TGGGAATGGC AGGAATCAACTGACAAGTCC CAGGTAGGCC AGCTGCCAGA GTGCCACACA GGGGCTGCCA GGGCAGGCAT GCGTGATGGC AGGGAGCCCC GCGATGACCT CCTAAAGCTC CCTCCTCCAC 320

ACGGGGATGG TCACAGAGTC CCCTGGGCCT TCCCTCTCCA CCCACTCACTCCCTCAACTG TGAAGACCCC AGGCCCAGGC TACCGTCCAC ACTATCCAGC ACAGCCTCCC CTACTCAAAT GCACACTGGC 450

CTCATGGCTGCCCTGCGCCA ACCCCTTTCC TGGTCTCCACAGCCAACGGGAGGAGGCCAT GATTCTTGGG GAGGTCCGCA GGCACATGGGCCCCTAAAGC CACACCAGGC TGTTGGTTTC ATTTGTGCCT 580 TTATAGAGCT GTTTATCTGC TTGGGACCTG CACCTCCACC CTTTCCCAAG GTGCCCTCAG CTCAGGCATA CCCTCCTCTAGGATGCCTTT TCCCCCATCC CTTCTTGCTC ACACCCCCAA CTTGATCTCT 710 CCCTCCTAAC TGTGCCCTGC ACCAAGACAG ACACTTCACAGAGCCCAGGA CACACCTGGG GACCCTTCCT GGGTGATAGGTCTGTCTATC CTCCAGGTGT CCCTGCCCAA GGGGAGAAGCATGGGGAATA 840 CTTGGTTGGG GGAGGAAAGG AAGACTGGGG GGATGTGTCA AGATGGGGCT GCATGTGGTG TACTGGCAGA AGAGTGAGAGGATTTAACTT GGCAGCCTTT ACAGCAGCAG CCAGGGCTTGAGTACTTATC 970

TCTGGGCCAG GCTGTATTGG ATGTTTTACA TGACGGTCTC ATCCCCATGTTTTTGGATGA GTAAATTGAA CCTTAGAAAG GTAAAGACAC TGGCTCAAGG TCACACAGAG ATCGGGGTGG GGTTCACAGG 1100

GAGGCCTGTC CATCTCAGAG CAAGGGTTCG TCCTCCAACT GCCATCTGCT TCCTGGGGAG GAAAAGAGCA GAGGACCGCT GCGCCAAGCC ATGACCTAGA ATTAGAATGA GTCTTGAGGG GGCGGAGACA 1230 -19

AspSer Val PheSer Ser Ser

AGACCTTCCCAGGCTCTCCC AGCTCTGCTT CCTCAGACCC CCTCATGGCC CCAGCCCCTC TTAGGCCCCT CACGAAGGTGAGCTCCCCTC CCTCCAAAAC CAGY AC TCA GTG TTC TCC AGC AGC 1353 -1 +1

Gin ArgAla His Gin Val LeuArg Ilie Arg Lys ArgAia Asn Ser Phe LeuGiu Giu LeuArg His Ser SerLeuGiu ArgGiu Cys IlieGin Giu Ile CysAsp

GAG CGT GCC CAC CAG GTG CTG CGG ATC CGC MAACGT GGC AAC TCCTIC CTG GAG GAG CTC CGT GAG AGC AGC CTGGAG CGG GAG TGG ATA GAG GAGAIG TGT GAC 1458 37

Phe Giu GiuAia LysGi Ilie PheGin Ann Vai Asp Asp Thr

TTC GAG GAG GGC MAG GAAATT TIC GMAMI GTGGAT GAG ACAVYGTMAGGCCAC CATGGGTCGA GAGGATGAGG CTCAGGGGCG AGCTGGTMAC CAGGAGGGGCCTCGAGGAGC 1570

AGGTGGGGAC TCMATGCTGAGGCGCTGTTA GGAGTTGTGG GGGTGGGTGA GTGGAGCGAT TAGGATGGTGGCCCTATGAT GTCGGGGGAGG GAGATGTGAG TGCAAGMAAC AGMATTCAGGAAGMAGCTCC 1700

AGGAAAGAGT GTGGGGTGAC GCTAGGTGGG GACTGGGACA GGGAGAGTGT AGGTGGITGAGTGGAGCGTG GAGGGACTGC TGAGGACCAC TGCCTCCGGG TGCGAGGTCA CAMAGAGGGG ACCTAMAGAC 1830

GACGCTGCTT CCAGGCATGC CTCTGCTGAT GAGGGTGTGTGTGTGAGGGA AAGTGACTTG TGTGGAGATA MAATCGCTCAGIGTGTGGGT CACATCAMAG GGAGAAMATC TGATTGITGA GGGGGTGGGA 1960 AGACAGGGTC TGTGTGGTATTTGTGTAAGG GTCAGAGTCC TTTGGAGGGG CCAGAGTGCT GTGGACGTGG GGGTAGGTAGTAGGGTGAGG TTGGTMACGGGGCTGGCTTG CTGAGACMAG GCTCAGACCC 2090

