The plasminogen activator family from the salivary gland of the vampire bat Desmodus rotundas: cloning and expression

Gene, 105 (1991) 229-237 0 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/91/$03.50

229

GENE 05094

The plasminogen activator family from the salivary gland of the vampire bat Dew&us and expression (Recombin~t

rotundus: cloning

DNA; cDNA cloning; nucleotide sequence; ~brinolysis; serine protease; transient expression)

J&n KrPtzschmar”, Bernard Haendler”, Gernot Langer”, Peter Donnera and Wolf-Dieter Schleuning”

Werner Boidola,

Peter Bringmann”,

Alejandro Alagonb,

a ~se~~c~ Laborafories o~Sc~e~ng~G Be&n, ~000 Berlin 4.5 {F,R. G.); and’ Centro de Inve~tigaci~~sobre Ingenieria Genkticay Biotecnolo~~a, Universidad National Authoma de Mt!xico, Cuernavaca, MoreIos 62270 (Xf&xico) Tel. (52-73) 172399 Received by F. Bolivar: 19 February 1991 Revised/Accepted: 29 March/7 April 1991 Received at publishers: 12 June 1991

SUMMARY

Compfementary DNAs coding for four Desmodus ro~~~~u~salivary plasminogen activators (DSPAs) were isolated and characterized. The predicted amino acid sequences display structural features also found in tissue-type plasminogen activator. The largest forms (DSPAal and -1x2)contain a signal peptide, a linger (F), an epidermal growth factor (EGF), a kringle, and a serine protease domain, whereas DSPAP and - y lack the F and F-EGF domains, respectively. Additional differences between the four forms suggest that distinct genes code for the members of the DSPA family. Transfection of DSPA-encoding cDNAs, placed under the control of the simian virus 40 late promoter, into COS-1 cells resulted in the secretion of highly fibrin-dependent PAS.

Plasminogen activators (PAS) are involved in various physiological processes such as fibrinolysis, inflammation and tissue remodelling. The tissue type serine protease t-PA catalyses the conversion of plasminogen into plasmin, a t~psin-lye enzyme which digests fibrin, the solid meshwork of coagulated blood. The proteolytic activity of t-PA is enhanced in the presence of fibrin. Urokinase (u-PA), is Correspondence to: Dr. W.-D. Schieuning, Institute of Cellular and Molecular Biology, Schering AG, Postfach 6503 11, D-1000 Berlin 65 (F.R.G.) Tel. (49-30)468 1390; Fax (49-30)463 16707.

Abbreviations: aa, amino acid(s); bp, base pair(s); D., Desmodus;DSPA, D. rorundus salivary PA; DSPA, gene (DNA) encoding DSPA; EGF, epidermal growth factor; F, finger; K, kringle; kb, kilobase or 1000 bp; nt, nucleotide(s); oligo, olig~eoxy~bonucleotide; PA, plasminogen activator; re, recombinant; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/0.015 M Na, . citrate pH 7.6; SV40, simian virus 40; t-PA, tissuetype PA; t-PA, gene (DNA) encoding t-PA; rsp, transcription start point(s); u-PA, urokinase; u-PA, gene (DNA) encoding u-PA.

mainly implicated in extravascular plasminogen activation (for reviews, see Blasi et al., 1986; Saksela and Rifkin, 1988). In recent years, PAS have been used in the treatment of thromboembolic diseases but side effects such as systemic fibrinogen breakdown and bleeding were observed (Cohen et al,, 1988; Haber et al., 1989). Intensive efforts to obtain safer PA variants with increased clot specificity have so far not led to a substantially improved thrombolytic agent (Harris, 1987; Higgins and Bennett, 1990). PA activity has previously been described in the saliva of the vampire bat D. ro~~d~s (Hawkey, 1966; Cartwright, 1974) where it is probably essential to support the feeding habits of this exclusively haematophagous animal. A characterization of this activity and the sequence of a single cDNA clone have recently been described (Garde11 et al., 1989). We now report the cloning and expression in COS-1 cells of cDNAs corresponding to four distinct, fibrin-dependent vampire bat PAS.

230 RESULTS AND DISCUSSION

(a) Isolation and characterization of DSPA cDNA clones Candidate clones were isolated after screening the cDNA library using human t-PA cDNA as a probe. Since none of them was full-length, a second cDNA library was constructed and screened with a probe derived from the 5’ moiety of the longest incomplete cDNA. Hyb~dizing clones were partially sequenced and found to correspond to four distinct forms (al, a2, ,B and y). The nt sequence of the longest insert for each type was determined (Fig. 1). The cDNA sequences of the two largest forms (a1 and a2) are closely related (80 differences for a total of 2245 nt). DSPAd cDNh exhibits six nt differences with a sequence published previously (Garde11 et al., 1989). DSPAB cDNA is shortened by an internal 13%ntlong deletion but displays otherwise only one nt difference when compared to DSPAd cDNA. DSPAy cDNA is even shorter (249-nt-Iong deletion) and differs from DSPAal and -PcDNAs in 54 and 23 positions, respectively (Fig. 1). Among the 32 DSPA cDNA clones analysed by restriction enzyme mapping and partial sequencing, eight belonged to the al, three to the a2, 19 to the /? and two to the y form. (b) Comparison with humab t-PA cDNA and gene sequences The regions missing in the j3 and y forms of DSPA cDNAs precisely span exons IV (F domain) and V (EGF domain) in the human t-PA gene (Degen et al., 1986; Fig. 1). Likewise, the region encoding the K2 domain in the human t-PA cDNA, which is altogether absent from DSPA cDNAs, corresponds exactly to t-PA exons VIII and IX. A case of domain deletion due to the absence of exon IV has also been reported for a t-PA cDNA isolated from Detroit 562 cells (Kagitani et al., 1985). These observations strongly suggest that the exon/intron organisation of DSPA coding sequences is analogous to that of the human t-PA gene. Considering the numerous nt differences found between the four sequences it can be inferred that a minimum of 25 discrete exon variants must exist in the D. mundw genome: one for the signal peptide exons (t-PA exons II and III), two for the 5’-untranslated region (I), the F (IV) and EGF (V) domains as well as for most of the protease domain exons (X, XII, XIII, and XIV) and three for the K domain exons (VI and VII) and exon XI of the protease domain (Fig. 1). As only those assemblies of exons corresponding to the four DSPA cDNAs described here were found, it is likely that the corresponding genes exist as distinct entities. A second form of DSPAal cDNA (DSPAal*) harbouring a 3 l-bp insertion in the 5’-untranslated region was also

