valent complex with 1251-urokinase that could be im- munoprecipitated with anti-PAI. The cDNA insert of the longer isolate (XPAI-75.15) consisted of 1909 base.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc
Vol. 262, No. 8, Issue of March 15, pp. 3 7 1 M 7 2 5 1987 Printed in d..S.A.
cDNA Cloning and Expression in Escherichia coli of a Plasminogen Activator Inhibitor from Human Placenta* (Received for publication, September 8, 1986)
Richard D. YeS6, Tze-Chein Wunll, and J. Evan SadlerSII From the $Howard Hughes Medical Institute Laboratories and Departments of Medicine and Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110 and the llMonsanto Company, St. Louis, Missouri 63198
Two nearly full-length cDNAs for placentalplasmin- 65,000)’ and urokinase-type plasminogen activator (u-PA,M , ogen activator inhibitor (PAI) have been isolated from 55,000) (reviewedin Refs. 1 and 2). a human placenta Xgtll cDNA library. One positive Both u-PA and t-PA areregulated by specific plasminogen (XPAI-75.1) expressed a protein thatcould adsorb and activator inhibitors (PAI). These inhibitors appear to function purify anti-PA1 antibodies. The expressed protein in- like other serine protease inhibitors, forming an inactive hibited the activity of human urokinase in a fibrin covalent complex with the target protease. Several kinds of autography assay, and formed a 79-kDa (reduced)co- PA1 have been distinguished. Endothelial cell-type PAI, or valent complex with 1251-urokinasethat could be im- PAI-1,’ is a glycoprotein of M , 54,000 that rapidly inactivates munoprecipitated with anti-PAI. The cDNA insert of both t-PA and u-PA. It is synthesized by endothelial cells (3, the longer isolate(XPAI-75.15)consisted of 1909 base 4) and certain hepatoma (5) and fibrosarcoma cell lines (6). pairs, includinga 5”noncoding regionof 55 base pairs, PAI-1 is found in platelets (4, 7) and it probably consititutes an open reading frameof 1245 base pairs, a stop codon, the major PA1 of plasma (4, 8), except perhaps during prega 3’-noncoding region of 581 base pairs, anda poly(A) nancy. tail. The size of the mRNA was estimated to be 2.0 Placenta-type PAI, or PAI-2, is a distinct protein of M , kilobases by Northern blot analysis. The translated 47,000 (9, 10) or 70,000 (11).The different forms may repreamino acid sequence consisted of 415 amino acids, sent different degrees of glycosylation. It appears to be imcorresponding to a 46.6-kDa protein. The sequence munologically and biochemically identical to a similar activity was related to members of the serpin gene family, of human monocytes (12, 13) and monocytic cell lines (14, particularly ovalbumin and the chicken gene Y pro- 15). PAI-2 reacts more rapidly with u-PA than with t-PA (15, tein. Like these avian proteins, placental PA1 appears 16). It is undetectable in the plasma of men and nonpregnant during late to lack a cleavable NHz-terminal signal peptide. Resi- women (11, 17), but rises to veryhighlevels dues 347-376 were identical to the partial sequence pregnancy (11,18-20). It is stimulated in monocytes by endoreported recently for a PA1 isolated from the human toxin (12,21) andin U-937 cells by tumor-promoting phorbol monocytic U-937 cell line. Placental PA1 mRNA was esters (15). apparently expressed at low levels in human umbilical Additional distinct protease inhibitors have PA1 activity. These include protease nexin-I (22),an inhibitor synthesized vein endothelial cells, but was not detectableHepG2 in hepatoma cells. It was present in U-937 cells and was by fibroblasts that inactivates u-PA. However, unlike the inducible at least 10-fold by phorbol 12-myristate13- endothelial cell or placental PAI, protease nexin-I is stimuacetate. Thus placental PA1is a unique member of the lated by heparin and it also inactivates plasmin and thrombin serpin gene family, distinct from endothelial-type PAI. (23, 24). Other PA1 activities have been described but have It is probably identical to monocyte-macrophage PAL not been characterized in detail. The fibrinolytic system plays a role in a wide variety of processes, and the system maybe regulated by changes in plasminogen, plasminogen activators, or PAI. Normal fibriPlasminogen activators are serine proteases that catalyze nolysis is critical for the prevention and removal of blood PA1 activity may be associated the conversion of the serine protease zymogen, plasminogen, clots. Elevated levels of plasma with increased risk of coronary artery (25, 26) and venous to the active protease, plasmin. Plasmin plays an important thrombosis (27, 28) and may accompany disseminated intrarole in fibrinolysis and otherlocalized extracellular proteolytic processes. Two distinct types of plasminogen activator have The abbreviations used are: t-PA, tissue-type plasminogen actibeen identified tissue-type plasminogen activator (t-PA, M , vator; u-PA, urokinase-type plasminogen activator; PAI, plasminogen
* The costs of publication
of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in thispaperhns been submitted
to the GenBankTM/EMBLDataBankwith
accession number(s)
502685. § Supported by the Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO. 11 To whom correspondence should be addressed 660 S. Euclid, Box 8045,Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110.
