Cathepsin substrate specificity

Cleavage occur s at the scissile bond. Residues in the substrate towards the N-terminus are numbered PI, P2, P3, etc, whereas residues towards the C-terminus are numbered PI, P2, P3 etc. Cleavage occurs between PI and P1. For a peptidase with limited specificity, only the residue in PI or PI is important for specificity. A peptidase with an extended substrate binding site will have a preference for residues in other positions. For example cathepsin L prefers substrates with phenylalanine in P2 and arginine in PI. However, this is a preference only, and cathepsin L cleaves substrates after other amino acids. Caspase-3 has a preference for Asp in both P4 and PI, but it is unusual for substrate specificity to extend much further from the scissile bond. The peptidase with the most extended substrate specificity may be mitochondrial intermediate peptidase that removes an octopeptide targeting signal from the N-terminus of cytoplasmically synthesized proteins that are destined for import into the mitochondrial lumen. 

This peptidase family Cl enzyme [EC 3.4.22.27] is a lysosomal cysteinyl-dependent endopeptidase with substrate specificity similar to cathepsin L. 

The reaction of human leukocyte elastase has been studied with a number of azapeptide p-nitrophenyl esters and some of the results are listed in Table VII. All of the azapeptides acylate elastase except Ac-Ala-Aphe-ONp, which reacts very slowly. However this azapeptide will react with cathepsin G as expected from the differing substrate specificity of the two enzymes. The kinetics of the reaction are described in detail elsewhere (36), but with most of the inhibitors, kCSLt is equal to the deacylation (or reactivation) rate of the acylated enzyme. Azapep-... 

Aibe, K. et al. 1996. Substrate specificity of recombinant osteoclast-specific cathepsin K from rabbits. Biol. Pharrn. Bull. 19, 1026-1031. 

Arnold D, Keilholz W, Schild H-J, Dumrese T, Stevanovic S, Rammensee H-G (1997) Substrate specificity of cathepsins D and E determined by N-lerminal and C-terminal sequencing of peptide-pools, Eur J Biochem 249 171-179. 

Cathepsins. Cathepsins are intracellular proteases of animal origin. The occurrence of several such enzymes has been demonstrated in various tissues, including spleen, pituitary gland, kidney, thymus, etc. It is obvious that there is no reason to anticipate that all cathepsins will have similar properties to each other or to any other proteases. Cathepsins have been designated by both Roman numerals and by letters. Some of these enzymes have been identified with enzymes purified independently, as cathepsin III with leucine aminopeptidase. Several are activated by sulfhydryl compounds, some by metals. The isolation of the various cathepsins and studies of their substrate specificities are subjects currently under investigation, but because of the lower concentration of enzyme in the source materials and the number of related enzymes present, this area of investigation has not reached the development of the study of digestive enzymes. 

It may be concluded that cathepsin D is quite similar to pepsin in its substrate specificity, two adjacent hydrophobic groups providing the most susceptible linkage, and rates of hydrolysis increasing markedly as peptide chain length grows up to about six residues. Cathepsin D, however, has a much more restricted series of di- and tri-peptides that it can hydrolyze, and must have obvious differences in its binding site. 

Additional serine proteases have been found in chronic wounds and neutrophils probably are also the primary source of these enzymes. These include cathepsin G, another cationic serine protease with broad substrate specificity, urokinase-type plasminogen activator (itfA), and protease 3 (3,32-35). In all probability, when looked for, elevated levels of additional neutrophil proteases will be found in chronic dermal ulcers. 

At least four proteolytic enzymes, known as kidney cathepsins I, II, III, and IV, were found in crude kidney extracts. In their substrate specificity these intracellular proteinases show similarities to pepsin, trypsin, aminop tidase, and carboxypeptidase, respectively (11). Plentl and Page (139) showed that cathepsins I and IV are not identical with renin. Schales, Holden, and Schales (160) differentiated renin from all four cathepsins. The experimental proof for the nonidentity of renin and kidney cathepsins is summarized in Table IV. 

