- Email: [email protected]
Binding of inactive mutant tissue plasminogen activator to human umbilical vein endothelial cells
POSTER SESSION - 1 varied in presence or absence of ffnger domaii (F) and in the following site mutalions: Lys277-Val (V277) and Asn448Gln (Q448). Thus, the mutants are denoted, K2P, K2P-V277, K2PXM48, FKZ!P, FK2PV277 and FK2PXM48. The mutants were expressed in Cl 27 cells and purifiedby immuno affinity chromatography. The specitic activity was determined and compared with a calibrated standard of single chain melanoma t-PA. Fibrin plate assay: The mutants were 1.5 - 5 f&f more active than melanoma t-PA in this assay. The three fingerless mutants had the highest values (3.5-5 fokf). Coupled chromogenic s&&rate assay (ptasmimgen + S2251 t CNBr fibrincgen): Mutants K2P and K2PV277 showed low relative values (30%) whereas K2w448 and the three fingercontaining mutants had a specific activity which was 90 - 100% of the melanoma t-PA. Lysis of human plasma clots in vitmz All mutants were less active than melanoma t-PA. The mutants K2P-V277 and FK2P-V277 displayed about 8Q%, whereas K2P and FK2P had &out 60% of the specific activity of the melancxna t-PA. The non-glycosylated varfant K2P-Q448 showed an actMy of about 50% while FK2PU448 was essentially inactive in this assay. The variable performance of the mutants in vitro complicates predictions of their activii In vlvo.
35 BINDING OF INACTIVE MUTANT TISSUE PLABMINOGEN ACTIVATOR TO HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS
34 EXPRESSKIN OF PLABMINOGEN ACTlVATORS AND THEIR
INHIBITORS IN TUMOR CELL LINES IS REGULATED AT TRANSCRlPTlONAL AND TRANSLATIONAL LEVEL P&4. afta& R.TJ. van Leeuwen, J.H Vwih?fen GaubiusInstituteRVO,Leiden, The Netherands Plasmlnogen activators and their inhibitors are thought to have an important function in the regulation of the protedytic events associated with metastasis and tumor invasion. Many tumor cell lines produce (t-PA) or urokinase-type plasminogen activators, either tissu&ype (u-PA) orbothtypes. Cell linescan alsoptcduceplasminogen activator inhibitorsPAl-1 andor PAI-2. However, the quantities of plasminogen activators and their inhibitors proc&ed by various cell liies differ widely. The aim of our study is to determine the level of regulation causinc these ckfferencesin PA and PAl production: is this at mRNA level, Gy transuiption regulation or mRNA stabilii, or at the level of translation efficiency or post-translational events? Twenty-two human tumor cell lines frcm a variety of malignancies were analyzed for the presence of PA and PAI antigen and mRNA. t-PA, u-PA, PAI-I and PAI- antigens were measured quantitatively both in cell extracts and conditioned media andthe PA and PAI mRNA contents of the cells were quantified on Northern blots and dot blots using %P labeled cDNA’s as probes. Quanfcatkm was done by comparison to standard curves of SPgfl7 in vitro transcripts of t-PA, uPA, PAI- and PAl-2 cDNA fragments, respectively. The cell lines were fcund to diier very much in their expression of plasminogen activators and plasmincgen activator inhibitors. A dear correlation in the expression of these various fibrfnolytic components cannot be observed. In the indiidual cell lines there is in general a relation between the mRNA level and the antigen level, indicating that regulation the mRNA level plays a major role in the regulation of PA and PAI prod&&n. The variation in antigen content is however much greaterthan thevariation in mRNA content. In theTable (below) some typical results are summarized.
of
I-PA
antiQm
m/W
8CWeS
11400 1480 773
47 25 0.44
HTlO80 HeLaSd
antgM?MI
UPA
280 80 1750
A431 HL80 SW&la
anb$an mt?M anlg~+71RN4 10200 1260 71
6.0 0.2 2.0
1700 8300 36
The great dfferences in the antigen/mRN
A ratio in the cell lines indicate that the post-transcriptionalregulation of PA and PAI producticn may contribute significanti y tothe differences of plasminogen activators and plasminogen adivator inhibitors produced by the human tumor cell lines. These differences in translation efficiency must be taken into account especially in experiments where localiiation of synthesis of plasmincgen activators and their inhibitors is studied by looking at the mRNA eyntMats.