GGTCTGTCGC TGGGGATCGC TTGAGCCACC AGGACGTGAAMATTGTGGAC GCGTGGGGGG CCTTCCAAGG GATGGAGGGAIGGCTTGGAG TGGAGGGTTT GAGGGGAGGA GACCGTGTGG CCTGCACCCT 2220

GTGTTGGGGI CAGCCTGCAC CTCCTTGACT GGAGCGGCAT GIGGAGCCGG ATCCGGAGCACCTCGTTIGGG GAGTGGCCTG GGIGGGAGAG ACCACAGTGA GITTIGCGAG GCACATATCT GATCACATCA 2350

AGTCCCCACC GTGCTGGCAG CTGAGGGATG GTGTGTGAGG CGCAGCAGGC TTGGGTGGGG TCTCTGATGG AGCAGGCATC AGGCAGAGGC CGTGGGTGTCAACGTGGGCT GGGTGGTGCC GGAGGAGCAG 2480

GAGGCGCCGC AGGAGCMACG GIGGTAGGIG GTTAGGAAGG GAGACCCTCT GCGCGCATGC GCCCAACTGI GAAAMAGCAT GGGTTAGGGAAAGGGCGGAT GCTGAGGGGT CCCCCAMAGC CGGGAGGCAG 2610 AGGGAGTGATGGGAGTGGAA GGAGGGCGAG IGACTTGGTGAGGGATTCGG GTGCCTTGGA TGGAGAGGGT GGIGTGGGAG CGGACAGTGG GGAGAGGAGG ACGCGAGGIG GATGGGGAGA GGGTGTTGCT 2740

GGAGGGAGGT GGGATGGAGGGTGGGGGGGG GCGGGGTGGCG GTGGAGGGCG GGGGAGGGGC AGGGAGCACC AGGTGGIAGC AGGGAACGAG GATCGGGGGTGGATGGCGTG TTGTGTGGAA GCCCTCCGCC 2870

38 45

GLen Ala PheTrp Ser LysHis VaI

GCCCTGCCCG GIGACCGCGG GGGGIGCGGG AGGGGGGGGG GGCGCTCGCG AGAGGGGCTG GAGGAGGGIG AGGGTGGGCGGITGITCGGG AGYCTG GCG TIC TGG TCCAAGGAG GIG GYGTGAGT 2993

46

AspGiy Asp GinGys Len Val Len Pro LeuGin

GGGIIGIAGA IGGGGGGCIG GACIACGGGG GGGGGGGCGCC CIGGGGAIGI GIGCGGGGGG AGGGGGIAGG GGGCCTIGIG IGGGAGV AG GGI GAG GAG IGG IIGGIG IIG CCCIIGGAG 3111 91

His Pro CysAla Ser Leu CysCys Gly His Gly ThrCys Ile Asp Gly Ile Gly Ser Phe Ser CysAsp CysArg Ser Gly TrpGlu Gly Arg Phe Cys Gln Arg

CAC CCG TGC GCC AGC CTG TGC TGC GGGCAC GGCACG TGC ATC GAC GGC ATC GGC AGC TTCAGC TGC GAC TGC CGC AGC GGC TGGGAG GGC CGCTICIGCGAG CGC 3216 92

GluVal Ser Phe LeuAsn

GYGTGAGGGGGAGAGGTGG ATGCTGGCGGGCGGCGGGGGC GGGGCTGGGGCCGGGTTGGG GGCGCGGCAC CAGCACCAGC TGCCCGCGCC CTCCCCTGCC CGCAGV AGGTGAGC TTC CTCAAT 3336

Biochemistry: Foster et al. Proc. Natl. Acad. Sci. USA 82 (1985) 4675

Cys Ser Leu Asp Asn Gly GlyCys ThrHis Tyr Cys Leu Glu Glu Val Gly Trp Arg Arg Cys Ser CysAla Pro Gly Tyr Lys Leu Gly Asp Asp Leu Leu Gln

TGC TCTCTG GAC AACGGC GGC TGCACG CAT TAC TGC CTA GAG GAG GTGGGC TGG CGG CGC TGT AGC TGTGCG CCT GGCTACAAG CTG GGG GAC GAC CTC CTG CAG 3440 136

CysHis Pro Ala

TGTCAC CCC GCAGYGTGAGAAGCC CCCAATACAT CGCCCAGGAA TCACGCTGGG TGCGGGGTGGGCAGGCCCCT GACGGGCGCG GCGCGGGGGG CTCAGGAGGG TTTCTAGGGA GGGAGCGAGG 3564 AACAGAGTTG AGCCTTGGGG CAGCGGCAGA CGCGCCCAAC ACCGGGGCCACTGTTAGCGCAATCAGCCCG GGAGCTGGGC GCGCCCTCCG CTTTCCCTGC TTCCTTTCTT CCTGGCGTCC CCGCTTCCTC 3694

CGGGCGCCCC TGCGACCTGG GGCCACCTCC TGGAGCGCAA GCCCAGTGGT GGCTCCGCTC CCCAGTCTGAGCGTATCTGG GGCGAGGCGT GCAGCGTCCT CCTCCATGTA GCCTGGCTGC GTTTTTCTCT 3824

