Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L.
Cathepsins V and L have high identity and few structural differences. In this paper, we reported a comparative study of the hydrolytic activities of recombinant human cathepsins V and L using fluorescence resonance energy transfer peptides derived from Abz-KLRSSKQ-EDDnp (Abz = ortho-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine). Five series of peptides were synthesized to map the S3 to S2' subsites. The cathepsin V subsites S1 and S3 present a broad specificity while cathepsin L has preference for positively charged residues. The S2 subsites of both enzymes require hydrophobic residues with preference for Phe and Leu. The S1' and S2' subsites of cathepsins V and L are less specific. Based on these data we designed substrates to explore the electrostatic potential differences of them. Finally, the kininogenase activities of these cathepsins were compared using synthetic human kininogen fragments. Cathepsin V preferentially released Lys-bradykinin while cathepsin L released bradykinin. This kininogenase activity by cathepsins V and L was also observed from human high and low molecular weight kininogens.
Puzer
Luciano
L
Department of Biophysics, Escola Paulista de Medicina, UNIFESP, Rua Três de Maio, 100 São Paulo 04044-020, Brazil.
Cotrin
Simone S
SS
Alves
Marcio F M
MF
Egborge
Tobore
T
Araújo
Mariana S
MS
Juliano
Maria Aparecida
MA
Juliano
Luiz
L
Brömme
Dieter
D
Carmona
Adriana K
AK
D002384:Catalysis; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007704:Kininogens / Q000378:metabolism; D009994:Osmolar Concentration; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D055672:Static Electricity; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D007704:Kininogens; D011994:Recombinant Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C117627:CTSV protein, human