activator inhibitor; PMA, phorbol 12-myristate 13-acetate; PBS. phosphate-buffered saline; DME, Dulbecco’s modified Eagle’s me. dium; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec. trophoresis; pfu, plaque-forming unit; IPTG, isopropyl-P-D-thiogalac. topyranoside; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; kb, kilobase pair(s) of DNA or kilobase(s) of RNA; BSA, bovine serum albumin. The term PAI-1 refers to the plasminogen activator inhibitor of endothelial cells and platelets, whereas PAI-2 refers to the plasminogen activator inhibitor of placenta, monocytes, and U-937 cells, in accordance with the recommendations adopted at the 32nd annual meeting of the International Committee on Thrombosis and Haemostasis, June 8, 1986, Jerusalem, Israel.
3718
Human Placental Plasminogen Activator Inhibitor
Additions: BSA Xgtll
3719
MW
XT
X,R
~
1
1 6
2 ~
3
4
5
6
7
8
88, 79‘ Additions: X75.I 175.2
X89.2 X89.3
+Anti-PA1 FIG. 1. Urokinase inhibitory activities in X g t l l fusion proteins. Phage lysates were preincubated with anti-PA1antibody (+unti-PAn or with preimmune serum (“anti-PAn and then tested for PA1 activity. Urokinase activity isindicated by the appearance of a clear lytic zone, and PA1 activity is shown by abolishment of the lytic zone. Lysates tested were from XPAI-75.1 (X75.1), XPAI-75.2 (X75.2),XPAI-T ( A T ) , XPAI-R (XR), XPAI-89.2(X89.2),XPAI-89.3 (X89.3), Xgtll without insert (Xgtll),and a bovine serum albumin control (BSA).
FIG. 2. SDS-PAGE analysis of the complex formation between lz51-urokinaseand the P A inhibitors in the phage lysates. Phage lysates containing fusion proteins from six positive clones were incubated with ’251-urokinase,immunoprecipitated by anti-placenta PA1 antibodies, and electrophoresed on a 7.5% polyacrylamide gel, as described under “Experimental Procedures.” Gel lanes were as follows: lune 1, PBB solution; lune 2, Xgtll lysate; lune 3, XPAI-T lysate; lune 4, XPAI-R lysate; lune 5,XPAI-75.1 lysate; lune 6,XPAI-75.2 lysate, lane 7, XPAI-89.2 lysate; lune 8, XPAI-89.3 lysate. Molecular weights of complexes are indicated a t the left. The bund a t the bottom of lune 5 represents the light chain of urokinase (M, 20,000).
