THE
JOURNAL
OF
BloLooIciu.
CHEMISTRY
0
1994
by The Ame ican Socie y
o
Biochemis y and Molecula
Biology,
Inc
Vol.
269,
No.
24,
Issue
o
June
17,
pp.
16766-16773, 1994
P in ed
in
U.S.A.
Molecula Cloning and Exp ession
o
Collagenase-3, a No el Human
Ma ix Me allop o einase P oduced
by
B eas Ca cinomas*
(Recei ed o publica ion, Ap il
1,
1994, and in e ised o m, Ap il 14, 1994)
Jose
M.
P.
F eijeS, I ene Diez-I za, Milag os Balbin, Luis
M.
Sanchez$, Ra ael Blascon,
Jo ge Toli iall, and Ca los Lopez-0 h””
F om he Depa amen o de Biologia Funcional and IWo ologia
y
Biologia Celula , Uniue sidad de Ouiedo,
33006
Ouiedo,
and ICen o de In es igacion
en
Sanidad Animal, INIA-MAPA, Valdeolmos,
28130
Mad id, Spain
A
cDNA coding o a new human ma ix me allop o-
einase
(M”)
has been cloned om a cDNA lib a y de-
i ed om a b eas umo . The isola ed cDNA con ains
an open eading ame coding o a polypep ide o 471
amino acids. The p edic ed p o ein sequence displays
ex ensi e simila i y o he p e iously known
MMPs
and
p esen s all he s uc u al ea u es cha ac e is ic o he
membe s
o
his p o ein amily, including he well con-
se ed PRCGXPD mo i , in ol ed in he la ency o he
enzyme and he zinc-binding domain
(HEXGHXXXX-
XHS).
In addi ion, his no el human
MMP
con ains in i s
amino acid sequence se e al esidues speci ic o he col-
lagenase sub amily
(m-214,
Asp-235, and Gly-237) and
lacks he 9- esidue inse ion p esen in he s ome-
lysins. Acco ding o hese s uc u al cha ac e is ics, he
MMP desc ibed he ein has been en a i ely called colla-
genase-3, since i ep esen s he hi d membe o his
sub amily, composed a p esen o ib oblas and neu o-
phil collagenases. The collagenase-3 cDNA was ex-
p essed in a accinia i us sys em, and he ecombinan
p o ein was able o deg ade ib illa collagens, p o id-
ing suppo o he hypo hesis ha he isola ed cDNA
codes o an au hen ic collagenase. No he n blo anal-
ysis o RNA om no mal and pa hological issues dem-
ons a ed he exis ence in b eas umo s o h ee di e -
en mRNA species, which seem o be he esul o he
u iliza ion o di e en polyadenyla ion si es p esen in
he 3’-noncoding egion
o
he gene. By con as , no col-
lagenase-3 mRNA was de ec ed ei he by No he n blo
o
RNA
polyme ase chain eac ion analysis wi h RNA
om o he human issues, including no mal b eas ,
mamma y ib oadenomas, li e , placen a, o a y, u e us,
p os a e, and pa o id gland. On he basis o he in-
c eased exp ession o collagenase-3 in b eas ca cino-
mas and he absence o de ec able exp ession in no mal
issues, a possible ole o his me allop o einase in he
umo al p ocess is p oposed.
A
dis inc i e cha ac e is ic o malignan umo s is hei abil-
i y o in ade no mal issues and sp ead o dis an si es gi ing
*
This wo k
was
suppo ed
by
G an
SAL91-0617
om
he
Comision
In e minis e ial
de
Ciencia
y
Tecnologia
and
a
g an
om
Plan FEDER-
Eu opean Communi y.
The
cos s
o
publica ion
o
his
a icle
we e
de-
ayed
in
pa
by
he paymen
o
page
cha ges.
This
a icle mus he e-
o e be he eby
ma ked
“adue isemen ”
in
acco dance
wi h
18
U.S.C.
Sec ion
1734
solely
o
indica e his ac .
The nucleo ide sequence(s) epo ed in his pape has been submi ed
o he GenBankTMIEMBL Da a Bank wi h accession numbe ($
X75308.
Recipien
o
a
ellowship
om
Fundaci6n pa a
la
In es igacion
Cien i ica
y
T6cnica-As u ias.
5
Recipien
o
a
ellowship
om
Asociacih
Lucha con a
el
Cance -
As u ias.
**
To
whom
co espondence
should
be
add essed. Tel.:
34-85-104201;
Fax: 34-85-103534
o
34-85-232255.
ise
o
me as asis. These p ocesses in ol e deg ada ion
o
he
di e en componen s
o
he ex acellula ma ix and appea o
equi e he ac ion o p o eoly ic enzymes p oduced ei he by he
umo cells hemsel es
o
by he su ounding s omal cells
(Lio a
e
aE.,
1991; Migna i and Ri kin,
1993).
Among he
a ie y o p o einases wi h po en ial in ol emen in acili a ing
in asion and me as asis,
a
la ge numbe o s udies ha e o-
cused on ma ix me allop o einases (MMPs),’
a
g oup o highly
ela ed enzymes ha a e in ol ed in he emodeling o he
connec i e issue du ing many no mal
o
pa hological condi-
ions (Woessne , 1991; Ma isian, 1992; Mu phy and Doche y,
1992).
Based on s uc u al and unc ional conside a ions, he
MMPs cons i u e
a
single e olu iona y p o ein supe amily
ha can be classi ied in o
a
leas h ee di e en amilies o
closely ela ed membe s: collagenases, gela inases, and s ome-
lysins.
All
o
hem a e simila in ha hey a e sec e ed in
a
la en o m, con ain
a
zinc-binding si e, and can be inhibi ed by
chela ing agen s and issue-speci ic inhibi o s. Howe e , hey
di e wi h espec o hei subs a e speci ici y (Nagase
e
al.,
1991). Thus, collagenases clea e he na i e helix o ypes
I,
11,
and
I11
ib illa collagens
a
a
single pep ide bond, gene a ing
agmen s app oxima ely 314 and 1/4 he size
o
he o iginal
molecule (Welgus
e
al.,
1981; Has y
e
al.,
1987). Gela inases
deg ade ypes
IV,
V,
VII,
and
X
collagens and elas in and may
ac syne gis ically wi h in e s i ial collagenases in he deg a-
da ion o ib illa collagens (Fessle
e
al.,
1984; Collie
e
al.,
1988; Wilhelm
e
al.,
1989; Senio
e
al.,
1991). Finally, s ome-
lysins ha e
a
b oad subs a e speci ici y and a e able o de-
g ade many ex acellula p o eins, including p o eoglycans,
laminin, and ib onec in (Chin
e
al.,
1985; Wilhelm
e
al.,
1987;
Quan in
e
al.,
1989; Mu phy
e
al.,
1991).
A p esen nine human MMPs ha e been isola ed and cha -
ac e ized, including he ecen ly desc ibed elas oly ic me allo-
p o einase p oduced by human al eola mac ophages (Shapi o
e
al.,
1993).
Two
o hese human MMPs belong o he collagen-
ase subg oup and ha e been named ib oblas and neu ophil
collagenases (Goldbe g
e
al.,
1986; Has y
e
ai.,
1990). The
gela inase subclass is composed o wo membe s, 72- and
92-
kDa ype
IV
collagenases (Collie
e
al.,
1988; Wilhelm
e
al.,
1989), whe eas he emaining ou human MMPs, s ome-
lysins-1, -2, and -3 and ma ilysin (Whi ham
e
al.,
1986; Mul-
le
e
al.,
1988; Basse
e
al.,
19901, ha e been ini ially included
in he s omelysin class. Howe e , he subs a e speci ici y o
s omelysin-3 has no ye been clea ly de ined and acco ding o
s uc u al compa isons and ch omosomal loca ion o he gene
(Le y
e
al.,
1992), his enzyme does no appea o belong
o
any
~
APMA,
4-aminophenylme cu ic
ace a e;
bp,
base
paids);
kb,
kilobase
The abb e ia ions
used
a e:
MMP, ma ix me allop o einase;
pai (s);
TBS,
T is-bu e ed
saline;
PAGE, polyac ylamide
gel
elec o-
pho esis;
PCR, polyme ase chain eac ion.
