Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis

Tate, Keri; Higgins, Deborah L.; Holmes, William E.; Winkler, Moritz; Heyneker, Herbert L.; Vehar, Gordon A.

doi:10.1021/bi00376a002

Cited by 152 publications

(99 citation statements)

References 34 publications

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“…Quantitative conversion of the enzymes into their two-chain forms was confirmed by SDS-PAGE and Western blotting. As previously demonstrated (27), variants containing the mutation R275E were not cleaved by plasmin-Sepharose (data not shown).…”

Section: Resultssupporting

confidence: 81%

“…It was therefore essential to assay both the single-and two-chain form of each variant of t-PA containing a mutation in the autolysis loop. Accurate measurement of the enzymatic activity toward plasminogen of the single-chain form of these variants proved difficult, however, because plasmin produced during the assay rapidly and efficiently converted the enzymes into their mature, two-chain form by cleaving the Arg 275 -Ile 276 bond of the single-chain t-PA. Consequently, to overcome this technical difficulty, we also constructed noncleavable forms of the nine mutated enzymes by introducing the additional mutation R275E into the new variants, a strategy that was first described by Tate and co-workers (27).…”

Section: Resultsmentioning

confidence: 99%

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Identification of a Hydrophobic Exosite on Tissue Type Plasminogen Activator That Modulates Specificity for Plasminogen

Tachias

Lamba

et al. 1997

Journal of Biological Chemistry

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A wide variety of important biological processes, including both the formation and dissolution of blood clots, depend on specific cleavage of individual target proteins by serine proteases. For example, tissue type plasminogen activator (t-PA), a trypsin-like enzyme that catalyzes the rate-limiting step of the endogenous fibrinolytic cascade, has only one known substrate in vivo, a single peptide bond (Arg 561 -Val 562 ) in the proenzyme plasminogen. We have previously suggested that the specificity of t-PA for plasminogen is mediated in part by direct protein-protein interactions between the protease domain of t-PA and plasminogen that are distinct from those occurring within t-PA's active site. We demonstrate in this study that residues 420 -423 of t-PA, which form a fully solvent-exposed, hydrophobic region of a surface loop mapping near one edge of the active site of t-PA, form, or are essential for the integrity of, an important, secondary site of interaction between t-PA and plasminogen that significantly modulates the rate of plasminogen activation in the absence, but not the presence, of fibrin. Identification of this secondary site of interaction between t-PA and plasminogen provides new insight into molecular details of the evolution of stringent substrate specificity by t-PA and suggests a novel strategy to enhance the fibrin dependence of plasminogen activation by t-PA. While the activity of wild type t-PA is stimulated by fibrin by a factor of approximately 650, the activity of two variants characterized in this study, t-PA/R275E,P422G and t-PA/R275E,P422E, is stimulated by a factor of approximately 39,000 or 61,000, respectively. It is therefore possible that, compared with wild type t-PA, the two variants would display enhanced "clot selectivity" in vivo due to reduced activity in the circulation but full activity at a site of fibrin deposition.Tissue type plasminogen activator (t-PA) 1 has been widely and successfully used as a therapeutic agent to treat acute myocardial infarction (1). t-PA does not, however, dissolve blood clots directly; instead, the enzyme catalyzes conversion of the zymogen plasminogen into the active protease plasmin, which efficiently degrades the fibrin mesh forming the core of a thrombus (2). Both of these critical fibrinolytic enzymes, t-PA and plasmin, are members of the chymotrypsin family of serine proteases (3, 4). Unlike plasmin, however, t-PA is a remarkably specific enzyme. Because such highly restricted substrate specificity is in striking contrast to the broad specificity of well studied serine proteases such as trypsin, chymotrypsin, and elastase (5), the molecular basis of the selectivity of t-PA for plasminogen is of considerable interest. A detailed understanding of the mechanisms employed by t-PA to ensure selectivity would provide new insight into the evolution of the endogenous fibrinolytic cascade and might suggest effective new strategies for the rational design of novel, highly selective proteases with unique specificities as well as novel plasminogen...