endothelial cells. Therefore, both placenta and endothelial cell cDNA libraries were employed. Initially, 100,000 plaquevascular coagulation (29, 30). Fibrinolysis is thought to be forming units from the placenta cDNA library were screened important in tissueremodeling and tumormetastasis (31,32), using the affinity purified antibody to human placental PAI. tumor angiogenesis (33), and macrophage function in inflam- Two positive clones, XPAI-75.1 and XPAI-75.2, were found. matory reactions (12). Screening of the endothelial cell cDNA library yielded four The primary structure of human endothelial cell-type PA1 positives among 80,000 recombinants: XPAI-T, XPAI-R, has recently been determined by cDNA cloning and sequenc- XPAI-89.2, and XPAI-89.3. The proteins induced by isopropyling (34-36). We report hereinthe characterization and expres- @-D-thiogalactopyranosidefrom these isolates were screened sion in Escherichia coli of human placenta-type PAI, a unique for PA1 activitywithafibrin-agarspot assay (Fig. 1). A member of the serpin gene family that is notably similar to standard amount of human u-PA was spotted onto a gel chicken ovalbumin. An mRNA of identical size is inducible containing fibrinand plasminogen. In theabsence of any PAI, in thehuman monocytic U-937 cell line by phorbol myristate the added u-PA activatesplasminogen to plasmin, which then acetate. degrades the fibrin to produce a cleared lytic zone. In the presence of PAI, lysis is prevented. Inhibition of fibrinolysis EXPERIMENTAL PROCEDURES3 was observed only with the protein expressed by isolate XPAI75.1. This inhibition was relieved by affinity-purified antibody RESULTS to placenta PAI, but not by preimmune y-globulin. The protein expressed by each of these isolates was also Screening of cDNA Libraries and Characterization of Retested for ability to form a covalent complex with u-PA (Fig. combinant Proteins-The cDNA libraries in Xgll were constructed using adaptors that avoid the need for methylation 2). Aliquots of X-phage lysate were incubated with lZ5I-urokiof EcoRI sites and digestion of the cDNA with EcoRI. Fur- nase (two-chain), and any complexes formed were immunothermore, the cDNA inserts canbe released by digestion with precipitatedwith affinity-purified anti-placentalPAI. The either EcoRI or SalI. When these studies were initiated, the immunoprecipitates were subjected to SDS-PAGE after rerelationship between placenta and endothelial cell PA1species duction, and the mobility of the lZ5I-labeledcomplexes was was not understood. Also, the cell source of placenta PA1 was determined by autoradiography. A complex of M , -79,000 not known, and placenta contains substantial quantities of was formed with the protein expressed by XPAI-75.1 (Fig. 2, lane 5 ) .The band atM , -20,000 represents the light chain of Portions of this paper (including “Experimental Procedures” and two-chain urokinase. The heavy chain of urokinase has M , Fig. 5) are presented in miniprint at the end of this paper. Miniprint 33,000, suggesting that the protein expressed by XPAI-75.1 is easily read with the aid of a standard magnifying glass. Full size had contributed M , -46,000 to the major complex detected. photocopies are available from the Journal of Biological Chemistry, By Western blotting of XPAI-75.1 lysate proteins, the recom9650 Rockville Pike, Bethesda, MD 20814. Request Document No. binant PA1 wasestimated to have M , -45,000, slightly smaller 86M-3102, cite the authors, and include a check or money order for placental PA1 of M , -47,000: This difference $3.20 per set of photocopies. Full size photocopies are also included than the natural in the microfilm edition of the Journal that is available from Waverly Press.
R. D. Ye, T.-C. Wun, and J. E. Sadler, unpublished results.
Human Placental Plasminogen Inhibitor Activator
3720
3
833 8
@
3
@j
I
I d
