Impaired secretion of the elongated mutant of protein C (protein C Nagoya) Molecular and cellular basis for hereditary protein C deficiency

Impaired secretion of the elongated mutant of

protein C (protein C-Nagoya). Molecular and

cellular basis for hereditary protein C

deficiency.

K Yamamoto, … , J Takamatsu, H Saito

J Clin Invest.

1992;

90(6)

:2439-2446.

https://doi.org/10.1172/JCI116135

.

Genetic analysis of a heterozygous protein C-deficient patient revealed a novel deletion of a

single guanine residue (8857G) among four consecutive guanine nucleotides

[380Trp(TGG)-381Gly(GGT)] in exon IX, which encodes the carboxyl-terminal region of

protein C. This deletion results in a frameshift mutation and substitution of the last 39 amino

acids (381Gly-419Pro) with 81 abnormal amino acid residues, and we have designated this

elongated variant as Protein C-Nagoya. A mutagenic primer was designed which replaced

the third guanine residue upstream from the deletion with cytosine, thereby creating a new

AvaI site in an otherwise normal allele. Analysis of the polymerase chain reaction products

derived from this mutagenic primer showed that the abnormal allele has been inherited in

this family. To elucidate how this molecular abnormality leads to protein C deficiency, an

expression plasmid containing this mutation was transfected into COS 7, BHK, and psi-2

cells, and the secretory process of the expressed Protein C-Nagoya was analyzed. ELISA

and immunoprecipitation analysis with [35S]methionine labeling indicated that the mutant

protein C, which was larger in size than normal, was mostly retained within the cells, and

only a small portion of it was secreted into the medium. These results suggest that most of

Protein C-Nagoya undergoes degradation within the producing cells, and this frameshift

mutation apparently leads to protein C deficiency by impairment of […]

Research Article

Find the latest version:

Impaired Secretion

of the

Elongated

Mutant of Protein C

(Protein C-Nagoya)

Molecular and Cellular

Basis for

Hereditary

Protein

C

Deficiency

Koji Yamamoto,* MitsuneTanimoto,* NobuhikoEmi,*TadashiMatsushita,* Junki Takamatsu, * and HidehikoSaito**

*First Department of Internal Medicine, Nagoya UniversitySchoolof Medicine, Showa-ku, Nagoya466,Japan;and tAichi Blood Disease Research Foundation, Oaza-Moriyama, Moriyama-ku, Nagoya 463, Japan

Abstract

Geneticanalysis ofaheterozygous proteinC-deficientpatient

revealed a novel deletion ofa single guanine residue

("'7G)

among four consecutive guanine nucleotides

[3Trp(TGG)-31Gly(GGT)j

inexonIX, which encodes the carboxyl-termi-nal region of protein C. This deletion results in aframeshift

mutation and substitution of the last 39 amino acids

("1Gly-419Pro) with 81 abnormal amino acid residues, and we have

designated this elongated variantasProteinC-Nagoya.A

mu-tagenic primerwasdesigned which replacedthe thirdguanine

residue upstream from the deletion with cytosine, thereby creatinganewAval siteinanotherwisenormal allele.Analysis

of the polymerase chain reaction products derived from this. mutagenic primer showed that the abnormal allele has been inherited in this family. To elucidate how this molecular abnor-mality leadstoprotein C deficiency,anexpression plasmid

con-taining this mutationwastransfected into COS 7, BHK, and P-2 cells,and thesecretory process of theexpressed Protein

C-Nagoyawasanalyzed. ELISA and immunoprecipitation

anal-ysis with I35Slmethionine labeling indicated that the mutant

protein C, which was larger in size than normal, was mostly

retained within the cells, and only a smallportion of it was

secreted into the medium. These results suggestthatmostof Protein C-Nagoya undergoes degradation within theproducing cells, and this frameshift mutation apparently leadstoprotein Cdeficiency by impairmentof secretion of theelongated

pro-tein C intoplasma. (J.Clin. Invest. 1992.90:2439-2446.) Key words:thrombophilia-frameshiftmutation_*familydiagnosis.

in vitromutagenesis * expression study

Introduction

Protein C isavitamin K-dependent plasma glycoprotein with anestimated molecular massof 62 kD, consisting ofaheavy

chainof 41 kDandalight chain of 21 kD linked by disulfide

bridges ( 1, 2). It isa precursor ofa plasma seine protease,

activatedprotein C, whichactsas ananticoagulant by

inacti-vating FactorsVa and VIlla(3,4), andstimulatesfibrinolysis

by reducing the activity of tissue-type plasminogen activator inhibitor (5). Insomefamilies, acompelling relationship

be-AddresscorrespondencetoDr.Koji Yamamoto, FirstDepartmentof

Internal Medicine, NagoyaUniversity School of Medicine, 65

Tsu-ruma-cho, Showa-ku,Nagoya466,Japan.

Receivedfor publication 14 October 1991 andinrevisedform26

May1992.

tween

thromboembolic disease and

heterozygosity for protein

C

deficiency has been found (6, 7), but the

vast

majority of

heterozygotes are

asymptomatic (8). The prevalence of

a

het-erozygous

protein C

deficiency

associated with

a

predisposition

towards

thrombosis has been estimated in about

1 out

of

16,000 individuals (9). Homozygous

protein C-deficient

pa-tients, who have little

or no

detectable

plasma levels of

protein

C,

suffer from

very severe

thromboembolic disease and

pur-pura

fulminans from

the

perinatal period

onwards

(

10).

