Acyl-Enzyme Complexes between Tissue-type Plasminogen Activator and Neuroserpin are Short-lived in Vitro

The serine protease tissue-type plasminogen activator (t-PA) initiates the fibrinolytic protease cascade and plays a significant role in motor learning, memory, and neuronal cell death induced by excitotoxin and ischemia. In the fibrinolytic system, the serpin PAI-1 negatively regulates the enzymatic activity of both single-chain and two-chain t-PA (sct-PA and tct-PA). In the central nervous system, neuroserpin (NSP) is a serpin thought to regulate t-PA enzymatic activity. We report that although both sct-PA and tct-PA rapidly form acyl-enzyme complexes with NSP in vitro, the interactions are short-lived, rapidly progressing to complete cleavage of NSP and regeneration of fully active enzyme. All NSP molecules appear to transit through the detectable acyl-enzyme intermediate and progress to completion of cleavage; no subpopulation that functions as a pure substrate was detected. Likewise, all molecules were reactive, with no evidence of a latent subpopulation. The interactions between NSP and t-PA were distinct from those between plasmin and NSP, wherein the same peptide bond was cleaved but there was no evidence of a detectable plasmin-NSP acyl-enzyme complex. The interactions between t-PA and NSP contrast with the formation of long-lived, physiologically irreversible acyl-enzyme complexes between t-PA and PAI-1, suggesting that the physiologic effect of t-PA-NSP interactions may be more complex than previously thought. The serine protease tissue-type plasminogen activator (t-PA) initiates the fibrinolytic protease cascade and plays a significant role in motor learning, memory, and neuronal cell death induced by excitotoxin and ischemia. In the fibrinolytic system, the serpin PAI-1 negatively regulates the enzymatic activity of both single-chain and two-chain t-PA (sct-PA and tct-PA). In the central nervous system, neuroserpin (NSP) is a serpin thought to regulate t-PA enzymatic activity. We report that although both sct-PA and tct-PA rapidly form acyl-enzyme complexes with NSP in vitro, the interactions are short-lived, rapidly progressing to complete cleavage of NSP and regeneration of fully active enzyme. All NSP molecules appear to transit through the detectable acyl-enzyme intermediate and progress to completion of cleavage; no subpopulation that functions as a pure substrate was detected. Likewise, all molecules were reactive, with no evidence of a latent subpopulation. The interactions between NSP and t-PA were distinct from those between plasmin and NSP, wherein the same peptide bond was cleaved but there was no evidence of a detectable plasmin-NSP acyl-enzyme complex. The interactions between t-PA and NSP contrast with the formation of long-lived, physiologically irreversible acyl-enzyme complexes between t-PA and PAI-1, suggesting that the physiologic effect of t-PA-NSP interactions may be more complex than previously thought. Tissue-type plasminogen activator (t-PA) 1The abbreviations used are: t-PA, tissue-type plasminogen activator; sct-PA, single-chain t-PA; tct-PA, two-chain t-PA; scu-PA, single chain urokinase plasminogen activator; tcu-PA, two chain urokinase plasminogen activator; NSP, neuroserpin; PAI-1, plasminogen activator inhibitor type-1 is one of two mammalian serine proteases that activate plasminogen to plasmin. Unlike the zymogen form of most serine proteases, the single-chain form of t-PA (sct-PA) retains roughly 10–25% of the enzymatic activity of the mature two-chain form (tct-PA) (1Boose J.A. Kuismanen E. Gerard R. Sambrook J. Gething M.J. Biochemistry. 