16766
This is an Open Access a icle unde he CC BY license.
Human Collagenase-3 16767
o he p e iously desc ibed subclasses and may ep esen he
i s membe o a new MMP sub amily (Mu phy
e
al.,
1991;
Basse
e
al.,
1993).
A
compa ison o he amino acid sequence
o
he di e en
membe s o he MMP amily e eals ha hese p o einases
sha e se e al domains wi h appa en speci ic unc ions: a p e-
domain encoding he leade sequence ha a ge s hese p o-
einases o sec e ion, a p o-domain o abou 80 amino acids
in ol ed in he main enance o he la ency
o
he enzymes, a
ca aly ic domain o app oxima ely 170 esidues which con ains
he zinc-binding egion, and inally, a COOH- e minal ag-
men o abou
200
amino acids, which is absen in ma ilysin
and ha shows sequence simila i y
o
hemopexin, a heme-
binding se um p o ein. In hese domains, he e a e wo sho
sequence mo i s ha a e pa icula ly well conse ed in all
MMPs cha ac e ized
o
da e (Van Wa and Bi kedal-Hansen,
1990). Thus, he p opep ide egion con ains in all cases he
sequence PRCG(V/N)PD, in which he cys eine esidue seems
o
be essen ial o main aining enzyme la ency. On he o he
hand, he ca aly ic domain o all hese p o einases con ains a
sho sequence
HEXGHXXEYHS
ha is hough
o
be in-
ol ed in he coo dina ion o he zinc a om a he ac i e si e
(Sanchez-L6pez
e
al.,
1988; Sp ingman
e
al.,
1990). The
oc-
cu ence o hese highly conse ed sequences opens he possi-
bili y
o
iden i y pu a i e addi ional membe s
o
he MMP gene
amily by PCR-based homology cloning, using degene a e oli-
gonucleo ides encoding hese
wo
s uc u al mo i s.
In his wo k, and as pa o ou s udies di ec ed
o
in es i-
ga e he in ol emen o p o eoly ic enzymes in b eas cance
(Sanchez
e
al.,
1992a, 1993; Diez-I za
e
al.,
19931, we ha e
used his cloning s a egy
o
sea ch pu a i e no el MMPs p o-
duced by b eas ca cinomas. We desc ibe he cloning o a gene
coding o a no el membe
o
he MMP gene amily, he e des-
igna ed collagenase-3. We also epo he exp ession o he gene
by a accinia i us ecombinan ha p oduced an ac i e colla-
genase-3. Finally, we show ha his gene is exp essed by hu-
man mamma y ca cinomas bu no by he no mal es ing mam-
ma y gland
o
by a numbe o di e en human issues.
Acco ding
o
hese esul s, he exp ession o his p o eoly ic
enzyme may be o impo ance in he malignan ans o ma ion
o he mamma y issue.
EXPERIMENTAL PROCEDURES
Ma e ialsSpecimens o human b eas umo s we e ob ained om
women who had unde gone su ge y o p ima y b eas ca cinoma; hu-
man placen a was ob ained immedia ely a e deli e y; o he issue
specimens we e om au opsies pe o med wi hin 15 h a e dea h.
Tissue samples we e ozen in liquid ni ogen and s o ed a -70 "C un il
used. The RNA-PCR ki used o he e e se ansc ip ion o o al RNA
and cDNA ampli ica ion was om Pe kin-Elme . Oligonucleo ides we e
syn hesized by he phospho amidi e me hod in an Applied Biosys ems
DNA syn hesize (model 381A) and pu i ied by polyac ylamide gel elec-
opho esis acco ding o s anda d p ocedu es (Mania is e
al.,
1982)
o
used di ec ly a e syn hesis. The poly(A)' RNA pu i ica ion and cDNA
syn hesis ki s we e om Pha macia Bio ech Inc. (Uppsala, Sweden).
Res ic ion endonucleases and o he eagen s used o molecula clon-
ing we e pu chased om Boeh inge (Mannheim, Ge many). Double-
s anded DNAp obes we e adiolabeled wi h [32PldCTP (3000 Ci/mmol)
using
a
comme cial andom-p iming
ki
om Pha macia Bio ech Inc.
Reagen s o amino acid sequencing we e om Applied Biosys ems.
Syn he ic pep ide
(Dnp-P o-Gln-Gly-Ile-Ala-Gly-Gln-D-A g-OH)
o en-
zyme ac i i y assays was om Bachem (Bubendo , Swi ze land),
whe eas adioac i ely labeled Type I collagen was kindly p o ided by
D . M. A. Liza be (Uni e sidad Complu ense, Mad id, Spain).
PCR Ampli ica ion
o
B eas Ca cinoma RNA-To al RNA om
a
b eas ca cinoma was isola ed by guanidinium
hiocyana e-phenol-chlo-
o o m ex ac ion (Chomczynski and Sacchi, 1987). cDNA syn hesis was
ca ied ou wi h he RNA-PCR
Ki
om Pe kin-Elme . The e e se
ansc ip ion was pe o med o
1
h a 42 "C wi h
1
pg o o al RNA and
andom hexame s
as
p ime . The whole mix u e was used o PCR wi h
wo degene a e oligonucleo ides co esponding o he highly conse ed
cys eine swi ch and zinc-binding domains o MMPs (B'-CCN(AC)GNT-
G(CT)GGNGTNCC and
5'-TGNCC(AG)AA(TC)TC(AG)TGNGC,
espec-
i ely) (100 pmol/ eac ion). PCR eac ion was ca ied ou in
a
Techne
PHC-3 The mal Cycle o 40 cycles o dena u a ion (94 "C,
1
nin),
annealing (45 "C,
1
min), and ex ension (72 "C, 2 min). The PCR p od-
uc s we e phospho yla ed wi h T4 polynucleo ide kinase, and he DNA
band o he expec ed size (app ox 0.4 kb) was gel-pu i ied and liga ed in
he SmaI si e o pEMBL19. DNA om 30 independen clones was iso-
la ed and sequenced by he dideoxy chain e mina ion me hod (Sange
e al., 1977) using he Sequenase Ve sion 2.0 ki (U.
S.
Biochemical
Co p.). All nucleo ides we e iden i ied in bo h s ands. Sequence ambi-
gui ies we e sol ed by subs i u ing dITP o dGTP in he sequencing
eac ions. Compu e analysis o DNA and p o ein sequences was pe -
o med wi h he GCG so wa e package o he Uni e si y o Wisconsin
Gene ics Compu e G oup (De e eux e al., 1984).
Cons uc ion and Analysis
o
a
B eas %mo cDNA Lib a y-B eas
ca cinoma poly(A)+ RNA was selec ed by oligo(dT)-cellulose ch oma og-
aphy using
a
comme cial ki om Pha macia. Double-s anded cDNA
was syn hesized wi h he You-P ime cDNA syn hesis ki (Pha macia)
using oligo(dT) as p ime and liga ed in o he EcoRI si e o Ag ll. Abou
3
x
lo5
plaque- o ming uni s o he esul ing lib a y (wi hou p e ious
ampli ica ion) we e pla ed using Esche ichia coli Y1088 as hos and
analyzed acco ding o he me hod o Ben on and Da is (1977) using he
pa ial MMP cDNA cloned by RNA-PCR
as
p obe. Hyb idiza ion o he
adiolabelled p obe was ca ied ou o
18
h in
6
x
SSC
(1
x
SSC
=
150
mM NaC1,15 mM sodium ci a e, pH 7.0),
5
x
Denha d 's
(1
x
Denha d 's
=
0.02% bo ine se um albumin, 0.02% poly inylpi olidone,
0.02%
Fi-
coll), 0.1% SDS, and 100 pg/ml dena u ed he ing spe m DNA. Subse-
quen ly, he il e s we e washed wice o
1
h
a
60
"C in
1
x
SSC, 0.1%
SDS and subjec ed
o
au o adiog aphy. Following plaque pu i ica ion,
he cloned inse was excised by No I diges ion, epai ed wi h Klenow,
subcloned in o he SmaI si e o pEMBL19, and sequenced
as
p e iously
desc ibed.