Hered-itary

protein C deficiency has been

classified

into

two

major

groups: type I

deficiency, which

has

equivalent

reductions in

enzymatic activity

and in

antigenic concentration,

and

type II

deficiency, wherein only protein C activity is reduced ( 11).

A

full-length

cDNA

and

a

genomic

copy

of

protein

C have

been

cloned

and

sequenced

(12-15),

and the

gene

has been

mapped

to

chromosome

2

(16). Several abnormalities in the

DNA

have been

found

to

underlie protein

C

deficiency;

these

include

gross

deletions,

nonsense

mutations, missense

muta-tions,

and

a

frameshift mutation

(

17-22).

In

the

present

study,

we

studied the molecular

basis of

a type I

protein

C-deficient

heterozygote and

characterized

a

single

nucleotide

deletion,

which

causes a

frameshift mutation.

To

elucidate the

mecha-nism

by

which

the

frameshift mutation

causes

reductions of

protein C levels in plasma, transient

and

stable

expression

ex-periments

were

performed using COS 7, BHK, and

4-2

cells.

Functional

aspects

of

recombinant protein

C molecules have

been

previously

analyzed

using in vitro mutagenesis

tech-niques (23-25).

However,

expression

study

of

the abnormal

protein C, which is

supposed to be

responsible for

the

protein

C-deficient

state

clinically,

has

notyet

been

performed. Only

a

small

amount

of

the

recombinant

mutant

protein C

designated

as

Protein C-Nagoya with

anapparent

elongated molecular

mass wassecreted

into

the cell culture

media, suggesting

that

this mutation

may be the molecular

basis

for protein

C

defi-ciency

in

this pedigree.

Methods

Patient

profile.

Thepatient is a 46-yr-old man who has had recurrent

episodesof thrombosis. His first episode was right leg deep vein throm-bosis diagnosed by venography at the age of 42. 3 mo later, he was admittedto ahospitalbecauseof chest pain and dyspnea. Pulmonary

embolismwasconfirmedby lung perfusion scintigraphy using

[99m-Tc] macroaggregated albumin.I wkafter admission, a lowdensity area was notedin the lateral lobe of the left cerebral hemisphere in a com-putertomographyscan of the brain, whichwasprobably related to

vascular occlusion. Since then, the patient has taken warfarin (3-5 mg

daily)and has hadnootherthromboembolicepisodes.

DNAsamples.Blood samples were taken from the patient and his

familymemberswith informed consent. Nine parts blood were mixed with one part 3.8% trisodium citrate, and the plasma wasthen sepa-rated bycentrifugation at 3,400 g for 15 min at4VCand stored at -80°C untiluse.GenomicDNAwasisolated fromperipheral blood leukocytes by phenol extraction, as previously described (26).

ImpairedSecretionoftheMutantProtein C CausedbyaFrameshiftMutation 2439 J.Clin.Invest.

©TheAmerican Society for Clinical Investigation, Inc. 0021-9738/92/12/2439/08 $2.00

TableI.Oligonucleotide Primers and

Amplified

Protein C Gene Fragments

Nucleotide positions*

PCR

Primer 5'--- -3' products Region

bp

A-1 AGGCAGAATTCGGCTTCGGGCAGAACAAGC (- 1641 to-1612) ₂₀₆

A-2 TCATAGAATTCCCTGGAGGGGGACTCACAG (-1449 to -1420) Exon I

B-i ACCCTGAATTCCAGCTTCCGCCTGACGGCC (-149 to- 10) 309 _ExonII

B-2 ACCCAGAATTCAGAGAGATGGTGGAAGCTG (146 to 175)

C-1 CCTCAGAATTCCTCATGGCCCCAGCCCCTC (1260 to 1289) _{Exon III}

C-2 CCCTGGAATTCATCCTCTGGACCCATGGTG (I508 to 1537) 262

D-1 TGCAGGAATTCGAGCCTGCCCGCTCTCTCC (2928 to 2957)

D-2 AGCGTGAATTCTGGGCGATGTATTGGGGGC (3462 to 3491) 548 Exons IV, V, andVI

E-i GGGAGGAATTCCTGGCAGGCCCCTCACCAC (6078 to 6107) ₂₃₂ Exon VII

E-2 AGGATGAATTCAGTGATCCCGGACCCAGCA (6297 to 6326)

F-1 GACTGGAATTCGTCAGGAGGCAGCCCTGTG (7084 to 7113) ₃₈₆

F-2 TGCCCGAATTCGAAGAGGGCACCAGAGAAC (7457 to 7486) Exon VIII

G-1 CTCAGGAATTCGCCACTGGGGAGAGGCTCC (8321 to 8350) ₃₇₃

G-2 CCACGGAATTCTTGATGAAGTTGAGGACGA (8681 to 8710) Exon IX

H-1 CTCGTGAATTCCTGGGGCTACCACAGCAGC (8600 to 8629) ₄₄₅ Exon IX

H-2 AGAACAGAATTCCGGTGTGCTTGTTACATG (9032 to 9061)

* Primer sequences and numbers are according to the nucleotide sequence of Foster et al. (14) except for the underlined EcoRI restriction sites.