GACGTTGTCCGGCGTGCATC GCATTTCCCT CTTTACCCCC TTGCTTCCTT GAGGAGAGAACAGAATCCCG ATTCTGCCTT CTTCTATATT TTCCTTTTTA TGCATTTTAA TCAAATTTAT ATATGTATGA 3954

AACTTTAAAA ATCAGAGTTT TACAACTCTT ACACTTTCAG CATGCTGTTC CTTGGCATGG GTCCTTTTTT CATTCATTTT CATAAAAGGT GGACCCTTTT AATGTGGAAA TTCCTATCTT CTGCCTCTAG 4084

GGCATTTATC ACTTATTTCT TCTACAATCT CCCCTTTACT TCCTCTATTT TCTCTTTCTG GACCTCCCAT TATTCAGACC TCTTTCCTCT AGTTTTATTG TCTCTTCTAT TTCCCATCTC TTTGACTTTG 4214

TGTTTTCTTT CAGGGAACTTTCTTTTTTTT CTTTTTTTTT GAGATGGAGT TTCACTCTTG TTGTCCCAGG CTGGAGTGCA ATGACGTGAT CTCAGCTCAC CACAACCTCC GCCTCCTGGATTCAAGCGAT 4344

TCTCCfTtdGCAGdtt:GAGTAGCTGGGATTACAGGCA TGCGCCACCA CGCCCAGCTA ATTTTGTGTT TTTAGTAGAGAAGGGGTTTCTCCGTGTTGG TCAAGCTGGT CTTGAACTCC TGACCTCAGG 4474

TGATCCACCT GCCTTGGCCT CCTAAAGTGC TGGGATTACA GGCGTGAGCC ACCGCGCCCGAGGGTqTT~ GG.GMTN.TACAACTTTA TAATTCAATT CTTCTGCAGAAAAATTTT TGGCCAGGCT 4604

CAGTAGCTCA GACCAATAAT TCCAGCACTT TGAGAGGCTG AGGTGGGAGG ATTrrTTrAr CTTGGGAGTTTGAGACTAGC C*TGGGCAACArArTrArArr CTGTCTCTATTTTTAAAAAA AGTAAAAAAA 4/34

LzbP~iILAX IRCALLARIRIM I,LLAbALRII I bAbAbli bMlXIbblbbbib AIbllXIIab CIIllbbAGII IbbAAI~bCl6XIbbbLMAALR lbibAbRCXLLLblXXIAIII IIIIMAMAAAbIMMARARR SQ/'

GATCTAAAAA TTTAACTTTTTATTTTGAAA TAATTAGATA TTTCCAGGAA GCTGCAAAGA AATGCCTGGTGGGCCTGTTG GCTGTGGGTT TCCTGCAAGGCCGTGGGAAGGCCCTGTCAT TGGCAGAACC 4864

CCAGATCGTG AGGGCTTTCC TTTTAGGCTG CTTTCTAAGA GGACTCCTCC AAGCTCTTGGAGGATGGAAGACGCTCACCC ATGGTGTTCG GCCCCTCAGA GCAGGGTGGG GCAGGGGAGC TGGTGCCTGT 4994 GCAGGCTGTG GACATTTGCA TGACTCCCTG TGGTCAGCTA AGAGCACCAC TCCTTCCTGA AGCGGGGCCT GAAGTCCCTA GTCAGAGCCT CTGGTTCACC TTCTGCAGGCAGGGAGAGGG GAGTCAAGTC 5124 AGTGAGGAGG GCTTTCGCAG TTTCTCTTAC AAACTCTCAA CATGCCCTCC CACCTGCACT GCCTTCCTGG AAGCCCCACA GCCTCCTATG GTTCCGTGGT CCAGTCCTTC AGCTTCTGGGCGCCCCCATC 5254

ACGGGCTGAG ATTTTTGCTT TCCAGTCTGC CAAGTCAGTT ACTGTGTCCA TCCATCTGCT GTCAGCTTCT GGAATTGTTG CTGTTGTGCC CTTTCCATTC TTTTGTTATG ATGCAGCTCC CCTGCTGACG 5384 ACGTCCCATT GCTCTTTTAA GTCTAGATAT CTGGACTGGG CATTCAAGGC CCATTTTGAG CAGAGTCGGG CTGACCTTTC AGCCCTCAGT TCTCCATGGA GTATGCGCTC TCTTCTTGGC AGGGAGGCCT 5514

CACMACATGCCATGCCTAT TGTAGCAGCT CTCCAAGAAT GCTCACCTCC TTCTCCCTGT AATTCCTTTC CTCTGTGAGG AGCTCAGCAG CATCCCATTA TGAGACCTTA CTAATCCCAG GGATCACCCC 5644

CAACAGGCCTGGGGTACAAT GAGCTTTTAA GAAGTTTAAC CACCTATGTA AGGAGACACAGGCAGTGGGC GATGCTGCCT GGCCTGACTC TTGCCATTGG GTGGTACTGT TTGTTGACTGAGTGAGTGAG 5774

TGACTGGAGGGGTTTGTAA TTTGTATCTC AGGGATTAGG GGGAAGAGG GTGGGGTAGA ATGAGGGTTG AAGAAGTTTAAGCAATATG TAAGGACACA CAGCCAGTGG GTGATGCTGC CTGGTCTGAC 5904

