The specificity of bovine spleen cathepsin S. A comparison with rat liver cathepsins L and B

Brὂmme, Dieter; Steinert, Albert; Friebe, Sieglinde; Fittkau, S.; Wiederanders, Bernd; Kirschke, Heidrun

doi:10.1042/bj2640475

Cited by 126 publications

(82 citation statements)

References 25 publications

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“…3E). Similar P 2 specificity for hydrophobic residues has been observed in single substrate studies for bovine and human cathepsin S (58,71). The diminished acceptance of P 2 Val residues relative to cathepsin V and cathepsin L may be attributed to the presence of a Val residue that more greatly restricts the entrance to the S 2 pocket in cathepsin S relative to cathepsin V (which contains a Leu residue at this position) and Cat L (which has a Met residue at this position).…”

Section: Resultsmentioning

confidence: 51%

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Gosalia

Salisbury

Ellman

et al. 2005

Molecular & Cellular Proteomics

152

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Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format AcAla-X-X-(Arg/Lys)-coumarin was synthesized (X ‫؍‬ all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXa␤, factor XIa and factor ␣ XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates ؋ 24 different proteases ؋ 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P 2 preference for Leu, Val, Phe, and Tyr in both the P 1 ‫؍‬ Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage. Molecular & Cellular Proteomics 4:626 -636, 2005.Because of their critical roles in biological pathways like hormone activation, proteasomal degradation, and apoptosis, proteases are essential for cellular function and viability. Proteases regulate hormonal activation, cellular homeostasis, apoptosis, and coagulation and play an important role in the pathogenicity and progression of many diseases (1). Proteases comprise one of the largest protein families in organisms from Escherichia coli to humans (2-4). Improved understanding of proteases will provide insight into biological systems and will likely provide a number of important new therapeutic targets (1).To properly function, proteases must preferentially cleave their target substrates in the presence of other proteins. While many factors impact protease substrate selection, one of the key aspects is the complementary nature of the enzymeactive site with the residues surrounding the cleaved bond in the substrate. As such, determination of the residues that comprise the preferred cleavage site of a protease provides critical information regarding substrate selection. Furthermore, determination of substrate specificity also provides a framework for the design of potent and selective inhibitors.Here we exploit solution-phase substrate nanodroplet microarrays (5), in which fluorogenic substrates suspended in glycerol droplets are treated with aerosolized aqueous enzyme solutions, to provide protease substrate specificity profiles (6). These arrays allow high throughput characterization of the preferred residues on the P side (7) of the substrate in a highl...

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Section: Resultsmentioning

confidence: 51%

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Gosalia

Salisbury

Ellman

et al. 2005

Molecular & Cellular Proteomics

152

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“…Both the F: hepatica cathepsins L1 and L2 have a high affintiy (k,,, /Km) for the substrate Z-Phe-Arg-NHMec and low affinity for the substrates Z-Arg-Arg-NHMec and Bz-ArgNHMec. The preference for a hydrophobic phenylalanine residue in the P2 position is consistent with the classification of both I;: hepatica proteinases as cathepsin L species [2] or cathepsin S proteases [26,271. Both enzymes, however, have a threefold greater affinity for the substrate Boc-Val-LeuLys-NHMec over the substrate Z-Phe-Arg-NHMec ; the cathepsin L2 has greater than twice the affinity for these two substrates when compared to the cathepsin L1.…”

Section: Kinetic Studies Of E Hepatica Cathepsins L1 and L2supporting

confidence: 54%

“…Bromme et al [26] showed that bovine cathepsin L had a tenfold preference for the substrate Z-Phe-Arg-NHMec Other strips were incubated in 10 pM fluorogenic peptide substrate. The substrates used were, Z-Phe-Arg-NHMec (FR) ; Boc-Val-LeuLys-NHMec (VLK); Tos-Gly-Pro-Arg-NHMec (GPR) and Tos-GlyPro-Lys-NHMec (GPK).…”

Section: Kinetic Studies Of E Hepatica Cathepsins L1 and L2mentioning

confidence: 99%

Purification and characterisation of a second cathepsin L proteinase secreted by the parasitic trematode Fasciola hepatica

