A 1.6-kilobase full-length cDNA of a novel human cysteine protease has been isolated and sequenced. The nucleotide sequence encodes a polypeptide of 329 amino acids composed of a 15-residue N-terminal signal peptide, a 99-residue propeptide, and a mature protein of 215 amino acids. The deduced amino acid sequence contains two potential N-glycosylation sites, one located in the proregion and one in the mature enzyme. Comparison of the amino acid sequence of cathepsin O2 with that of known human lysosomal cysteine proteases revealed a substantial degree of similarity to cathepsins L and S. Northern blot analysis indicates predominant levels of expression in osteoclastomas and ovary and therefore the enzyme was named cathepsin O2. The extremely high expression levels of human cathepsin O2 in osteoclastomas suggest a major role of this novel enzyme in bone remodelling and bone related diseases.

Cathepsin O2, a human cysteine protease predominantly present in osteoclasts, has been functionally expressed in Spodoptera frugiperda Sf9 cells using the Autographa californica nuclear polyhedrosis virus. Following in vitro activation at pH 4.0 with pepsin, active enzyme with an apparent molecular weight of 29,000 was obtained. N-terminal sequencing revealed the typical processing site for cysteine proteases of the papain family with a proline in the position adjacent to the N-terminal alanine residue. The S 2 P 2 subsite specificity of human cathepsin O2 is similar to cathepsin S but distinguished from cathepsins L and B. Similar to cathepsin S, cathepsin O2 is characterized by a bellshaped pH activity profile and is stable at pH 6.5 for 30 min at 37°C. Cathepsin O2 is further distinguished by its potent collagenolytic activity against Type I collagen between pH 5 and 6, and elastinolytic activity against insoluble elastin at pH 7.0.Its capacity to efficiently degrade Type I collagen and its high expression in osteoclasts suggest that cathepsin O2 may play a major role in human osteoclastic bone resorption.

Peptidyl vinyl sulphones are a novel class of extremely potent and specific cysteine protease inhibitors. They are highly active against the therapeutically important cathepsins O2, S and L. The highest k inact \K i values exceed 10( M −" :s −" for cathepsin S and 10& M −" :s −" for cathepsins O2 and L. To study the primary specificity site of the novel human cathepsin O2 and the effectiveness of this novel class of inhibitors, a series of peptidyl vinyl sulphones with variations in the P # residue was synthesized. Leucine in the P # position was proven to be the most effective residue for cathepsin O2 and also for cathepsins S and L.

Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a functional cysteine protease of the papain family which is expressed after infection in Spodoptera fruglperda Sf9 cells. The protease displays an inhibition profile typical for cysteine proteases and is highly active against synthetic peptide substrates. The pH optimum of the bell-shaped pH-activity curve is between 5.0 and 5.5. The best substrate tested is Z-Arg-Arg-MCA which is specific for cathepsin B. The specificity constant (Kcat/Km) of AcNPV protease for this substrate is approximately two times higher than for human cathepsin B. In contrast to human cathepsins, AcNPV protease does not exhibit a discriminating specificity towards neutral hydrophobic residues in the P2 position. These substrates are hydrolysed at a ten-fold lower rate than the P2 arginine containing substrate. The pH activity profile against the Z-Arg-Arg-MCA substrate reveals a pK of 5.35 which can be assigned to a glutamate residue in the S2 subsite pocket. Like in cathepsin B, this residue facilitates the binding of positively charged P2 residues in the primary binding pocket. In this respect, the AcNPV protease resembles cathepsin B more than cathepsins L and S.

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