isolated (Fig. 1; nt 191-221). This insertion is precisely located between the sequences corresponding to exons I and II of the human t-PA gene (Degen et al., 1986). Interestingly, it is closely related to a DNA stretch present in the first t-PA intron that is flanked by sequences reminiscent of splice sites (Fig. 2). An AP-2 recognition sequence which has been identified as a regulatory element of the human t-PA gene (~edc~f et al., 1990) was found to be conserved in the DSPAg cDNA clone, which has the longest 5’-untranslated region of all sequenced clones (Fig. 1; nt 60-74). (c) Deduced aa sequences The deduced aa sequence of DSPAa 1 is in full agreement with sequencing data obtained for the N-terminal 15 aa of mature high-M= DSPA isolated from D. rotundus (P-D., unpublished results; Duong et al., 1990). DSPAa2, which displays three aa substitutions in this region, is probably less represented since sequencing data failed to identify the corresponding N terminus. DSPAal and -a2 are related but distinct proteins: both are 477 aa long, but differ in fifty aa (89% identity; Figs. 3 and 4). DSPAP and -y are smaller proteins composed of 43 1 and 394 aa, respectively. As expected of secretory proteins, a signal peptide (36 aa) is present in all four forms of DSPA (Fig. 3). The close relationship between DSPAP and -a2 (only two substitutions in the conserved aa sequences) was established by complete sequencing of two independent BcDNA clones. DSPAy, in contrast, seems more distantly related since 13 and 32 differences in the aa sequence are found between this form and DSPAa2 and -al. The variations observed between the conserved domains of DSPAs imply that the smaller p and yforms are not mere deletion derivatives of DSPAa2, as stated by Garde11 et al. (1989) and Duong et al. (1990). The protein corresponding to the sole cDNA sequence previously reported (Garde11 et al., 1989) exhibits three discrepancies, all located in the protease domain, in comparison to DSPAa2: Asn367 -+ Lys, Tyr381 -+ His and Met399 --+Arg (Fig. 3). These aa are conserved among DSPAal, -a2, -p and -y. Comparison of the aa sequences of DSPAal and -a2 with that oft-PA (Pennica et al., 1983; Edlund et al., 1983) reveals that the F, EGF, K and serine protease domains are conserved (Figs. 3 and 4). The single DSPA K motif resembles t-PA Kl more than K2. DSPAj.l displays the same structural features except for the noticeable absence of the F domain. DSPAy lacks the F as well as the EGF domain. The plasmin-sensitive site of t-PA (between aa 189 and 190 of the alignment in Fig. 3) is conspicuously absent in all DSPA forms, making them the only natural PAS active exclusively as single-chain molecules. The previously described activity of t-PA in its uncleaved

c-c

tg

c

IV

tg___- a c

t

ag_a_a

ta

t a_c-g--c exon IV >< exon v * *

t -C-C_

Ca_CCg t

C ______ tt

tc

g

c

*

a

ca

g_c_a_ca_g

a

ca

***

*

g

tg g-c-c

g-c-c

g_g_g_c_ccyc_c

tg -__ at

a

gcgt_g_a_a_c

t -gcL---g-c

g-t-a-

*+

_a-.-gg

g-g-g

*

ca

a-t t

a_t_g_c-c--

Beta

GWlW3

t-PA

*, -al,

__c_ t

a

(Fig. I continued on page 232)

were washed

under low-stringency

exon VII 2-c excln VIII

kit; Pharmacia),

following the protocol were transferred

y0 SDS/l

x Denhardt’s/lOO

onto nylon membranes

salmon

an

of screening.

As no full-length

sperm DNA per ml (Maniatis

clone

et al.,

of 5-methyl dCTP and size selection and screened with a

vector

glands was used first to construct

TM, Du Pont/NEN) after two rounds

pg denatured

Plus

in the presence (GeneScreen

(4 x SSC/O.l y0 SDS; 42°C) and eleven positive clones were detected

was for 14 h at 42°C in 6 x SK/l

library

D. rotundus salivary

(1983). A second library was made in the Uni-ZAPTM

from Mexican and Hoffman

virus reverse transcriptase

of Gubler

1000

. . .. .. . .. . .. . .. . . .. . .. . .. .. . . .. . . .. . . . .. . . .. . . .. . .. . .. . .. . .. . .. . . .. .. . .. . ... . .. .. . .. .. . .. .. . .. . . .. .. . .. . .. .. . .. .. . . . .. . . ..