Protein Cassays. Protein C activitywas determinedusing protein C-deficient plasmaas asubstrate and Protac® (Boehringer Mannheim

GmbH,Mannheim, Germany)as anactivator ofproteinC(Staclot

Protein CO;Boehringer Mannheim)(27).

Twodifferent ELISAs were usedtodetermineproteinCantigen

levels. Totalprotein C antigen levelwasfirst measuredusinga polyclo-nalrabbit serumagainsthumanproteinC(Asserachrom Protein CO;

BoehringerMannheim).The level ofcalcium-dependent proteinC

an-tigenwasalsomeasuredusingarabbitmonoclonalantibodythat recog-nizesaconformationalchange in the gammacarboxyglutamicacid ofa

glutamate residue(Gla)(TD-82;Teijin, Tokyo, Japan).

ProteinCactivityandantigenlevelsin thesampleswereexpressed

aspercentages of those observed inpooled plasmafrom 20 normal

individuals (28). The levelsofproteinCactivity andantigen in the

pooled plasmawerebothdefinedas 100%.

Westernblotanalysis.200Mil ofthe testplasmawasdilutedwith 800 td of 0.32% sodium citrate in 50 mMTris-HCl, pH7.5. Barium citrate

adsorptionwasthen carriedoutbyadding40 td of 1 MBaCl2tothe dilutedplasmaandchillingitonice for 60 min. After 5 min

ofcentrifu-gationat 10,000g, the supernatantwas

discarded,

and the

pellet

was

redissolved in 100

,d

of 0.25 MEDTA,50

,M

Tris-HCl,

pH7.5

(29).

Thesamplesweredialyzedagainstdistilled water,

dried,

andsubjected

toSDS-PAGE, and thefractionated

proteins

were

electrically

trans-ferredtonitrocellulose paperfor

immunological

detection.

PlasmaproteinCwasthen immunolabeled witharabbit

anti-pro-tein C polyclonalantibodyprovidedbyDr. M.Matsuda(JichiMedical School,Tochigi,Japan), and immunodetectedbyanenhanced chemi-luminescence detection kit(Amersham, Bucks,UK).Human

protein

C,purified byimmunoaffinity chromatographyon acolumn of

Ca2+-dependentanti-proteinC monoclonal

antibody

immobilizedon

Seph-arose,wasusedaspositivecontrol.

Preparationofoligonucleotide primers. Eight pairsof

oligonucleo-tidepolymerase chain reaction

(PCR)' primers

coveringthe5'

un-translated(presumedpromoter)region,all nine exons, and the

exon-1. Abbreviationusedinthispaper:

PCR,

polymerase

chain reaction.

intron boundaries of the protein C gene (Table I), have restriction endonucleasecleavage sitesconvenient for subsequent cloning and

se-quencing.Theprimerswere prepared on a DNA synthesizer (Applied Biosystems, Inc., Foster City, CA) (28).

PCR. PCRwasperformed as previously described by Saiki et al. (30) with minor modifications. The PCR reaction mixture (100

,u)

contained 1

1Ag

ofgenomicDNAin 50 mM KCI, 10 mM Tris-HCI (pH

8.3),1.5 mMMgCl2,0.01% (wt/vol) gelatin,

200,uM

each dNTP,100 pmol of eachprimer,and2.5 U of recombinant Taq DNA polymerase

(PerkinElmer-Cetus, Norwalk, CT). The mixture was overlaid with mineral oiltoavoidcondensationandsubjected to 35 cycles ofamplifi-cation. Thesamplesweredenatured at 94°C for 1 min,annealed at

60°Cfor 2 min, and subsequently heated to polymerize at 72°C for 2 min inanautomaticthermal cycler (Perkin Elmer-Cetus).

DNAsequencing. The PCR products were digested with EcoRI

en-donuclease, then subcloned intoMi3 vectors to obtain single-strand DNAfragments.Thesefragmentsweresequenced by thedideoxy chain termination methodasdescribedpreviously(28, 31). Thepossibility ofmispolymerization byTaq polymerasewasruled out bysequencing

multipleclones withdistinctorientation.

Construction

of

expression vectors. An expression vectorforthe normalproteinC(pEUK-PC)wasconstructed asfollowsbyinserting a cDNAfragment codingforproteinCintopEUK-C (Clonetech, Palo

Alto, CA).The cDNAfragment encodingnormalproteinC,provided by Dr. Earl W. Davie(University of Washington, Seattle, WA) in pUC 19, containedtwoEcoRIsites,betweenwhich lay 69noncoding bases 5'of the ATG initiationsite, 126 bases of leader sequence, 1,383 basesencodingtheentireproteinC molecule, and 202noncoding bases 3'of the TAG stop codon; the latterregion includingtwopoly(A)

signals.This fragmentwasexcisedfrom pUC19by EcoRIdigestion

andligatedinto themulticloningsite of thepBluescriptKSII(+).The direction of insertionwasconfirmedbydigestionwith NaeI. For inser-tioninto the mammalian cellexpressionvector(pEUK-C), which has the SV40 late promoter upstream of the XbaI site,the cDNAwas

excised frompBluescript KSII(+) by XbaI-XhoIdigestion and in-serted intothesesamerestriction sites inpEUK-C.

sites inpLXSN(32), which containstheNeo-resistance

(Neoe)

gene undercontroloftheSV40earlypromoter.Thedirection of insertion

wasconfirmedbydigestion with SalIandXhoI.

