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 P2 position when tested with constant arginine at P1. The enzyme activity expressed in CHO cell extracts was sensitive to inhibition by E-64 and cystatin with IC50s 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.
Guay
J
J
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada.
Riendeau
D
D
Mancini
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000201:enzymology; D056657:Cathepsin K; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003374:Coumarins / Q000378:metabolism / Q000494:pharmacology; D006224:Cricetinae; D015891:Cystatins / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
C112287:Ac-aspartyl-glutamyl-valyl-aspartyl-aminomethylcoumarin; D003374:Coumarins; D015891:Cystatins; D017931:DNA Primers; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D009842:Oligopeptides; D010446:Peptide Fragments; D011062:Polynucleotide Adenylyltransferase; D002403:Cathepsins; C534130:CTSK protein, human; D056657:Cathepsin K; D007930:Leucine; C024974:E 64