No he n Blo Analysis-Samples
o
abou 40 pg o o al RNA we e
sepa a ed by elec opho esis in 1.4% aga ose- o maldehyde gels. A e
assessing RNA in eg i y and equal loading by obse ing he appea ance
o he ibosomal RNAs, blo ing on o Hybond N nylon il e s was ca ied
ou . Fil e s we e p ehyb idized a 42 "C o 3 h in
50%
o mamide,
5
x
SSPE
(1
x
SSPE
=
150 mM NaCl, 10 mM NaH,PO,,
1
mM EDTA, pH 7.41,
2
x
Denha d 's, 0.1% SDS, and 100 pg/ml dena u ed he ing spe m
DNA and hen hyb idized o 48 h unde he same condi ions. Fil e s
we e washed wi h 0.2
x
SSC,
0.5%
SDS o 2 h
a
65
"C and exposed o
au o adiog aphy.
Exp ession in E. coli-Plasmid pNo 3a, which con ains he ull-
leng h cDNA o human collagenase-3, was diges ed consecu i ely wi h
No I, HindIII, and nuclease
S1,
and he 1.5-kb blun -ended agmen
con aining he en i e coding sequence was pu i ied and liga ed
o
he
exp ession ec o pET3c (Rosenbe g e al., 1987), p e iously ea ed
wi h NdeI and nuclease
S1.
The esul ing plasmid, called pETI9, was
ans o med in o E. coli s ain BL21(DE3). BL21(DE3) cells ans-
o med wi h he exp ession plasmid pETI9
o
wi h pET3c wi hou in-
se we e g own in LB b o h con aining 200 pg/ml ampicillin a 37 "C
o abou 16 h, dilu ed 1/100 wi h he same medium, and g own o aA,,,
o 1.0. Then,
isop opyl-1- hio-P-o-galac opy anoside
was added o a inal
concen a ion o
1
m
and he incuba ion was con inued o ano he 3
h. Cells we e collec ed by cen i uga ion, esuspended in
0.05
olume o
TBS (50
m
T is/HCl, pH
8,
150 mM NaCl), lysed by using a F ench
p ess, and cen i uged a 20,000
x
g
o 20 min a 4 "C. The insoluble
ac ion o he ex ac was washed wi h he same olume o 2
M
u ea in
TBS and inally solubilized
wi h
1
olume o
8
M
u ea in TBS and
cen i uged
as
be o e.
Amino Acid Sequencing-Di ec sequencing o ecombinan collagen-
ase-3 was ca ied ou by he me hod
o
Ma sudai a (1987). P o eins
p esen in he
8
M
u ea ex ac we e sepa a ed by SDS-polyac ylamide
gel elec opho esis, blo ed on o an Immobilon ans e memb ane (Mil-
lipo e), and s ained
wi h
Coomassie Blue, and he memb ane ca ying
he ecombinan p o ein was placed di ec ly in o he eac ion chambe
o
a
model 477A Sequence (Applied Biosys ems). Edman deg ada ion
was pe o med acco ding o he Blo p og am indica ed by he manu-
ac u e . The aniliao hiazolinones we e con e ed o phenyl hiohydan-
oin de i a i es in he au oma ic con e sion lask o he Sequence and
quan i ied wi h an on-line phenyl hiohydan oin analyze (model 120A
Applied Biosys ems).
An ise um P oduc ion and Wes e n Blo ing-1 ml
o
8
M
u ea ex ac
was elec opho esed h ough
a
12% polyac ylamide gel, and he po ion
o
he gel con aining he ecombinan p o ein was excised, g ound, and
incuba ed wi h 2 ml o deionized wa e a 37 "C o abou 20 h wi h
spo adic o exing. 1 ml o SDS-PAGE-pu i ied p o ein was used o
immunize a New Zealand Whi e abbi acco ding o he me hod de-
sc ibed by Vai ukai is (1981). The abbi was bled 6 weeks a e he
injec ion, and IgGs we e pu i ied by ch oma og aphy h ough a DEAE-
cellulose column (Wha man DE52) equilib a ed and elu ed wi h 20 mM
phospha e bu e , pH 7.2. Finally, he ob ained an ibodies (dilu ed
l/1000) we e used o Wes e n blo analysis as p e iously desc ibed
(Sanchez e al., 1992a, 1992b).
Gene a ion
o
Vaccinia Vi us Recombinan s-Vaccinia i us exp ess-
ing human collagenase-3 was ob ained using a plaque selec ion sys em.2
Plasmid pRB-co13 was ob ained by inse ing an EcoRIIHindIII ag-
men con aining he gene downs eam o a accinia i us syn he ic
ea ly/la e p omo e , in plasmid pBR21. Con luen monolaye s o CV-1
cells in T25 lasks we e in ec ed wi h 1 plaque o ming uni /cell accinia
i us RBl2 clone a4 and ans ec ed wi h 10 pg o plasmid pRB-~013.
A 2 days pos -in ec ion, he p ogeny i us was ha es ed. The ecom-
binan , e med W-~013, was selec ed by wo consecu i e ounds o
plaque pu i ica ion on BSC-1 cell monolaye s. Fo p oduc ion o he
ecombinan p o ein, p econ luen BSC-1 cells in 900-cm2 olle bo les
we e in ec ed wi h wild- ype accinia i us (s ain WR) o W-co13 a a
mul iplici y o in ec ion o abou 5 plaque o ming uni s/cell. Ex acel-
lula medium and cell ex ac s we e ha es ed a 24 h pos -in ec ion.
Enzyme Ac i i y Measu emen s-lo-yl aliquo s o medium ha es ed
om cells in ec ed wi h collagenase-3 ecombinan i us o wi h wild
ype i us we e incuba ed in he p esence o 1 nM APMA. A e 4 h a
oom empe a u e, samples we e dilu ed wi h 90 pl o assay bu e (50
mM T is, pH 7.5, 150 nM NaCl, 10 nM CaCl,, 0.05% B ij 35 ( / ), 0.02%
sodium aside) and incuba ed 24 h ei he wi h 0.1 nM syn he ic pep ide
(D ip-P o-Gln-Gly-Ile-Ala-Gly-Gln-n-A g-OH) o wi h econs i u ed in-
soluble ib ils o “‘C- adiolabeled ype I collagen. Hyd olysis o he pep-
ide was e alua ed by measu ing he abso bance a 365 nm a e ex-
ac ion wi h e hyl ace a e as desc ibed (Masui
e al.,
1977).
Deg ada ion o ib illa collagen was de e mined by liquid scin illa ion
coun ing o he solubilized ma e ial.
Immunohis ochemical S aining-Immunohis ochemical assays we e
pe o med on 6-pm o malin- ixed pa a in-embedded issue sec ions
using he a idin-bio in p ocedu e (Hsu e al., 1981). Endogenous pe -
oxidase and nonspeci ic binding we e blocked by sequen ial incuba ion
o he sec ions in 10% hyd ogen pe oxide solu ion and in no mal se um.
Incuba ion wi h an ise um agains ecombinan collagenase-3 (dilu ed
1:500 in 20 nM phospha e bu e , pH 7.21 was pe o med a 4 “C o 16
h. Then, he slides we e incuba ed wi h he second bio inyla ed an i-
body ob ained om Dako (Dako, Denma k) and he a idin-bio in com-
plex eagen (Vec o Labo a o ies, Bu lingame, CA). A e 30 min a
oom empe a u e, he eac ion was de eloped wi h 0.06% diaminoben-
zidine and 0.01% hyd ogen pe oxide. Finally, he sec ions we e coun-
e s ained wi h a modi ica ion o he o maldehyde- hionine me hod
(Toli ia and Toli ia, 1985), dehyd a ed, clea ed in eucalyp ol, and
moun ed wi h Euki . Speci ici y o s aining was de e mined by using
con ols ha in ol ed incuba ion o issue sec ions alone o wi h an
equal amoun o IgG om nonimmunized abbi s.
