- Email: [email protected]
194 Subcellular distribution of urokinase-type plasminogen activator and uPA-receptor in neutrophils determined by immunogold labeling and electron microscopy
56
Late Abstracts
195
194 Subcellular distribution nf urnkinase-type plasminngen activator and uPAreceptor in neutrnphils determined by immunognld labeling and electron microscopy. Trine Lindhardt Pedersen ~. Thomas Horn2, Niels Ebbe Hansent, Keld Dano3 and Torban Plesner ~. tDepartment of Hematology, ~Department of Pathology, Herlev
Hospital, Herlev and JFinsen Laboratory, Rigshospitalet, Copenhagen, Denmark. By subcellular ffactionafion and flow cytometry we have previously demonstrated uPAR and uPA in easily mobilizable intracellular granules and vesicles of human neutrophils (Blood 83,808-815, 1994). We have now used immuno-gold labeling ofuPAR and uPA in human neutrophils fixed for electron microscopy to determine more precisely the co-localization of uPAR, uPA and markers of well defined subsets nf neutrophil granules. In contrast to our previous results immunoelectron microscopy provides evidence for the presence of uPAR and uPA in I;rimo~ granules as shown by a significant degree of co-localization ofuPAR and uPA with myeloperoxidase (MPO) and neutrophil elastase. This discrepancy may be explained by proteolytie degradation of uPAR and uPA from primary granules by particularly aggressive proteinases when unfixed cells are disrupted for subsequent subcellular fractionafion. Immuno-alectron microscopy also demonstrates uPAR and uPA to be frequently present in gelatinase-containing granules (GELA), but less DitCh present in laetoferrin-containing specific granules (LF). As expected a significant degree of co-localizafion was found between uPAR and uPA. The finding in this study ofuPAR and uPA in primary granules is of particular interest, since this type of granule is not easily mobilized by activation of neutrophils and has not been shown to be implicated in functions generally associated with uPAR and uPA such as pl; sminosen activation, cell adhesion and misration. % MPO + uPAR +
60
% GELA +
% LF +
51
15
uPA + 64 55 5 Table 1. For each set of markers the numberof single and double labeledgranuleswere counted andthe degree of co-localizationwas estimatedas the numberof double-stained granules relative to the total numberof either uPAR or uPA positivegranules.The result is given as the mean valuefor twenty neutrophils.
196 EXPRESSION OF " R E L A X E D " C O N F O R M A T I O N OF PAI-2 AND L R P IN H U M A N T E R M G E S T A T I O N A L T I S S U E S D. Tsatas, M. S. Baker ~, D. Saunders ~ and G. E. Rice Dept. of Perinatal Medicine,RoyalWomen's Hospital, Victoria,and ~Dept.of Biological Sciences, Universityof Wollotlgong,Wollongong,New South Wales, 2522.AUSTRALIA The relaxed conformation of plasminogen activator inhibitor-2 (PAL 2r) occurs after interaction of active PAI-2 with bovine thrombin, the plasminozen activators (ul'A tPA) or insertion of a P~. 4 reactive site loop peptide i s - ~ ~ ~., ~,,~,~l The presence of PAI-2r in tissues, determ ned using a MAb specific for PAI-2r (2H5), may be taken as indicative of in situ inhibition of serine protease-mediated proteolysis by PAI-2. In addition, it is known that relaxed serpins function as ligands for the clearance receptor, low density lipoprotein receptor-related protein (LRP), promoting the intemalisatinn of uPA-PAI complexes from the cell surface. Intemalisation of receptor-bound proteases may be an important criterion determining the invasive cell phenotype. This study characterises the immunohistochemical lncalisation of PAI2r in human term gestational tissues (amninn, choriodecidua and placenta) and establishes its expression with other components of the uPA cascade. The results obtained indicate that PAI-2r immunnreactivity was exclusively Iocalised to amnion epithelial cells, with only minimal staining in the underlying chorion. Although PAI-2 antigen is present (using AD1 '~3750), no PAI-2r immunoreactivity was detectable in any of the trophoblastic tissues examined (villous and extravillous). Immunoreacfive LRP was confined to trnphoblasts of the chorion, villous and extravillous tissue, whereas staining in the amnion was absent. For the first time, locallsation of PAI-2r at the tissue level has been identified. In addition, the data obtained are consistent with the hypothesis that cells of invasive phenotype, although expressing all components of the uPA cascade, do nnt accumulate immunoreactive FAL2r, since it is rapidly intcmalised frnrn the cell surface. Conversely, cells of non-invasive phenotype may only accumulate PAI-2r immunoreactivity in the absence of LRP expression. We propose, therefore, that the presence of PAI-2r combined with the absence of LRP at the cell surface are putative markers of ~.JHt h non-invasive phenotypes in cells that express PAI-2tT~ t' .... i. . . h. . . Cy ,...... i, press]
LYSINE-DEPENDANT GLU-PLASMINOGEN BINDING TO a - E N O L A S E INDUCES C O N F O R M A T I O N A L C H A N G E S R E S U L T I N G IN H I G H E R A F F I N I T Y I N T E R A C T I O N S Nicholas Andronicos, Marie Ranson, Martin Laekman t and Mark S. Baker.