. . .. .. . .. . .. . .. . . .._...................................................................................................... gaaacagtgactgctactilgggaatgggtcagcctaccgtggcacgcacagcctcaccgagtcgggtgcctcctgcctcccgtggaattccatgatcctgataggcaaggtttacacagca

clones of the first cDNA conditions

g

g_aaca

g-cexon VI >c exon VII 800

t

*

tgt

. .. . .. . .. . .. . .. . . . .. .. . .. . .. . . .. . . .. . . . .. . . . .. .. . .. . .. . .. . . .. . .. . .. . .. . .. .. .. .. . .. .. . .. .. . .. . .. . .. . .. . .. . .. .. .. . .. . . . . .. ..

of the first strand with the Moloney murine leukaemia

synthesis

(Fisher et al., 1985). Hybridization

50000 primary

XC _C-tg-

g-c-c

Ca

gt600 ****

a aa

400

aa

t--a_

. . .. .. . .. . .. . . .. . . .. .. . . .. .. . . .. . . . .. . . .. . . . .. . .. . .. . .. . .. . .. . . .. . .. .. . .. . .. .. .. .. .. . .. . .. .. . .. . . .. .. . . .. . .. .. .. . . .. . . . . ..

-aZ,-a -y and human r-PA cDNAs. Poly(A) + RNA prepared

La Jolla, CA) after synthesis

than 500 bp. About

human r-PA cDNA

greater

1982). The membranes

nick-translated

for cDNAs

kit; Stratagene,

a

t

(cDNA synthesis

of DSPAal

tt _-t

tt

cDNA library in the Igt 10 vector (cDNA

of the nt sequences

tt _g-a_g_a

a

a-c-

ac --

oligo(dT)-primed

Fig. 1. Comparison

at

CCCCAAAACCTTGGTGCTATGTCATCMGGCAGGCAGTTCGG..........................................................................................................................

Alpha-2

Alpha-l

Alpha-l*

l

ag

a

exon v >< exon VI

a

t-PA

C

. .. .. . .. ..._c_a

Gamna

Beta

Alpha-2

Alpha-l

*

GTGTGAAGTAGATACCCGTGCCACCTGCTATGAGGGCCAGGGTGTCACCTACAGGGGCACATGGAGCACAGCA~GTAGGGTTGAGTGTATC~CTGG~CAGCAGCCTTCTGACCCGGAGGACCTAC~TGGGCGGATGCCAGATGCCTTC~CCTGGGCCTTGG~TCAC~TTACTGCAG~CCC~TG~G

tt

Alpha-l'

_~~t_t_c-----c___

_a_tc

t-PA

Beta

.. .. . ... .. . ... .. .. .. . . .. .._.............................................................................................................................................................................

c

tg

won

Gam

xxx

t a att a t a-a a .. .. . .. . .. .. .. .. . .. . .. . .. . .. . .. .. . .. . .. .. .. . .. .. .. . . ... . .. .. . .. .. . .. .. . ... . .. . .. .. . .. . .. . .. . . . .. . .. .

start

Alpha-2

Alpha-l

Alpha-l*

c exon II

t c tgcc_g_cag_a_g~t_t_ccaa_at~g_t__a_g~ --11 >c excm III . exon III >< em”

. . .. .. . .. . . .. . .. . .. . .. .. . .. . .. . . . .. . . . gtt_c_c

.. .. . .. .. . .. .. .. .. .. ._~ a

t-PA g_gg_c_gc-g-g

..

... .. ... . . ... . .. .. .. .

Gamm

g_tgt_a_c_ca_g_g_

t 4-a--a_ . . .. . .. . .. . .. . .. . .. . .. .. . .. . .. . . . .. . . .

.. ..

. .. .. .. .. .. .. . .. .. .. .

200

Beta

I >

..

exon

. .. .. ... .. . .. . .. .. .. .

.

.. . .. . .. .. . .. .. . .. .. .

I[

Alpha-2

TTCTGTTCAAGAAGAGAGGTTTTAAGGGACACC..GCAGAAATGGTGAATACAATGAAGACAAAGCTGTTGTGTGTACTGCTGCTTTGTGGAGCAGTCTTCTCGTTGCCCAGGCAGGAAACCTACAGGCAATTGGCATGACATACCGG

I[

. .-CC_.

. . .. .. . . .. . . .. . . . . .. .. . . .. . . . . ..

. . . . .. . .. .

. . . . .. .. . .

Alpha-l

Alpha-l*

start I##

tc a g

w_a_a_g_g_aa_gp

atggccctgtccactgagcatcctcccgccacacagaaacccgcccagccggggccaccgaccccaccccctgcctggaaacttaaaggaggccggagctgt..ggggagctcagagctg_gat _______________ 1

t-PA

---

.-..

..

.. . .. . .. .. .. . . .. ... . .. .. . ..gctacagagaagcccgcccactgtgggccactgaccccaccccctgctttgaaatacaggggaggccgaggctgtgcggagagattggcgctg

GamM g

.-..

..

.. . .. .. . .. . .. .. . ... . .. .. . .. . .. . .. .. . .. ... . .. . .. .. . . .. .. .. .. .. .. . .. .. . .. .. . .. .. . .. .. . . .. . ..ggccaaggctgtgcggagagattggcgctg

Beta a

.-_.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

.-..

.. ..