RESULTS
Iden i ica ion and Cloning
o a
cDNA
o
Human Colla-
genase-3-To iden i y new membe s o he human
MMP amily
p oduced by b eas umo s, wo degene a e oligonucleo ides
we e designed om wo domains highly conse ed among he
di e en membe s o his p o einase amily. A e RNA-PCR o
o al RNA isola ed om a mamma y ca cinoma (I-91, a band o
he expec ed size (abou 0.4 kb) was ob ained and cloned in he
plasmid ec o pEMBL19. DNA om 30 independen clones
(19-l o 19-30) was isola ed and sequenced. Analysis o he
nucleo ide sequence o hese clones e ealed ha 12 o hem
co esponded o a cDNA wi h a high deg ee o simila i y o
MMPs p esen in he da a bases bu dis inc om all p e iously
cha ac e ized p o einases belonging o his gene amily.
In o de o ob ain a ull-leng h cDNA o his pu a i e no el
MMP, a cDNA lib a y was p epa ed using, as s a ing ma e ial,
poly(A)’ RNA om he same b eas ca cinoma used o he
RNA-PCR expe imen desc ibed abo e. Upon sc eening o ap-
p oxima ely 3 x lo5 plaque- o ming uni s using he PCR gen-
e a ed cDNA as p obe, h ee posi i e clones we e iden i ied.
’ R. Blasco and B. Moss, manusc ip in p epa a ion.
16768
Human Collagenase-3
One o hem, named 19c9, had an inse o 2.7 kb, which could
be la ge enough o con ain he comple e coding in o ma ion o
a MMP. The nucleo ide sequence o he cloned cDNA (Fig. 1)
e ealed an open eading ame 1413 bp long, s a ing wi h an
ATG codon a posi ion 5 and ending wi h a TAA codon a posi-
ion 1418. This open eading ame codes o a p o ein o 471
amino acids con aining all he cha ac e is ic ea u es o MMPs
and wi h a p edic ed molecula weigh o 53,759 ha is e y
simila o hose co esponding o o he MMPs belonging o he
collagenase and s omelysin subg oups (Mu phy and Doche y,
Human Collagenase-3
16769
A
FIBROBLAST
FIBROBL BOV
FIBROBL
PIG
FIBROBL
RAB
OSTEOBL RAT
OSTEOBL MOU
NEUTROPHIL
COLLAGENASE-3
STROMELYSIN-1
STROMELYSIN-2
STR-1 RAT
STR-2 RAT
B
FIBROBLAST
FIBROBL BOV
FIBROBL PIG
OSTEOBL RAT
FIBROBL
RAB
OSTEOBL
MOU
NEUTROPHIL
COLLAGENASE-3
STROMELYSIN-1
STROMELYSIN-2
STR-1 RAT
STR-2 RAT
2
68 284
RSQNPVQ---------PIGPQTPKAC
PSQNPTQ---------PVGPQTPEVC
PSENPVQ---------PSGPQTPQVC
PSQNPSQ---------PVGPQTPKVC
PGDEDPN-------”PKHPKTPEKC
LSSNPIQ-------”PTGPSTPKPC
PGDEDPN-------”PKHPKTPEKC
pGDEDpN””--”- PlQCIPKTPDKC
PPPDSPETPLVPTEPVPPEPGTPANC
PPPASTEEPLVPTKSVPSGSEMPAKC
PPTESPDVLWPTKSNSLDPETLPMC
ARP-SSDATWPVPSVSPKPETPVKC
213 238
EYNLHRVAAHELGHSLGLSHSTDIGA
DYNLYRVAAHEFGHSLGLAHSTDIGA
DYNLYRVAAHELGHSLGLSHSTDIGA
NYNLYRVAAHELGHSLGLSHSTDIGA
GYNLFIVAAHELGHSLGLDHSKDPGA
GYNLFIVAAHELGHSLGLDHSKDPGA
NYNLFLVAAHEFGHSLGLAHSSDPGA
GYNLF’LMAAKEFGASLGLDRSKDPGA
GTNLFLVAAHEIGHSLGLFHSANTEA
GTNLFLVAAHELGHSLGLFHSANTEA
GTNLFLVAAHELGHSLGLFHSANAEA
GTNLFLVAAHELGHSLGLFHSNNKES
*+
FIG.
2.
Compa ison
o
p o ein sequences
om
collagenases
and s omelysins a ound he p oposed c i ical egion
o
sub-
s a e speci ici y.
The a ailable amino acid sequences
o
collagenases
and s omelysins we e ex ac ed om he SwissP o da a base and used
o mul iple alignmen . Numbe ing co esponds o collagenase-3. A,
amino acid sequences a ound he 9- esidue inse ion cha ac e is ic
o
s omelysins;
B,
egion con aining dis inc i e esidues be ween collag-
enases and s omelysins. Residues ha a e conse ed in all collag-
enases and which a e dis inc in s omelysins a e indica ed wi h an
as e isk.
1993). Amino acid sequence compa ison wi h he emaining
human MMPs showed ha he simila i y anges om 50.5%
(neu ophil collagenase) o 36% (s omelysin-3). Howe e ,
when he compa ison was pe o med wi h all sequences con-
ained in he da a bank, he highes amino acid sequence simi-
la i y
(86%)
was ound wi h a and mouse collagenases p o-
duced by u e ine smoo h muscle cells, os eosa coma cells, and
os eoblas s and whose p ima y s uc u e has been ecen ly
elucida ed (Quinn
e al.,
1990; Hen ie
e al.,
1992). O e he
las yea s, i has been widely assumed ha hese p o einases
a e he mu ine coun e pa s o human ib oblas in e s i ial
collagenase and consequen ly, hey ha e been designa ed
as
MMP-1 (Clohisy
e al.,
1992; Sco
e al.,
1992). Howe e , he
inding ha he human MMP desc ibed he ein is mo e closely
ela ed o hese enzymes han ib oblas collagenase s ongly
sugges s ha hese mu ine collagenases a e enzymes dis inc
om ib oblas collagenase.
A mo e de ailed amino acid sequence compa ison o his
no el human MMP wi h he emaining MMPs cha ac e ized o
da e p omp ed us o include i in he subclass o collagenases.
Thus, acco ding
o
ecen s uc u e- unc ion ela ionship s ud-
ies, he speci ic ac ion o in e s i ial collagenases on iple hel-
ical collagen is de e mined by he p esence o
a
16-amino acid
sequence in hei COOH- e minal domain (Hi ose
e al.,
1993).
In s omelysins, his egion con ains an inse ion o 9 amino
acids, whose in oduc ion in he co esponding sequence o neu-
ophil collagenase esul s in comple e loss o he collagenoly ic
ac i i y o his chime ic enzyme (Hi ose
e al.,
1993). A com-
pa a i e examina ion o his domain in he iden i ied open
eading ame shows he p esence o his c i ical egion o
subs a e speci ici y agains ib illa collagens and he absence
o he 9 esidues cha ac e is ic o s omelysins (Fig.
2A).
In
addi ion, his no el human MMP also con ains in i s p edic ed
20
14.4
FIG.
3.
P oduc ion
o
collagenase-3 in
E.
coli
BL21(DE3).
5-pl
aliquo s
o
soluble
(SF:
)
and insoluble
(13
)
ac ions
o
he bac e ial
ex ac s, as well as
o
p o eins solubilized wi h he indica ed concen-
a ions o u ea, and
1
pl
o pu i ied collagenase-3
(Ag)
we e analyzed by
SDS-PAGE. The size in kilodal ons o he molecula size ma ke s
(MWM)
is shown a
igh
o
igu e.
amino acid sequence he 3 esidues (Ty -214, Asp-235, and
Gly-237) ha a e conse ed in all collagenases cha ac e ized o
da e and which a e ne e p esen in s omelysins (Fig. 2B).
