A mechanism of TNFR type II (75 kDa) "shedding" in macrophages

Adherent macrophage populations derived from monocytes isolated from peripheral blood were evaluated for their ability to "shed" the membrane-associated receptor for TNF-alpha (TNFR) following exposure to a calcium ionophor (A23187) and a synthetic chemotactic peptide (fMLP) reagent. A soluble fraction of TNFR was detected in "cell-free" supernatant produced by stimulated macrophage populations applying 125I-TNF-alpha and biotinylated TNF-alpha ligand-binding analysis (96-well format) in combination with conventional autoradiographic techniques. Approximate molecular weight of the shed TNFR glycoprotein fraction was estimated to be 75 kDa based on interpretation of nondenaturing PAGE gels transferred laterally onto sheets of nitrocellulose membrane subsequently probed by ligand-binding analysis applying 125I-TNF-alpha and biotinylated TNF-alpha as detection modalities. Immunorecognition techniques were also employed to detect TNFR fragments shed from macrophages using biotinylated anti-TNFR Type II (75 kDa) monoclonal antibody in combination with conjugated strepavidin:HRPO and a chemiluminescent substrate reagent. In an effort to identify the class of enzyme directly mediating TNFR Type II (75 kDa) shedding, a spectrum of carboxyl- (e.g., aspartate), hydroxyl- (e.g., serine), thiol (e.g., cysteine), and metalo- (e.g., Ca2+, Mg2+) protease-inhibiting agents were evaluated. Experimental findings implied that a carboxy (aspartate) peptidase, and possibly to a lesser extent, serine (hydroxyl), and thiol (cysteine) peptidases participate in macrophage TNFR Type II (75 kDa) shedding phenomena. Subsequent investigations demonstrated that the carboxy (aspartate) peptidase cathepsin-D promoted liberation of TNFR Type II (75 kDa) in unactivated populations of adherent macrophages. In an effort to complement these observations, a protein fraction with presumed carboxy (aspartate) protease activity was isolated from the cell-free supernatant generated by activated populations of adherent macrophages using immobilized pepstatin-A beaded agarose. Exposure of unstimulated populations of adherent macrophages to the partially purified pepstatin-A binding protein fractions resulted in the liberation of a soluble TNFR Type II (75 kDa) fragment based on interpretation of ligand-binding and immunorecognition analysis of samples developed by SDS-PAGE/PAGE format and transferred onto sheets of nitrocellulose membrane. The molecular weight of the macrophage pepstatin-A binding protein fraction was estimated to be 47-52 kDa with lesser bands also visible at approximately 26-32 kDa, and 100 kDa based on SDS-PAGE analysis. Nondenaturing hemoglobin-PAGE substrate gel analysis of protein fractions possessing pepstatin-A binding-avidity detected a protease with a molecular weight of approximately 47-52 kDa that proteolytically digested hemoglobin, in addition to a synthetic cathepsin-D specific peptide substrate. Collective interpretation of these experimental findings directly corresponds with many of the physical (molecular) and functional (biochemical) characteristics known to be associated with the leukocyte carboxy (aspartate) peptidase cathepsin-D, which is a non-metaloprotease known to exert relatively limited proteolytic activity.