.. .. . . .. .. .. . .. .. .. . .. . . . .. . .. .. . .. . .. .. .. .. .. . .. . .. . .. .. .. .. .. . .. . .. .. .. . .. .. . .. .. . . .. . .. . .. . .. . .. . .. .. . .. . . . .. .ggcgctg

Alpha-2

.. .. . . .. .. . .. .. .. .. . .. .. . .. . .. . .. .. . .. .. .. .. . .. .. . .. . .. .. .. .. . .. .. .. . .. .. . .. .. . .. .. . . .. . .. . .. . .. . .. . .. .. . .. . . . .. . . .. . . . .AAGCCCTGCAAGAG..CTGAGCTGACGGGAAATCCTCT.CCAGG..AGAGAAGG~GGCAAGGAGTGGCGGTATAAACAG

Alpha-l

Alpha-l"

CCACCTGTGGCCTGAGAAAGTACAAGGAGCCACAGCTTCACAGTACAG~G~CTCTTCACA~~ ..............................................I......................................................................................... ....................................................._.......................................................................~.......... ~.....................~..~....~......~~.....~....~.~.....,.~..............~~.....~........~..~..................~..~..........f......... ............~~...............~..~...~..~......~.......~.~...........~...~.........................~.............~.........~............~ ................*.................................................................................................*..................... gcc_._t___t__g_tc_a_g_ g-c: cagaaccccagtgcccaggcactgggcctgggcaaacataattactgccggaatcctgatggggatgccaagccctggtgccacgtgctgaagaaccgcaggctgacgtgggagtactgtgatgtgccctcCtgct C C 1200 ex0n IX >< em" x . exm VIII >< exm IX *

g-g-g _~g-a---g-g-g-g -a-g___-g-

t

-.----gg-a..-.--9 c-t-g g exm XI >< emn XII

--

g-g___ g-g--g-gt_gt_c_gc 4

cag_gtg ~_

a tg_g_c_t_cccg_gg

g_g_g--

c c 1600

AGACATTTAAAGTCAAAAAATACATCGTCCATMGGAATTTGATGACGACACTTAC~CMTGACATTGCACTGCTGCAGCTGAAATCGGACTCACCACAGTGTGCCCAAGAGAGTGACAGTGTCCGCGCCATCTGTC~CCCGG~GCC~CCTGCAGCTGCCC~CTG~CAG~TGTGAGCTGTCTGGCTACGGC

g----g~

!Lct_-_-_a_e

3-t-g

a--t ac am _t_ac_g-_a__._cg

a-t-g

t--___.__g_cc_c_gg_ct~c~

c_t_g~c_ em" XIII >< exon XIV

1800

(Fig. t conr~~uedotzpage 233 1

AluI-BanzHl fragment (nt 269-344) derived from the 5’ end of the longest positive clone was used to screen 120000 independent clones plated from the second unamplified library. Hybridization was carried out for 16 h at 50°C in 6 x SSC/l y0 SDS/l x Denhardt’s/lOO pg denatured salmon sperm DNA per ml, and washing for 1.5min at the same temperature in 6 x SSC/l% SDS, then 4 x SSC/O.l y0 SDS, and eventuaJly 2 x SSC. Out of 200 positive clones which were thus found, 35 were subjected to in vivo excision after superinfection with the R408 helper phage, following the supplier’s instructions. Sequencing was performed using the dideoxy chain-termination method (Sanger et al., 1977) after subcloning into the Ml3 phage (Messing, 1983) or directly on double-stranded plasmid with LISPA-specific oligo primers. Sequence analysis was done using the University of Wisconsin GCG package (Devereux et al., 1984). The complete sequence of DSPAal * cDNA is shown in capital letters. Only the nt that differ are given, in lower-case letters, for the other cDNAs. Gaps are indicated by dots. The

was obtained,a nick-translated 76-bp

#X# stop (9) Alpha-l* GTGTATGAATGACAACCACATGACTTTGCTTGGCATCATCAGTTGGGGTGTTGGCTGTGGG~G~GACGTTCCAGGTGTATACACC~GGTTACT~TTACCTAGGCTGGATTCGAGAC~CATGCACCTGT~CC~G~CACAC~CTCCCTGGCAGCCCCTG.......CCTTCCTCCAGCCCAGAAGAAAC.. Alpha-l . . .. .. . . ‘_ Alpha-2 _ a ..*.... c g mg_Ce . ‘_ a Beta . .. . .. . c g__ca 4 -_-. *I_ Gam a . .. . .. . c g_ca-_--g~_ .._ t~ t-PA c_g_tcaaa_aaa_agatcccg_c_t_ttctt c tgc _c_c_t!i~ ~c_%._t_~_g_g w_c_g_~_ -~9 K.g c_.._-cc-g_ C--Cpa -RX# stop 2000

-g-gx_tg_ exon XII >( exm XIII

-9

Gail@a

t-PA

-P

Beta

(cl (41 (fJ (a) Alpha-l* CATAAGTCATCTTCTCCTTTCTATTCTGAGCAGCTGAAGG~GGGCATGTCAGGCTGTACCCCTCCAGCCGCTGCGCACCC~G~TTC~GTTT~C~CCGTCAC~C~CATGCTGTGTGCTG~GACACGCGGAGCG~GAGATCTATCC~TGTGCAC~TGCCTGCCAGGGTGACTCAG~GGCCCCTTGGT Alpha-l Alpha-2 g --a-t-g_-_

Alpha-l* Alpha-l Alpha-f Beta Gamna t-PA

Alpha-l* CATCACCTCTCATCCATGGCAGGCTGCCATCTTTGCCCA~CAG~GGTCATCAG~~GGTTCTTGTGTGGGGG~TATTGATCAGTTCCTGCTGGGTCCTGACTGCTGCCCACTGCTTCCAGGA~GCTATCTTCCT~CCAGCTT~GGTGGTTTTGGGCA~CATACCGGGTG~CCTG~~G~~GC Alpha-l Alpha-Z a-_.---g c cc_g_t_g __a Beta g_c___cc_g_t_g a Gam a g_c__cc_g_t_g -a_t_ct_c @c_~_%..__ t-PA a--gc g_t_Cg_~C_c_g_~-~_c g~_~_g~~~_~-.~_~_~_~~ __-g-~_c-c 1400 e7.m X zc exm XI x

Alpha-l* Alpha-l Alpha-Z Beta Gama t-PA

11,

..

ctt ______

c

g

. cat -_

ca

t

t-PA

stop codons

(# ), as well as the polyadenylation

ac

nt. The nt sequence

M63990 ( y).

are aligned with the corresponding

with an asterisk.