TGTTGGGATT-AGAGgCT GTTTGTTGAC TGACTGACTG ACTGACTGGCTGAGTGGAGG GGGTTCATAG CTAATATTAA TGGAGTGGTC TAAGTATCAT TGGTTCCTTG AACCCTGCAC TGTGGCAAAG 6034 137

VJal Lys Phe Pro Cys Gly Arg ProTrp Lys Arg TGGCCCACAGGCTGGAGGAG GACCAAGACAGGAGGGCAGT CTCGGGAGGA GTGCCTGGCA GGCCCCTCAC CACCTCTGCC TACCTCAGVTG AAG TTC CCT TGT GGG AGG CCC TGGAAG CGG 6154

MetGlu Lys Lys Arg Ser His LeuLysArgfAspThr G1uAsp Gin GiUAsp Gin Val Asp Pro ArgkLeu Ile Asp Gly Lys Met ThrArg Arg Gly Asp Ser Pro

ATG GAG AAG MGCGC AGT CAC CTG AMCGA GAC ACA GAA GACCM GMGAC CAA GTA GAT CCG CGG CTC ATT GAT GGG MG ATG ACCAGG CGGGGA GAC AGC CCC 6259

184

Trp Gin

TGG CAGYGTGGGAGGCGAGGCAGCACC GGCTCGTCAC GTGCTGGGTC CGGGATCACT GAGTCCATCC TGGCAGCTAT GCTCAGGGTGCAGMACCGA GAGGGAAGCGCTGCCATTGC GTTTGGGGGA 6385

TGATGMGGT GGGGGATGCTTCAGGGAMG ATGGACGCMCCTGAGGGGA GAGGAGCAGCCAGGGTGGGT GAGGGGAGGG GCATGGGGGC ATGGAGGGGTCTGCAGGAGG GAGGGTTACA GTTTCTAMA 6515

AGAGCTGGMAGACACTGCT CTGCTGGCGG GATTTTAGGC AGAAGCCCTG CTGATGGGAG AGGGCTAGGA GGGAGGGCCG GGCCTGAGTA CCCCTCCAGC CTCCACATGG GMCTGACAC TTACTGGGTT 6645

CCCCTCTCTG CCAGGCATGG GGGAGATAGGAACCMG MG TGGGAGTATT TGCCCTGGGG ACTCAGACTC TGCMGGGTC AGGACCCCM AGACCCGGCA GCCCAGTGGG ACCACAGCCA GGACGGCCCT 6775

TCMGATAGGGGCTGAGGGA GGCCMGGGG MCATCCAGG CAGCCTGGGGGCCACMAGT CTTCCTGGM GACACMGGC CTGCCAAGCC TCTMGGATG AGAGGAGCTC GCTGGGCGATGTTGGTGTGG 6905

CTGAGGGTGACTGAAACAGT ATGMCAGTG CAGGMCAGC ATGGGCAMG GCAGGMGAC ACCCTGGGAC AGGCTGACAC TGTAAMTGG GCAAAAATAGMAACGCCAG AAAGGCCTAAGCCTATGCCC 7035

185

Vai Val Leu LeuAsp Ser Lys

ATATGACCAG GGAACCCAGG AAAGTGCATA TGAMCCCAG GTGCCCTGGA CTGGAGGCTG TCAGGAGGCA GCCCTGTGAT GTCATCATCCCACCCCATTC CAGVGTG GTC CTG CTG GAC TCAMG 7159

0 223

Lys Lys Leu AiaCys GlyAia Vai Leu IleHis Pro SerTrp Vai Leu Thr Aia Ala HisCys MetAsp Glu SerLys LysLeu Leu Val Arg Leu

MG MGCTG GCC TGC GGGGCA GTG CTC ATC CAC CCC TCCTGG GTGCTG ACA GCG GCC CAC TGC ATG GAT GAGTCC AAGAAGCTC CTT GTC AGG CTT G V GTATGGGCTG 7266

GAGCCAGGCA GAAGGGGGCT GCCAGAGGCCTGGGTAGGGG GACCAGGCAG GCTGTTCAGG TTTGGGGGAC CCCGCTCCCC AGGTGCTTM GCMGAGGCT TCTTGAGCTC CACAGAAGGT GTTTGGGGGG 7396

AAGAGGCCTA TGTGCCCCCA CCCTGCCCAC CCATGTACAC CCAGTATTTT GCAGTAGGGG GTTCTCTGGT GCCCTCTTCG AATCTGGGCA CAGGTACCTGCACACACATGTTTGTGAGGG GCTACACAGA 7526

CCTTCACCTC TCCACTCCCA CTCATGAGGA GCAGGCTGTG TGGGCCTCAG CACCCTTGGGTGCAGAGACC AGCMGGGCCT GGCCTCAGGG CTGTGCCTCC CACAGACTGA CAGGGATGGA GCTGTACAGA 7656

GGGAGCCCTA GCATCTGCCA MGCCACMGCTGCTTCCCTAGCAGGCTGG GGGCTCCTATGCATTGGCCC CGATCTATGG CAATTTCTGG AGGGGGGGTC TGGCTCMCT CTTTATGCCA AAAAGAAGGC 7786

