Kallikrein hK14 a proteolytic enzyme highly expressed in cancer cells: The effect of glycosaminoglycans on its pH x activity profile.
Bruna Visniauskas† Tadaki Kishi§; Loyse M. Felber§; Eleftherios P. Diamandis Christoph Kündig§ ;Ivarne L.S.Tersariol†* ;David Deperthes§ ;Jair Ribeiro Chagas†*
§Med Discovery SA, CH-1066 Epalinges, Switzerland; ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada; †Centro Interdisciplinar de Investigacao Bioquimica, Universidade de Mogi das Cruzes, Brazil; *Universidade Federal de São Paulo – UNIFESP.
The human tissue kallikrein family is composed of 15 secreted serine proteases (denoted hK), encoded by 15 highly similar genes (denoted KLK) in terms of structure and regulation. Several genes of the kallikrein family are aberrantly expressed in various cancers but especially in hormone-dependent cancers such as prostate, breast, ovarian or testicular cancers. One gene, KLK14, encoding the human kallikrein 14 protein (hK14) is expressed in various biological fluids and tissues, including central nervous system and in endocrine-related tissues such as breast, prostate, thyroid and uterus. Recently, we characterized the enzymatic activity of human kallikrein 14 using phage display technology and identified trypsin and chymotrypsin-like activities with a preference for an arginine residue in position P1. Here we present data showing evidences of hk14 interactions with glycosaminoglycans, heparin and chondroitin sulphate, at 20uM final concentration. pH activity curves, in absence and presence of glycosaminoglycans, using the fluorogenic substrate Abz-TFRSAK(Dnp)-NH2 at 5 uM show a 1 to 1,5 pH value displacement and 1.5 to 2.0 fold increase on the kcat/Km values. These data are important to planning of selection methodologies to find substrates and/or inhibitors for proteolytic enzymes as they represent more closely the true physiological conditions.
|