Acco ding o hese s uc u al compa isons wi h o he MMPs,
he pu a i e new amily membe iden i ied in his wo k has
been en a i ely called collagenase-3, since i ep esen s he
hi d membe o his subclass composed a p esen o ib oblas
and neu ophil collagenases. Fu he mo e, his p oposed name
a emp s o e lec he pa allelism o his enzyme wi h s ome-
lysin-3,
a
ecen ly desc ibed MMP ha has been also ound
associa ed wi h b eas ca cinomas (Basse
e al.,
1990, 1993).
On he o he hand, and ollowing he nomencla u e sys em
p oposed by Okada
e al.
(1986), we would assign numbe
13
o
he no el human MMP he e desc ibed, numbe 12 being he
mu ine and human me alloelas ases ecen ly isola ed by
Shapi o
e al.
(1992, 1993).
Exp ession
o
he Collagenase-3
cDNA
in E. coli and in Vac-
cinia Vi us-As
a
p e ious s ep o examine he unc ional el-
e ance o collagenase-3 and
o
ob ain speci ic an ibodies ha
could aid in he iden i ica ion o he na i e p o ein in human
issues, s udies we e unde aken o exp ess collagenase-3
cDNAin
E.
coli.
Fo his pu pose,
a
1.5-kb agmen con aining
he en i e open eading ame was subcloned in he exp ession
ec o pET3c (Rosenbe g
e al.,
1987). The esul ing plasmid,
called pETI9, was ans o med in o
E.
coli
BL21(DE3), and he
ans o med bac e ia we e induced
o
p oduce he ecombinan
p o ein. Ex ac s we e p epa ed om he induced bac e ia and
analyzed by SDS-PAGE (Fig.
3).
The insoluble ac ion o he
ex ac showed a majo band, co esponding o a polypep ide o
abou 40 kDa, which was no p esen in he bac e ia ca ying
he con ol plasmid. A e washing wi h 2
M
u ea, he ecombi-
nan p o ein was solubilized wi h
8
M
u ea, elec opho esed,
blo ed on o a poly inylidine di luo ide memb ane, and sub-
jec ed o di ec amino acid sequencing. The ob ained pa ial
sequence (MNLTYRIVNYTPDMT) ma ches
o
esidues
116-
130 o he deduced amino acid sequence o collagenase-3, sug-
ges ing he occu ence o
a
p o eoly ic p ocessing e en . By
analogy wi h o he MMPs, which unde go au oca aly ic ag-
men a ion a ound he cys eine-swi ch sequence o he p o-do-
main, ecombinan collagenase-3 could be sel -p ocessed du ing
cell cul u e and pu i ica ion o igina ing he 40-kDa o m de-
ec ed in he p esen wo k. Howe e , he pa icipa ion o some
bac e ial p o eoly ic ac i i y wi h
a
ypsin-like speci ici y e-
qui ed o clea e he Lys-Me pep ide bond ound o be clea ed
in ecombinan p ocollagenase-3, canno be excluded. This
16770
Human Collagenase-3
APMA
-
APMA
+
0.5h 4h 0.5h 4h
MW
(kDa)
Ag
C-
C+
C-
C+
C-
C+
C-
C+
""
106-
80
-
49.5
-
0
32.5-
.*
-
28.5
-
18.5
-
i ely glycosyla e collagenase-3 in some
o
all o he h ee po-
en ial N-glycosyla ion si es con ained wi hin he p esumed
ac i e o m o he molecule (Fig.
1).
In o de
o
examine he enzyma ic ac i i y
o
his ecombi-
nan p o ein, bo h collagenase-3-con aining medium and ex a-
cellula medium ha es ed om cells in ec ed wi h wild ype
accinia i us we e ea ed wi h
APMA
and incuba ed wi h
di e en subs a es o MMPs. As can be seen in Fig.
5,
colla-
genase-3-con aining medium deg aded Type I collagen, as well
as he syn he ic pep ide
(Dnp-P o-Gln-Gly-Ile-Ala-Gly-Gln-
D-A g-OH) commonly used as
a
subs a e o assaying e e-
b a e collagenases (Masui e
al.,
1977; Kleine e
al.,
1993). In
addi ion, his p o eoly ic ac i i y was ully abolished by EDTA,
an inhibi o o me allop o einases (Fig. 5). By con as , we
FIG.
4.
P oduc ion
o
collagenase-3 in accinia
i us.
Samples
o
ailed o obse e any deg ading ac i i y on gela in
o
casein
medium ha es ed om cells in ec ed wi h he collagenase-3 ecombi-
zymog ams. Taken oge he , hese esul s indica e ha colla-
nan
i us
(lanes
C+)
o
wi h wild ype
i us
(lanes
c-)
we e incuba ed
genase-3
is
a
bona
ide ma ix me allop o einase wi hadeg ad-
a oom empe a u e in he p esence o absence
o
1
mM
APMA.
A e
ing ac i i y on ib illa collagen clea ly in acco dance wi h ha
he indica ed imes, 5-pl aliquo s we e emo ed and analyzed by Wes -
an icipa ed
om
i s
amino acid sequence (Fig. 2).
e n blo wi h an ibodies agains pu i ied collagenase-3 p oduced in
E.
Exp ession
Analysis
,nollagenase-3
in
~~~~~l
and
n-
coli. Lane
Ag
is ecombinan collagenase-3 om
E.
coli.
mo al Human Tissues-To s udy he exp ession o he collagen-
unca ed collagenase-3 was hen used o ob aining polyclonal
an ibodies,
as
well as o pe o ming unc ional s udies. How-
e e ,
all
a emp s o de ec any p o eoly ic ac i i y o he e-
combinan p o ein on collagen, casein,
o
gela in subs a es
we e unsuccess ul (da a no shown). In ela ion o his,
i
is
ema kable ha simila nega i e esul s ha e been ecen ly
desc ibed o ecombinan s omelysin-3 p oduced in
E.
coli by
using he same T7 phage RNA polyme ase sys em employed in
his wo k (Mu phy e
al.,
1993).
Since he abo e esul s sugges ed ha he bac e ially p o-
duced p o ein was inco ec ly olded, s udies we e unde aken
o p oduce human collagenase-3 in an euka yo ic exp ession
sys em. To do ha , he comple e cDNA coding o human col-
lagenase-3 was cloned in
a
plasmid designa ed pRB21, which
con ains
a
s ong syn he ic accinia
i us
ea ly/la e p omo e
and he comple e coding egion o p37, he majo p o ein in
he ex e nal en elope o ex acellula in ec ious accinia
i i-
ons ha is essen ial in he p ocess o plaque o ma ion in cell
monolaye s (Blasco and Moss, 1991). The esul ed plasmid
pRB-col3 was used o ans ec CV-1 cells in ec ed wi h RB12,
a
accinia
i us
lacking he p37 gene and he e o e unable o
o m plaques. A e selec ion o he ecombinan i uses by
successi e ounds o plaque pu i ica ion on BSC-1 cell mono-
laye s,
a
accinia i us ecombinan designa ed W-col3 was
ob ained and used o examine he p oduc ion o human colla-
genase-3. In his way, p o eins p esen in medium ha es ed
om cells in ec ed wi h w-col3
o
wi h wild ype accinia i us
(s ain
WR),
we e sepa a ed by SDS-PAGE and analyzed by
Wes e n blo wi h an ibodies agains pu i ied collagenase-3
p oduced in
E.
coli.
As
shown in Fig. 4,
a
single band o abou
65 kDa was de ec ed in he ex acellula medium om cells
in ec ed wi h he ecombinan
i us
bu no in ha ob ained
om cells in ec ed wi h wild ype accinia
i us.
Fu he mo e,
when he medium was ea ed wi h 4-aminophenylme cu ic
ace a e
(APMA),
an o ganome cu ial agen known o speci i-
cally ac i a e MMPs, an addi ional immuno eac i e band o
abou 55 kDa was de ec ed (Fig.
4).