The six discrepancies

are marked

by brackets.

-j and -y cDNAs

vector are indicated

t

The restriction data reported

found between

. .

. . .. . .

regulatory

cDNA

ctt.......

the GenBank

tgg-

accession

..

aa

a-

2200

2400

. .a_a

tga_gc_ca_t_g_t_ag_t

g_ca_at_t

are between

Nos. M63986 (al *), M63987 (al), M63988 (LYE),M63989 (/I), and

Last digits of numerals

into the expression

The nt differences

(converging

t

arrowheads)

. .. . . . .. . . . ... .

. .. . .

g_catgag

as well as the EcoRI site used for subcloning

et al., 1990) is underlined.

by Garde11 et al. (1989) are shown in parentheses.

used for screening

t

at the tsp and their boundaries

agtgtgtaa_g_c_

.. .. .. . . . . . *... . . .. . .. . . . .. . . .. ..

in the first t-PA exon (Medcalf published

fragment and the nt sequence

a...

2600

. .. . .

AGTATAAATCTTTTTCCTTTATAAACTTTATAGTAG.....AAGAACTGTATCATTCTGAT

g_a_a_tL._g_c_g_tg

beginning

2124

c_gtttttcc

r-PA exon sequences

.

_ at ~

sequence

in this paper have been assigned

DSPAd

sites flanking the AhI-BumHI

The AP-2-like

The complete

gt_....t

signal (colon), are marked.

_____~

Degen et al. (1986). The insert found in DSPAaZ * cDNA is overlined.

DSPAd,

from

start and

(Fig. I, conclusion)

gt_gtttttactttct

ctt.......

Garmm

a

ctt.......

a

Beta

ctt.......

a

. .. . .. . ... .. . .. . .. . .. .. .. . .. . .. .. . .. ...

atg

. .. .. .. .. .. .

cc_ctccctg

Alpha-2

c

c

I[

aa

. .. . . .. .. .. ..

. .._aac

g_c_c_ag_ct_ct

c

c c c c

c

gc

GTTGCCACGACTCTGTATTATACTACACTGGAAAAATAAATTCAGGCATATTTTTCA..........

a aag_c_

. .

c

..

Alpha-l

AACTGCTAAACTAGGCTTTAGCATTTTGATATCAATCCATTGTA.............

c

Alpha-l*

g

_g_.........ct_a_gtt_ggca

t-PA

Gamaa

Beta

Alpha-2

a

. .. . . . . . . . . .._ t . . . . . . ._ t .. t

a

CAGCGACCTGGGCCTCCTAACAAGGAATGGGCTGTCTGGCCAGATTGCGTTCCTC.AGGCCATCCTTTAAGT.ATTTGACTATCCTCTTTCTACA.......GCTCTTGGAAGGAATTCCTTTTGTGTAC.........

ca

Alpha-l*

Alpha-l

cg

_aagtggccatgcc_cctgtt_tc_gct

g_t_tc_ct

t-PA g-c --

g

.. . . .. . . .. .. .

GamM

..

Beta

..

9

. .. .. .. . .. . ..

Alpha-Z g-

...............

.............

Alpha-1

ACCACCATCTAGGGCAG.............ACAAGAGGTAGAATAAAAGCCCAACCTCCTAATCTGTCAAGTATCTTTC..TAGTAAAAAATAGCTAGATCCTTCAGTAAAGAAGTATCACATTA~T~TGTCCATGTATAGTCAC~G~CGCCC~

t_tgg

Alpha-l"

..a_a_gc_t_t_c_a-

a-gcg-

t-PA

.g_c_ac-c_c_ga_c_ac_c_c_c-

a-9

GamM

tct_..gac_

..

a-g-

Beta

..

.. a-g-

Alpha-2

CAAGGCAGAATGCTTCCTGACACCAGTTTCTCCACAAGCCTGCACACAGTACTAGTGGGGGGAG~CGTATAGGA~GGGGAGAGGGTATACTTCCACAGGTACTTCCCACTTCAT~GTTTTCAGGGGAT~GGTCT~TTT.AGAATCCATTTCTGTCAGACAAWV\GACA..W\TAAATGCCAACCCCTCCCAGAAC

Alpha-l

Alpha-l*

234 221

191 :~TATAAACAGTTCTGTTCAAGAAGAGAG~:

DSPAal

: IIIIIIIIIIIIIlII

8406

III

III1

t_PA

~TCTCTTCTC~C~AG:TTATAAACAGTT~TGTT.AAG.AGAGGG~:AT~~~~

splice

TTTTTTTTTTT

ccccccccccc

consensus

C

C : N AG:G...........................

junction

T

A

:

intron :

Fig. 2. Comparison (nt 191-221

of

DSPAal

in Fig. 1) with a stretch

(nt 8406-8456;

Degen

et al.,

sequences,

with N standing

exon-intron

boundaries

cDNA

of the human

1986).