11
anithaRWnabisitn#tn, Jo& B. Baker Dspt of Cardiovai.wlar Q4080, USA
K&hen Rmrch,
R&chto4 Genent&,
WaRwCfWmnneand Inc., So&
San Fran&co,
CA
Cutturw of human umbilical vein endothelial cells (HUVECs) have soeci5c bindina sites for ‘25-l-tPA (Haiiar et al. 11887) J. Clin. Invest. 8b:i712-1719,Baranathan et al.‘(l688) J. Bal. C’hern. 283 (10): 7782-7799). In our experiments employing ‘?-tPA in the amcentration range between l-25 nM, the specific biisof ‘l-tPA in HUVECcultureswaseccountedforbytheinteradfonof I-tPAwith PAI-1 and with the plastic culture s&sbatum. SDS-gels showed a DlT-stable corrplex which c+migrated wfth the tPA-PACl ccrrpfex. Treatment of tPA with d&opmpyffl~ata (DFP) resulted in a QQ%reduction of binding. However, a catalytically inactive mutant form of tPA, S478A, in which the active site Ser is replaced by Ala, exhibited 1550 fold more binding to HUVECs than did wild-type tPA, corresponding to approximately 12 x ld S478A tPA molecules bound per cell. This strikingbirding interacticn (a) was specitic, as itwas blodcedby excess native tPA or S478A tPA, (b) was not observed in cell-free cultures or cultures containing human fib&lasts; (c) was profoundly inhibited by arnonodcnal antibo&thatrecognizestheproteasedcmainoftPA; and (d) was significantlyinhibitedby an&PAl-1 antibody. ThesedataareconsistentwiththepossibilitythatS478AtPAbinds to the large amount of PAI- that is known to be present in the extracellular matrices of cultured endothelial cells. The vast amount of binding observed with S478A tPA, relative to the amount of binding observed with native tPA, could refled the dissociation of tPA-PAl-1 con-@exas (Knudsen and Nadrman (lQ88) J. Bid. Chem. 283 (la), 94768481) but not of S478A tPA-PAI- complexes, from matrIces.
A RECOMW4ANT PRWJROKINASE DERIVED MUTANT MISSING THE GROWTH FACTOR LlKE-DOMAlN DOE9 NOT BIND TO ITS RECEPTOR F. RobbM/, hl.L No/i4 A. SofM?fin4 E Smbbi, W? Stoppelri Ii), 0
t23ssan4F.Pm~andF.Blesl~2)
Ltpefk Research Centq Me&Dow Res. lnst, Gwenzano (VA), fta&; (1) Ml. lnst of Gene@ andBkphysks, Napfes, lta&; (2) MkrotMgisk Inst, UniveMy of Cpnhagen, Denmark Both u-PA and pro-u-PA bind to an extracellular receptor present on the metirane of several cellular types of both malignant and normal origin, in&ding andothelial cells. While the growth factor domain (GFD) of u-PA has bean shown to be involved in receptor binding, no of other parts of the information is available as to the cxntribnion mdecule. In order to test for this possbilii, we constructed a mutant U-PA gene in which *k&on C and intron D are fused together, thus deleting exon IV that codes for the GFD (a.a. Q45). Mutant g-45 was placed under the transcriptional control of a u-PA derived promoter missing negative regulatory elements and expressed in different cell types. Mutant Q-45 codas for a single chain, pm-u-PA derived protein with an apparent Mr of &out 43 kD. t&on activation by plasmin to the two chain fom~, mutant Q-45 has a specific actii 18% higher than urinary U-PA Mutant 9-45 is recognized by an anti-human uPA serum, by a mcnodonal antibody directed against the ktingle domain but not by a monodonal raised against the GFD. N-terminal and C-terminal amino-acid sequencing of the purified protein confimrs that : a) the GFD is absent; b) the difference in M.W. is not due to a truncated protein; c) the missing a.a. Q-45 are substituted by a novel tyrosine joining the last amino acid in exon Ill to the first amino acid of exon V. Mutant 845 does not bind to tha u-PA receptor as shown by its inabilii to compete with ‘251-labelledDFP-treated uPA for binding to the receptor of human cells, even at a concentration a thousand fold higher than that of contrd pro-u-PA. These data de5niiely prove that all racaptor bindingdeterminants of pu-PA (and u-PA) reside in the GFD.