Since he ac i a ion o
MMPs in ol es he p o eoly ic emo al o he co esponding
p o agmen , he abo e esul s s ongly sugges ed ha he ac-
cinia
i us
exp ession sys em was able o p oduce and sec e e
o he medium
a
ecombinan collagenase-3 ha could be use ul
o pe o m he equi ed unc ional s udies. In addi ion, he ac
ha he obse ed immuno eac i e bands we e o
a
highe size
han hose calcula ed om he amino acid sequence seemed o
indica e ha he accinia
i us
sys em was also able o e ec-
ase-3 gene in bo h no mal and pa hological human issues,
samples om se e al issues (u e us, placen a, li e , p os a e,
pa o id gland, b eas , ib oadenomas, and mamma y ca cino-
mas) we e collec ed. To al RNA was isola ed om he samples
and analyzed by No he n blo , using he ull-leng h collagen-
ase-3 cDNA
as
p obe. Th ee hyb idizing bands we e ecognized
by he p obe in he lane co esponding
o
RNA om a b eas
ca cinoma (Fig. 6A), while none o hem was p esen in any
o
he examined issue specimens. These bands co espond o
mRNA species o app oxima ely
2,
2.5,
and 3 kb, espec i ely.
Since se e al pu a i e polyadenyla ion signals can be ecog-
nized in he 3"noncoding sequence o he cloned collagenase-3
cDNA (Fig. 11, he h ee RNA bands could be he esul o
u iliza ion o di e en polyadenyla ion si es. To examine his
possibili y, he same il e was s ipped and ehyb idized using
as p obe
a
D aI-EcoRI agmen 232 bp long, co esponding o
he 3'-end o he cDNA(Fig.
6A).
This p obe only ecognized he
la ges mRNA species, demons a ing ha he h ee mRNA
bands de ec ed wi h he ull-leng h p obe di e in hei 3'-
un ansla ed egions.
In an a emp o inc ease he sensi i i y o de ec ion o pu-
a i e collagenase-3 exp ession in bo h no mal and pa hological
human issues, we pe o med PCR-RNA analysis wi h RNAs
ob ained om
a
wide a ie y o samples including b eas ca -
cinomas, b eas ib oadenomas and no mal issues, as well as
wi h RNAs ob ained om b eas cance cell lines. Oligonucle-
o ides 5'-TCATGACCTCATCTTC and 5"GAACAGCTGCACT-
TAT we e used as he p ime pai in
a
RNA-PCR expe imen
o
ampli y
a
134-bp segmen co esponding o nucleo ides 1,030-
1,163 o he collagenase-3 cDNA. RNA-PCR omi ing he e-
e se ansc ip ase s ep was used as a con ol o RNA-depend-
en ampli ica ion. The quali y o he s udied RNAs was checked
by PCR ampli ica ion
o
he e e sed ansc ibed RNAs using a
pai o p ime s
(5'-CGGCGAGTACAACAAAGCCA
and 5'-CA-
CAGCGTAGATCTGGAAAG) ha di ec ed he ampli ica ion o
a 219-bp segmen co esponding o he cDNA sequence o he
human cys a in C gene, a housekeeping gene ha is exp essed
in all he issues s udied
so
a (Ab ahamson e
al.,
1990; F eije
e
al.,
1991). The esul s ob ained indica ed ha ampli ica ion
o
a
cDNA segmen om collagenase-3 RNA was de ec ed in
RNA om eigh di e en b eas ca cinomas bu no in RNA
om no mal es ing mamma y gland, h ee di e en b eas
ib oadenomas, li e , placen a, o a y, u e us, p os a e,
o
pa-
o id gland (Fig. 6B and da a no shown), suppo ing he e-
sul s ob ained by No he n blo analysis. In addi ion, no colla-
genase-3 exp ession could be de ec ed by PCR in RNA om
Human Collagenase-3
16771
A
B
-.
EDTA
-
EDTA
+
EDTA
-
EDTA
+
RG.
5.
Enzyma ic ac i i y
o
ecombinan collagenase-3. Hyd olysis o me allop o einase subs a es incuba ed wi h medium ha es ed
om cells in ec ed ei he wi h ecombinan collagenase-3 accinia
i us
o
wi h wild
ype
i us.
Assays we e pe o med in he p esence
o
absence
o
50 m~
EDTA.
A,
hyd olysis o
Dnp-P o-Gln-Gly-ne-Ala-Gly-Gln-D-kg-OH.
One
uni
o
ac i i y co esponds o
1
pol
o hyd olyzed pep ide
x
min".
B,
hyd olysis
o
insoluble ype
I
[14Clcollagen. Ac i i y
is
measu ed as 14C-solubilized hyd olyzed ma e ial.
A
B
pa hological
human
issues.
A,
abou
40
l.lg
o
o al
RNA
om
a
FIG.
6.
Exp ession
analysis
o
collagenase-3
in
no mal
and
b eas ca cinoma we e sepa a ed by aga ose gel elec opho esis, blo ed
on o nylon il e s, and analyzed by hyb idiza ion wi h he 111-leng h
cDNA o collagenase-3
o
wi h a DmI-EcoRI agmen co esponding
o
he 3'-end
o
he cDNA.
The
in eg i y o he RNA was asce ained by
di ec isualiza ion o he s ained gel and he nylon memb ane unde
UV
ligh .
The
posi ions o
28
and
18
S
RNA a e shown.
B,
RNA-PCR
was pe o med
on
1
pg o RNA om he samples indica ed
in
a olume
o
100
pl.
COL3
lunes
indica e RNA-PCR ampli ica ion o a segmen
o
collagenase-3 cDNA.
C+
lanes
indica e RNA-PCR ampli ica ion
o
a
segmen
o
cys a in
C
cDNA.
5
pl
(C+
lunes)
o
20
pl
(COG'
lunes)
o he
inal p oduc we e sepa a ed on
a
2% aga ose gel
un
in T idbo a el
EDTA. pBR322 diges ed wi hHueII1 (Ma ke
V,
Boeh inge Mannheim)
was used as a
size
ma ke .
h ee di e en b eas cance cell lines: T47-D, MCF-7, and
The p oduc ion
o
collagenase-3 by b eas ca cinomas was
also obse ed a he p o ein le el by immunohis ochemical
analysis
o
issue sec ions om b eas ca cinomas. Rep esen -
a i e examples o hese immunohis ochemical s udies a e p e-
sen ed in Fig. 7.
As
can be seen, a s ong collagenase-3
immu-
no eac i i y was de ec ed in he cy oplasm
o
b eas cance
cells, al hough in some cases, a sligh immuno eac i i y could
also be obse ed in he su ounding s omal cells.
ZR75-1.
DISCUSSION
In his wo k we desc ibe he molecula cloning, unc ional
s udies, and exp ession analysis
o
human collagenase-3, a
no el
MMP
p oduced by b eas ca cinomas bu no by ei he
he no mal es ing mamma y gland
o
a numbe
o
examined
no mal human issues. The iden i ica ion and cloning o he
cDNA
o
his
new umo p o einase was pe o med by sc een-
ing o a b eas ca cinoma
cDNA
lib a y wi h a p obe ob ained
FIG.
7. Immunohis ochemical
s aining
o
collagenase-3
in
hu-
man
b eas
cance .
Tissue
sec ions we e incuba ed wi h an i-colla-
genase-3 dilu ed
1:500
in
phospha e bu e
(A)
o
wi h phospha e bu e
alone
(B).
Sec ions we e coun e s ained wi h o maldehyde- hionine.
O iginal magni ica ion,
x
270.
by using
a
PCR-based homology cloning s a egy wi h p ime s
deduced om conse ed sequences among
"Ps.