Mutagenesis.

Site-directed

mutagenesis

of the

protein

C

coding

se-quences wasperformed forasingleGdeletionatnucleotide8857using

anoligonucleotide-directed in vitro Mutagenesis System@, version 2.0

(AmershamCorp.,ArlingtonHeights, IL) (33).Wepreparedthe

fol-lowing mutant oligonucleotide, 5'-CCCACAGCCCTCACCCAGCT-CACCAGGCCC-3'. After invitro

mutagenesis,

themutant

protein

C

cDNAwassequencedbythe

dideoxy

chain termination

technique

as describedearlier,usingdouble-stranded DNAas atemplate,toverify

themutation and thefidelity oftheresultingsequence.The fragment

containing the mutationwastheninserted intopEUK-CorpLXSN, creating the mutated protein C expression vectors, pEUK-mPC or

pLXSN-mPC.

Transientorstable expressionofrecombinantproteinC. COS7and BHKcells were grown in Iscove's MEMcontaining10%FCS and500 ng/mlof vitamin K in 35-mm wells. COS7cells(3X 105perwell)

were transfected with either pEUK-PC or pEUK-mPC using the DEAE-dextrantechnique (34).BHKcellswerealsotransfected with bothexpressionvectorsbycalcium phosphate coprecipitationmethod. Theculturemediumwasremovedfromthe cellsforproteinC

measure-ment48 hafter transfection. Afterwashing with PBS,thecellswere

lysedwith 0.1% NonidetP-40(SigmaChemicalCo.,St.Louis, MO) containing10 mMPMSF,and the cellextracts werecollected.

Forthe stableexpression system, t-2cells(3 x 105 perwell),the

ecotropicretroviruspackagingcellline(35),werealsotransfected with eachretroviral vector,pLXSN-PCorpLXSN-mPC,by calcium phos-phatecoprecipitationmethod, andsomeclonesstablyexpressing

nor-malor mutant proteinC were selected inamediumcontainingthe

neomycinanalogG418 (500

,g/ml).

The levelsofproteinCin the culture media and in the cellextracts

ofCOS 7, BHK and{-2cellsweremeasuredbyELISA (Asserachrom ProteinC®;BoehringerMannheim). We alsoperformedWestern blot

analysis ofthe cell extractsof4-2cellstransfected withtheretroviral vectors under reducedconditions.

Northern blot analysis.Tomeasure the transcript levelsofthe

intro-ducedtransientexpressionvectors,cytoplasmic RNA samples (2, 4, or 20

Ag)

fromCOS 7 cellstransfected with pEUK-PC or pEUK-mPC werequantitatedbyabsorbance at 260 nm, subjected to 1.2% formalde-hyde-agarose gel electrophoresis, and transferred toanylon membrane for RNA blotanalysis.Membraneswerethenhybridized for16 h toa

protein C cDNA (1.8 kb) or a 778-bpPstI-XbaIfragment of human GAPDH cDNAasinternal control (36).

Immunoprecipitation

of[

5S] methionine-labeled recombinant

pro-tein C.

i-2

cells(3 x 105 per well) transfected with each retroviral vector,pLXSN-PC or pLXSN-mPC, were prepared, washed with PBS, andincubatedwith 2 ml of methionine-free MEM containing 2% dia-lyzed FCS.After1 h of incubation at 37°C, the cells were labeled with 40

,Ci

of [35S]methioninein 2 mlofthemethionine-freemediumfor 1 h,followedbychasing with an excess of unlabeled methionine (250

,gg/ml)

for0 min, 15 min, 30 min, 1 h, 2 h, and 4 h. Cell extracts and mediawere harvested at appropriate intervals, after which

recombi-nantproteinC wasimmunoprecipitatedfrom each sample with rabbit

anti-proteinC polyclonal antibody as described above. The

immuno-precipitates were eluted from formaldehyde-fixed Staphylococcus

au-reusCowan strain II (Pansorbin; Calbiochem-Behring, La Jolla, CA) (37) byboiling, electrophoresed on 12% SDS-PAGE, and autoradio-graphed.

Results

Protein

C

in

the

plasma

of

the patient and his

family.

As

shown

in

Fig.

1,

the

patient (I-1; 46

yr

old),

a

sister

(1-2;

41 yr

old),

a

brother

(1-3;

39 yr old), and a

nephew

(11-1;

14 yrold) had

nearly

half the normal levels

of

both

protein

C

activity

and

441 50 47

51 48 45

II

132

55

90

63 82

Figure 1. The pedigree ofaprotein C-deficient patient. The proband is indicated byan arrow.Blacksquareindicates clinicalthrombotic disease. Heterozygotesareindicated byadot in the middle of the cir-cleorsquare.Squares and circlesrepresentmales and females, re-spectively. Protein Cenzymeactivity (PC.Ac) and antigen(PC.Ag)

levelsweremeasured for each individual in the pedigreeas

percent-agesofthose found in pooled plasma from 20 normal individuals. Each individual is referredtoasfollows: I-l, the patient; I-2, the

pa-tient'ssister;I-3, the patient's brother;II-],thepatient's nephew; and 1I-2, the patient's niece.

antigen in their plasma. No thromboembolic episodes had been previously observed inanyof thefamily members except the

patient.

Incontrasttotheother family members,

however,

both protein C activity and antigen levels in the patient's niece (11-2; 10yrold)werealmost normal.