quence. The first ATG triplet isfollowed by a n open reading frame of 1245 nucleotides, astop codon, a 3”noncodingregion of 581 nucleotides, and a poly(A) tail. The sequence surrounding the initiator codon, ACAATGG, differs only at position -1 from the proposed optimal sequencefor initiation by eukaryotic ribosomes, ACCATGG (60). There are five potenI I XPAI-75.15 tial polyadenylation or processing signals with the sequence AATAAA (61). Isolates XPAI-75.1 and XPAI-75.15 and a third partial cDNA clone (not shown) all share the same3’ termi1 . 1 . 1 , 1 . 1 , 1 , 1 . 1 . 1 , 1 , ) nus, 25-26 nucleotides after the pairof overlapping AATAAA -01 0 0.2 0.4 0.6 OB 10 1.2 1.4 1.6 1.8 2.0K1bhn sequences a t residue 1804. FIG. 3. Partial restriction map and sequencing strategy for Protein Sequence of Placental PAI and Homology to Other the XPAI-75.1 and XPAI-75.15 inserts. The scale at the bottom indicates the nucleotide positions relative to the protein initiation Serpins-The cDNA sequence encodes aprotein of 415 amino codon. The thick bar represents the coding region for the 415 amino acid residues with a calculated molecular weight of 46,600, acids of the placental PAI. The restriction endonuclease sites shown close to the M , 47,000 of placental PA1 determined by SDSwere confirmed by digestion. PAGE. The calculated amino acid composition agrees well with that determined for purified naturalplacental PAI.5 may reflect the lack of glycosylation of the protein expressed There are three potential N-glycosylation sites. Comparison in E. coli. A faint band atM , -88,000 was detected for isolate of this protein sequence with that of other serine protease XPAI-89.3 (Fig. 2, lane 8). This may represent a distinct inhibitors clearlyshows thattheplacental PA1 is a new urokinase inhibitor which is recognized by the anti-placental member of the serpin gene family (Fig. 5). The most variable PA1 antibody. Preliminarysequence data for this cDNA insert sequences among the placental PA1 and other serpins map shows that it is different from both the endothelial cell PA1 primarilytothesurface of the molecule, as predicted by (34-36) and the placental PA1 (Fig. 4).4 A minor band of comparison to the3-A crystal structureof al-antitrypsin (55, similar mobility was also seen for XPAI-75.1 (Fig. 2, lane 5). 57). Placental PA1 contains a 23-32-residue peptide inserted In the course of cDNAlibrary screening, the strongest between helix C and helix D that is not present in any other signals with the anti-PA1 antibodywere exhibited by isolates serpin. From the alignment of Fig. 5, the position of the XPAI-75.1 and XPAI-T. The immunological relationship be- reactive site is predicted to between be Arg-380 (Pl)and Thrtween the proteins expressed by these isolates and authentic 381 (Pl’), which is consistent with the substrate specificity placental PA1 was tested by the methodof “epitope selection” of the target proteases, u-PA and t-PA. (53). Proteins inducedby isopropyl-0-D-thiogalactopyrano- The sequence of endothelial cell PA1 has recently been sidein E. coli infected by these X-phage or by Xgtll were determined by cDNA cloning (34-36) and it is quite different bound to nitrocellulose filters and used for affinity purifica- from placental PAI. A phylogenetic tree based on the aligntion of the rabbit anti-human PA1 antibody.Onlyisolate ment of Fig. 5 is shown in Fig. 6. Placental PA1 is most closely XPAI-75.1 yielded antibody that could recognize natural pla- related to ovalbumin and the chicken gene Y protein. Like cental PA1 (datanotshown), suggesting that the protein these avian proteins, placental PA1 lacks a typical cleavable expressed by this isolate and placentalPA1 share at least one NH2-terminal signal peptide(68, 69). The hydrophilicity and epitope. hydropathy profiles did not suggest a particularly good interThe cDNA insert of XPAI-75.1 did notcross-hybridize with nal signal sequence (data not shown). any of the other positivesidentified with the antibody to Kruithof et al. (15) have reported 30 residues of protein placental PAI, and this insert was used to isolate additional sequence for a PA1 isolated from the human monocytic cell clones by hybridization. Among 