This lysosomal enzyme [EC 3.4.22.1], also known as cathepsin Bl, is a member of the peptidase family Cl. The catalyzed reaction is the hydrolysis of peptide binds with a broad specificity. The enzyme prefers the ArgArg—Xaa bond in small peptide substrates (thus distinguishing this enzyme from cathepsin L). The enzyme also exhibits a peptidyl-dipeptidase activity, releasing C-terminal dipeptides from larger polypeptides. 

Substrates for hepatic metabolism include insulin, glucagon, and t-PAs [89,90]. For insulin, an acidic endopeptidase (termed endosomal acidic insulinase ) appears to mediate internalized insulin proteolysis at a number of sites [91]. Specifically, the endosomal activity results from cathepsin D, an aspartic acid protease [92]. Similarly, proteolysis of glucagon has also been attributed to membrane-bound forms ofcathepsins B and D [93]. 

Abz was combined with a broad variety of non-fluorescent acceptors such as p-nitrobenzyl for leucine aminopeptidase (Carmel et al., 1977), pNA for trypsin (Bratanova and Petkov, 1987), 4-ni-trophenylalanine [Phe(NC>2)] for HIV protease (Toth and Marshall, 1990), and V-(2,4-di n itrophenyl) ethylenediamine (EDDnp) for thermolysin and trypsin (Nishino et al., 1992). Lecaille et al. (2003) described a FRET quench assay based on a specific substrate for cathepsin K labeled with Abz and EDDnp. This substrate is not cleaved by the other Cl cysteine cathepsins and serine proteases in contrast to methoxycoumarin (Mca)-based substrates described earlier (Aibe et al., 1996 Xia et al., 1999) and merely covered the non-primed site of the scissile bond. The 5-[(2-aminoethyl)amino] naphthalene-l-sulfonic acid (EDANS) compound is a second example of a fluorescence donor historically used for many FRET quench-based protease assays, e.g., in combination with tryptophan as a quencher in an ECE activity assay (Von Geldren et al., 1991). The FRET-1 example in Table 2.2 shows the typical dynamic range that can be achieved with an EDANS/DABCYL-based assay. 

Studies of the cleavage specificity of cathepsin L demonstrated that it prefers to cleave on the NH2-terminal side of dibasic residue processing sites of enkephalin-containing peptide substrates BAM-22P and Peptide F (22) and to cleave at the N-terminal sides of dibasic residues within peptide-MCA substrates (32). The cleavage specificity of cathepsin L results in enkephalin intermediate peptides with NH2-terminal basic residue extensions, which are then removed by Arg/Lys aminopeptidase. Secretory vesicles from adrenal medullary chromaffin cells (33) and from pituitary (34) contain Arg/Lys aminopeptidase activity for neuropeptide production. 

Recent molecular cloning studies have identified aminopeptidase B as an appropriate Arg/Lys aminopeptidase (35). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that provided production of specific antisera to demonstrate localization of AP-B in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and NPY. AP-B was also found in several neuroendocrine tissues by western blots. Recombinant bovine AP-B (35) and rat AP-B were compared. Recombinant bovine AP-B showed preference for Arg-MCA substrate compared with Lys-MCA. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin and Lys-(Met)enkephalin neuropeptide substrates to generate (Met)enkephalin, whereas rat AP-B preferred Arg-(Met)enkephalin. Furthermore, AP-B possesses an acidic pH optimum of 5.5-6.5 that is similar to the internal pH of secretory vesicles. The significant finding of the secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides. 

Like other proteases, cathepsins are synthesized as high molecular weight precursors that require processing for activation. Cathepsin B (CB) is a thiol-dependent protease normally found in lysosomes, and is activated by cathepsin D (CD) and matrix metahoproteinases. Activated CB can in turn activate uPA and specific metalloproteinases. Cathepsin L (CL) is similar in specificity to that of CB however, it has little activity toward small molecular substrates. Cathepsin D, like CB, is a lysosomal protease however, CD belongs to the aspartyl group of proteases. 

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