Western

blot

analysis of

the

patient's plasma.

Plasma

sam-ples taken from the patient, his niece, andanormalindividual

were analyzed for protein C by Western blot under reduced

conditions (Fig. 2). A band of 62 kD in size, which

corre-spondstonormalprotein C, and doublet heavy chains of 39-42 kDwere detected in both normal plasma and the

sample

takenfrom the protein C-deficient heterozygote. Doublet light chains of 22-25 kD were notshown in this analysis. Bands larger than normal protein C werenot detected in the

pro-band's plasma.

Analysis of

the protein C

gene.

Southern blot

analysis of

genomic DNA samples taken from the patient and his family members revealed neithergrossdeletionnorrearrangementof

theprotein Cgene(datanotshown). Eight regionsofthe

pro-tein C genomicsequence,amplified by PCR, totaled - 1.8 kb

in length; this

included

the 5' untranslated region, all nine

exons, all exon-intron boundaries, and the proximal 3'

un-translated region ofthe protein C mRNA. We obtainedatleast fourindependent clones forsequenceanalysis of each of the eight regions. All sequencesthus analyzedwere foundto be identicaltothe normal protein Cgene,exceptforasingle

gua-nine residue (8857G) inastretch of four consecutive guanines,

whichwasdeleted from the lastexonofoneallele(Fig. 3). This

resultwasconfirmed by sequencing fivemorecloneswith

dif-ferent orientations.

The deletion of this single guanine nucleotide apparently

results in aframeshift mutation, which permits transcription

pastthe normal termination codon throughtothenextchain termination signal. This is expectedtocausethereplacementof the last 39 amino acidsatthe carboxyl terminus with 81 abnor-malamino acid residues (Fig. 4), generatinganabnormal

pro-tein C withamolecularmass 4kDlargerthan normal. We

have designated this elongated mutantas ProteinC-Nagoya.

Computer analysis bytheGENETYXprogram(Software

De-velopment Co., Tokyo, Japan) further showed thata

{kDw

Figure2.Western blot

(kD)analysis

ofproteinCin plasma samples taken

97_

from thepatient, his

niece,

andanormal

in-66

-dividual.The

samples

45

- _ were

subjected

to 12%

45 _{SDS-PAGE under}

re-ducedconditions after

31

bariumcitrate

adsorp-tion. Each lane is re-ferredtoasfollows: 1,

1

2

3

4

thepatient; 2,the pa-tient'sniece;3,anormal individual; and 4, purified human protein C. Thepositions of molec-ularmassmarkers areshownattheleft.

phobic

stretch of 17 amino acid residues was created in the carboxyl-terminal portionofProtein

C-Nagoya

(Fig.

4).

Analysis

offamily

members.

To

determine

the

heredity of

this mutation, we examined the genomic DNA ofthis patient's

family

members

using

the

following

mutagenic primers (28,

38,

39):

5'-CCTGGTTCCTGGTGGGCCTGGTGAGCTC*GG-3'

5'-AGAACAGCAGGCCGGTGTGCTTGTTACATG-3'

After

amplifying

the

protein

C

genomic

sequence

by

PCR

us-ing this

mutagenic

primer, digestion

of the

product

of the

nor-mal

allele with Aval yielded

twosizes of

fragments,

207 and 27 bp in length. This restriction

site,

however,

wasnotpresentin

the mutant

allele,

thus Aval

digestion

of the PCR

product

de-5' 5 rG, T L_G A G --C 0 G G -G

G_{<lde et'Or}

Val _T

-G A A-g G -G Alai -C

A G C T A G C T

.6.U.i v _

W...ho

,tHfS

i

__{_lar}

^

_NEW

:-

we.tr

_EA_ A...U

_ S

_#_

9,4 ,,

._

.,<b.lP,,et, w s .'

>mu- 'w ,..:.Y

*it .. A:

5 s:7

_ 4pS,.,eEf'

..A;...

^I':...

' ^':.\bW _.

^*_:z and,SS<

.__ X.

_

mutant allele normal allele

0- G- A-G Ser' C-T -G Tro"r G G G GIl`:.

T-G A Gi so`

G--G G Gly 3

-C

3'

Figure 3.ADNAsequencecomparisonbetween the normal and the

mutantproteinCallele, both isolated froma

protein

C-deficient pa-tient. Normal andmutantsequences of thecodingstrandnearthe 3' endof theproteinCcoding regionareshown in the

right

andthe left

panels,respectively.The normal nucleotide and the deduced amino acid sequences with

corresponding

residue numbersareindicatedat

therightside.Aguaninenucleotide has been deleted from themutant

allele inastretchof four consecutive G

residues, causing

aframeshift mutation.