300,000 recombinants line U-937. These exactly match the sequence of placental screened from the endothelialcell cDNA library, no positives PA1 at residues 347-376. Thus, placentalPA1 may be identical were found. Among the same number of recombinants from to the monocyte-macrophage PAI. the placental cDNA library, 15 positives were isolated. Occurrence of Placental P A I mRNA in Cultured Cells-The Nucleotide Sequence of Placental P A I cDNA Isolates-The distribution of mRNA for placental PA1 was studied by cDNA insert of XPAI-75.1 was 1.9 kbinlengthand was Northernblotting (Fig. 7).Asingle bandat 2.0 kb was sequencedby the strategy shown in Fig. 3. The entiresequence detected in placental RNA with the cDNA insert ofXPAIwas determined on both strands and corresponds to nucleo- 75.1 as the probe. A faint signal at the same position.was tides -52-1829of Fig. 4, except that nucleotide -52 was G detected in human endothelial cells. The absence of a signal instead ofA, and nucleotide 1829 was A instead ofC. An at 3.0 kb indicates that the 2.0-kb band detected in endothelial oligonucleotide corresponding to the complementof residues cells is not due to mRNA for the endothelial PAI, since that -42--25 (Fig. 4) was used to screen14 isolates obtainedfrom protein isencoded by RNAs of 2.0 and 3.0 kb that are present the placental cDNA library with the cDNA insert of XPAIin equal amounts in cultured endothelial cells (34-36). Fur75.1 as probe. One of these, XPAI-75.15, wasalsopositive thermore, thereis not sufficient similarity between the cDNA with the oligonucleotide probe. A single sequencing reaction sequences of the placental and endothelial PAIS to permit from each endof this cDNA insert confirmed that XPAI-75.15 cross-hybridization under the conditionsemployed. No signal overlapped with XPAI-75.1, with three additional nucleotides was observed with RNA from HepG2 cells, which do syntheat the 5’-end, and a poly(A) tail. Although isolated by hybrid- size the endothelialcell-type PAI.‘ The humanmonocytic cell ization, XPAI-75.15 was subsequently shown to express im- line U-937 exhibited a weak band a t 2.0 kb thatwas stimulated munoreactive PA1 of the same size as that of isolate XPAI- by treatment of the cells with phorbol12-myristate13-acetate. 75.1 by Western blotting.* The experiment was not intended tobe quantitative, but the The combinedsequence of XPAI-75.1 and XPAI-75.15 is degree of stimulation appeared to beat least 10-fold. Shorter shown in Fig. 4. The first ATG codon is preceded by a 5’- autoradiographic exposure demonstrated that no additional noncoding region of 55 nucleotides that containsa stop codon in-frame atresidue -27. For isolate XPAI-75.1, this stopcodon T.-C. Wun, and E. Reich, unpublished results. T.-C. Wun, unpublished results. is also in-frame with the vector0-galactosidasecodingse-
-- - -
L
I
I ,
I
XPAI-75.1
Human Placental Plasminogen Activator Inhibitor
3721
-55 G T T A CC C G T C A G A C AG C M C T C A G AG M T M C C A GA G M C M C C AG A T T G W C A
-1
A T 0 GAG GAT C T T TOT GTG OCA M C ACA CTC TTT GCC C T C M T T T A T T C M G CAT CTG OCA AAA OCA AGC CCCACC CAG M E C I C T I C C Z C Mot G l u Asp L o u C y 1 V a l A l a A m Thr L o u Pbo A l a L a u A s n Lou Pha L y s His L o u A l a L y s A l a Sor Pro Thr G l n A a n L o u Pbo L o u 10 20 30 T C C CCATOG
AGC A T C T C G T C C ACC ATG GCC ATG G T C TAC A T 0 GGC T C C AGO GGC AGC ACC G M GAC CAG ATG GCC
M G GTGCTT
CAG ZTT
Sor Pro Trp Sor 11. Sor Sor Thr Mot A l a N o t V a l Tyr N o t G l y Sor Arg G l y S a c Tbr G l u Asp G l n N o t A l a L y s V a l L o u G l n 50
40
90
180
Pho 60
ACC CCC ATG A C T CCA GAG M C TTT ACC AGC T G T GGG T T C ATG CAG CAG A T C CAG M G GOT AGTTAT Thr P C 0 Mot Thr P r o G l u A a n Phm Thr S a c Cy. G l y Pho Mot G l n G l n 110 G l n L y s G l y Sor T y r 70 80 90
270
C C T GAT GCG AT? T T G CAG OCA C M GCT GCA GAT AAA A T C CAT T C A T C C T T C CGC T C T C T C AGC T C T OCAATC M T O C AT C CA C A GGG GAT P r o ' A a p A l a I l o L o u G l n A l a G l n A l a A l a Asp L y a 110 Iiia Sor S o l Pho A r g S o r Lau s a c s a c A l a I l e A s n A l a Sac Thr G l y Asp 100 110 120