Dowd¹,

Smith²,

McGonigle³

et al. 1994

European Journal of Biochemistry

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A 29.5-kDa cysteine proteinase was purified from medium in which mature Fasciola hepatica parasites were maintained. The N-terminal sequence (14 residues) of the purified protein is similar to known cathepsin L proteinases, including a 27-kDa cathepsin L proteinase, also secreted by this parasite, which had been isolated previously in our laboratory [Smith, A. M., Dowd, A. J., Mc Gonigle, S., Keegan, P. S., Brennan, G., Trudgett, A. & Dalton, J. P. (1993) Mol. Biochem. Parasitol. 62,. The N-terminal sequences of the 29.5-kDa and 27-kDa cathepsin L proteinases differ only in residue number seven (arginine and proline, respectively). Immunoblot studies, using antiserum that reacts with both cathepsin L proteinases, rule out the possibility of both enzymes arising from a higher molecular sized parent molecule. The reaction kinetics of the two F. hepatica cathepsin L proteinases on a variety of peptide substrates revealed that the two enzymes differ in their substrate specificity. Five peptide substrates that are cleaved with high affinity by the 29.5-kDa cathepsin L isolated in this study are not cleaved by the previously purified 27-kDa cathepsin L. The protein-modifying reagent, tetranitromethane, affected the 29.5-kDa cathepsin L proteinase only, causing inactivation of the enzyme and changing its migration in polyacrylamide gel electrophoresis. Our studies suggest that the two F. hepatica cysteine proteinases represent two distinct subclasses within the cathepsin L class.Cathepsin L proteases are lysosomal cysteine proteinases which, along with cathepsins B, H and S, belong to the papain superfamily [ l -31. Cathepsin L can hydrolyse a variety of substrates such as extracellular matrix proteins, collagen and elastin [l, 41 and type-IV collagen which is a major component of basement membranes [l]. Cathepsin B can indirectly cause the degradation of collagen since it is capable of converting inactive procollagenase IV to its enzymically active form [5]. It is suggested that where certain malignant tissues secrete cathepsin-like proteinases, these enzymes may be involved in extracellular matrix destruction, in the facilitation of cellular detachment from primary tumour masses, and in reinvasion of tissue [4].Cathepsins L and B are synthesised as inactive, alkalistable, precursors (proenzymes) of 39 kDa prior to their targeting to the lysosomes [6, 71. Cathepsin H is synthesised as a 41-kDa proenzyme [7]. The proenzymes are subseCorrespondence to J. P. Dalton, School of Biological Sciences,

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“…According to previously reported studies, the specificity of CatS is mainly determined by the amino acid at the P-2 position (9,(32)(33)(34). In those studies, conclusions on the CatS specificity were often drawn from the cleavage of fluorogenic substrates containing the fluorophore at the P-1Ј position.…”

Section: Discussionmentioning

confidence: 99%

Quantifying Cathepsin S Activity in Antigen Presenting Cells Using a Novel Specific Substrate

Lützner

Kalbacher

2008

Journal of Biological Chemistry

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Cathepsin S (CatS) is a lysosomal cysteine protease belonging to the papain superfamily. Because of the relatively broad substrate specificity of this family, a specific substrate for CatS is not yet known. Based on a detailed study of the CatS endopeptidase specificity, using six series of internally quenched fluorescent peptides, we were able to design a specific substrate for CatS. The peptide series was based on the sequence GRWHTVGL-RWE-Lys(Dnp)-DArg-NH 2 , which shows only one single cleavage site between Gly and Leu and where every substrate position between P-3 and P-3 was substituted with up to 15 different amino acids. The endopeptidase specificity of CatS was mainly determined by the P-2, P-1, and the P-3 substrate positions. Based on this result, systematically modified substrates were synthesized. Two of these modified substrates, Mca-GRWPP-MGLPWE-Lys(Dnp)-DArg-NH 2 and Mca-GRWHPMGAPWELys(Dnp)-DArg-NH 2 , did not react with the purified cysteine proteases cathepsin B (CatB) and cathepsin L (CatL). Using a specific CatS inhibitor, we could further show that these two peptides were not cleaved by endosomal fractions of antigen presenting cells (APCs), when CatS was inhibited and related cysteine proteases cathepsin B, H, L and X were still active. Although aspartic proteases like cathepsin E and cathepsin D were also present, our substrates were suitable to quantify cathepsin S activity specifically in APCs, including B cells, macrophages, and dendritic cells without the use of any protease inhibitor. We find that CatS activity differs significantly not only between the three types of professional APCs but also between endosomal and lysosomal compartments.Lysosomal cysteine proteases of the papain family were long believed to be exclusively involved in nonspecific proteolysis within the lysosome. However, the use of specific protease inhibitors and the study of gene knock-out mice suggested that some of them are involved in many other processes, such as cellular homeostasis, autophagy, apoptosis, and antigen presentation (1-3). Antigen presentation by MHC 2 class II molecules requires the entry of antigens into the endosomal-lysosomal compartment. These antigens are then processed by proteolytic enzymes, of which the lysosomal cysteine proteases of the papain family constitute an important subset. The generated peptides bind to MHC class II molecules, which are then displayed at the surface of professional APCs including macrophages, dendritic cells (DCs), and B cells. The variety of MHC class II peptide products that can activate CD4 ϩ T cells is on the one hand determined by allelic variation in MHC molecule binding specificity and on the other by the identity and level of activity of processing proteases present in APCs. Although CatS is one of the major proteases involved in antigen processing (4 -6), specific determination of its activity in antigen presenting cells is currently complicated because a specific substrate is not yet known. One reason for this is that CatB, CatL, and CatS show relati...

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The specificity of bovine spleen cathepsin S. A comparison with rat liver cathepsins L and B

Cited by 126 publications

References 25 publications

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Purification and characterisation of a second cathepsin L proteinase secreted by the parasitic trematode Fasciola hepatica

Quantifying Cathepsin S Activity in Antigen Presenting Cells Using a Novel Specific Substrate

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