The deduced
amino acid sequence
o
collagenase-3 displays signi ican se-
quence simila i y
o
he p e iously
known
membe s
o
his
p o einase amily, including he h ee domains ha a e con-
se ed among
all
o
hem: he p e-domain encoding a hyd o-
phobic leade sequence, he p o-domain con aining he well
conse ed PRCGXPD mo i in ol ed in main aining he la ency
o
hese enzymes, and he ca aly ic domain wi h he
HEXGHXXXXXHS
mo i con aining he His and
Glu
esidues
16772
Human Collagenase-3
conside ed o be he c i ical ca aly ic zinc-binding si es. The
iden i ied open eading ame also con ains he hemopexin-like
domain ound in he COOH- e minal egion in all amily mem-
be s wi h he excep ion o ma ilysin.
The inclusion o his no el MMP in he collagenase subclass
o MMPs was ini ially based on s uc u al compa isons, since
collagenase-3 con ains in i s amino acid sequence a numbe o
ea u es cha ac e is ic o his speci ic sub amily o MMPs.
Thus, collagenase-3 lacks he 9-amino acid inse ion p esen in
all s omelysins and he ib onec in-like domain cha ac e is ic
o he gela inases bu con ains se e al esidues speci ic
o
he
collagenase sub amily (Ty -214, Asp-235, and Gly-237) (Fig. 2).
Since hese esidues ha e been p oposed as undamen al de-
e minan s o collagenase speci ici y (Sanchez-L6pez
e
al.,
19931, i s p esence in collagenase-3 s ongly sugges ed ha
his enzyme belonged
o
he collagenase sub amily o MMPs.
Func ional analysis o collagenase-3 p oduced in
an
euka yo ic
exp ession sys em p o ided de ini i e suppo o ou p oposal
ha his enzyme is an au hen ic collagenase. Thus, by using a
accinia i us exp ession sys em, we we e able
o
p oduce a
ecombinan collagenase-3 which was ac i e agains Type I
collagen as well as agains a syn he ic pep ide used o assay-
ing in e s i ial collagenases. Acco ding
o
hese da a, he ac-
cinia i us exp ession sys em he ein used appea s
o
be app o-
p ia e o p oduc ion o ac i e human MMPs, hus opening he
possibili y
o
ex end i s use in he unc ional exp ession
o
o he
membe s o his p o ein amily.
In his wo k, we ha e also pe o med an analysis
o
colla-
genase-3 exp ession in a numbe o no mal human issues,
benign and malignan b eas issue specimens, and b eas can-
ce cell lines. Acco ding
o
he ob ained esul s, collagenase-3
exp ession was de ec ed by PCR analysis in all examined
b eas ca cinomas bu no in o he issues including no mal
b eas , mamma y ib oadenomas, li e , placen a, o a y, u e us,
p os a e, and pa o id gland, no in T47-D, MCF-7, and ZR75-1
b eas cance lines. This exp ession analysis also e ealed he
occu ence in b eas ca cinomas o h ee di e en mRNA spe-
cies, which seem
o
be he esul o he u iliza ion o di e en
polyadenyla ion si es p esen in he 3'- lanking egion o he
collagenase-3 gene. Al e na i e u iliza ion o h ee polyadeny-
la ion si es has also been epo ed o ca hepsin
B,
a lysosomal
cys eine p o einase ha is o e p oduced in malignan umo s
including b eas ca cinomas (Qian
e
al.,
1989; Sloane, 1990).
In e es ingly, he p esence
o
mul iple ansc ip s
is
de ec ed in
umo cells bu no in no mal issues, sugges ing ha he pos -
ansc ip ional p ocessing pa hway o he ca hepsin
B
gene
may be modi ied in malignan umo s (Qian
e
al.,
1991).
A
simila si ua ion could also occu in he case o collagenase-3,
al hough he lack o de ec ion o signi ican collagenase-3 ex-
p ession in any examined no mal issues p ecludes a p esen
u he s udies di ec ed
o
p ecisely es ablish he possible as-
socia ion o mul iple collagenase-3 ansc ip s wi h malignancy.
A
inal ques ion ega ding he occu ence
o
human collagen-
ase-3 in b eas ca cinomas makes e e ence
o
i s possible pa -
icipa ion in he umo igenic p ocess. In ela ion
o
his, he
inding he e desc ibed o inc eased collagenase-3 exp ession in
b eas umo s compa ed
o
no mal issues
o
benign umo s is
consis en wi h he hypo hesis ha his enzyme may be in-
ol ed in he ly ic p ocesses associa ed wi h in asi e b eas
cance lesions. In his ega d, ou ecen inding in b eas ca -
cinomas o a no el issue inhibi o
o
me allop o einases des-
igna ed TIMP-3 (U ia
e
al.,
1994) aises in e es ing ques ions
abou he possibili ies o collagenase-3 inhibi ion in his spe-
ci ic ype o umo . S udies a e in p og ess
o
elucida e his
ques ion as well as
o
es ablish he unc ional signi icance o
collagenase-3 and i s p ecise ole among he inc easing numbe
o p o einases wi h po en ial in ol emen in he malignan
ans o ma ion o human mamma y issue.
Acknowledgmen s-We hank D .
S.
Gasc6n
o
suppo , D .
M.
A.
Liza be
o
p o iding collagen samples, D .
F.
Vizoso o supplying
hu-
man issue specimens, and D .
S.
G.
G anda (Scien i ic Compu e Cen-
e , Uni e sidad de O iedo)
o
compu e acili ies.
REFERENCES
Ab ahamson, M., Ola sson,
I.,
Palsdo i ,
A,,
Ul sback, M., Lundwall, A,, Jensson,
Basse ,
P.,
Bellocq, J.-P., Wol , C., S oll,
I.,
Hu in, P., Limache ,
J.
M., Podhajce ,
0.
Basse , P.,
Wol ,
C., and Chambon, P.
(1993)
B eas Cance Res. nea .
24,185-193
Ben on,
W.
D., and Da is, R. W.
(1977)
Science
196, 180-182
Blasco, R., and Moss, B.
(1991)
J.
i ol.
65, 5910-5920
Chin,
J.
R., Mu phy,
G.,
and We b,
Z.
(1985)
J.
Biol.
Chem.
260,12367-12376
Chomczynski,
P.,
and Sacchi, N.
(1987)Anal.
Biochem.
162, 156-159
Clohisy,
J.
C., Sco , D.
K.,
B akenho ,
K.
D., Quinn, C.
O.,
and Pa idge, N. C.
(1992)
Mol. Endoc inol.
6,1834-1842
Collie ,
I.
E., Wilhelm,
S.
M., Eisen,
A.
Z.,
Manne , B. L., G an ,
G.
A,, Sel ze ,
J.
L., K onbe ge , A., He, C., Baue , E.
A.,
and Goldbe g,
G.
I.
(1988)
J.
Biol. Chem.
De e eux,
J.,
Haebe li,
P.,
and Smi hies,
0.
(1984)
Nucleic Acids Res.
12,387-395
263,6579-6587
Diez-I za,
I.,
Me ino,
A.
M., Toli ia,
J.,
Sbnchez, L. M., and L6pez-O in, C.
(1993)
Fessle , L.
I.,
Duncan,
K
G., Fessle ,
J.
H., Salo, T., and T ygg ason,
K.
(1984)
J.
F eije,
J.
P.,
Ab ahamson, M., Ola sson,
I.,
Velasco, G., G ubb, A,, and Mpez-O in,
Goldbe g, G.
I.,
Wilhelm,
S.
M., K onbe ge ,
A,,
Baue , E. A,, G an , G.
A.,
and
Has y,
K.
A.,
Je ey,
J.
J.,
Hibbs, M.
S.,
and Welgus, H. G.
(1987)
J.
Biol.
Chem.
Has y,
K.
A., Pou mo abbed,
T.
E,
Goldbe g, G.
I.,
Thompson,
J.
P., Spinella, D. G.,
S e ens,
R.
M., and Maina di, C. L.
(1990)
J.
Biol.
Chem. 285, 11421-11424
Hen ie , P., Rousseau,
G.
G., and Eeckou ,
Y. (1992)
FEBS Le .
310,175-178
Hi ose, T., Pa e son, C., Pou mo abbed, T., Maina di, C. L., and Has y,
K.
A.
Hsu,
S.
M., Raine, M. L., and Fange ,
H.