AAAGCATATT GAGAAAGGCCAMATTCACAT TTCCTACAGC ATAATCTATG CCAGTGGCCC CGTGGGGCTT GGCTTAGMT TCCCAGGTGC TCTTCCCAGGGMCCATCAG TCTGGACTGAGAGGACCTTC 7916 TCTCTCAGGT GGGACCCGGC CCTGTCCTCC CTGGCAGTGC CGTGTTCTGG GGGTCCTCCT CTCTGGGTCT CACTGCCCCT GGGGTCTCTC CAGCTACCTT TGCTCCATGT TCCTTTGTGG CTCTGGTCTG 8046

TGTCTGGGGT TTCCAGGGGT CTCGGGCTTC CCTGCTGCCC ATTCCTTCTC TGGTCTCACG GCTCCGTGACTCCTGMAAC CAACCAGCAT CCTACCCCTT TGGATTGACA CCTGTTGGCC ACTCCTTCTG 8176

GCAGGMAAG TCACCGTTGA TAGGGTTCCA CGGCATAGAC AGGTGGCTCC GCGCCAGTGC CTGGGACGTG TGGGTGCACA GTCTCCGGGT GAACCTTCTT CAGGCCCTCT CCCAGGCCTG CAGGGGCACA 8306

224

GlyGlu TyrAsp LeuArg Arg TrpGlu Lys Trp Glu LeuAsp GCAGTGGGTG GGCCTCAGGA MGTGCCACT GGGGAGAGGC TCCCCGCAGC CCACTCTGACTGTGCCCTCT GCCCTGCAGYGA GAG TATGAC CTG CGGCGC TGG GAGMG TGG GAGCTG GAC 8426

0

LeuAsp Ile Lys Glu Val Phe Val HisProAsn Tyr Ser Lys SerThr ThrAspAsn Asp IleAla Leu LeuHis Leu Ala Gln ProAla Thr Leu SerGln Thr

CTG GAC ATC MG GAG GTC TTC GTC CACCCCAAC TAC AGC MGAGC ACC ACC GAC MT GAC ATC GCA CTG CTG CAC CTG GCC CAGCCC GCC ACC CTC TCGCAG ACC 8531 Ile Val Pro Ile Cys Leu Pro Asp SerGly LeuAla Glu Arg GluLeu AsnGln Ala Gly Gin Glu Thr LeuVal Thr Gly Trp Gly Tyr His Ser Ser Arg Glu

ATAGTG CCCATC TGC CTC CCG GAC AGC GGC CTT GCA GAG CGC GAG CTC MTCAG GCC GGC CAG GAGACC CTC GTGACG GGC TGG GGC TAC CAC AGC AGC CGA GAG

Lys Glu AlaLys Arg Asn ArgThr Phe Val LeuAsn Phe Ile Lys Ile ProVal Val Pro His Asn Glu Cys SerGlu Val Met SerAsn MetVal Ser GluAsn AAG GAG GCC AAG AGA MC CGC ACC TTC GTC CTC MC TTC ATC AAG ATT CCC GTGGTC CCGCACAAT GAG TGCAGC GAG GTCATG AGC AACATG GTGTCT GAG AAC

0

Met Leu Cys Ala Gly IleLeu GlyAsp Arg GlnAspAla Cys Glu Gly AspSer Gly GlyProMet Val Ala Ser Phe His Gly Thr Trp PheLeuVal Gly Leu

ATG CTG TGT GCG GGCATC CTC GGG GAC CGG CAGGAT GCC TGC GAGGGC GAC AGTGGG GGG CCC ATGGTC GCC TCC TTC CACGGC ACC TGG TTC CTG GTGGGC CTG

8636

8741

8846

Val Ser Trp Gly Glu Gly Cys Gly Leu Leu HisAsn Tyr Gly Val Tyr Thr Lys Val SerArg Tyr LeuAsp Trp Ile His Gly His Ile Arg Asp Lys GluAla GTG AGC TGG GGT GAG GGC TGT GGGCTC CTTCAC MC TAC GGCGTT TAC ACCMA GTC AGC CGC TAC CTC GAC TGGATC CAT GGGCAC ATCAGAGAC AAG GAAGCC 8951

419

Pro Gin LysSer Trp Ala ProSTOP

CCC CAGMG AGC TGG GCACCT TAG CGACCCTCCCTGCAGGGCTG GGCTTTTGCA TGGCMTGGA TGGGAGATTA':GGGACATG TAACAAGCAC ACCGGCCTGC TGTTCTGTCC TTCCATCCCT 9075

CTTTTGGGCTCTTCTGGAGG GMGTAACAT TTACTGAGCA CCTGTTGTAT GTCACATGCC TTATGMTAG MTCTTAACT CCTAGAGCAA CTCTGTGGGG TGGGGAGGAGCAGATCCAAG TTTTGCGGGG 9205