The

and corresponds to the Asn448 glycosylation site of t-PA (Pohl et al., 1987). A further potential glycosylation site, corresponding to the Asn ‘17 oligomannose site of t-PA, is

sequence

f-PA gene intron

consensus

splice

for any nt, are from Mount

are marked

AGT G

intron

:

insertion

(Fig. 3, aa 391), are conserved. A common potential N-glycosylation site is present in the protease domain of all four forms of DSPA (Fig. 3, Asn362)

A

: AG:GT :

exon

the

Lys4’6 of t-PA (Fig. 3), as well as the Asp477 counterpart

8456

I/II:

A

junction

found at aa position

(1982). The

117 in the K region of DSPActl

and

a previously undescribed site, with no equivalent in t-PA, at aa position 149 in DSPAa2 and -/I The DSPAy K region lacks any N-glycosylation signal. Comparison of DSPAcrl and -a2 with human (Pennica et al., 1983; Edlund et al., 1983), mouse (Rickles et al.,

(colons).

single-chain form seems to depend on the formation of a salt bridge involving Lys416 (but not LYS~~~) and Asp477 (adjacent to the active site Ser478 residue) (Petersen et al.,

1988) and rat (Ny et al., 1988) t-PAS reveals similar levels of aa identity in the shared domains: between 72.0 and

1990). The DSPA aa sequence data are compatible with these findings, as only the LYS~~’ residue corresponding to

72.5% with DSPAcrl, and 74.4x, respectively, with DSPAa2.

73.2%,

and 72.5x,

Alpha-l Alpha-2 Beta GamM Human Rat Mouse

-36 1 . 106 MVNTMKTKLLCVLLLCGAVFSLPRQETYRQL~GSRAYG~A~KDEI~QM~YRRQESWLRPEVRSKRVEH~Q~DRGQAR~H~VPVNS~SEPR~FNGG~~WQA~YFSDFV~Q~PAGY~GKR~EVD~RA~~YEGQGV~YRG~W r-- lq -k 1 asp-q-h_kdk r_k_i qq ...........T..................................g_l as k q__h_kd ..................................................................................._ph_kd .mda_rg_~ c vs_s_iharfr_a_s_q_i_r_k__i_qqhq~-- vl n y_~_ns_r_q_s_k efa c i d is_ q-' ~_ ...._ge di-f---i va_t_d_gih_rfr_a_s_rat_r_q_t_qqhq~ml_gn_y_r_ns_l_q_s_r q-l-.------ d fv---. . . ._re d fv di fe i la_p_d_gihgrfr_a_s_rat_r P t qqhq ml s Y r_ns_lvq_s_r q-1 ____-

Alpha-l Alpha-2 Beta Gamma HUmall Rat Mouse

+ 107 + . 175 ~ESRVECINWNSSLLTRRTYNGRMPDAFNLGLGNHNYCRNPNGAPKPW~YVIKAGKFTSESCSVPVCS~...................................................................... rs it dnns s ilf ......................................................................... s_gaq-n rs it dnns s il f ................................................................_........ s_gaq-n ~n i~e vk-~ds r ......................................................................... s_gaq drds f ys_f_t_a_egns.dcyfgngsayrgthsltesgasclpwnsmiligkvytaqnpsaqalglgkhnycrnpdgdakpwchvlk _ga_t-a_aqkp_s_r__+ a sqkp_sa_r_n_ik drdv-f y_t_f_t_a_p_gptedcyvgkgvtyrgthsfttskasclpwnsmiligktytawransqalglgrhnycrnpdgdakpwchv~ _nga -v slkp_a_r_n_ik drdl~f y_t_f_t_a_p_gksedcyvgkgvtyrgthslttsqasclpwnsivl~ksytawrtnsqalglarhnycrnpdgdarpwchv~ -ga --

>< 176 I. . # 303 Alpha-l ..............~TCGLRKYKEPQLHSTGGLFTDITSHPWQ~IFAQNRRSSGERFLCGGILISSCWVLT~HCFQESYLPDQLKVVLGRTYRVKPGEEEQTFKVKKYIVHKEFDDDTYNNDIALLQLKSDSPQCAQES~ Alpha-2 .............. k-e-e r_p_qh_r g c e .............. Beta k-e-e-c-i-e r_p_qh_r g .............. GamM k-__-eeec r_p_qh_r g HUllIan nrrltweycdvpscs- q_sq_frik_a_a-..____ is d sr_s kh_p _ -rfp_hh_t_i-v ~-- kee Rat drkltweycdmspcs q q_frik v k eie d-r--s-s vk_k_p_v _s-v_rfp_hh

Mouse

drkltweycdmspcs

Alpha-l Alpha-2 Beta GamM Human Rat Mouse

304 :# (r) 441 .+ (k) (h) S~RAICLPEANLQLPD~~ECELSGYGKHKSSSPFYSEQLK~GH~RLYPSSR~APKFLFNK~V~NNML~AGD~RSGEIYPN~HDA~QGDSGGPLV~MNDNHM~LLGIISWGVGCGEKDVPGVY~KV~NYLGWIRDNMH~ ts i rp ts i rp ts i v eal ra tsqh 1 r d d-rp v_tv__p_d gpqa_l l_gr_v_ 1-q a tsqh-_is ~_ t gnq.d i_~ kr _gta_dpdv ea- fdr _ 'q n q kq - -1 ea_f_dr_atsqh inkq--_t lq --d--h kq _#a_@ _gnq.dl