(1981)
J.
His ochem. Cy ochem.
29,
Kleine, T., Ba sch,
S.,
Blase ,
J.,
Schnie e ,
S.,
T iebel,
S.,
Valen in, M.,
Go e,
T.,
Le y, A., Zucman,
J.,
Dela e,
O.,
Ma ei, M.-G., Rio, M.-C., and Basse , P.
(1992)
Lio a, L. A,, S eeg, P.
S.,
and S e le -S e enson, W. G.
(1991)
Cell
64,
327-336
Mania is,
T.,
F i sch, E. F., and Samb ook,
J.
(1982)
Molecula C1oning:A
Lubo-
a o y Manual,
Cold Sp ing Ha bo Labo a o y, Cold Sp ing Ha b ,
NY
Masui,
Y.,
Takemo o,
T.,
Sakakiba a,
S.,
Ho i, H., and Nagai,
Y.
(1977)
Biochem.
Med.
17,215221
Ma isian, L. M.
(1992)
BioEssays
14,455-463
Ma sudai a, P.
(1987)
J.
Biol. Chem.
262,10035-10038
Migna i, P., and Ri kin, D. B.
(1993)
Physiol.
Reu.
73,161-195
Mulle , D., Quan in, B., Gesnel, M.-C., Millon-Colla d, R., Abecassis, J., and
Mu phy, G., and Doche y,
A.
J.
P.
(1992)
Am.
J.
Respi :
Cell.
Mol. Bid.
7, 120-125
Mu phy, G.
J.
P., Mu phy,
G.,
and Reynolds,
J.
J.
(1991)
FEBS Le .
289,4-7
Mu phy, G., Segain,
J.
P.,
O'Shea, M., Cocke , M., Ioannou, C., Le ebwe,
O.,
Nagase, H., Oga a,
Y.,
Suzuki,
K.,
Enghild,
J.
J.,
and Sal esen, G.
(1991)
Biochem.
Okada,
Y.,
Nagase, H., and Hams, E. D., J .
(1986)
J.
Biol.
Chem.
261, 14245-
O.,
and G ubb, A.
(1990)
Biochem.
J.
268,287-294
L.,
Chena d, M. P., Rio, M. C., and Chambon,
P.
(1990)
Na u e
348,699-704
B :
J.
Cance
68,637-640
Bid. Chem.
259,9783-9789
C.
(1991)
J.
Biol. Chem.
266,20538-20543
Eisen,
A.
Z.
(1986)
J.
Biol. Chem.
261,6600-6605
262,10048-10052
(1993)
P oc. Na l. Acad. Sci.
U. S.
A.
SO,
2569-2573
577-580
and Tschesche, H.
(1993)
Biochemis y
32, 14125-14131
Genomics
13,881-883
B ea hnach, R.
(1988)
Biochem.
J.
253,
187-192
Chambon,
P.,
and Basse , P.
(1993)
J.
Bwl.
Chem.
268, 15435-15441
SOC. Dans.
19, 715-718
Qian, F., Bajkowski,
A.
S.,
S eine , D. F., Chan,
S.
J., and F ank a e ,
A.
(1989)
Qian, F., F ank a e , A,, S eine , D. F., Bajkowski,
A.
S.,
and Chan,
S.
J. (1991)
Quan in, B., Mu phy, G., and B ea hnach, R.
(1989)
Biochemis y
28, 5327-5334
Quinn, C.
O.,
Sco , D.
K.,
B incke ho , C. E., Ma isian, L. M., Je ey,
J.
J.,
and
Rosenbe g, A. H., Lade, B. N., Chui,
D.,
Lin,
S.,
Dunn,
J. J.,
and S udie , F. W.
Shchez, L. M., F eije,
J.
P., Me ino,
A.
M.,
VIZOSO,
F., Fol mann, B., and Mpez-
Sbnchez, L. M.,
VIZOSO,
F., Diez-I za,
I.,
and Mpez-O in, C.
(1992b)
Cance Res.
52,
Sbnchez, L. M., Fe ando,
A.
A,,
Diez-I za,
I.,
Vizoso, F., Ruibal,A., and Mpez-0 h.
Sbnchez-Mpez, R., Nicholson, R., Gesnel, MA., Ma isian, L.
M.,
and B ea hnach,
Sbnchez-Mpez,
R.,
Alexande , C. M., Beh end sen,
O.,
B ea hnach, R., and We b,
Sange , F., Nicklen,
S.,
and Coulson,
A.
R.
(1977)
P oc. Na l. Acad. Sei.
U.
S.
A.
74,
14255
Cance Res.
49, 4870-4875
An icance Res.
11, 1445-1452
Pa idge, N. C.
(1990)
J.
Biol.
Chem.
265, 22342-22347
(1987)
Gene (Ams .)
66,
125-135
O in, C.
(1992a)
J.
Biol. Chem.
267,24725-24731
95-100
C.
(1993)
B .
J.
Cance
67, 10761081
R.
(1988)
J.
Biol. Chem.
263,11892-11899
Z.
(1993)
J.
Biol. Chem.
268,7238-7247
Sco , D.
K.,
B akenho ,
K.
D., Clohisy,
J.
C., Quin, C.
O.,
and Pa idge, N.
C.
Senio , R. M., G i in,
G.
L., Flisza , C. J., Shapi o,
S.
D., Goldbe g, G.
I.,
and
Shapi o,
S.
D., G i in, G. L., Gilbe , D.
J.,
Jenkins, N. A,, Copeland, N. G., Welgus,
5463-5467
(1992)
Mol. Endoc inol.
6, 2153-2159
Welgus, H. G.
(1991)
J.
Biol. Chem.
266, 7870-7875
Human Collagenase-3
16773
Shapi o,
S.
D., Kobayashi,
D.
K.,
and
Ley,
T.
J.
(1993)
J.
Biol. Chem.
268,23824-
Sloane, B. F.
(1990)
Semin.
Cance
Biol.
1,137-152
Sp ingman, E. B., Angle on, E. L., Bi kedal-Hansen, H., and Van Wa ,
H.
E.
(1990)
P oc.
Na l. Acad. Sci.
U.
S.
A.
87, 364-368
Toli ia,
J.,
and lbli ia,
D. (1985)
J.
Neu osci. Me hods
13,
30L311
U ia,
J.
A,,
Fe ando,
A. A.,
Velasco,
G.,
F eije,
J.
P.,
and L6pez-O in, C.
(1994)
Vai ukai is,
J.
L.
(1981)
Me hods Enzymol.
73,
46-52
Van Wa , H.
E.,
and Bi kedal-Hansen, H.
(1990)
P oc.
Na l. Acad. Sci.
U.
S.
A.
87,
H. G., Senio , R. M., and Ley,
T.
J.
(1992)
J.
Biol. Chem.
267,4664-4671
23829
Cance
Res.
64,2091-2094
557g5582
Welgus, H. G., Je ey,
J.
J.,
and Eisen,
A.
Z.
(1981)
J.
Biol. Chem.
266,9511-9515
Wilhelm,
S.
M., Collie ,
I.
E., K onbe ge ,
A,,
Eisen,
A.
Z.,
Manne , B. L., G an , G.
A,,
Baue , E.
A,,
and Goldbe g, G.
I.
(1987)
P oc. Na Z. Acad. Sci.
U.
S.
A.
84,
6725-6729
Wilhelm,
S.
M., Collie ,
I.
E., Manne , B. L., Eisen,
A.
Z.,
G an , G.
A.,
and
Whi ham,
S.
E., Mu phy, G., Angel,
P.,
Rahmsdo , H.
J.,
Smi h,
B.
J.,
Lyons,
A.,
Goldbe g, G.
I.
(1989)
J.
Bid. Chem.
264, 17213-17221
Hams,
T.
J.
R.,
Reynolds,
J.
J.,
He lich,
P.,
and Doche y,
A.
J.
P.
(1986)
Biochem.
J.
240,913-916
Woessne ,
J.
F.
(1991)
FMEB
J.
5,2145-2154