35 BINDING OF INACTIVE MUTANT TISSUE PLABMINOGEN ACTIVATOR TO HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS
34 EXPRESSKIN OF PLABMINOGEN ACTlVATORS AND THEIR
INHIBITORS IN TUMOR CELL LINES IS REGULATED AT TRANSCRlPTlONAL AND TRANSLATIONAL LEVEL P&4. afta& R.TJ. van Leeuwen, J.H Vwih?fen GaubiusInstituteRVO,Leiden, The Netherands Plasmlnogen activators and their inhibitors are thought to have an important function in the regulation of the protedytic events associated with metastasis and tumor invasion. Many tumor cell lines produce (t-PA) or urokinase-type plasminogen activators, either tissu&ype (u-PA) orbothtypes. Cell linescan alsoptcduceplasminogen activator inhibitorsPAl-1 andor PAI-2. However, the quantities of plasminogen activators and their inhibitors proc&ed by various cell liies differ widely. The aim of our study is to determine the level of regulation causinc these ckfferencesin PA and PAl production: is this at mRNA level, Gy transuiption regulation or mRNA stabilii, or at the level of translation efficiency or post-translational events? Twenty-two human tumor cell lines frcm a variety of malignancies were analyzed for the presence of PA and PAI antigen and mRNA. t-PA, u-PA, PAI-I and PAI- antigens were measured quantitatively both in cell extracts and conditioned media andthe PA and PAI mRNA contents of the cells were quantified on Northern blots and dot blots using %P labeled cDNA’s as probes. Quanfcatkm was done by comparison to standard curves of SPgfl7 in vitro transcripts of t-PA, uPA, PAI- and PAl-2 cDNA fragments, respectively. The cell lines were fcund to diier very much in their expression of plasminogen activators and plasmincgen activator inhibitors. A dear correlation in the expression of these various fibrfnolytic components cannot be observed. In the indiidual cell lines there is in general a relation between the mRNA level and the antigen level, indicating that regulation the mRNA level plays a major role in the regulation of PA and PAI prod&&n. The variation in antigen content is however much greaterthan thevariation in mRNA content. In theTable (below) some typical results are summarized.
of
I-PA
antiQm
m/W
8CWeS
11400 1480 773
47 25 0.44
HTlO80 HeLaSd
antgM?MI
UPA
280 80 1750
A431 HL80 SW&la
anb$an mt?M anlg~+71RN4 10200 1260 71
6.0 0.2 2.0
1700 8300 36
The great dfferences in the antigen/mRN
A ratio in the cell lines indicate that the post-transcriptionalregulation of PA and PAI producticn may contribute significanti y tothe differences of plasminogen activators and plasminogen adivator inhibitors produced by the human tumor cell lines. These differences in translation efficiency must be taken into account especially in experiments where localiiation of synthesis of plasmincgen activators and their inhibitors is studied by looking at the mRNA eyntMats.