Dept ofBiologicalSci#nces University ofWollongong Wollongong N ' ' SW, & t Walter& Eliza Hall Insirute of Medical Research, Parkville," Victoria, AUSTRALIA. Human glu-plasminogen (Glu-Plg) is a single chain trypsin-like serine endopeptidase zymogen, composed of both a heavy chain (65 kDa), containing an N-terminal acidic domain with basic hinge region (pmactivation domain), five triple-disulfide-bonded kringle structures on which the lysine binding sites (kringles 1,4 and 5) are located, and a light chain (25 kDa) containing the protease domai.n. Glu-Plg exhibits a closed conformation with a radius of gyration of 39 A. The acidic pro-activation domain assists in maintaining the closed conformation of the molecule. Removal of this domain results in the formation of lys-Plg which has a more open conformation. Pig interacts with proteins such as ct-enolase in a lysine-dependent manner. This is mediated by the lysine binding sites on the kringles of Pig which contain a hydrophobic pocket. This study describes the binding of Glu- and Lys-Plg to immobilised dimeric ct-enolase using BIAcore analysis. Binding nf Lys-Plg to ¢t-enolase was saturable at 4 mM with an apparent KDI=3.3xl0-8M and KD2=I.lxl0" 7M. The binding of Glu-Plg to ct-enolase was ant saturable at 4 mM and had dissociation constants of KDI=4.0xl0"6M and KD2=8.8x10"8M. Titration of Glu-Plg with e-amino caproic acid and ct-enolase in the presence of the hydrophnbic flurosecence probe 8-anilinn-l-napthalene sulfonic acid (ANS) resulted in an initial lysine-dependent ANS fiurnsecence decrease followed by an increase in ANS fluroscence, as well as a concomittant lysine-dependent increase in its intrinsic fluroscence which was comparable to that of Lys-Plg. Our data suggest that the binding of Glu-Plg to ct-enolase may be a multi-step event that involves lysine-mediated conformational changes in Glu-Plg resulting in higher affinity interaction(s) between Glu-Plg and ct-enolase.
197 DO PLASMINOGEN RECEPTORS PLAY A ROLE IN BREAST CANCER MALIGNANCY? Marie Ranson, Nicholas M. Aadronicos and Mark S. Baker.
Dept. of Biological Sciences, Wollongong Uni., NSW, 2522. AUSTRALIA. The metastatic capacity of cancer cells has been strongly associated with increased activity of the plasminogen activation cascade. This results n the conversion of cell-bound plasminogen (pig) by specific pig activators to the active, broad spectrum protease, plasmin. However, wh le the inappropriate expression of pig activator and their receptors (viz., uPA and uPAR respectively) are well recognised to contribute to breast cancer malignancy, whether cellular pig receptors are also increased and the importance of these molecules in intracellular and cxtracellular events related to metastasis remain unclear. To date several candidate pig binding proteins have been identified and we have recently shown that the glycolytie enzyme, ct-enolase, is an authentic pig binding protein ~. Tumnurogcnic and metastatic breast cancer cell lines are EGF-R(+), p 185HER2/neu (+), estrogen receptor positive, ER(-) and uPAjuPAR(+). We will present data to show that t! -se cell hnes also have a greater p~g 2 binding capacity (by flow cytometry, h stochem stry and hgand blnttmg) and that a -enolase may contribute to this finding. Of note is the fact that a enolase is a cytoplasmic protein without secretion signals nor hydrophobic domains. However, that a-enolase is a known substrate for EGF-R and other kinases and possesses fatty acylation sites. We are currently investigating the intracellular signalling processes that target a-anolase to the cell surface where it may act as a pig receptor. Andronicos, N.M., Ranson, M., Bognacki, J., and M.S. Baker. (1997)
Biochim. Biophys. Acta. 1337, 27-39. 2 Ranson, M., Andronicos, N.M., and M.S. Baker. (1997) Br. J. Cancer,
submitted.