1987; 28: 635-643Google Scholar, 2Ranby M. Bergsdorf N. Nilsson T. Thromb. Res. 1982; 27 (suppl.): 175-183Google Scholar, 3Tachias K. Madison E.L. J. Biol. Chem. 1997; 272: 28-31Google Scholar). Because both sct-PA and tct-PA are active enzymes, inhibition of t-PA activity, rather than maintenance of the protease in zymogen form, is necessary to prevent undesired proteolysis. In plasma, the physiologic or cognate inhibitor of t-PA appears to be the serine protease inhibitor (serpin) plasminogen activator inhibitor type-1 (PAI-1) (4Kruithof E.K. Tran-Thang C. Ransign A. Bachmann F. Blood. 1984; 64: 907-913Google Scholar). In addition to its role in maintaining vascular hemostasis, t-PA is a key extravascular protease in the central and peripheral nervous systems. t-PA can be detected on the axonal growth cone of developing neuroblasts (5Pittman R.N. Ivins J.K. Buettner H.M. J. Neurosci. 1989; 9: 4269-4289Google Scholar) and is involved with motor learning and memory (6Calabresi P. Napolitano M. Centonze D. Marfia G.A. Gubellini P. Teule M.A. Berretta N. Bernardi G. Frati L. Tolu M. Gulino A. Eur. J. Neurosci. 2000; 12: 1002-1012Google Scholar). Studies in t-PA null mice have shown that t-PA enzymatic activity mediates neuronal cell death associated with both seizure kindling and ischemia and is involved with myelinated nerve regeneration following nerve crush injury (7Tsirka S.E. Gualandris A. Amaral D.G. Strickland S. Nature. 1995; 377: 340-344Google Scholar, 8Tsirka S.E. Rogove A.D. Strickland S. Nature. 1996; 384: 123-124Google Scholar, 9Akassoglou K. Kombrinck K.W. Degen J.L. Strickland S. J. Cell Biol. 2000; 149: 1157-1166Google Scholar). To date, the mechanism by which t-PA activity in the nervous system is regulated is unknown, although several studies have suggested that the recently described serpin, neuroserpin (NSP), may serve this function (10Hastings G.A. Coleman T.A. Haudenshild C.C. Stefansson S. Smith E.P. Barthlow R. Cherry S. Sandkvist M. Lawrence D.A. J. Biol. Chem. 1997; 272: 33062-33067Google Scholar, 11Osterwalder T. Cinelli P. Baicia A. Pennella A. Krueger S.R. Schrimpf S.P. Meins M. Sonderegger P. J. Biol. Chem. 1998; 273: 2312-2321Google Scholar). NSP is an ∼45-kDa glycosylated serpin that is expressed almost exclusively in the nervous system throughout embryologic development and adulthood (12Krueger S.R. Ghisu G.P. Cinelli P. Gschwend T.P. Osterwalder T. Wolfer D.P. Sonderegger P. J. Neurosci. 1997; 17: 8984-8996Google Scholar). NSP inhibits the enzymatic activity of t-PA in vitro (10Hastings G.A. Coleman T.A. Haudenshild C.C. Stefansson S. Smith E.P. Barthlow R. Cherry S. Sandkvist M. Lawrence D.A. J. Biol. Chem. 1997; 272: 33062-33067Google Scholar), and pharmacologic administration of NSP to rats that have undergone middle cerebral artery occlusion decreases the volume of the ischemic penumbral area concomitant with inhibition of t-PA enzymatic activity (13Yepes M. Sandkvist M. Wong M.K. Coleman T.A. Smith E. Cohan S.L. Lawrence D.A. Blood. 2000; 96: 569-576Google Scholar). Also, transgenic mice that overexpress NSP show a decreased volume of neuronal cell death following middle cerebral artery occlusion compared with their wild type counterparts (14Cinelli P. Madani R. Tsuzuki N. Vallet P. Arras M. Zhao C.N. Osterwalder T. Rulicke T. Sonderegger P. Mol. Cell. Neurosci. 2001; 18: 443-457Google Scholar). It is presumed that NSP inhibits t-PA in a manner analogous to PAI-1 to mediate these effects. Serpins inhibit the enzymatic activity of their cognate serine proteases via initial formation of a Michaelis complex in which the enzyme is reversibly bound and inhibited by the serpin (15Ye S. Ceah A.L. Belmares R. Bergstom R.C. Tong Y. Corey D.R. Kanost M.R. Goldsmith E.J. Nat. Struct. Biol. 2001; 8: 979-983Google Scholar). The protease initiates cleavage of the scissile peptide bond between the P1 and P1′ residues of the reactive center loop, which leads to the formation of an acyl-enzyme intermediate, with the active site serine covalently bound to the P1 residue (16Lawrence D.A. Ginsburg D. Day D.E. Berkenpas M.B. Verhamme I.M. Kvassman J.O. Shore J.D. J. Biol. Chem. 1995; 270: 25309-25312Google Scholar, 17Higazi A.A. Upson R.H. Cohen R.L. Manuppello J. Bognacki J. Henkin J. McCrae K.R. Kounnas M.Z. Strickland D.K. Preissner K.T. Lawler J. Cines D.B. Blood. 