11
anithaRWnabisitn#tn, Jo& B. Baker Dspt of Cardiovai.wlar Q4080, USA
K&hen Rmrch,
R&chto4 Genent&,
WaRwCfWmnneand Inc., So&
San Fran&co,
CA
Cutturw of human umbilical vein endothelial cells (HUVECs) have soeci5c bindina sites for ‘25-l-tPA (Haiiar et al. 11887) J. Clin. Invest. 8b:i712-1719,Baranathan et al.‘(l688) J. Bal. C’hern. 283 (10): 7782-7799). In our experiments employing ‘?-tPA in the amcentration range between l-25 nM, the specific biisof ‘l-tPA in HUVECcultureswaseccountedforbytheinteradfonof I-tPAwith PAI-1 and with the plastic culture s&sbatum. SDS-gels showed a DlT-stable corrplex which c+migrated wfth the tPA-PACl ccrrpfex. Treatment of tPA with d&opmpyffl~ata (DFP) resulted in a QQ%reduction of binding. However, a catalytically inactive mutant form of tPA, S478A, in which the active site Ser is replaced by Ala, exhibited 1550 fold more binding to HUVECs than did wild-type tPA, corresponding to approximately 12 x ld S478A tPA molecules bound per cell. This strikingbirding interacticn (a) was specitic, as itwas blodcedby excess native tPA or S478A tPA, (b) was not observed in cell-free cultures or cultures containing human fib&lasts; (c) was profoundly inhibited by arnonodcnal antibo&thatrecognizestheproteasedcmainoftPA; and (d) was significantlyinhibitedby an&PAl-1 antibody. ThesedataareconsistentwiththepossibilitythatS478AtPAbinds to the large amount of PAI- that is known to be present in the extracellular matrices of cultured endothelial cells. The vast amount of binding observed with S478A tPA, relative to the amount of binding observed with native tPA, could refled the dissociation of tPA-PAl-1 con-@exas (Knudsen and Nadrman (lQ88) J. Bid. Chem. 283 (la), 94768481) but not of S478A tPA-PAI- complexes, from matrIces.
A RECOMW4ANT PRWJROKINASE DERIVED MUTANT MISSING THE GROWTH FACTOR LlKE-DOMAlN DOE9 NOT BIND TO ITS RECEPTOR F. RobbM/, hl.L No/i4 A. SofM?fin4 E Smbbi, W? Stoppelri Ii), 0
t23ssan4F.Pm~andF.Blesl~2)
Ltpefk Research Centq Me&Dow Res. lnst, Gwenzano (VA), fta&; (1) Ml. lnst of Gene@ andBkphysks, Napfes, lta&; (2) MkrotMgisk Inst, UniveMy of Cpnhagen, Denmark Both u-PA and pro-u-PA bind to an extracellular receptor present on the metirane of several cellular types of both malignant and normal origin, in&ding andothelial cells. While the growth factor domain (GFD) of u-PA has bean shown to be involved in receptor binding, no of other parts of the information is available as to the cxntribnion mdecule. In order to test for this possbilii, we constructed a mutant U-PA gene in which *k&on C and intron D are fused together, thus deleting exon IV that codes for the GFD (a.a. Q45). Mutant g-45 was placed under the transcriptional control of a u-PA derived promoter missing negative regulatory elements and expressed in different cell types. Mutant Q-45 codas for a single chain, pm-u-PA derived protein with an apparent Mr of &out 43 kD. t&on activation by plasmin to the two chain fom~, mutant Q-45 has a specific actii 18% higher than urinary U-PA Mutant 9-45 is recognized by an anti-human uPA serum, by a mcnodonal antibody directed against the ktingle domain but not by a monodonal raised against the GFD. N-terminal and C-terminal amino-acid sequencing of the purified protein confimrs that : a) the GFD is absent; b) the difference in M.W. is not due to a truncated protein; c) the missing a.a. Q-45 are substituted by a novel tyrosine joining the last amino acid in exon Ill to the first amino acid of exon V. Mutant 845 does not bind to tha u-PA receptor as shown by its inabilii to compete with ‘251-labelledDFP-treated uPA for binding to the receptor of human cells, even at a concentration a thousand fold higher than that of contrd pro-u-PA. These data de5niiely prove that all racaptor bindingdeterminants of pu-PA (and u-PA) reside in the GFD.