Late Abstracts
195
194 Subcellular distribution nf urnkinase-type plasminngen activator and uPAreceptor in neutrnphils determined by immunognld labeling and electron microscopy. Trine Lindhardt Pedersen ~. Thomas Horn2, Niels Ebbe Hansent, Keld Dano3 and Torban Plesner ~. tDepartment of Hematology, ~Department of Pathology, Herlev
Hospital, Herlev and JFinsen Laboratory, Rigshospitalet, Copenhagen, Denmark. By subcellular ffactionafion and flow cytometry we have previously demonstrated uPAR and uPA in easily mobilizable intracellular granules and vesicles of human neutrophils (Blood 83,808-815, 1994). We have now used immuno-gold labeling ofuPAR and uPA in human neutrophils fixed for electron microscopy to determine more precisely the co-localization of uPAR, uPA and markers of well defined subsets nf neutrophil granules. In contrast to our previous results immunoelectron microscopy provides evidence for the presence of uPAR and uPA in I;rimo~ granules as shown by a significant degree of co-localization ofuPAR and uPA with myeloperoxidase (MPO) and neutrophil elastase. This discrepancy may be explained by proteolytie degradation of uPAR and uPA from primary granules by particularly aggressive proteinases when unfixed cells are disrupted for subsequent subcellular fractionafion. Immuno-alectron microscopy also demonstrates uPAR and uPA to be frequently present in gelatinase-containing granules (GELA), but less DitCh present in laetoferrin-containing specific granules (LF). As expected a significant degree of co-localizafion was found between uPAR and uPA. The finding in this study ofuPAR and uPA in primary granules is of particular interest, since this type of granule is not easily mobilized by activation of neutrophils and has not been shown to be implicated in functions generally associated with uPAR and uPA such as pl; sminosen activation, cell adhesion and misration. % MPO + uPAR +
60
% GELA +
% LF +
51
15
uPA + 64 55 5 Table 1. For each set of markers the numberof single and double labeledgranuleswere counted andthe degree of co-localizationwas estimatedas the numberof double-stained granules relative to the total numberof either uPAR or uPA positivegranules.The result is given as the mean valuefor twenty neutrophils.
196 EXPRESSION OF " R E L A X E D " C O N F O R M A T I O N OF PAI-2 AND L R P IN H U M A N T E R M G E S T A T I O N A L T I S S U E S D. Tsatas, M. S. Baker ~, D. Saunders ~ and G. E. Rice Dept. of Perinatal Medicine,RoyalWomen's Hospital, Victoria,and ~Dept.of Biological Sciences, Universityof Wollotlgong,Wollongong,New South Wales, 2522.AUSTRALIA The relaxed conformation of plasminogen activator inhibitor-2 (PAL 2r) occurs after interaction of active PAI-2 with bovine thrombin, the plasminozen activators (ul'A tPA) or insertion of a P~. 4 reactive site loop peptide i s - ~ ~ ~., ~,,~,~l The presence of PAI-2r in tissues, determ ned using a MAb specific for PAI-2r (2H5), may be taken as indicative of in situ inhibition of serine protease-mediated proteolysis by PAI-2. In addition, it is known that relaxed serpins function as ligands for the clearance receptor, low density lipoprotein receptor-related protein (LRP), promoting the intemalisatinn of uPA-PAI complexes from the cell surface. Intemalisation of receptor-bound proteases may be an important criterion determining the invasive cell phenotype. This study characterises the immunohistochemical lncalisation of PAI2r in human term gestational tissues (amninn, choriodecidua and placenta) and establishes its expression with other components of the uPA cascade. The results obtained indicate that PAI-2r immunnreactivity was exclusively Iocalised to amnion epithelial cells, with only minimal staining in the underlying chorion. Although PAI-2 antigen is present (using AD1 '~3750), no PAI-2r immunoreactivity was detectable in any of the trophoblastic tissues examined (villous and extravillous). Immunoreacfive LRP was confined to trnphoblasts of the chorion, villous and extravillous tissue, whereas staining in the amnion was absent. For the first time, locallsation of PAI-2r at the tissue level has been identified. In addition, the data obtained are consistent with the hypothesis that cells of invasive phenotype, although expressing all components of the uPA cascade, do nnt accumulate immunoreactive FAL2r, since it is rapidly intcmalised frnrn the cell surface. Conversely, cells of non-invasive phenotype may only accumulate PAI-2r immunoreactivity in the absence of LRP expression. We propose, therefore, that the presence of PAI-2r combined with the absence of LRP at the cell surface are putative markers of ~.JHt h non-invasive phenotypes in cells that express PAI-2tT~ t' .... i. . . h. . . Cy ,...... i, press]
LYSINE-DEPENDANT GLU-PLASMINOGEN BINDING TO a - E N O L A S E INDUCES C O N F O R M A T I O N A L C H A N G E S R E S U L T I N G IN H I G H E R A F F I N I T Y I N T E R A C T I O N S Nicholas Andronicos, Marie Ranson, Martin Laekman t and Mark S. Baker.