381

mutantallele F L V G L V S W V R A V G S F T T.T

TTCCTGGTGGGCCTGGTGAGCTGQ TGAGGGCTGTGGGCTCCTTCACAACTAC

normal allele F L V G L V S Wd En C G L L H N Y (8857)

400

A F T P K S A A T S T G S M G T S E T R K P

GGCGTTTACACCAAAGTCAGCCGCTACCTCGACTGGATCCATGGGCACATCAGAGACAAGGAAGCC

G V Y T K V S R Y L D W H G H R D K E A

(8900) (8950)

420

P R R A G H L S D P P C R A G L L H G N G W

CCCCAGAAGAGCTGGGCACCTTAGCGACCCTCCCTGCAGGGCTGGGCTTTTGCATGGCAATGGATG

P 0 K S W A P ₍₉₀₀₀₎

440

D K G T C N K H TGL FC PSI PL G

GGACATTAAAGGGACATGTAACAAGCACACCGGCCTGCTGTTCTGTCCTTCCATCCCTCTTTTGGG

(9050) 460

CTCTTCTGCAGGGAAGTAACATTTACTGAGCACCTGTTGTATGTCACATGCCTTATGAATAGAA (9100)

Figure 4. Theeffect of a single nucleotide deletion on the deduced amino acid sequence of the carboxyl-terminal region of protein C. Thenucleotidesthat codefor amino acids are numbered beginning with theinitiation codon, and are shown in parentheses. The deduced amino acid sequence from the mutant allele is written with the amino acid numberabove the nucleotide sequence, while the amino acid sequence of normalproteinCis shown beneath the nucleotide se-quence.Thetermination codons are underlined, and the GGG direct repeats are doubleunderlined. The deletion of a single guanine

nu-cleotide, indicatedbyawhite letter, results in a frameshift mutation that causesthe replacementof the last 39 amino acids at the carboxyl terminus with 81 abnormal amino acid residues. 17 amino acid resi-dues that correspondtotheadditionalhydrophobicstretch are dotted.

rived from the mutant allele

produced only

one

234-bp

frag-ment.

The PCR

products

from 20

normal

individuals

produced

twosizes offragments (207 and 27 bp in length) when digested with AvaI,

suggesting that they

were

homozygous

for

the nor-mal allele (data not

shown).

AvaI

digestion of the PCR

prod-uctfrom the niece

(II-2),

who hadnormal levels of

protein

C

antigen

and

activity, produced

two

bands

(only

the 207-bp fragmentwasdetected) (Fig. 5). On the other hand, the PCR products from the

family members who

were

deficient for

pro-tein C

(I-

1,

I-2,

1-3 andII-1 ) yielded three bands (234 bp, 207 bp, and the smaller 27-bp fragment) after Aval digestion (Fig. 5),

indicating

that each

of

these

family members carried

one

Figure5. Analysisof

proteinC alleles in the

familymembersby

_2234 Bp PCRamplification

us---207

Bp

ing

the

mutagenic

primer followed by AvaI

digestion.

After PCR

1-1 1-2 I-3 11-1 11-2

amplification,

the

frag-ments weredigested

with AvaI,subjectedto

9%PAGE, and stained with ethidium bromide.Fragmentsof 27

_bp

in lengthwere notdetected in thisgel.The PCRproductsobtained from the individualswhoaredeficient forproteinC

(I-i,

I-2, I-3,

andII-I,asdescribed in

Fig.

1)

yielded

twobandsof 207 and 234

bp

in length, after Avaldigestion.Incontrast, Aval

digestion

of the PCR product obtained from the individual who isnotdeficient for

protein

18S-

ii-

I

28S-AB C D E F G H I J K

¶IbtalRNA

20 2 2 4 4

20 20

4

4

20 20

(:4)

Figure 6.RNAblotanalysisof thetranscription levelsofpEUK-PC

andpEUK-mPC transfected into COS7 cells.TotalRNA(2, 4,or

20 Mg)fromeachconstructwasseparatedby 1.2%

formaldehyde-agarose gel electrophoresis, transferredtoanylon

membrane, andhybridizedfor 16 hto32P-labeled protein CcDNA probe(lanes A-G)orhuman GAPDH cDNA(lanes H-K). LaneA

representsRNAisolated from COS 7cells into which plasmidswere

nottransfected. Lanes B, D, F, H, and JcorrespondtoRNAsamples isolatedfromCOS7cellstransfectedwithpEUK-PC, while lanes C, E,G.I,andKrepresentRNAisolated from COS7cells transfected with pEUK-mPC.Thepositions of ribosomalRNAsizemarkersare

indicatedattheleft margin (285 and 185).

mutantandonenormalallele. The 27-bp

fragments

werealso

undetected in these lanes.

Expression

ofthe normal and

mutantprotein Cgenes. To

verify whether the frameshift mutation identified in the patient couldindeed result in protein C deficiency, expressionvectors

containing either the normalorthemutantprotein C cDNA,

wereprepared and transfected into COS 7, BHK, and ,6-2 cells, as described in Methods. RNA blot analysis of cytoplasmic

RNA isolated from

COS

7 cells was performed using three

different

amountsof

RNA,

andthe results did notrevealany

significant

difference in the transcript levels obtained from thesetwocDNAs

(Fig. 6).

The levels of normal protein C in the culture media of COS 7 and BHK

cells,

asmeasured

by

ELISA, were 63.0±9.6 ng/ml and 20.8±3.3 ng/ml,

respec-tively.

On the other

hand,

the level of normal protein C in the cellextractsof COS 7 was6.0±0.9

ng/ml,

andBHK 1.9±0.3

ng/ml. Evidently, "90%

of normal protein C synthesized in each cell line wassecretedinto the culture

medium,

whereas theamountofmutantprotein C secretedwaslower than the limit

of detection

byELISA (<1 ng/ml). The cellularcontent

ofmutant

protein C, however,

didnotdiffer

significantly (COS

7, 5.6±0.9 ng/ml; BHK, 1.9±0.3 ng/ml). Similar resultswere

obtained in the stable

expression

experiments using

ik-2

cells

(data

notshown). Protein Cwas notdetected in theextractsof COS 7 and

i/-2

cells transfected with the mutant protein C cDNA, under reduced conditions by Western blot analysis (datanot

shown).

