Cynomolgus monkey (Macaca fascicularis) cathepsin K: cloning, expression, purification, and activation.
Methodology for the production of recombinant active cynomolgus monkey (Macaca fascicularis) cathepsin K (EC 3.4.22.38) was elucidated. The cDNA encoding the cathepsin K was cloned from female M. cynomolgus monkey mRNA. The deduced amino acid sequence of M. cynomolgus preprocathepsin K from the cDNA sequence showed 94.2% identity to human preprocathepsin K. Sequence differences occurred only in the prepro- domains; the mature domains were identical. The recombinant M. cynomolgus cathepsin K was expressed as a secreted proenzyme using baculovirus-infected SF21 insect cells having the predicted N-terminus (LYPEEILDTH ellipsis ), indicating proper cleavage of the secretion sequence. Purified monkey procathepsin K was activated under autocatalytic conditions at pH 4.0. The mature enzyme was composed of mixture of enzymes having N-termini of Gly113 and Arg114. The molecular weight was determined to be 23,668.3 Da by MALDI-TOF-MS which is consistent with the absence of carbohydrate on the mature enzyme. These results indicate that monkey procathepsin K is able to autoactivate and produces a mature enzyme which is identical to that of human cathepsin K. Since the sequence of monkey and human mature cathepsin K are identical and the in vitro activation mechanisms appear to be indistinguishable, monkeys are predicted to be a good animal model for evaluating cathepsin K inhibitors in vivo as therapeutic agents for diseases characterized by excessive bone loss, such as osteoporosis.
McQueney
M S
MS
Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania, 19406, USA. Michael_S_McQueney@sbphrd.com
Feild
J
J
Hanning
C R
CR
Brun
K
K
Ramachandran
K
K
Connor
J
J
Drake
F
F
Jones
C S
CS
Amegadzie
B Y
BY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016723:Bone Remodeling / Q000187:drug effects / Q000502:physiology; D056657:Cathepsin K; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008252:Macaca fascicularis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D018411:Spodoptera / Q000166:cytology
D004791:Enzyme Inhibitors; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D002403:Cathepsins; C534130:CTSK protein, human; D056657:Cathepsin K