Identification of SAF-2, a novel siglec expressed on eosinophils, mast cells, and basophils.

BACKGROUND: Eosinophils, basophils, and mast cells are believed to be the central tenet cells in allergic conditions including allergic rhinitis, asthma, and eczema. The molecular mechanisms underlying the recruitment of these cells to sites of allergic inflammation are poorly understood. OBJECTIVES: Our aim was to identify a common adhesion molecule that could potentially be responsible for mediating the recruitment of the allergic cell types to the lungs and other sites of allergy. METHODS: We have cloned a sialoadhesin molecule from a human eosinophil library with the use of expressed sequence tag technology and characterized its expression on allergic cells by the use of flow cytometry and specific mAbs. RESULTS: With the use of expressed sequence tag sequencing, we have identified a novel siglec molecule, SAF-2. SAF-2 has homology with other sialoadhesin family members (CD33 and siglec-5) and belongs to a subgroup of the Ig superfamily. SAF-2 is a 431-amino acid protein composed of 3 Ig domains with a 358-amino acid extracellular domain and a 47-amino acid tail. SAF-2 is highly restricted to eosinophils, basophils, and mast cells. Antibodies to SAF-2 do not modulate Ca(++) mobilization or chemotaxis of human eosinophils induced by eotaxin. CONCLUSION: SAF-2 is a highly restricted sialoadhesin molecule, which may be useful in the detection and/or modulation of allergic cells.

Potent dipeptidylketone inhibitors of the cysteine protease cathepsin K.

Cathepsin K (EC 3.4.22.38) is a cysteine protease of the papain superfamily which is selectively expressed within the osteoclast. Several lines of evidence have pointed to the fact that this protease may play an important role in the degradation of the bone matrix. Potent and selective inhibitors of cathepsin K could be important therapeutic agents for the control of excessive bone resorption. Recently a series of peptide aldehydes have been shown to be potent inhibitors of cathepsin K. In an effort to design more selective and metabolically stable inhibitors of cathepsin K, a series of electronically attenuated alkoxymethylketones and thiomethylketones inhibitors have been synthesized. The X-ray co-crystal structure of one of these analogues in complex with cathepsin K shows the inhibitor binding in the primed side of the enzyme active site with a covalent interaction between the active site cysteine 25 and the carbonyl carbon of the inhibitor.

Expression in Escherichia coli, refolding, and purification of human procathepsin K, an osteoclast-specific protease.

We have constructed and optimized a high yielding Escherichia coli expression system to produce glycosylation-free human procathepsin K and have developed conditions for refolding this enzyme. Recombinant human procathepsin K (EC 3.4.22.38) was expressed in E. coli, refolded from inclusion bodies, and further purified by Superdex 75 size-exclusion chromatography. Purified procathepsin K had a [MH]+ of 35,063 Da which is in agreement with the predicted mass of the construct. Amino-terminal sequence analysis matched the predicted sequence with no secondary sequence detected. Purified procathepsin K activated under autocatalytic conditions to a final specific activity of 23 micromol 7-amido-4-methylcoumarin liberated/min/mg of enzyme using the fluorescent peptide substrate benzyloxycarbonyl-phenylalanine-arginine-7-amido-4-methylcoumarin. This expression and refolding procedure yielded 50 mg of purified, glycosylation-free human procathepsin K from 1 liter of E. coli cell culture and enabled the determination of the structure of human procathepsin K at 2.6 A resolution.

The crystal structure of human procathepsin K.

Cathepsin K is a cysteine protease present in human osteoclasts that plays an important role in bone resorption. Cathepsin K is synthesized as an inactive proenzyme and activated under conditions of low pH. Autoproteolytic processing of the N-terminal 99 amino acid propeptide produces the active, mature form of cathepsin K. It is presumed that the activation of procathepsin K in vivo occurs in the bone resorption pit, which has a low-pH environment. We have determined the structure of human procathepsin K at 2.8 A resolution. The structure of the mature enzyme domain within procathepsin K is virtually identical to that of mature cathepsin K. The fold of the propeptide of procathepsin K is similar to that observed in procathepsins B and L despite differences in length and sequence. A portion of the propeptide occupies the active site cleft of cathepsin K. Hydrophobic interactions, salt bridges, and hydrogen-bonding interactions are observed in the structure of the propeptide and between the propeptide and the mature enzyme of procathepsin K. These interactions suggest an explanation for the stability of the proenzyme. The structure of procathepsin K contributes to an understanding of the molecular basis of inhibition by the propeptide portion of the molecule and activation of this important member of the cysteine protease family.

Design of potent and selective human cathepsin K inhibitors that span the active site.

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.