The Role of Gly-4 of Human Cystatin A (Stefin A) in the Binding of Target Proteinases. Characterization by Kinetic and Equilibrium Methods of the Interactions of Cystatin A Gly-4 Mutants with Papain, Cathepsin B, and Cathepsin L

Estrada, Sergio; Nycander, Maria; Nj, Hill; Cj, Craven; Jp, Waltho; Björk, Ingemar

doi:10.1021/bi980026r

Cited by 38 publications

(68 citation statements)

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“…This is the first known report of the functional expression of recombinant human stefin A in mammalian cells. The expression of functional recombinant human stefin A in E. coli has been previously reported (18)(19). However, in most cases the proteins expressed were stefin A fusion protein containing N-terminal extensions.…”

Section: Discussionmentioning

confidence: 99%

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Functional expression of recombinant human stefin A in mammalian and bacterial cells

Calkins

Dosescu

Day

et al. 2007

Protein Expression and Purification

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Recombinant human cysteine protease inhibitor, stefin A, was expressed in both E. coli and BSC-1 monkey kidney cells utilizing pET and recombinant Vaccinia virus systems, respectively. The expressed protein was purified and analyzed by SDS-PAGE and western blot analysis utilizing a polyclonal antibody against rat cystatin α. In both cases the purified protein appeared as a single band corresponding to the molecular weight of stefin A (~10 kDa). Viability of the expressed stefin A was determined by the inhibition of the plant cysteine protease, papain. Recombinant human stefin A expressed in both E. coli and BSC-1 cells was shown to almost completely inhibit papain. The expression of a fully functional recombinant human stefin A in the bacterial system provides a highly efficient tool for the production of large quantities of the protein. This can be an important tool in kinetic studies as well as in production of antibodies for other analytical studies (immunoblot, immunohistochemical studies, etc.). Expression in the mammalian cells on the other hand, can provide a significant research tool to study the functional roles of stefin A in the mammalian systems such as the regulation of cysteine proteases.

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Section: Discussionmentioning

confidence: 99%

“…The expression of functional recombinant human stefin A in E. coli has been previously reported (18)(19)(20). However, the proteins expressed were fusion proteins containing N-terminal extensions or tags.…”

Section: Introductionmentioning

confidence: 99%

Functional expression of recombinant human stefin A in mammalian and bacterial cells

Calkins

Dosescu

Day

et al. 2007

Protein Expression and Purification

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“…Cathepsins are strongly inhibited by cystatins in cells and in extracellular fluids (32). We investigated whether DNA influenced the reaction between cathepsin V and cystatin A (33), since the mode of interaction between cathepsins and cystatins is entirely different from that seen with serpins. In particular, cystatins do not interact directly with the catalytic residues of cysteine proteases and instead occlude the active site, preventing access to substrates.…”

Section: Involvement Of a Templating Mechanism In The Increase Of Catmentioning

confidence: 99%

DNA Accelerates the Inhibition of Human Cathepsin V by Serpins

Ong

McGowan

Pearce

et al. 2007

Journal of Biological Chemistry

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A balance between proteolytic activity and protease inhibition is crucial to the appropriate function of many biological processes. There is mounting evidence for the presence of both papain-like cysteine proteases and serpins with a corresponding inhibitory activity in the nucleus. Well characterized examples of cofactors fine tuning serpin activity in the extracellular milieu are known, but such modulation has not been studied for protease-serpin interactions within the cell. Accordingly, we present an investigation into the effect of a DNA-rich environment on the interaction between model serpins (MENT and SCCA-1), cysteine proteases (human cathepsin V and human cathepsin L), and cystatin A. DNA was indeed found to accelerate the rate at which MENT inhibited cathepsin V, a human orthologue of mammalian cathepsin L, up to 50-fold, but unexpectedly this effect was primarily effected via the protease and secondarily by the recruitment of the DNA as a "template" onto which cathepsin V and MENT are bound. Notably, the protease-mediated effect was found to correspond both with an altered substrate turnover and a conformational change within the protease. Consistent with this, cystatin inhibition, which relies on occlusion of the active site rather than the substrate-like behavior of serpins, was unaltered by DNA. This represents the first example of modulation of serpin inhibition of cysteine proteases by a cofactor and reveals a mechanism for differential regulation of cathepsin proteolytic activity in a DNA-rich environment.The papain-like cysteine protease, cathepsin L, has long been known as a degradative enzyme of endocytic organelles of mammalian cells, participating in the important role of protein turnover and antigen processing in these compartments (1, 2). Cathepsin L is also associated with an endosomal processing step during invasion of the cell by the Ebola virus (3), the SARS coronavirus (4), and the murine hepatitis coronavirus (5). Gene duplication in primates has given rise to two isoforms of this protein, termed cathepsin L and cathepsin V (6); an abnormal skin phenotype exhibited by mice lacking the cathepsin L gene was rescued by a transgenic strain bearing human cathepsin V but not human cathepsin L (7), suggesting specific and distinct roles for these isoenzymes.Recently, an internally translated form of murine cathepsin L with a shortened preproregion was shown to traffic to the nucleus (8). Nuclear murine cathepsin L was able to cleave the transcription factor CDP/Cux and thereby influence progression through the cell cycle (8). These studies and others (9 -12), have begun to uncover a hitherto unsuspected role for cathepsins outside endocytic compartments. Further, the observation that these cathepsins are unstable at neutral pH and hence that their proteolytic activity should be confined to acidic organelles of the cell has been shown to be an artifact of buffer choice in in vitro experiments (11)(12)(13)(14). It is clear that in conditions mimicking physiological pH and ionic strength, th...