360
TAT TTA CTG G M AGT G T C M T M G CTG TTT GGT GAG M G T C T GCG AGC T T C COG G M G M TAT AT? CGACTCTOT CAG AAA TAT T A CT C C T y r T y r Sor T y r L m u L o u G l u S a c V a l A s n L y s L e u Pho G l y G l u L y a S o r A l a S a c Pho A r g G l u G l u T y r I l o A r g L o uC y sG l nL y s 130 140 150
450
T C A G M CCC CAG GCA GTA GAC T T C C T A G M T O T OCA G M G M GCT AGA AAA M G AT? M T T C C TOG GTC M G A C T C M A C C AAA GGC AAA S a c G l u Pro G l n A l a V a l A a p Pho L a u G l u Cy. A l a G l u G l u A l a A r g L y aL y a I14 A s n S o r Trp V a lL y a Thr G l n Thr L y sG l yL y s 160 170 180
540
A T C CCA M C TTG T T A C C T GAA GOT T C T GTA GAT 000 GAT ACC AGO AT0 G T C CTG GTG M T GCT GTC T A CT T C AAA GOA M G TGG W ACT 110 Pro A a n L o u L o u Pro G l u G l y Sor V a l Asp G l y A a p Thr Arg N o t V a l Leu V a l Asn A l a V a l T y r Pho Lys G l y L y s Trp L y s Thr 190 200 210
630
CCA TTT GAG M G AAA C T A M T GGG C T T TAT C C T T T C COT GTA M C TCG GCT CAG CGC ACA C C T GTA CAG AT0 ATG T A CT T GC O T G M M G P r o Pha G l u L y a L y a L o u Asn G l y L o u T y r P r o Pho A r g V a l A a n Sor A l a G l n Arg The Pro V a l G l n not N o t Tyr L o u Arg G l u L y s 240 220 230
720
C T A M C AT? GGA TAC ATA G M GAC CTA M G G C T CAG AT? CTA G M C T C CCA TAT GCT GGA GAT G T T AGC ATG T T CT T GT T GC T TC C AG A T Lou A a n 11. G l y T y r Ilm G l u Asp L o u L y a A l a G l n 11. L o u G l u LOU Pro Tyr A l a G l y Asp V a l s o r mot Pha LOU L ~ UL o u Pro Asp
810
M T G M GTG GOA GCC M T G C AG T T Aan G l u V a l G l y A l a Aan A l a V a l
~
250
270
260
G M AT? GCC GAT GTG T C C A C T GGC T T G GAG CTG CTG G M AGT G M ATA ACC TAT GAC AAA C T C M C M G TQG ACCAGC AAA GAC AAA ATG G l u 110 A l a Asp V a l Sor Thr G l y L o u G l u L a U LOU G l u S o r G l u 11. Thr Tyr Asp L y s L o u A m L y s T r p Thr S o r L y s Asp L y s Mot 280 290 300
900
ATA CCC CAG T T C A M ??A G M GAG CAT T A T G M C T C AGA T C C AT? CTG AGA AGC G C T O M GAT G M GTT GAG G T AT A C ATGGGC ATG GAG A l a G l u Aap G l u V a l G l u V a l Tyr 110 Pro G l n Pho L y a Lou G l u G l u Pis T y r G l u L o u Arg Sor I l o L o u A r g Sor Mot G l y Mot G l u 310 320 330
990
GAG AGG M T GAC CTG TTT C T TT C T G M GTGCTCCAC C M GCCATGGTG A s n Aap Lou Pho Lou Sor G l u V i 1 Pho Iiia G l n A l a N o t V a l
1010
G A CG C CT T C M C M G GOA COG GCC M T TTCTCAGGGATGTCG Aap A l a Pha Asn L y s G l y A r g A l a Asn Pho Sor G l y N e t
Sor G l u Arg
350
340
360
OCA GCC GCT GGC ACA GOA GGT GTT ATG ACA GGG AGA'ACT G a l CAT OQA GGCCCACAG GAT GAG GAG G G c ACT GM T I T GTGOCA Aap V a l Asn G l u G l u G l y Thr G l u A l a A l a A l a G l y Thr G l y G l y V a l N o t Thr G l y ArgATbr G l y Pis G l y G l y Pro G l n Pho V a l A l a
380
3 70
1170
390
T M M C T M GCG TGC GAT CAT CCG TTT CTT TTT C T T AT? ATG CAT M G ATA ACC M G TGC AT? T T A TTT T T C GGC AGA TTT T G CT C AC C C Asp nia Pro Pho L o u Pho L o u 11. Mot Pia Lys 11. Thr L y s Cy. 110 L o u Pha Pha G l y Arg Pho C y s Sac Pro End 400 410
1260
T G C T T C T G C AA A A G A T T T I TG T A G A T G A G CT G T G T G C C T CA G M T T G C T AT T T C A A A T T G
1370
CTGCTACCCA CTAAAT-
CC-TTT
AGAGATGTTT T C T A C A T A T TT C T G C T C T T CT G M C M C T T
ACACAGAAAT M T T A G A C MT T G T C T A T T AZ M C A T G A C AA C C C T A T I MT C A T T T G G T C
T T C C T T A C T AT C A G T T T A T TT T T A T M C A TT M C T T T T A C
TTTGTTATTT ATTATTTTAT A T M T G G T G AG T T T T T A A A T
TAAAGTTATA G M G C A G A T GA T C T G T T M TT T C C T A T C T AA T A A A T G C C TT T M T T G T T CT C A T M T G MG M T M G T A G TCTGGMAAAACATTAAACAATAGGCAAAT
-
AAA CC C ATAGACCTMTC A
ATATGTTATG T G C A T T T C T A GAAATACATA ACACATATATATGTCTGTAT
"A
TTCTAMATGGGATCATGCCCATTTAGATT
1480
T A T T G C T C A CT G C C T A T T T AA T G T A G C T M
1590
GTATCCCTCCATGCCCTTC?
ATAATAAATA
C T T A T A T T C AA T T G C M G T A
TATMIAM?
