title: Characterization of SPINK9, a KLK5-specific inhibitor expressed in palmo-plantar epidermis.
abstract: SPINK9, a Kazal-type serine protease inhibitor, is almost exclusively expressed in the palmo-plantar epidermis. SPINK9 selectively inhibits kallikrein-related peptidase 5 (KLK5), no other target enzyme is known at present. In this study, we defined the reactive loop to residues 48 and 49 of SPINK9 and characterized the inhibition and binding of different SPINK9 variants towards KLK5, KLK7, KLK8 and KLK14. Substitutions of single amino acids in the reactive loop had a large impact on both inhibitory efficiency and specificity. Binding studies showed that it is mainly the dissociation rate that is affected by the amino acid substitutions. The inhibitory effect of wild-type SPINK9 was clearly pH-dependent with an improved effect at a pH similar to that of the outer layers of the skin. Modeling of the enzyme-inhibitor complexes showed that the reactive loop of SPINK9 fits very well into the deep negatively charged binding pocket of KLK5. A decrease in pH protonates His48 of the wild-type protein resulting in a positively charged residue, thereby explaining the observed decreased dissociation rate. Interestingly, substitution with a positively charged amino acid at position 48 resulted in a more efficient inhibitor at higher pH.
authors.affiliation: Umeå University, Department of Medical Biochemistry and Biophysics, Sweden.
authors.forename: Kristoffer
authors.initials: K
authors.lastname: Brännström
authors.forename: Anders
authors.initials: A
authors.lastname: Ohman
authors.forename: Ulrich
authors.initials: U
authors.lastname: von Pawel Rammingen
authors.forename: Anders
authors.initials: A
authors.lastname: Olofsson
authors.forename: Maria
authors.initials: M
authors.lastname: Brattsand
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004817:Epidermis / Q000201:enzymology*; D005528:Foot; D005786:Gene Expression Regulation; D006225:Hand; D006801:Humans; D007610:Kallikreins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D053491:Proteinase Inhibitory Proteins, Secretory / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000074162:Serine Peptidase Inhibitors, Kazal Type; D013379:Substrate Specificity
chemical_list: D007610:Kallikreins; D011480:Protease Inhibitors; D053491:Proteinase Inhibitory Proteins, Secretory; D000074162:Serine Peptidase Inhibitors, Kazal Type; C542011:SPINK9 protein, human; C400147:KLK5 protein, human