1996; 88: 542-551Google Scholar). However, presumably before this intermediate can be hydrolyzed to yield cleaved serpin and active protease (i.e. completion of the cleavage reaction), the protease, bound to the P1 residue of the serpin, is translocated to the opposite pole of the serpin (18Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Google Scholar). The tertiary structure of the translocated protease active site is sufficiently disrupted to prevent hydrolysis of the acyl-enzyme complex (19Huntington J.A. Read R.J. Carrell R.W. Nature. 2000; 407: 923-926Google Scholar), making the rate of deacylation extremely slow (i.e. weeks). The complexes display new molecular determinants that allow for cellular internalization and degradation in a matter of minutes (20Strickland D.K. Kounnas M.Z. Argraves W.S. FASEB J. 1995; 9: 890-898Google Scholar). Therefore, protease-serpin acyl-enzyme complex formation can usually be viewed as biologically irreversible. The work presented here demonstrates that the interaction of NSP with its putative cognate protease, t-PA, differs from the currently held paradigm in that the acyl-enzyme intermediates between t-PA and NSP are much less stable than other cognate protease-serpin complexes. Indeed, t-PA seems to handle NSP more like a substrate than a suicide inhibitor, with the acyl-enzyme complex being a detectable intermediate. These findings may provide insights into the physiologic interaction between NSP and t-PA, which may differ from certain functions inferred from studies with pharmacological concentrations of NSP in animal models. Sct-PA (greater than 95% single-chain t-PA) isolated from Bowes melanoma cells was purchased from Biopool and from Calbiochem. Tct-PA was generated by treating sct-PA with plasmin linked to Sepharose beads (plasmin-Sepharose) at 37 °C for times determined in preliminary experiments, to yield complete cleavage of sct-PA by that batch of plasmin-Sepharose as determined by silver staining of SDS-polyacrylamide gels run under reducing conditions. Human scu-PA was a generous gift from Dr. Jack Henkin of Abbott Laboratories. Human tcu-PA (Winkinase) was obtained from Dr. Gene Murano, Monsanto, St. Louis, MO. The cDNA for human NSP was obtained from Human Genome Sciences, Rockville, MD. Recombinant human NSP was expressed and purified from a baculovirus-based system. PAI-1 (14-1b mutant) was expressed and purified from Escherichia coli as described previously (10Hastings G.A. Coleman T.A. Haudenshild C.C. Stefansson S. Smith E.P. Barthlow R. Cherry S. Sandkvist M. Lawrence D.A. J. Biol. Chem. 1997; 272: 33062-33067Google Scholar, 21Lawrence D. Strandberg L. Grundstrom T. Ny T. Eur. J. Biochem. 1989; 186: 523-533Google Scholar) and was ∼50% active as measured by stable inhibition of tct-PA. Polyclonal antibodies against NSP were generated in rabbits (10Hastings G.A. Coleman T.A. Haudenshild C.C. Stefansson S. Smith E.P. Barthlow R. Cherry S. Sandkvist M. Lawrence D.A. J. Biol. Chem. 1997; 272: 33062-33067Google Scholar). Plasminogen was radioiodinated as described previously (22Deutsch D.G. Mertz E.T. Science. 1970; 170: 1095-1096Google Scholar, 23Schwartz B.S. Espana F. J. Biol. Chem. 1999; 274: 15278-15283Google Scholar). Plasmin, aprotinin, and spectrozyme t-PA were purchased from American Diagnostica, Greenwich, CT. Bovine serum albumin, obtained from Sigma, was treated with 100 μm diisopropyl fluorophosphate to inhibit potentially contaminating serine proteases, followed by exhaustive dialysis. Horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody was purchased from Pierce. Kodak 1-D Image software was used to digitize and quantify bands on autoradiograms and Western blots. All electrophoresis and