title: Activity of human kallikrein-related peptidase 6 (KLK6) on substrates containing sequences of basic amino acids. Is it a processing protease?
abstract: Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R↓R-ACC and Z-R↓R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
authors.forename: Roberta N
authors.initials: RN
authors.lastname: Silva
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
authors.forename: Lilian C G
authors.initials: LCG
authors.lastname: Oliveira
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
authors.forename: Carolina B
authors.initials: CB
authors.lastname: Parise
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
authors.forename: Juliana R
authors.initials: JR
authors.lastname: Oliveira
authors.affiliation: Dipartimento di Farmacia, Università degli Studi di Napoli "Federico II", Via D. Montesano, 49, 80131 Napoli, Italy.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Severino
authors.affiliation: Dipartimento di Farmacia, Università degli Studi di Napoli "Federico II", Via D. Montesano, 49, 80131 Napoli, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Corvino
authors.affiliation: Dipartimento di Farmacia, Università degli Studi di Napoli "Federico II", Via D. Montesano, 49, 80131 Napoli, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: di Vaio
authors.affiliation: The Wohl Institute, King's College London, 5 Cutcombe Rd, London SE5 9RT, UK; Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Comp. Univ. Monte Sant'Angelo Via Cintia 21, 80126 Naples, Italy.
authors.forename: Piero A
authors.initials: PA
authors.lastname: Temussi
authors.affiliation: Dipartimento di Farmacia, Università degli Studi di Napoli "Federico II", Via D. Montesano, 49, 80131 Napoli, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Caliendo
authors.affiliation: Dipartimento di Farmacia, Università degli Studi di Napoli "Federico II", Via D. Montesano, 49, 80131 Napoli, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Santagada
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil. Electronic address: map.juliano@gmail.com.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
mesh_terms: D024361:Amino Acids, Basic / Q000737:chemistry / Q000235:genetics; D004745:Enkephalins / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D045683:Furin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007610:Kallikreins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D053511:Matrix Metalloproteinase 14 / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D020932:Nerve Growth Factor / Q000737:chemistry / Q000378:metabolism; D009414:Nerve Growth Factors / Q000737:chemistry / Q000378:metabolism; D020933:Neurotrophin 3; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D059748:Proteolysis; D013379:Substrate Specificity
chemical_list: D024361:Amino Acids, Basic; D004745:Enkephalins; C000595808:NTF3 protein, human; D009414:Nerve Growth Factors; D020933:Neurotrophin 3; D010455:Peptides; D011498:Protein Precursors; C029992:proenkephalin; D020932:Nerve Growth Factor; D010447:Peptide Hydrolases; C103551:KLK6 protein, human; D007610:Kallikreins; D045683:Furin; D053511:Matrix Metalloproteinase 14; C073922:neurotrophin 4
keywords: Enkephalins; Matrix metalloproteases; Neurotrophins; Plasma kallikrein; Proteolytic enzymes; Tissue kallikrein