-
1700 1810
1855
U
FIG. 4. Nucleotide sequence and translated amino acid sequence of the human placenta PA1 cDNA. Nucleotides are numbered on the right-hand side. Nucleotide 1was assigned to theA of the ATG that encodes the first methionine. Negative numbers refer to the 5'-noncoding region. Amino acids are numbered underneath the sequence. Potential N-linked glycosylation sites are marked by asterisks. The proposed reactive center is indicated by arrowheads. Potential polyadenylation signals are underlined. Sequence from nucleotides -52-1829 was derived from XPAI-75.1 and was determined on both strands of the cDNA. The first three nucleotides (GTT) and the poly(A) tail were determined on one strand from the XPAI-75.15 insert.
bands were concealed under this intensesignal. Hybridization of the same blot to a cDNA probe for human y-actin gave comparable signals in all lanes (data not shown). DISCUSSION
Two nearly full-length cDNA isolates for human placental PA1 (PAI-2)have been characterized.The longest cDNA insert was 1.9 kb in length, compared to the 2.0-kb mRNA identified by Northern blotting (Fig. 7). The identity of the isolates was confirmed by showing thatrecombinant PA1 expressed in E . coli inhibited u-PA, and that this activity was itself inhibited by affinity-purifiedrabbitantibodyraised against thehomogeneous placental PA1 (Fig. 1).Like natural
placental PAI, the recombinantPA1 formed a covalent complex with u-PA(Fig. 2). Furthermore, the recombinant protein andnaturalplacental PA1 hadepitopes incommon. The coding sequence of XPAI-75.1 was in-frame with the ,@-galactosidase coding sequence of the cloning vector, Xgtll. However, the isolation with an antibody probe of XPAI-75.1 was probably fortuitous, since the presence of an ochre stop codon in-frame between the ,@-galactosidase coding sequence and the openreadingframe for the PA1 (Fig. 4) should preclude synthesis of a fusion protein containing both@-galactosidase and PA1 sequences. The complex between the recombinant PA1 and u-PA hadM , 79,000, whereas the heavy chain of uPA hasM , 33,000. The contributionof the PA1 to thiscomplex can be estimated at M , -46,000, whereas the unglycosylated
3722
E
-8
70
-
Human Placental Plasminogen Activator Inhibitor
I
I
_”“_ _I
I
-1.
fa-
2 50
-
40
-
FIG. 6. Phylogenetic tree for the serpins. The tree shown is based on the alignment of Fig. 5. The divergence of individual proteins is represented as the average percentage of different amino acids (Percent Difference) over the segment corresponding to thebeginning of helix A of cy,-antitrypsin through the carboxyl terminus of the proteins compared. Terminal gaps were ignored, but internal gaps not common to both sequences were treated as differences. Similar trees were calculated with different indices of divergence. For the region enclosed in the dashed ellipse (- - -), the precise order of branching was sensitive to the index of divergence employed, as described under “Experimental Procedures.”
I
2
3
4
5
28 S, 4.4-
2.32.018s-
FIG. 7. The distribution of human placenta PA1 messenger in different cell types as detected by RNA blot hybridization. The cDNA insert of APAI-75.1 was used to probe a Northern blot as described under “ExperimentalProcedures.” Each lane contains5 pg of poly(A)+RNA from the indicated source: lane 1, human placenta; lane 2, human umbilical vein endothelial cell; lane3, HepG2 hepatoma cells; lane 4, U-937 cells; lane 5, U-937 cells treated with phorbol 12myristate 13-acetate. The mobility of 28 S and 18 S RNA standards and denatured Hind111 fragments of X-phage DNA, is shown at the left.
PA1 has a calculated M , of 46,600(Fig. 4). By Western blotting, the recombinant PA1 expressed by both XPAI-75.1 and XPAI-75.15 had M , -45,000, compared to -47,000 for the protein isolated from placenta? This difference may reflect the absence of carbohydrate in the recombinantproteins.