TCTAAAGCTG TGTGTGTTGA GGGGGATACT CTGTTTATGA AAAAG~AAACAGAACCACGMGCCACTAGAGCCTT TTCCAGGGCT TTGGGAAGAG CCTGTGCAAG CCGGGGATGC TGMGGTGAG 9335 GCTTGACCAG CTTTCCAGCT AGCCCAGCTA TGAGGTAGAC ATGTTTAGCT CATATCACAG AGGAGGAAAC TGAGGGGTCT GMAGGTTTACATGGTGGAGCCAGGATTCAMTCTAGGTC TGACTCCAM 9465

ACCCAGGTGC TTTTTTCTGTTCTCCACTGT CCTGGAGGACAGCTGTTTCGACGGTGCTCA GTGTGGAGGC CACTATTAGC TCTGTAGGGA AGCAGCCAGA GACCCAGAAA GTGTTGGTTCAGCCCAGAAT 9595

FIG. 2. Nucleotide sequence for the gene for humanproteinC. The first base of the methionine codon wheretranslation is initiated is numbered+1. Arrowheads indicate intron-exonsplicejunctions.ThetwoAlu sequences in intron Ehave been underlined withasolidline; the 18-base repeatsflankingthe first Alu sequence and the 8-base repeatsflankingthesecond Alu sequence have been underscored with dots. Thehighly conserved sequences of C-C-A-G-C-C-T-G-G have been underlined withaheavysolidline, contrastingwith thetwohomologous 160-bp repeats in intron E which have been lightly underlined. The polyadenylylation or processing sequences of A-T-T-A-A-A and A-A-T-A-A-Aatthe3' endareboxed. TheconsensusofC-T-T-T-G,which also may be involved inpolyadenylylationorcleavageof mRNA at the 3'end, is underlined withawavyline. *, Potential carbohydrate bindingsitestoasparagine residues; ,apparent cleavage sites for processing of theconnecting dipeptide; I, siteofcleavageintheheavychainwhenproteinCisconvertedtoactivatedprotein C;o, active site asparticacid,histidine,andserineresidues;*, sites ofpolyadenylylation.

DNA sequenceoverlappingthetwoEcoRIjunctionsbetween the three fragments, twoBglIIfragmentsof 3.3 and 7.0 kb wereisolatedand subcloned into the BamHI siteofpUC9. These twoclones span the EcoRI sites.

A detailed restriction map as well as approximate place-ment ofthe exon regions within the subcloned fragments wereestablishedby further restrictionanalysisandSouthern

blotting (Fig. 1). When the 5' and3' ends of the genewere established, thenucleotide sequence of the gene was

deter-mined by the dideoxy chain-termination method using

nucleaseBAL-31toprovide overlappingsequences between theendsoflargerestrictionfragments.

Thenucleotidesequenceforthe geneforhuman

protein

C

spans -11 kbofDNA(Fig. 2). Comparison ofthe

genomic

sequence with that ofthe cDNA (9) revealed that the gene

consists of eight exons

ranging

in size from 25 to 885 nucleotides andsevenintronsranginginsize from 92to2668

nucleotides. An additional intron(s) in the 5'

noncoding

region cannot be ruled out because a cDNA

covering

this regionwasnotavailable for

comparison

with thegene.

Also,

LG D Y L 44 G L IntronE P~A ~c (137) GG 5CCHP AVKFPCGRPWKRMEKKR DtL H H Lp- 155 LH .8 97-LL9L V L T IDNH2 O_GWE_{R S}F _RE T H tonGE S F V E(5D) D CCR A LR G r ACTIVATION SL 020N p E (15/16) E A C.C A A T L vV PE C rSF S L K L L W 0 P5G 0 H R K L F V Hr H E L R K 0 R D K G T Pr8L e CS LVL 71TIDP KCMK (Thrombnb T 0 L 4c L T 0 GD T D 9C8 MKG H (46) S K L V NH? K S K v L H -42 M w E ON79 V Sp H H D0 0 EK PHVF K L A w E T

TL OnfoB K CATALYTIC DOMAIN ADH1F~A

D(37/38)OR VAS HG W L F~ L D N144 M V Y 0 L v RNM V p 0G R K L N T S s G L S S F F M K . V v w V 0 mG011VEN K A A I L E SG E W r29 N-5 L E GCOOH G5

A FIKIPVVPHNEC AGILGDRODD LNY4ts

F Int~~~~~~~~~~~~PonA p' 4

C78RrLSSHRLFFLFSNo.AKRIRLVQAESFS

r "mass" ~~~~~PRE-POO LEADER

Protein

C

Proc. NatL. Acad. Sci. USA 82(1985)