Fig. 3. Comparison

q

r_frik_y

of the aa sequences

letters. Only the differences

vk_k_p_v

of DSPArl,

-12, -a -y with human,

found in other PAS are shown, in lower-case

with dots. The active site residues (#) and potential site in t-PA are also shown (converging salt bridge. The three discrepancies

N-glycosylation

arrowheads)

"

~s.____rfp_nh

eie

rat and mouse t-PA. The entire aa sequence

letters. Conserved

regions

sites (plus symbols) are marked.

are indicated The positions

e

d

of DSPAal

with dashes

rqk_s

is given in capital

and the deleted regions

ofthe aa flanking the plasmin cleavage

as well as the LYS~~”and Asp391 residues (colons) possibly involved in the formation with the aa sequence published by Garde11 et al. (1989) are shown in parentheses.

of an alternative

Fig. 4. Schematic representation of DSPAal, -a2, -p and -7. Position 1 corresponds to the N terminus of the mature protein. Lines joining Cys residues were drawn in analogy to putative disulfide bridges in human t-PA. The active site residues are shown with blackened triangles. Short zig-zag lines indicate potential N-linked glycosylation sites. The shape of the missing domains in DSPAfi and -y is indicated by a dotted outline. The aa differences between the different forms are shown in blackened circles. Arrows point to positions mutated in DSPAfior -y but not -a2, and crosses indicate positions conserved in DSPAy

but not in DSPAa2

or -A when compared

to DSPAal.

235

.. .

-. _***_

236 of DSPA in COS-1 cells Supernatants of COS-1 cells transfected with expression plasmids for DSPAal, -a2, -band -y exhibited PA activity. Comparison with the activities of re-u-PA (Fig. 5A) and re-t-PA (not shown) revealed that the ratio of fibrinolytic vs. caseinolytic activity was higher for all re-DSPAs. Fibrinenhanced PA activity has also been reported for derivatives of t-PA depleted of the K2 region (with a domain organisation analogous to that of DSPAa) or of the F and K2 regions (analogous to DSPAB) (Gething et al., 1988; Stern et al., 1989; Stern and Weidle, 1990). Secreted re-DSPAs from the COS-1 supernatants were electrophoresed on an SDS/polyacrylamide gel and subjected to zymographic analysis (Fig. 5B). In the case of re-DSPAal and -a2, PAS of A4,s distinguishable from that of re-t-PA were observed. As expected, re-DSPA/? and -y had lower M,.s than reDSPAcll and -a2 proteins. An additional band probably corresponding to a high-Mr covalent complex between DSPA and a PA inhibitor was also detected.

(d) Expression

A

al

a2

a1

P

a2

P

$ u-PA

Y

C

y

u-PA

C

(e) Conclusions

Four different DSPA cDNAs have been cloned and characterized. The corresponding proteins can be divided into three groups: the large a-forms including the novel, abundant DSPAal and the related but significantly different DSPAa2, the intermediate DSPAB, which is the most represented at the cDNA level, and the smaller DSPAy. The differences between these four forms indicate that they are the products of discrete genes. Recombinant DSPAs secreted by COS-1 cells display a fibrin-dependent PA activity which makes them promising candidates for the development of safer and more effective agents in the treatment of thromboembolic diseases. Fig. 5. Transient secretion

expression

of DSPAal,

or casein (right) plates. ACKNOWLEDGEMENTS

included.

The authors wish to thank Dr. G. Siewert and B. Baldus for helpful discussions, Dr. L. Toschi for the gift of the u-PA cDNA clone and Dr. T. Petri for providing the COS-1 cells. We are indebted to J. Dieckmann for cell culture, to D. Schmidt for oligo synthesis and to A. Toben, C. Gonschorek, I. Kaehler, M. Mann, D. Rt)ben, A. Wegg and A. Olvera for further technical assistance. We acknowledge Dr. H. Dinter, Dr. E. Vakalopoulou and A. Shevack for critical comments and P. Haendler-Stevens for help with the preparation of the manuscript.

macia).

vector

For transfection

Lipofectin COS-1

A control

An EcoRI fragment

pSVL expression

of D.SPA cDNAs.

(Gibco-BRL)

plasmid

supernatant

experiments,

1Opg plasmid

using the fibrin/casein

gel as described

h, the supernatants lysis assay (Fisher

(3 and 7), CHO

re-DSPAb(9)

was carried

to MIX after were

et al.,

(lanes 1 and 8), bat re-t-PA

(4), COS-1

and control COS-1 supernatant

out on a 0.1% SDS/l

(Levin and Lostukoff,

(Gibco-BRL)

of 4 x lo5 cells/well

analysis ofre-DSPAct2

(2), re-DSPAal

(10). The analysis

for 24-48

(Phar-

and 2Opg

at 37°C in DMEM-NUT

tested for their activity

1985). (Panel B) Zymographic

into the

The medium was reolaced

incubation

DSPAa

DNA

were added in 1 ml Opti-MEM

16 h and, after further

re-t-PA (S), re-DSPAy(6),

(well C) is also

the SV40 late promoter

plate and incubated

cell

on fibrin (left)

which carries

cells seeded 24 h earlier at a concentration

in a six-well culture

A) COS-1

of the DSPA cDNA was subcloned

F-12/10”/,.” fetal calf serum (Gibco-BRL).

saliva

(Panel

-ct2, +I’, -y and u-PA was assessed

1% polyacrylamide

1982).

REFERENCES Blasi, F., Riccio, A. and Sebastio, G.: Human plasminogen Genes and protein structures. In: Blasi, F. (Ed.), Human Diseases.