Thus, translation of the recombinant PA1 in E. coli probably was initiated a t nucleotide 1(Fig. 4). There are three potential N-glycosylation sites in the sequence; however, glycosylation is not necessary for PA1 function, since the nonglycosylated protein expressed in E. coli was active. The apparentlyidentical PA1 synthesized by the humanmonocytic U-937 cell line was reported to have M , -47,000 and did not bind to concanavalin A (15). Thus, itmay not be N-glycosylated either. The 3”noncoding region of the cDNA contained five potential polyadenylation or processing signals, AATAAA (61). All of the cDNA isolates characterized to date (two in this report and one unpublished4)have the same 3‘-terminus, and only a single mRNA species has been identified by Northern blotting (Fig. 7). Therefore, only thelast two overlapping signals appear to be utilized. Comparison of thetranslated protein sequence for the placental PA1 to otherserine proteaseinhibitors clearly shows that it is a member of the serpin gene family (Fig. 5). It is especially similar to ovalbumin and the chicken gene Y protein, and placental PA1 is no more similar to endothelial cell PA1 (PAI-1) than it is to any other mammalian serpin (Fig. 6). If PAI-1 and PAI-2 do serve overlapping or homologous functions in theregulation of fibrinolysis, this is not reflected in a correspondingly conserved primary structure. Residues 347-376of placental PA1 exactly match the 30 residues of sequence reported for a PAI-purified from the human monocytic cell line U-937 (15). The PA1 proteins from placenta and monocyte (or U-937 cells) are biochemically and immunologically indistinguishible (12-15). They are also encoded by mRNAs of the same size (Fig. 7). Thus, thesePAIS (PAI-2) may be identical. The cell type in placenta that is responsible for the synthesis of PAI-2 is not known. However, monocytes comprise a significant fraction of the cells that can be recovered from placenta (62) and might account for some placental PAI-2. Examination of the proposed reactive center region fails to reveal a serine residue at anypossible P1’ position. The most likely site of cleavage by u-PA or t-PA is between Arg-380 (Pl) and Thr-381 (Pl’), based on the alignment of Fig. 6 and the substrate specificity of the target proteases. Cleavage at an Arg-Thr bond is also a featureof C1-esterase inhibitor (55, 63). The sequence of the placental PA1 is notable for the lack of a typical cleavable signal peptide. The amino terminus of the natural protein from placenta6 and U-937 cells (15) appears to be blocked, so the NH2-terminalresidue is not known. However, the sequence can be aligned precisely with that of ovalbumin and the chicken gene Y protein, which also lack cleavable signal peptides. All three proteins begin in thehelix A structural region of al-antitrypsin (Fig. 5), which is moderately conserved among other serpins as well. Finally, the most hydrophobic segmentnear the NH2-terminus of the placental PAI, residues 4-16, is interrupted by 2 asparagine residues. Thus, theamino-terminal sequence does not resemble any known cleavable signal peptide (68, 69). Hydrophilicity or hydropathy profiles did not reveal any particularly good candidate site for an internal signal sequence (data not shown). Placental PA1 is found in plasma during pregnancy (18), and the identical monocyte-macrophage PA1 is secreted from activated monocytes and from monocytic U-937 cells (15). However, the fraction of the total PA1 synthesized that is secreted is not known. Some other proteins that are apparently secreted by activated monocytes appear to lack NH2terminal signal sequences, including interleukins la and 1/3 (70-72), as well as acidic (73) and basic fibroblast growth factors (73, 74). The release of interleukin 1 from monocytes
Human Placental Plasminogen Activator Inhibitor has been proposed to reflect cell damage (70). However, the level of mRNA for the placental PA1 clearly increases manyfold in response to stimulation of U-937 cells with PMA (Fig. 7), so that cell damage alone cannot account for the increased release of this PAI. The structural basis for the apparent secretion of the placental PA1 and other proteins that lack cleavable signal sequences remains incompletely understood. The physiological role of placental PA1 is not known. A definite function in human reproduction has not yet been demonstrated. It is induced in monocytes or U-937 cells by stimuli that also induce the expression of tissue factor activity (75, 76). Thus, theactivation of monocytes may initiate blood clotting through the extrinsic cascade, and also inhibit fibrinolysis through the secretion of PAI-2. Both responses would lead to the deposition and persistence of fibrin, which is a prominent feature of cell-mediated immune reactions (77). In addition, activated monocytes may secrete plasminogen activators that would tend tooppose these procoagulant responses (14, 78, 79). Thus, understanding the regulation of PA1 expression may clarify the function of this direct link between macrophage activation and blood coagulation in cell-mediated immunity. Acknowledgments-We wish to thankDr. David Tiemeier for helpful discussions and encouragement, Dr. David Ginsburg for sharing sequence data prior to publication, Dr. Laurence Kedes for the yactin cDNA, and Dr. Robert L. Hill for advice on affinity purification of antibodies. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
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Human Placental Plasminogen Inhibitor Activator
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