R N G K IntronE Q ET -S (128) 14 FactorXI, R NGRCCS EPAVPFPGRVSVSQTSKLT&jEAVFPDVDYVN-12 KC. FO V CIT NF K A T E

<°FpFNNN DSC.CsEAAN EE N TTE EK1nfronn G EAACTIVATIONPEPTIDE

0 PC.CELDT L H AG A54 L W InfronD N v T v D 'IE (85) F pL aE V IntronF N*22 pLNy KL E K I VV05/16) T N SN K D R K t 0 F L N K GN w s ER SG E V G K T CK_ 6 S L RI v QF D A4Y 41 V A G FC DR LR LC - C C P N NHCN2 rvLOCVKp 8890 ID -46SD D-IFntronCS H H A G A F N G D T DO GIAI N~ rssc PE-PR5f R Dv R N TIFH G FactorXI R K V N N V I N F F IA KEN T 40 Hi T K CATALYTIC DOMAIN M T Int~ronB RGw KEA R 3/9 S VTEVEGTSF N K E *AL H V K3 N L FT P G R K FL T N G 5 1235 L V ~~~~~~SN 0)t85 S V COOH T 3 8 M -D WE K R L L F E5 T c A R_vP C-C QC-C L RT AGFHESGGRDS AMKGKYGSGL rF Y A s IntronA L Ci' C, 7 (~(-17) L~ KC3R3LNG0VFTrTLKG;SN®MKPRNLIKNANEHDLFVTCEA Q.5

DOMAIN PIOTEASE PRE-PRO LEADER

Factor IX

FIG. 3. Amino acid sequence and tentative structuresfor humanprepro-protein C and preprofactorIX. Protein Cis shownwithout the

Lys-Argdipeptide,whichconnectsthelightandheavychains.Locationsof the seven introns (Athrough G)for each gene areindicated by solid bars. Amino acidsflankingknownproteolytic cleavagesitesarecircled.Theactive-sitehistidine, aspartic acid,andserineresidues are also circled.*,Potential carbohydratebindingsites. Theproposeddisulfide bonds have beenplacedbyanalogytothosein bovine prothrombin and

epidermal growthfactor. Thefirstamino acids in thelight chain,activationpeptide,andheavychainstartwith number 1 anddifferfrom that shown inFig. 2. Thefactor IX structurewasthat ofYoshitake et al. (12). y, y-carboxyglutamic acid;A,

3-hydroxyaspartic

acid.

several

potential

intron/exon

splice

donor and acceptor sequenceswereidentified in the 5'

noncoding region.

All the

intron/exon splice junctions

were similarto the consensus

sequences

recently

summarized

by

Mount

(20)

andfollow the

G-T/A-G

rule ofBreathnach andChambon(21).

Several

potential

"TATA" sequences were found

up-streamfromthe

preproleader

sequence in thegenefor human

protein

C. The sequences of T-A-T-A-A-T-A

(starting

at

position -1785)

and T-A-T-A-A-T-T

(starting

at

position

-1853)

show the strongest homology with the consensus sequence of T-A-T-A- -A-A.

Both, however,

lack

nearby

"CAAT"sequencesupstream. If eitherof thesesequencesis associated with initiation of

transcription,

then

protein

C would have eitheraverylong 5'noncoding sequenceor an additional

intron(s)

in the 5'

noncoding region

of thegene.

Two

polyadenylylation

orprocessing sequences of

A-T-T-A-A-A and A-A-T-A-A-A (22) were found 47 and 276 nucleotides downstream from the translation stop codon

(nucleotides

starting

at9022 and9251). The second of these also has a sequence of C-T-T-T-G starting 37 nucleotides downstream. This latter sequencecorresponds to the

C-A-C-T-G

consensus sequence and also may be involved in

polyadenylylation

or

cleavage

atthe 3' end ofthemRNA(23).

The DNA sequence of

eight

separate cDNAsatthe 3' end indicates that polyadenylylation occurs with about equal

frequency

downstream from the two polyadenylylation or

processing

sites(datanot shown).

Thegene forprotein C contains twoAlu sequences (24), and bothare locatedinintronE (solid underline inFig. 2). Thefirst isa

complete

copywithanorientation of 3'to5'.It is flanked

by

the directrepeat sequenceof

T-C-T-T-T-C-A-G-G-G-A-A-C-T-T-T-C-T. The second Alu sequence is 30 nucleotidesafter the

flanking

repeatof

the

first and isapartial copyofanAlusequenceoriented5'to3'. This Alusequence

lacks the

right

half of the Alu consensus sequence and is flanked

by

the directrepeatofA-A-A-A-A-T-T-T. IntronE

also

contains

twodirectrepeatsof about 160nucleotides of

unknown significance (dashed underline in Fig. 2). These repeatsare about 93%homologous and start atnucleotides 5628 and 5800. They are separated by 10 nucleotides. A

computer comparison of this sequence with the National

Institutes of Healthsequencedata bank revealedno

signifi-canthomologywithpublishedsequences.

The cDNA sequence (9), along with that of the gene, provides the entire amino acid sequence for human

preproprotein

C(Fig.3

Left).

These dataindicate that human

protein C, like the other vitamin K-dependent coagulation

factors, is initially synthesized as a single-chain precursor

withapreproleadersequenceof42 aminoacids.This leader

sequenceshowsconsiderable amino acid sequencehomology

with thatrecentlydescribed forbovineproteinC (10). Based on

homology

with the leadersequenceof bovine proteinC and other

-y-carboxylated

coagulationproteasesintheregion

from -1 to -20, it is likely that this leader sequence is cleaved by a signal

peptidase

after the alanine residue at position -10. This wouldyield aprozymogenform witha highly basicpropeptide of nine residues. Processing tothe matureproteinthat circulatesinplasmainvolves additional

proteolytic cleavage after residues at -1, 155, and 157 to

remove the amino-terminal propeptide and the Lys-Arg

dipeptide

thatconnectsthelight and heavychains (9). The processing of the single chain is not complete, however,

because about 5-15% of the proteinC in human plasma is

presentas asingle-chain molecule(25).

