S3 to S3' subsite specificity of recombinant human cathepsin K and development of selective internally quenched fluorescent substrates

Abstract

We have systematically examined the S3 to S3' subsite substrate specificity requirements of cathepsin K using internally quenched fluorescent peptides derived from the lead sequence Abz-KLRFSKQ-EDDnp [where Abz is o -aminobenzoic acid and EDDnp is N -(2,4-dinitrophenyl)ethylenediamine]. We assayed six series of peptides, in which each position except Gln was substituted with various natural amino acids. The results indicated that the S3-S1 subsite requirements are more restricted than those of S1'-S3'. Cathepsin K preferentially accommodates hydrophobic amino acids with aliphatic side chains (Leu, Ile and Val) in the S2 site. Modifications at P1 residues also have a large influence on cathepsin K activity. Positively charged residues (Arg and Lys) represent the best accepted amino acids in this position, although a particular preference for Gly was found as well. Subsite S3 accepted preferentially basic amino acids such as Lys and Arg. A broad range of amino acids was accommodated in the remaining subsites. We further explored the acceptance of a Pro residue in the P2 position by cathepsin K in order to develop specific substrates for the enzyme. Two series of peptides with the general sequences Abz-KXPGSKQ-EDDnp and Abz-KPXGSKQ-EDDnp (where X denotes the position of the amino acid that is altered) were synthesized. The substrates Abz-KPRGSKQ-EDDnp and Abz-KKPGSKQ-EDDnp were cleaved by cathepsin K at the Arg-Gly and Gly-Ser bonds respectively, and have been shown to be specific for cathepsin K when compared with other lysosomal cysteine proteases such as cathepsins L and B and with the aspartyl protease cathepsin D.

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

“…3C), consistent with literature reports (68,69). We observed a minor preference for P 2 Lys (for the P 1 ϭ Arg sublibrary), consistent with a previous report of cathepsin K accepting P 2 basic residues (69).…”

“…Unlike the homologous cathepsins V, L, and H, cathepsin K also accepted Pro at the P 2 position. This P 2 Pro acceptance is required for the unique collagenase activity of cathepsin K (68) and has been noted previously in both PS-SCL (68) and fluorescence resonance energy transfer (FRET) studies (69). While cooperative interactions between substrate subsites cannot be detected using PS-SCLs, the study by Alves et al using FRET substrates had noted that the acceptance of P 2 Pro residues was dependent on the neighboring substrate residues.…”

“…While cooperative interactions between substrate subsites cannot be detected using PS-SCLs, the study by Alves et al using FRET substrates had noted that the acceptance of P 2 Pro residues was dependent on the neighboring substrate residues. Of the six P 2 Pro substrates tested in the FRET study, the most efficient cleavage was observed with substrates containing a P 3 Lys residue (69). We found that for our Ac-Ala-P 3 -Pro-(Arg/Lys)-ACC-NH 2 substrates, P 3 Lys substrates were among the most efficient (along with P 3 ϭ Pro and Gly for the P 1 ϭ Arg sublibrary and P 3 ϭ Gly, Ile, and Leu for the P 1 ϭ Lys sublibrary).…”

Proteolytic Excision of a Repressive Loop Domain in Tartrate-resistant Acid Phosphatase by Cathepsin K in Osteoclasts

“…Substrate specificity studies have shown that the primary specificity of papain-like cysteine proteinases is determined by S2-P2 interactions with a preference in cathepsin K for Pro or hydrophobic amino acids with aliphatic side chains (Leu, Ile, and Val) in the P2 position, whereas cathepsin L prefers bulky aromatic amino acids (Phe, Trp, and Tyr) or hydrophobic amino acids (67,68). In the P1 position, the substrate preference is similar in cathepsin L and K with a preference for amino acids with a hydrophilic side chain such as Arg, Lys, Gln, or Met (31,67,68), although amino acids with short side chains such as Gly or Ser can also be allowed (69,70 Both cathepsin K and L are expressed in osteoclasts (71), although cathepsin K is more abundant than cathepsin L at the mRNA (72,73) and activity (52) levels.…”

The use of Fluorescence Resonance Energy Transfer (FRET) peptidesfor measurement of clinically important proteolytic enzymes

“…Peptides up to 20 residues can provide significant increases in florescence (de Souza et al 2000), allowing the measurement of the enzymatic activity on continuous base. The FRET peptides introduced by Chagas et al (1991) was a breakthrough in the study of proteases' specificity, and the synthesis of different Abzpeptidyl-EDDnp sequences provided the opportunity for us to study the activity of various endopeptidases such as human renin (Oliveira et al 1992), kallikreins (Chagas et al 1995, Del Nery et al 1995, Portaro et al 1997, Angelo et al 2006), cathepsin G (Réhault et al 1999, Korkmaz et al 2008), cathepsin D (Pimenta et al 2000, pro hormone convertase (Johanning et al 1998), lysosomal cathepsins (Portaro et al 2000, Alves et al 2003, Puzer et al 2004) and neprilysin (Medeiros et al 1997).…”