Substrate specificity of human kallikrein 6: salt and glycosaminoglycan activation effects.
Human kallikrein 6 (hK6) is abundantly expressed in the central nervous system and is implicated in demyelinating disease. This study provided biochemical data about the substrate specificity and activation of hK6 by glycosaminoglycans and by kosmotropic salts, which followed the Hofmeister series. The screening of fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp resulted in the finding that Abz-AFRFSQ-EDDnp (where Abz is ortho-aminobenzoic acid and EDDnp is N-[2,4-dinitrophenyl]ethylenediamine)) is the best synthetic substrate described so far for hK6 (kcat/Km 38,667 s(-1) mm(-1)). It is noteworthy that the AFRFS sequence was found as a motif in the amino-terminal domain of seven human ionotropic glutamate receptor subunits. We also examined the hK6 hydrolytic activity on FRET peptides derived from human myelin basic protein, precursor of the Abeta amyloid peptide, reactive center loop of alpha1-antichymotrypsin, plasminogen, and maturation and inactivation cleavage sites of hK6, which were described earlier as natural substrates for hK6. The best substrates were derived from myelin basic protein. The hK6 maturation cleavage site was poorly hydrolyzed, and no evidence was found to support a two-step self-activation process reported previously. Finally, we assayed FRET peptides derived from sequences that span the cleavage sites for activation of protease-activated receptors (PAR) 1-4, and only the substrate with the PAR 2 sequence was hydrolyzed. These results further supported the hypothesis that hK6 expressed in the central nervous system is involved in normal myelin turnover/demyelination processes, but it is unlikely to self-activate. This report also suggested the possible modulation of ionotropic glutamate receptors and activation of PAR 2 by hK6.
Angelo
Pedro Francisco
PF
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-20 São Paulo, Brazil.
Lima
Aurelio Resende
AR
Alves
Fabiana M
FM
Blaber
Sachiko I
SI
Scarisbrick
Isobel A
IA
Blaber
Michael
M
Juliano
Luiz
L
Juliano
Maria Aparecida
MA
D000682:Amyloid / Q000737:chemistry; D016229:Amyloid beta-Peptides / Q000737:chemistry; D001665:Binding Sites; D002951:Citrates / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D006025:Glycosaminoglycans / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007610:Kallikreins / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D004676:Myelin Basic Protein / Q000737:chemistry; D009186:Myelin Sheath / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010958:Plasminogen / Q000737:chemistry; D011485:Protein Binding; D044464:Receptor, PAR-2 / Q000378:metabolism; D018092:Kainic Acid Receptors / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012492:Salts / Q000737:chemistry*; D000077559:Sodium Citrate; D013379:Substrate Specificity; D000514:alpha 1-Antichymotrypsin / Q000737:chemistry; D000097806:GluK2 Kainate Receptor
D000682:Amyloid; D016229:Amyloid beta-Peptides; D002951:Citrates; D006025:Glycosaminoglycans; D007610:Kallikreins; D004676:Myelin Basic Protein; D010455:Peptides; D010958:Plasminogen; D044464:Receptor, PAR-2; D018092:Kainic Acid Receptors; D011994:Recombinant Proteins; D012492:Salts; D000077559:Sodium Citrate; D000514:alpha 1-Antichymotrypsin; D000097806:GluK2 Kainate Receptor; C103551:KLK6 protein, human