Dept ofBiologicalSci#nces University ofWollongong Wollongong N ' ' SW, & t Walter& Eliza Hall Insirute of Medical Research, Parkville," Victoria, AUSTRALIA. Human glu-plasminogen (Glu-Plg) is a single chain trypsin-like serine endopeptidase zymogen, composed of both a heavy chain (65 kDa), containing an N-terminal acidic domain with basic hinge region (pmactivation domain), five triple-disulfide-bonded kringle structures on which the lysine binding sites (kringles 1,4 and 5) are located, and a light chain (25 kDa) containing the protease domai.n. Glu-Plg exhibits a closed conformation with a radius of gyration of 39 A. The acidic pro-activation domain assists in maintaining the closed conformation of the molecule. Removal of this domain results in the formation of lys-Plg which has a more open conformation. Pig interacts with proteins such as ct-enolase in a lysine-dependent manner. This is mediated by the lysine binding sites on the kringles of Pig which contain a hydrophobic pocket. This study describes the binding of Glu- and Lys-Plg to immobilised dimeric ct-enolase using BIAcore analysis. Binding nf Lys-Plg to ¢t-enolase was saturable at 4 mM with an apparent KDI=3.3xl0-8M and KD2=I.lxl0" 7M. The binding of Glu-Plg to ct-enolase was ant saturable at 4 mM and had dissociation constants of KDI=4.0xl0"6M and KD2=8.8x10"8M. Titration of Glu-Plg with e-amino caproic acid and ct-enolase in the presence of the hydrophnbic flurosecence probe 8-anilinn-l-napthalene sulfonic acid (ANS) resulted in an initial lysine-dependent ANS fiurnsecence decrease followed by an increase in ANS fluroscence, as well as a concomittant lysine-dependent increase in its intrinsic fluroscence which was comparable to that of Lys-Plg. Our data suggest that the binding of Glu-Plg to ct-enolase may be a multi-step event that involves lysine-mediated conformational changes in Glu-Plg resulting in higher affinity interaction(s) between Glu-Plg and ct-enolase.
197 DO PLASMINOGEN RECEPTORS PLAY A ROLE IN BREAST CANCER MALIGNANCY? Marie Ranson, Nicholas M. Aadronicos and Mark S. Baker.
Dept. of Biological Sciences, Wollongong Uni., NSW, 2522. AUSTRALIA. The metastatic capacity of cancer cells has been strongly associated with increased activity of the plasminogen activation cascade. This results n the conversion of cell-bound plasminogen (pig) by specific pig activators to the active, broad spectrum protease, plasmin. However, wh le the inappropriate expression of pig activator and their receptors (viz., uPA and uPAR respectively) are well recognised to contribute to breast cancer malignancy, whether cellular pig receptors are also increased and the importance of these molecules in intracellular and cxtracellular events related to metastasis remain unclear. To date several candidate pig binding proteins have been identified and we have recently shown that the glycolytie enzyme, ct-enolase, is an authentic pig binding protein ~. Tumnurogcnic and metastatic breast cancer cell lines are EGF-R(+), p 185HER2/neu (+), estrogen receptor positive, ER(-) and uPAjuPAR(+). We will present data to show that t! -se cell hnes also have a greater p~g 2 binding capacity (by flow cytometry, h stochem stry and hgand blnttmg) and that a -enolase may contribute to this finding. Of note is the fact that a enolase is a cytoplasmic protein without secretion signals nor hydrophobic domains. However, that a-enolase is a known substrate for EGF-R and other kinases and possesses fatty acylation sites. We are currently investigating the intracellular signalling processes that target a-anolase to the cell surface where it may act as a pig receptor. Andronicos, N.M., Ranson, M., Bognacki, J., and M.S. Baker. (1997)
Biochim. Biophys. Acta. 1337, 27-39. 2 Ranson, M., Andronicos, N.M., and M.S. Baker. (1997) Br. J. Cancer,
submitted.