protein transfers were carried out in the Bio-Rad Mini-protein 3 system. Electrophoresis reagents and precast 8–16% gradient SDS-polyacrylamide gels were purchased from Bio-Rad. 10% polyacrylamide gels with a 3% stacking gel or precast 8–16% gels with a 4% stacking gel were used as noted in each figure legend. The gradient gel system was selected for optimal separation of 40- and 45-kDa NSP species but does not allow for optimal detection of NSP·t-PA acyl-enzyme complexes. A 10% separating gel was used for experiments in which optimal detection of NSP·t-PA acyl-enzyme complexes was necessary. NSP was detected immunologically after electrophoresis by electroblotting proteins from the gel to polyvinylidene difluoride membrane for 1 h at 100 V (24Laemmli U.K. Nature. 1970; 277: 680-685Google Scholar, 25Gallagher S. Winston S.E. Fuller S.A. Hurrell J.G.R. Current Protocols in Molecular Biology. 2. John Wiley & Sons, Etobicoke, Ontario, Canada1997: 10.8.1-10.8.21Google Scholar). Membranes were then blocked with Tris-buffered saline, 0.01m Tris, 0.15 m NaCl, pH 7.2, containing 0.05% Tween and 0.25% gelatin and then blotted with a 1:5000 dilution of rabbit antibody against NSP (10Hastings G.A. Coleman T.A. Haudenshild C.C. Stefansson S. Smith E.P. Barthlow R. Cherry S. Sandkvist M. Lawrence D.A. J. Biol. Chem. 1997; 272: 33062-33067Google Scholar) (demonstrated in preliminary experiments to be specific for NSP and to detect intact, cleaved, and complexed NSP) in the blocking buffer followed by blotting with a 1:5000 dilution of horseradish peroxidase-conjugated goat antibody against rabbit IgG in the blocking buffer. Membranes were then rinsed several times in Tris-buffered saline with 0.05% Tween and subsequently treated with Pierce West Super Pico Enhanced Chemiluminescence Reagent, then exposed to film. Assays to monitor the cleavage of125I-plasminogen were as described previously L. Lawrence D. Thromb. Res. 1984; (suppl.): Scholar). Plasminogen was by the plasmin chain L. Lawrence D. Thromb. Res. 1984; (suppl.): Scholar). The of t-PA activity for each of inhibitor was determined by the of plasmin chain in with of plasmin chain in with 1 Assays of t-PA activity were in pH at t-PA and serpin were for the times followed by the addition of spectrozyme t-PA at a of The on a was used at 1 at for 1 were to substrate in The of active t-PA was determined by the of cleavage in the of of cleavage in the of were 3 NSP and the protease were at the and times in buffer containing m and 100 serum albumin, pH were by the addition of buffer followed by and Western as of bands as be for a complex between that protease and NSP was as evidence of acyl-enzyme formation (10Hastings G.A. Coleman T.A. Haudenshild C.C. Stefansson S. Smith E.P. Barthlow R. Cherry S. Sandkvist M. Lawrence D.A. J. Biol. Chem. 1997; 272: 33062-33067Google Scholar, D.A. Ginsburg D. Day D.E. Berkenpas M.B. Verhamme I.M. Kvassman J.O. Shore J.D. J. Biol. Chem. 1995; 270: 25309-25312Google Scholar, J.A. Read R.J. Carrell R.W. Nature. 2000; 407: 923-926Google Scholar). of an by antibody was with complete cleavage of NSP and of NSP and tct-PA or plasmin were in a volume for at 37 were by the addition of were by electrophoresis on a 3% 10% and separating SDS-polyacrylamide gel followed by to a polyvinylidene difluoride membrane G. Biochem. Scholar). peptide bands were then and to degradation a peptide In with NSP inhibited t-PA cleavage of the substrate spectrozyme t-PA (10Hastings G.A. Coleman T.A. Haudenshild C.C. Stefansson S. Smith E.P. Barthlow R. Cherry S. Sandkvist M. Lawrence D.A. J. Biol. Chem. 1997; 272: 33062-33067Google Scholar). However, NSP was a inhibitor of t-PA cleavage at a of serpin sct-PA or tct-PA was not of of the cleavage as PAI-1 inhibited both of t-PA in cleavage with the previously described of the single-chain and two-chain of t-PA to PAI-1 inhibition (4Kruithof E.K. Tran-Thang C. Ransign A. Bachmann F. Blood. 