Wiley, London,

1986, pp. 317-414.

activators. Genes and

Cartwright, T.: The plasminogen 43 (1974) 3 17-326. Collen, D., Stump,

activator

of vampire

DC. and Gold, H.K.: Thrombolytic

Rev. Med. 39 (1988) 405-423.

bat saliva. Blood therapy.

Annu.

237 Degen, S.J.F., Rajput, B. and Reich, E.: The human tissue plasminogen activator gene. J. Biol. Chem. 261 (1986) 6972-6985. Devereux, J., Haeberli, P. and Smithies, 0.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Duong, L.T., Jacobs, J.W., Friedman, P.A., Dixon, R.A.F., Garde& SJ., Mark, G.E. and Daugherty, B.L.: Vampire bat salivary plasminogen activators. European Patent Application EP-A 0 352 119(1990). Edlund, T., Ny, T., Ranby, M., Heden, L.-O., Palm, G., Holmgren, E. and Josephson, S.: Isolation of cDNA sequences coding for a part of human tissue plasminogen activator. Proc. Natl. Acad. Sci. USA 80 (1983) 349-352. Fisher, R., Wallet+, E.K., Grossi, G., Thompson, D., Tizard, R. and Schleuning, W.-D.: Isolation and characterization of the human tissue-type plasminogen activator structural gene including its 5’ flanking region. J. Biol. Chem. 260 (1985) 11223-l 1230. Gardell, S.J., Duong, L.T., Diehl, R.E., York, J.D., Hare, T.R., Register, R.B., Jacobs, J.W., Dixon, R.A.F. and Friedman, P.A.: Isolation, characterization and cDNA cloning of a vampire bat salivary plasminogen activator. J. Biol. Chem. 264 (1989) 17947-1’7952. Gething, M.-J., Adler, B., Boose, J.-A., Gerard, R.D., Madison, E.L., McGookey, D., Meidell, R.S., Roman, L.M. and Sambrook, J.: Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain. EMBO J. 7 (1988) 2731-2740, Gubler, LJ. and Hoffman, B.J.: A simple and very efficient method for generating cDNA libraries. Gene 25 (1983) 263-269. Haber, E., Quertermous, T., Matsueda, G.R. and Runge, M.S.: Innovative approaches to plasminogen activator therapy. Science 243 (1989) 51-56. Harris, T.J.R.: Second-generation plasminogen activators. Protein Eng. 1 (1987) 449-458. Hawkey, C.: Plasminogen activator in saliva of the vampire bat ~esmo~us rozmdus. Nature 211 (1966) 434-435. Higgins, D.L. and Bennett, W.F.: Tissue plasminogen activator: the biochemistry and pharmacology of variants produced by mutagenesis, Annu. Rev. Pharmacol. Toxicol. 30 (1990) 91-121. Kagitani, H., Tagawa, M., Hatanaka, K., Ikari, T., Saito, A., Bando, H., Qkada, K. and Matsuo, 0.: Expression in E. coli of finger-domain lacking tissue-type plasm~ogen activator with high llbrin affinity. FEBS Lett, 189 (1985) 145-149. Levin, E.G. and Loskutoff, D.J.: Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators. J. Cell Biol. 94 (1982) 63 l-636.

Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Medcalf, R.L., ROegg, M. and Schleuning, W.-D.: A DNA motif related to the CAMP-responsive element and an exon-located activator protein-2 binding site in the human tissue-type plasminogen activator gene promoter cooperate in basal expression and convey activation by phorbol ester and CAMP. J. Biol. Chem. 265 (1990) 14618-14626. Messing, J.: New Ml3 vectors for cloning. Methods Enzymol. 101 (1983) 20-79. Mount, SM.: A catalogue of splice junction sequences. Nucleic Acids Res. 10 (1982) 459-472. Ny, T., Leonardsson, G. and Hsueh, A.J.W.: Cloning and characterization of a cDNA for rat tissue-type plasminogen activator. DNA 7 (1988) 671-677. Pennica, D., Holmes, W.E., Kohr, W.J., Harkins, R.N., Vehar, G.A., Ward, C.A., Bennett, W.F., Yelverton, E., Seeburg, P.H., Heyneker, H.L., Goeddel, D.V. and Collen, D.: Cloning and expression of human tissue-type plasminogen activator cDNA in E. cofi. Nature 301 (1983) 214-221. Petersen, L.C., Boel, E., Johannessen, M. and Foster, D.: Quenching of the amidolytic activity of one-chain tissue-type plasminogen activator by mutation of lysine-416. Biochemistry 29 (1990) 3451-3457. Pohl, G., Kenne, L., Nilsson, B. and Einarsson, M.: Isolation and characterization of three diierent carbohydrate chains from melanoma tissue pl~minogen activator. Eur. J. Biochem. 170 (1987) 69-75. Rickles, R.J., Darrow, A.L. and Strickland, S.: Molecular cloning of complementary DNA to mouse tissue plasminogen activator mRNA and its expression during F9 teratocarcinoma cell differentiation. J. Biol. Chem. 263 (1988) 1563-1569. Saksela, 0. and R&in, D.B.: Cell-associated plasminogen activation. Annu. Rev. Cell. Biol. 4 (1988) 93-126. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Stern, A. and Weidle, U.H.: Kringle 1 domain of human tissue-type plasminogen activator is a functional module mediating fibrinogenstimulated plasminogenolytic activity. Gene 87 (1990) 305-308. Stern, A., Mattes, R., Buckel, P. and Weidle, U.H.: Functional topology of human tissue-type plasminogen activator: characterization of two deletion derivatives and of a duplication derivative. Gene 79 (1989) 333-344.