It is probable that themutant protein C is very unstable within the cells; therefore, even with the stable expressionsystem,it would beverydifficulttodetect the mu-tantprotein Cby Western blot analysis.

Pulse-chase analysis of recombinant protein C with

[35S]-methionine labeling.

To confirm further whether the

deletion

of 8857G really gave riseto the transport-deficient phenotype, recombinant protein Cwas

immunoprecipitated

fromcell ex-tracts or media of the transfected i1-2 cells, which had been pulse labeled forIh and then chased for varying periods upto4 h with unlabeled methionine (Fig. 7).

Immediately

after label-ing, the cells transfected with pLXSN-PC contained a62-kD form of protein C. During the4-hchase period, essentially all of this 62-kD formwassecretedinto the media. The specificity of this analysiswasverified by the absence ofacorresponding protein whenanormal rabbit IgG fractionwas used asnegative

62 ->

<

Normal

Protein C

>

0 Vin 0 00

chase

(min)

W- _{en~ '0e41}

- 69

-Zew

46

-<

Protein C

-

Nagoya

>

(kD)

-

92.5

0 in 0 0

V- en C e4 19

r-4 eq

69

46

Figure 7.Immunochemical analysis of pulse-chase la-beledprotein C in the

trans-fected

_4&-2

cells. After selec-tion of clones stably express-ing normal protein C or Protein C-Nagoya, the cells were pulse-labeledfor 1 h with [35S]methionine and chased for the indicated pe-riods with an excess of unla-66 beledmethionine. The

re-combinant protein Cwas

then immunoprecipitated

from the cellextracts or the

conditioned media with a rabbit anti-human protein C

immunoglobulinand Pan-sorbin. The

immunoprecipi-tates weresubjectedto

elec-trophoresison 12% SDS-PAGEand autoradiography.

66 The molecular mass

stan-dards used were phosphory-lase b(92.5 kD), BSA (69 kD), and ovalubumin(46 kD).

ImpairedSecretionofthe MutantProtein C Causedbya_FrameshiftMutation 2443

Cell

Extract

Medium

-

92.5

I -k- * *

control. In contrast, the recombinant Protein C-Nagoya

ap-peared in the cells as an apparently larger species (66 kD)

im-mediately after labeling. This difference in size between the

normal protein C and

Protein C-Nagoya is consistent with our

prediction that an

elongation ofthe amino acid sequence in the

mutant protein C might be caused by the frameshift mutation.

When chased with

unlabeled methionine, Protein C-Nagoya

remained within the cells for a prolonged period, as compared

with the normal protein C. Apparently, only a small portion of

the

synthesized mutant molecule had been secreted into the

medium even when the intracellular protein C had become

almost

undetectable. These observations indicate that most of

the mutant

protein

C molecules must have undergone

degrada-tion

within

the

cells while their transport was retarded in the

intracellular transport pathway.

Discussion

The molecular

basis

of protein C deficiency has recently been

studied

in a number

of

cases.

DNA

abnormalities

are

reported

in more than 50 cases, and most of them are caused by

mis-sense

mutations,

while the remainder are caused by nonsense

mutations,

frameshift

mutations, or

splice site

abnormalities.

Generally,

clinical

phenotype

appears

to

vary with the site at

which a missense

mutation

occurs.

However,

even

if

it occurs

at

the same

site,

a

different

clinical phenotype results

depend-ing

on

which

amino

acid

residue is substituted. For example,

replacement

of

'5Arg (CGG)

with Gln

(CAG)

or

Trp

(TGG)

causes

type

I

protein

C

deficiency (40),

while

replacement

of

'5Arg

(CGG)

with

Gly

(GGG)

results in

a

functional

abnormal-ity in

calcium binding,

thus abnormal

protein

C molecules

exist

in

plasma

in

this

case

(type

II

protein

C

deficiency)

(41).

More

recently,

Reitsma

et al.

(42) studied

40

protein

C-defi-cient

probands

and

identified 15 distinct

mutations,

six of

which were

found

in more than

one

unrelated

proband.

Three

mutations

(23OArg

--

Cys,

132Gln

--

Stop,

306Arg

->

Stop)

were

considerably

more

frequent

than others and occurred in nine,

nine, and five

distinct

pedigrees, respectively.

As to

frameshift

mutations,

Grundy

et

al.

(20) reported

a

single basepair

dele-tion (a

missing

G

residue

in

147Arg),

which

was

predicted

to

lead to premature

termination of

protein

synthesis nine

codons

downstream.

However,

further

details of

the

effects

of these

mutations

on

the

functional

aspects

or

impaired secretion

of

protein

C have not yet been

clarified.

The

mutation

responsible

for

the abnormal

protein

C

ana-lyzed in

this study is

a

deletion

of

one

residue in

a

sequence of

four consecutive

guanine

nucleotides.

The fact that the

muta-tion occurred

in a run

of like nucleotides is in accord with the

DNA

slippage hypothesis of

frameshift mutations

(43).

