Definition and Redesign of the Extended Substrate Specificity of Granzyme B

Harris, Jennifer L.; Peterson, Erin P.; Hudig, Dorothy; Thornberry, Nancy A.; Craik, Charles S.

doi:10.1074/jbc.273.42.27364

Cited by 182 publications

(165 citation statements)

References 45 publications

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“…Together with the previous Western blot analyses, these data suggest that rec.gzmB and rec.caspase 3 mainly cleave rec.c-gelsolin at the same AA residue. The finding that five out of six cleavage sites of rec.gzmB are processed after aspartic acid, is consistent with the known specificity of mouse gzmB (12). Five of the fragments generated resulted from cleavage after the sequence TAKED 240 , which is located between domain 2 and 3 and, thus, might represent another (dominant) gzmB cleavage site within c-gelsolin.…”

supporting

confidence: 66%

Granzyme B-induced and Caspase 3-dependent Cleavage of Gelsolin by Mouse Cytotoxic T Cells Modifies Cytoskeleton Dynamics

Martin

Pardo

Schill

et al. 2010

Journal of Biological Chemistry

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supporting

confidence: 66%

Granzyme B-induced and Caspase 3-dependent Cleavage of Gelsolin by Mouse Cytotoxic T Cells Modifies Cytoskeleton Dynamics

Martin

Pardo

Schill

et al. 2010

Journal of Biological Chemistry

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“…To precisely define the GB cleavage site(s) in B23, the B23 cDNA was used as a template for sitedirected mutagenesis of potential P 1 aspartic acid residues in B23 that contained the GB tetrapeptide consensus sequence recently described by Thornberry and colleagues (23,24). Cleavage of B23 by GB was completely abolished when the aspartic acid residue D 161 was mutated to alanine (Figure 3), defining LAAD 161 as the GB cleavage site.…”

Section: Resultsmentioning

confidence: 99%

Selective cleavage of nucleolar autoantigen B23 by granzyme B in differentiated vascular smooth muscle cells: Insights into the association of specific autoantibodies with distinct disease phenotypes

Ulanet¹,

Flavahan²,

Casciola‐Rosen³

et al. 2004

Arthritis & Rheumatism

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Objective. To investigate the association of specific autoantibodies with distinct disease phenotypes. The association of autoantibodies to nucleophosmin/ B23 with pulmonary hypertension in scleroderma, and the susceptibility of autoantigens to cleavage by granzyme B (GB), provided a focus for these studies.Methods. Intact cells were subjected to cytotoxic lymphocyte granule-induced death, and the susceptibility of autoantigens to cleavage by GB was addressed by immunoblotting and/or by a novel immunofluorescence assay.Results. B23 was cleaved efficiently by GB in vitro, but was highly resistant to cleavage by GB during cytotoxic lymphocyte granule-mediated death of many intact cell types. In contrast, this molecule was highly susceptible to GB-mediated proteolysis exclusively in differentiated vascular smooth muscle cells. Topoisomerase I and several other GB substrates did not show this striking change in cleavage susceptibility in different cell types.Conclusion. These data demonstrate that the cleavage of B23 by GB in intact cells is dependent upon both cell type and phenotype. The susceptibility of this autoantigen (which is associated with a distinct pulmonary vascular phenotype in scleroderma) to GBmediated proteolysis selectively in vascular smooth muscle cells suggests that the GB-cleavable conformation of autoantigens may occur selectively in the target tissue, and may play a role in shaping the phenotypespecific autoimmune response.

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“…The only difference seen in expression and purification of the two enzymes is the final yield of active enzyme with tryptase ␤I expressing 10-fold more than tryptase ␤II. The phenomenon of reduced expression upon the removal of a glycosylation site has been observed with other proteases and has been postulated to involve decreased stability or solubility of the enzyme lacking post-translational glycosylation (20).…”

Section: Resultsmentioning

confidence: 99%

Definition of the Extended Substrate Specificity Determinants for β-Tryptases I and II

Harris¹,

Niles²,

Burdick³

et al. 2001

Journal of Biological Chemistry

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Tryptases ␤I and ␤II were heterologously expressed and purified in yeast to functionally characterize the substrate specificity of each enzyme. Three positional scanning combinatorial tetrapeptide substrate libraries were used to determine the primary and extended substrate specificity of the proteases. Both enzymes have a strict primary preference for cleavage after the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine. ␤I and ␤II tryptase share similar extended substrate specificity, with preference for proline at P4, preference for arginine or lysine at P3, and P2 showing a slight preference for asparagine. Measurement of kinetic constants with multiple substrates designed for ␤-tryptases reveal that selectivity is highly dependent on ground state substrate binding. Coupled with the functional determinants, structural determinants of tryptase substrate specificity were identified. Molecular docking of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that involves interactions from two adjacent protomers, including P4 Thr-96, P3 Asp-60B and Glu-217, and P1 Asp-189. Based on the determined substrate information, a mechanism-based tetrapeptide-chloromethylketone inhibitor was designed and shown to be a potent tryptase inhibitor. Finally, the cleavage sites of several physiologically relevant substrates of ␤-tryptases show consistency with the specificity data presented here.Mast cells, mediators of inflammatory and allergic response, are found throughout the body concentrated near blood vessels in connective tissue and the mucous membranes of the respiratory and gastrointestinal tract. They play an important role in innate and acquired immune responses through the release of dense granules upon activation. Mast cell activation has also been implicated as a mediator of asthma and other inflammatory diseases. The major components of mast cell secretory granules are the tryptase serine proteases (1). Tryptases are secreted as catalytically active tetramers that are resistant to inactivation by plasma inhibitors. The 3-Å crystal structure has been solved and reveals a ringlike structure with the four active sites facing a central cavity (2). Several in vitro studies have identified multiple substrates for tryptase, including neuropeptides, fibrinogen, stromelysin, prourokinase, prothrombin, and protease-activated receptor-2 (3-6). Human chromosome 16 encodes several homologous tryptase genes, designated tryptase ␣, ␤, and ␥ (7, 8). The ␤-tryptases share greater than 99% sequence identity, with tryptase ␤I and ␤II differing by a single N-glycosylation site. It is unclear why so many highly similar tryptases are expressed by mast cells. One possibility is that they each perform different proteolytic functions that may be reflected in their substrate specificity preferences. Indeed, it has recently been shown that a single amino acid substitution between tryptase ␣ and tryptase ␤II account...

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Definition and Redesign of the Extended Substrate Specificity of Granzyme B

Cited by 182 publications

References 45 publications

Granzyme B-induced and Caspase 3-dependent Cleavage of Gelsolin by Mouse Cytotoxic T Cells Modifies Cytoskeleton Dynamics

Granzyme B-induced and Caspase 3-dependent Cleavage of Gelsolin by Mouse Cytotoxic T Cells Modifies Cytoskeleton Dynamics

Selective cleavage of nucleolar autoantigen B23 by granzyme B in differentiated vascular smooth muscle cells: Insights into the association of specific autoantibodies with distinct disease phenotypes

Definition of the Extended Substrate Specificity Determinants for β-Tryptases I and II

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