Improvement of cathepsin S detection using a designed FRET peptide based on putative natural substrates.
Cathepsin S is a lysosomal cysteine peptidase of the papain superfamily which is implicated in physiological and pathological states. The enzyme is highly expressed in antigen presenting cells and is thought to play an important role in the processing of the major histocompatibility complex (MHC) class II-associated invariant chain. In pathological processes, cathepsin S is associated with Alzheimer's disease, atherosclerosis and obesity and can be regarded as a potential target in related disorders. However, due to the broad substrate specificities of the lysosomal cathepsins, the specific detection of cathepsin S is difficult when other cathepsins are present. In an attempt to distinguish cathepsin S from other cathepsins we synthesized and tested fluorescence resonance energy transfer (FRET) peptides derived from two of its putative natural substrates, namely insulin beta-chain and class II-associated invariant chain (CLIP). The influence of ionic strength on the catalytic activity and the enzyme stability in neutral pH was also analyzed. Using data gathered from our study we developed a selective substrate for cathepsin S and establish the assay conditions to differentiate the enzyme from cathepsins L, B, V and K. The peptide Abz-LEQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp=N-[2,4-dinitrophenyl]ethylenediamine]) in 50mM sodium phosphate buffer, pH 7.4, containing 1M NaCl was hydrolyzed by cathepsin S with k(cat)/K(m) value of 3585mM(-1)s(-1), and was resistant to hydrolysis by cathepsins L, V, K and B. Thus, we developed a sensitive and selective cathepsins S substrate that permits continuous measurement of the enzymatic activity even in crude tissue extracts.
Oliveira
Marcela
M
Department of Biophysics, Federal University of São Paulo, Rua 3 de Maio 100, São Paulo, Brazil.
Torquato
Ricardo J S
RJ
Alves
Marcio F M
MF
Juliano
Maria A
MA
Brömme
Dieter
D
Barros
Nilana M T
NM
Carmona
Adriana K
AK
D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000235:genetics / Q000378:metabolism; D056657:Cathepsin K / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002403:Cathepsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D000949:Histocompatibility Antigens Class II / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014020:Tissue Extracts / Q000737:chemistry
D000944:Antigens, Differentiation, B-Lymphocyte; D000949:Histocompatibility Antigens Class II; D010455:Peptides; D011994:Recombinant Proteins; D014020:Tissue Extracts; C045084:invariant chain; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C028217:cathepsin S; D056657:Cathepsin K; C117627:CTSV protein, human