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J. Biol. Chem., Vol. 281, Issue 6, 3116-3126, February 10, 2006

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Substrate Specificity of Human Kallikrein 6

SALT AND GLYCOSAMINOGLYCAN ACTIVATION EFFECTS^*

Pedro Francisco Angelo, Aurelio Resende Lima, Fabiana M. Alves, Sachiko I. Blaber, Isobel A. Scarisbrick^¶1, Michael Blaber², Luiz Juliano, and Maria Aparecida Juliano³

From the Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-20 São Paulo, Brazil, the Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida 32306-4300, and the ^¶Departments of Neurology, Physical Medicine, and Rehabilitation, Mayo Medical and Graduate School, Mayo Clinic, Rochester, Minnesota 55905

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 (k_cat/K_m = 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 A amyloid peptide, reactive center loop of ₁-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.

Received for publication, September 14, 2005 , and in revised form, November 30, 2005.

^* This work was supported by Fundação de Amparo Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Científico e Tecnológico. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by National Multiple Sclerosis Society Grant RG 3406-A-2.

² Supported by National Multiple Sclerosis Society Grant RG-3367-A-2.

³ To whom correspondence should be addressed: Dept. de Biofísica Escola Paulista de Medicina-UNIFESP Rua Três de Maio, 100 São Paulo-04044-020 Brazil. Tel.: 55-11-5576-4455; Fax: 55-11-5575-9617; E-mail: juliano.biof{at}epm.br.

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