This

theory

predicts

that frameshift mutations

occur more

fre-quently

in runs

ofidentical bases

where

displacement

or

"loop-ing out" of bases from

either

the

template

strand

(causing

a

deletion)

or

the

growing

strand

(causing

an

insertion)

can be

stabilized by

normal

basepairing beyond

the

unpaired

base

during the

replication

process

(44).

Human gene deletions

are

often associated with direct repeat sequences,

an

observation

explained

by the

"slipped mispairing" hypothesis (45).

Consis-tently, in the

protein

C gene,

a

GGG direct repeat

occurs

flank-ing

the

site of

deletion

(Fig.

4).

ATGGGG

sequence, found

also in the

tandemly repeated

immunoglobulin

switch

(Sm)

regions (46),

corresponds

to

the

_f3-globin

deletion

hotspot,

co-don 41

(45), and is similar

to

both the known

polymerase

a

arrest

sites

(47)

and

the human

deletion hotspot

consensus sequence

(45).

The

single guanine

nucleotide deletion

we have found

changes

the chain

termination signal

and causes the

replace-ment

of the last 39 amino acids

atthe carboxyl terminus with

81 abnormal

amino acid

residues (Fig.

4).

To our knowledge,

this

type

of

frameshift mutation, which

causes elongation of

protein C,

has

notyet

been previously

reported. This elongated

protein

C

(Protein

C-Nagoya)

should be - 66 kD in size, but a

protein

C

species

with

a

molecular

masslarger than normal was

not

detected in the patient's

plasma by Western blot analysis

(Fig.

2), suggesting

that there

may

be

impaired

secretion or

rapid

removal

of the

mutant

protein

C from circulation.

Ap-proximately

30

amino acid residues beginning

with

176Gly

are

included

in the

highly

conserved

region

common to members

ofthe

serine

protease

family

(48).

38"Gly

is thought

to be one of

the

residues

(379Ser-38OTrp-381Gly)

opposite

the substrate

bind-ing pocket

which

interact with

the

side chains

of the substrate

in order

to

properly

orient the bond that is

to be cleaved (49).

Therefore,

it is

possible

that the catalytic domain

of protein C,

which

reacts

with

its

substrates, is destroyed

by this frameshift

mutation.

Although

a

comparison

between the

deduced amino

acid

sequences

from the normal and the

mutant DNA se-quence

of

protein

C

shows

an

additional hydrophobic

stretch

of

17

amino

acid

residues

at

the carboxyl

terminus

of the

mu-tant

protein

C

(Fig.

4),

wecannotsay atpresent

whether

this

hydrophobic

region

is

necessary

for

susceptibility

to

internal

degradation.

Our

expression

study strongly

suggests

impairment of

the

mutant

protein

C

secretion

in this

case.

Recently,

impaired

intracellular

transport

has

also

been reported

for

several other

naturally

occurring

or

genetically engineered

mutant

proteins

(50,

51

),

and

it has been

hypothesized

that

a

newly synthesized

secretory

or

membrane

protein

would

not

be transported

from

the

rough endoplasmic

reticulum

to

the

Golgi

apparatus

unless

it

folds

into

native

or

near-native conformation

(52, 53).

The

aberrant molecules

may

be removed

from the rough

endoplas-mic

reticulum

or

the

Golgi

apparatus

before

secretion through

a

mechanism

that

involves so-called "molecular chaperone"

proteins (54,

55).

In

the

case

of

Protein C-Nagoya,

a

significant

alteration in conformation of the native

protein

C molecule is

easily predictable,

as

described above.

This,

in

turn,

probably

causes

improper folding,

thereby affecting its posttranslational

transport

through

the

intracellular secretory

compartments.

The

expression study

also

suggests

that the

mutant

protein

C is

unstable within the

cells, possibly

attributed its

altered

confor-mation,

because

no

abnormal

intracellular accumulation

was

observed in

spite

of

its

apparently

retarded

secretion (Fig. 7).

Furthermore,

this mutation

doesnotappearto

have

any

signifi-cant

effect

on

transcription

asshown

in the Northern

blot

anal-ysis

of

our

transfection

system

(Fig. 6).

From these

data, it

seems

likely

that

the

production

of

themutant

protein

C

is

not

decreased within

the

cells,

but rather the mutant

protein

C

molecules

are

poorly secreted,

andmost

of

themare

degraded

in

the

producing

cells.

Moreover,

Protein

C-Nagoya

secretedat

avery low

level

might

be removed

from

the

circulation

in

vivo

more

rapidly

than normal

protein

C

molecule because

of

the

experimen-tal evidence for this

at

the

present

time. Further studies

are

requiredonthe

intracellular

transport

of

mutantsecretory

pro-teins, and

we

hope that Protein

C-Nagoya

will

be

a

useful

modelfor these

studies.

Acknowledgments

We thank Dr. Masanari Awaya (Sakuragaoka Branch of Toyohashi-Shimin Hospital,Toyohashi, Japan) for providing materials from his patients and Dr. Earl W. Davie (University of Washington, Seattle, WA) for the generous giftof the protein C cDNA. We also thank Dr. Michio Matsuda (Jichi Medical School, Tochigi, Japan) for providing anti-proteinC polyclonalantibody,andSayoko Sugiura for her

help-ful technicalassistance.

This work was partly funded by grant 63637003 for Scientific Re-search in Priority Areas from the Japanese Ministry of Education,

Science,and Culture.

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