The amino acid composition of the mature protein C

circulating

in plasma was calculated as follows:

Asp2sAsp

(OH)X Thr15

Ser30 Glu24 Gln13

Glag Pro18

Gly33

Ala2l

VaI26

Met7Ilel6Leu43Tyr8Phel3Lys22Hisl7Arg23Trpl3Cys24,

in

which Gla is

-carboxyglutamic

acid and

Asp(J30H)

is f8-hydroxyaspartic acid. The molecularweightfor theprotein wascalculatedtobe47,456 withoutcarbohydrateandabout

61,600with theaddition of23% carbohydrate (26). Four of thepotential carbohydratechains boundtoasparagineoccur

Proc. Natl. Acad. Sci. USA 82 (1985) 4677 atresidues 97 in thelight chain and at residues79, 144, and

160 in the heavy chain (Fig. 3).

The DNA sequence of the coding region for the genefor

human protein C agrees wellwiththatofthecDNAforhuman

protein C(9) except for thetriplet coding forAsp-214. Both

thegenomicsequence (GAT)andthe cDNA sequence(GAG) specify aspartic acid at this position. It is likely that the

discrepancy is due to either polymorphism or a cloning artifactat nucleotide 7228. Thegenomic DNA sequence and

thesequenceof longer cDNAmoleculeshaveshownthat the

amino acidatresidue64iscysteine rather thanglutamineas

previously reported (9). This discrepancy is likely to have resulted from anartifactualerrorintroducedinto thecDNA sequence adjacent tothe EcoRI linker used inconstructing

the Xgt1l cDNA library. This phenomenon has been ob-served in several other cDNAscharacterized inthis

labora-tory (unpublished results).

Protein C shows considerable amino acid sequence and structural homology with the other vitamin K-dependent

coagulationfactorsincludingprothrombin,factorVII, factor IX, and factor X. Factor IX, factor X, and protein C are

unusually similar in that they have common domain struc-tures throughout their molecules including a y-carboxyglu-tamic acid domain,twopotential growthfactordomains,an

activation peptide or connecting region, and a catalytic

domain (27). In prothrombin, the potential growth factor domains have been replaced bytwokringle structures. The

similarity between theseproteinsisalsoevidentatthe level

ofthe gene where protein C and factor IX show unusual

homology. This is illustrated in Fig. 3, which shows the

proposed domain structures and the seven introns in the genesfor these twoproteins.Inboth genes, the intronsoccur in essentially the samepositions throughout theamino acid

sequence ofthe two proteins. The similaritybetween these two genes is further reflected in the conservation ofsplice junction type. All seven introns in the gene for protein C

exhibit the same splice junction type as the intron in the correspondinglocationin the gene for factor IX(12). How-ever, a computer search of the DNA sequences within the

introns of thegenes in protein C and factor IX showed no significant homology, indicatingthat the sequences ofthese

regions ofthe genes are notconserved during evolution.

Thelocationsof the introns in the genes forproteinC and factorIX areprimarily between various functional domains

of the two proteins (Fig. 3). Exon II spans the highly conservedregionof the leadersequenceand the y-carboxy-glutamic acid domain. Exon III includes a stretch ofeight

amino acids whichconnect the y-carboxyglutamic acid and

growth factor domains. Exons IV and V each represent a potential growth factor domain, while exon VI covers a connectingregionthat includes the activationpeptide.Exons VIIandVIII coverthecatalytic domaintypicalofallserine proteases.

Thefirstthreeintronsin the gene for humanprothrombin (28) also occur in the same position in the amino acid sequenceasthose ofproteinC and factorIX.Inprothrombin,

however, the y-carboxyglutamic acidregion is followed

by

twokringlestructures,whichareunrelated in sequencetothe potential growthfactor domains ofproteinC and factor IX.

Afterthefirstthreeintrons,there appearstobenosimilarity in gene structurebetween thatof

prothrombin

and thoseof factorIXandprotein C.

Thealignmentof intron boundariesin thegenes for

protein

C,factorIX,andprothrombinprovidesadditionalevidence

for the evolution ofthese genes from a common ancestral

precursor. This could have resulted from the joining of

numerous fragments of similar DNA sequences by a translocationevent(s) between chromosomes during evolu-tion. This could leadtotheformation ofagenecodingfora seine protease containing additional domains such as the

potential growth factor domains, kringle domains, and y-carboxyglutamicacid domains(12).

The authorsthank Drs. DominicChung,StevenLeytus,Takehiko

Koide,and Kotoku Kurachi forhelpfuldiscussions andadvice,and Dr. TomManiatis forkindlyprovidingthe humangenomic library

constructed inXCharon 4Abacteriophage.This workwassupported in partbyaresearch grant(HL16919)from the National Institutes of Health. D.C.F. wassupported byNational Institutes of Health

TrainingGrant GM 07270.

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