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“…The ϩCys-cystatin A variant, having an extra Cys at the N-terminus, was expressed with a His-tag, and purified essentially as in previous work~Pol et al, 1995;Estrada et al, 1998!, although with minor modifications due to the presence of the free cysteine residue. As the formation of dimers, like those seen with cystatin B Turk et al, 1992!, might have interfered with the cleavage of the His-tag by enterokinase, the protein was treated with DTT before incubation with the enzyme.…”

Section: Expression and Labelingmentioning

confidence: 99%

“…However, such truncation does not affect the rate of complex formation with these proteinases, indicating that the N-terminal region is not necessary for the fast association of the two molecules~Lindahl et al., 1992b;Björk et al, 1994;Estrada et al, 1999!. The N-terminal region may thus bind to proteinases with accessible active sites subsequent to the other binding regions in a reaction that was not detected by the kinetics studies. However, interaction of the N-terminal region with cathepsin B appears to precede and facilitate the displacement of the occluding loop in the binding of cystatins to this enzyme~Björk et al, 1994enzyme~Björk et al, , 1995enzyme~Björk et al, , 1996Nycander et al, 1998!. In this work, we have used the efficient expression system developed for the production of human cystatin A~Pol et al, 1995;Estrada et al, 1998! to express a variant of the inhibitor with an extra cysteine residue before the N-terminal methionine residue of the wild-type protein.…”

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confidence: 99%

The N‐terminal region of cystatin A (stefin A) binds to papain subsequent to the two hairpin loops of the inhibitor. Demonstration of two‐step binding by rapid‐kinetic studies of cystatin A labeled at the N‐terminus with a fluorescent reporter group

et al. 2000

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The three-dimensional structures of cystatins, and other evidence, suggest that the flexible N-terminal region of these inhibitors may bind to target proteinases independent of the two rigid hairpin loops forming the remainder of the inhibitory surface. In an attempt to demonstrate such two-step binding, which could not be identified in previous kinetics studies, we introduced a cysteine residue before the N-terminus of cystatin A and labeled this residue with fluorescent probes. Binding of AANS-and AEDANS-labeled cystatin A to papain resulted in ;4-fold and 1.2-fold increases of probe fluorescence, respectively, reflecting the interaction of the N-terminal region with the enzyme. Observed pseudo-first-order rate constants, measured by the loss of papain activity in the presence of a fluorogenic substrate, for the reaction of the enzyme with excess AANS-cystatin A increased linearly with the concentration of the latter. In contrast, pseudo-first-order rate constants, obtained from measurements of the change of probe fluorescence with either excess enzyme or labeled inhibitor, showed an identical hyperbolic dependence on the concentration of the reactant in excess. This dependence demonstrates that the binding occurs in two steps, and implies that the labeled N-terminal region of cystatin A interacts with the proteinase in the second step, subsequent to the hairpin loops. The comparable affinities and dissociation rate constants for the binding of labeled and unlabeled cystatin A to papain indicate that the label did not appreciably perturb the interaction, and that unlabeled cystatin therefore also binds in a similar two-step manner. Such independent binding of the N-terminal regions of cystatins to target proteinases after the hairpin loops may be characteristic of most cystatin-proteinase reactions.Keywords: cystatin; fluorescence labeling; N-terminal region; papain; stefin; stopped-flow fluorescence; two-step binding Reprint requests to: Ingemar Björk, Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Box 575, SE-751 23 Uppsala, Sweden; e-mail: Ingemar.Bjork@vmk.slu.se.Abbreviations: AANS,~2-~49-acetamido!anilino!naphthalene-6-sulfonic acid; AANS-cystatin A, cystatin A labeled with an AANS group at a Cys residue introduced in the N-terminus; AEDANS, 5-~~~~2-acetyl!amino!ethyl!amino!naphthalene-1-sulfonic acid; AEDANS-cystatin A, cystatin A labeled with an AEDANS group introduced in the N-terminus; app, subscript denoting an apparent equilibrium or rate constant measured in the presence of an enzyme substrate; ϩCys-cystatin A, a variant of cystatin A with an extra Cys residue introduced before Met-1 of the wild-type inhibitor; DTT, dithiothreitol; His-tag, 10 consecutive histidine residues fused to an expressed protein; IAANS,~2-~49-iodoacetamido!anilino!naphthalene-6-sulfonic acid; IAEDANS, 5-~~~~2-iodoacetyl!amino!ethyl!amino!naphthalene-1-sulfonic acid; k ass , bimolecular association rate constant; K d , dissociation equilibrium constant; k diss , dissociation...

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The Role of Gly-4 of Human Cystatin A (Stefin A) in the Binding of Target Proteinases. Characterization by Kinetic and Equilibrium Methods of the Interactions of Cystatin A Gly-4 Mutants with Papain, Cathepsin B, and Cathepsin L

Cited by 38 publications

References 46 publications

Functional expression of recombinant human stefin A in mammalian and bacterial cells

Functional expression of recombinant human stefin A in mammalian and bacterial cells

DNA Accelerates the Inhibition of Human Cathepsin V by Serpins

The N‐terminal region of cystatin A (stefin A) binds to papain subsequent to the two hairpin loops of the inhibitor. Demonstration of two‐step binding by rapid‐kinetic studies of cystatin A labeled at the N‐terminus with a fluorescent reporter group

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