Cloning and expression of rhesus monkey cathepsin K

Cathepsin K is a cysteine protease involved in degradation of human type I collagen and plays a primary role in bone resorption. We have cloned rhesus monkey cathepsin K by reverse transcriptase-polymerase chain reaction (RT-PCR) from rhesus ovary poly A(+) RNA, The sequence for the rhesus enzyme is 98% identical to that of the human with 100 % identity within the mature active form of cathepsin K, Rhesus monkey cathepsin K was transiently expressed in Chinese hamster ovary (CHO) cells and found to be secreted as the proenzyme in the culture media and 50% activated to the mature form intracellularly, The substrate specificity preference of aminomethylcoumarin and rhodamine peptide substrates was Leu > Phe > Pro in the P-2 position when tested with constant arginine at P-1. The enzyme activity expressed in CHO cell extracts was sensitive to inhibition by E-64 and cystatin with IC(50)s of 3.5 nmol/L and 13 ng/mL, respectively, The apparent second order rate constants of inactivation by E-64 were 66,000 M-1 s(-1) and 130,000 M-1 s(-1) for the recombinantly expressed rhesus monkey and human cathepsin K, respectively. The high similarity between the sequences and the kinetic properties of rhesus monkey and human cathepsin K establishes this monkey species as a suitable animal model for development of novel cathepsin K inhibitors as antiresorptive agents. (C) 1999 by Elsevier Science Inc, All rights reserved.