1984; 64: 907-913Google Scholar) inhibition by to of NSP μm μm is a more t-PA inhibitor in a than in a that the cleavage of plasminogen to plasmin. The were as described under and in a new NSP is a more t-PA inhibitor in a than in a that the cleavage of plasminogen to plasmin. The were as described under and The cleavage of substrate differ not in the substrate being cleaved but in the of the cleavage for evidence of t-PA activity to be minutes are for the substrate The in t-PA inhibition by NSP in these two that the inhibition of t-PA by NSP may be a function of To the of t-PA-NSP each form of t-PA was with NSP at 37 and the of NSP was followed as the NSP and sct-PA or tct-PA a of acyl-enzyme complex formation However, at the of the acyl-enzyme and with this an was suggesting that NSP in complex with t-PA was completion of the cleavage the of acyl-enzyme complexes and their presumed hydrolysis much more rapidly with two-chain than with single-chain t-PA, with the of tct-PA in These to PAI-1, NSP inhibits t-PA via acyl-enzyme complex formation detected in However, in contrast to acyl-enzyme complexes are deacylation in a matter of minutes not detect this These that the acyl-enzyme complex between form of t-PA and NSP be as a a detectable intermediate in the of peptide bond cleavage by the is that the of the active site that of protease-serpin may not be as for as for other protease-serpin To acyl-enzyme complexes are a of NSP interactions with protease, between plasmin and NSP and between and NSP were demonstrates that plasmin NSP with no detection of plasmin-NSP complexes. demonstrates that scu-PA detectable complex formation with cleavage of tcu-PA NSP with a of acyl-enzyme complexes detected on autoradiograms the of a significant acyl-enzyme intermediate seems to be a of the does not form acyl-enzyme complexes with NSP of each or with scu-PA or tcu-PA was for the times at 37 In and NSP were for the at 37 °C for a of the of NSP cleaved by tct-PA and by tcu-PA the same and under the same conditions. were by and as described under and Image of the of the reactive center of NSP for cleavage by proteases and was that proteases peptide in NSP, with the one by t-PA a of the to allow for of the protease in a stable and the site by plasmin a that was for of the intermediate A. Carrell R.W. J.A. J. Biol. Chem. 2001; Scholar). However, for both enzymes, the bond was determined to be by of the cleavage peptide plasmin and t-PA both the same but the of the interaction of t-PA with NSP is a detectable plasmin NSP as a substrate evidence of the complex. It is not this a more rate of deacylation by plasmin that there is for (18Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Google Scholar) or the of the proteases in a molecular between NSP and plasmin that is for the active site for a detectable acyl-enzyme of the cleavage site in of of from residue and t-PA both NSP at the same residue of the reactive center NSP was with plasmin or tct-PA. The were by and to a polyvinylidene difluoride and the peptide of NSP generated by cleavage was to degradation in a new and t-PA both NSP at the same residue of the reactive center NSP was with plasmin or tct-PA. The were by and to a polyvinylidene difluoride and the peptide of NSP generated by cleavage was to degradation To that the of acyl-enzyme complexes was a of the protein and not a function of an P. P. E. J. Biol. Chem. Scholar, A. Thromb. 1998; Scholar), a of t-PA enzymatic activity was of NSP and t-PA were for t-PA enzymatic activity was via cleavage of substrate A and and of the same were by and Western blotting to the of the NSP demonstrates that with the enzymatic activity of t-PA that inhibited by NSP was A and enzymatic concomitant with the of cleaved NSP the of the cleavage deacylation experiments with t-PA inhibition by PAI-1 A and no of enzymatic activity of form of t-PA to their interaction with the of acyl-enzyme complexes is a of the and is not a function of the In these that t-PA is with deacylation of complexes. and that NSP cleavage by form of t-PA via the acyl-enzyme intermediate, with evidence for a NSP that as a pure t-PA the by out at °C that cleaved NSP was by the of acyl-enzyme complexes under that substrate by the the substrate of to of protease a with on L. L. J. K.W. Eur. J. Biochem. 