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Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Identification of a Hydrophobic Exosite on Tissue Type Plasminogen Activator That Modulates Specificity for Plasminogen

Tachias

Lamba

et al. 1997

Journal of Biological Chemistry

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“…Accurate measurement of the catalytic activity of the single-chain form of these variants toward the physiological substrate plasminogen was difficult, however, because plasmin produced during this assay rapidly and efficiently converted the variants into their mature, two-chain forms by cleaving the Arg-15-Ile-16 bond of the single-chain t-PA. Consequently, to overcome this technical difficulty, we also constructed noncleavable forms of the two mutated enzymes by introducing the additional mutation R15E into the new variants, a strategy that was first described by Tate et al (37).…”

Section: Resultsmentioning

confidence: 99%

Distinct Contributions of Residue 192 to the Specificity of Coagulation and Fibrinolytic Serine Proteases

Zhang

Hervio

Strandberg

et al. 1999

Journal of Biological Chemistry

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Archetypal members of the chymotrypsin family of serine proteases, such as trypsin, chymotrypsin, and elastase, exhibit relatively broad substrate specificity. However, the successful development of efficient proteolytic cascades, such as the blood coagulation and fibrinolytic systems, required the evolution of proteases that displayed restricted specificity. Tissue-type plasminogen activator (t-PA), for example, possesses exquisitely stringent substrate specificity, and the molecular basis of this important biochemical property of t-PA remains obscure. Previous investigations of related serine proteases, which participate in the blood coagulation cascade, have focused attention on the residue that occupies position 192 (chymotrypsin numbering system), which plays a pivotal role in determining both the inhibitor and substrate specificity of these enzymes. Consequently, we created and characterized the kinetic properties of new variants of t-PA that contained point mutations at position 192. These studies demonstrated that, unlike in coagulation serine proteases, Gln-192 does not contribute significantly to the substrate or inhibitor specificity of t-PA in physiologically relevant reactions. Replacement of Gln-192 with a glutamic acid residue did, however, decrease the catalytic efficiency of mature, two-chain t-PA toward plasminogen in the absence of a fibrin co-factor.Appreciation that thrombotic disorders are the major cause of morbidity and mortality in many countries sparked intense interest in the human fibrinolytic system, which normally provides a counterbalance to the blood coagulation cascade (1-5). The rate-limiting step in the fibrinolytic cascade, conversion of the circulating zymogen plasminogen into the active protease plasmin, is catalyzed by t-PA, 1 a member of the chymotrypsin family of serine proteases (4, 6, 7). Discovery of this important physiological role encouraged extensive scrutiny of t-PA, and the efforts of many investigators have placed this enzyme among the most thoroughly characterized human enzymes (6 -8). Administration of wild type t-PA has now become standard therapy for acute myocardial infarction (5, 9 -13), and several variants of the enzyme have either recently entered phase III clinical trials for testing as improved thrombolytic agents or already received approval by the Food and Drug Administration (14, 15).One of the most important and intriguing biochemical properties of t-PA is the highly stringent substrate and inhibitor specificity (6, 7), which is in stark contrast to that of archetypal chymotrypsin family enzymes such as chymotrypsin, trypsin, and elastase (16). The striking substrate specificity of t-PA is mediated in part by interactions of both the enzyme and its substrate with the co-factor fibrin (6, 7). However, even in the absence of a co-factor, t-PA maintains strict specificity for plasminogen, the primary physiological substrate, and we have shown that this specificity is an inherent property of the isolated protease domain of t-PA (17). Recent structural...

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“…In this case, only the >200 and the 65 kDa proteins were detectable (not shown). One other study describes the existence of a >200 kDa t-PA complex and suggests that this might be a t-PA-az-macroglobulin complex [39]. We have therefore purified this >200 kDa protein to homogeneity and probed it with a monoclonal antibody directed against human cua-macroglobulin in a radio-immuno-assay.…”

Section: Discussionmentioning

confidence: 99%

Heterogeneity of human tissue‐type plasminogen activator

et al. 1988

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Tissue-type plasminogen activator (t-PA) from human melanoma cells (Bowes) was purified by immunosorbent chromatography on affinospecific polyclonal antibodies and gel filtration in the presence of KSCN. The immunosorbent eluate contained three major components of >200, 85 and 65 kDa, respectively. The 65 kDa t-PA component could be separated by gel filtration on Ultrogel AcA44 in the presence of KSCN to a pure preparation yielding a unique N-terminal amino acid sequence. Immunoblot analysis, using affinospecific antibodies against t-PA, was a specific and sensitive method to identify different types of t-PA (I-IV), as well as t-PA-inhibitor complexes and degradation products in unstimulated melanoma cell culture fluids. Furthermore, the t-PA preparations, produced by phorbol ester-treated melanoma cells, were free of type IV and thus differed physicochemically from the constitutively produced t-PA preparations. The composition of t-PA from mammalian cell cultures is thus more complex than hitherto described.

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Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis

Cited by 152 publications

References 34 publications

Identification of a Hydrophobic Exosite on Tissue Type Plasminogen Activator That Modulates Specificity for Plasminogen

Identification of a Hydrophobic Exosite on Tissue Type Plasminogen Activator That Modulates Specificity for Plasminogen

Distinct Contributions of Residue 192 to the Specificity of Coagulation and Fibrinolytic Serine Proteases

Heterogeneity of human tissue‐type plasminogen activator

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