1996; Scholar), all NSP to cleavage via the acyl-enzyme intermediate. In all of the NSP is cleaved, evidence of a latent form of the serpin for tct-PA, not shown for Therefore, NSP appears to as a single of molecules that is cleaved by t-PA via through a detectable acyl-enzyme intermediate. The presented that the interactions between NSP and t-PA are more to cleavage of a substrate with a intermediate than the biologically irreversible inhibition usually associated with this is the of NSP in an that t-PA enzymatic activity a that deacylation to yield a more to that of a substrate than to the with To this the inhibition of and cleavage by PAI-1, NSP, and a physiologic were The NSP inhibition of both of t-PA are to the of inhibition by a and differ from the inhibition of PAI-1 is to be to in of t-PA for PAI-1 as the to and of protease inhibition by PAI-1 and NSP to be in the in It seems that NSP is a pure inhibitor a pure substrate of t-PA, as the rate of acyl-enzyme complex deacylation in vitro is to inhibition at but slow to a significant in the substrate of NSP and to of an substrate by an is with the with of NSP or with the administration of pharmacological concentrations of NSP into the central nervous system of mice with middle cerebral artery occlusion (13Yepes M. Sandkvist M. Wong M.K. Coleman T.A. Smith E. Cohan S.L. Lawrence D.A. Blood. 2000; 96: 569-576Google Scholar, D.A. Ginsburg D. Day D.E. Berkenpas M.B. Verhamme I.M. Kvassman J.O. Shore J.D. J. Biol. Chem. 1995; 270: 25309-25312Google Scholar). to be is the effect of plasmin on t-PA effect in the central nervous system is as much Strickland S. Cell. 1997; Scholar), the and cleavage of NSP by plasmin have to be and against the of plasmin by Likewise, NSP is cleaved by both t-PA and a but function for cleaved NSP may be of the to its NSP a of negatively residues in an to the residues on the of the in certain that with activity T. J. E.T. Sonderegger P. 1996; Scholar). there is the that a NSP and or its activity. a was by NSP with of to allow for interaction between NSP and a followed by the addition of was no evidence of of t-PA inhibition or of of complexes not The presented the of the physiologic interaction between t-PA and In other protease-serpin the acyl-enzyme complex molecular determinants not on the that allow cellular internalization of the via the protein A.A. Upson R.H. Cohen R.L. Manuppello J. Bognacki J. Henkin J. McCrae K.R. Kounnas M.Z. Strickland D.K. Preissner K.T. Lawler J. Cines D.B. Blood. 1996; 88: 542-551Google Scholar). The for complex internalization in in R. D. Strickland D.K. McCrae K.R. J. Biol. Chem. 1998; 273: Scholar) that this may not be to complexes before deacylation at 37 be of to acyl-enzyme complexes have a than other protease-serpin that complexes are than NSP may serve to or mediate of these to be that stable acyl-enzyme complexes are of cognate protease-serpin with complexes being in M. T. Biochemistry. Scholar), seems to NSP is a physiologic inhibitor of the in that with a of NSP, in in of NSP and presumably a in NSP, their of as M. M. J. M. P. F. R. P. D. R. M. J. 2000; Scholar), and the in of that is blocked by NSP M. M. Coleman T.A. E. D. Lawrence D.A. J. Scholar), seems that a t-PA-NSP interaction in as a of It be to the of NSP with on its in We and from the of for in peptide We like to for work on the interactions between urokinase and NSP and for and in of this

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