STUDIES ON A NOVEL MACROPHAGE-SPECIFIC CALMODULIN BINDING GLYCOPROTEIN

Abstract

The murine macrophage-like cell line J774 and peritoneal exudate cells elicited with thioglycollate or starch contain a major calmodulin-binding protein which is absent in trifluoperazine-resistant variants of J774, resident peritoneal macrophages and those elicited with concanavalin A, lipopolysaccharide, proteose peptone or Bacillus Calmette Guerin. Resident murine peritoneal cells maintained in tissue culture for 3 days begin to accumulate this protein as do human peripheral blood monocytes after 7 days of culture.;A specific competitive displacement radioimmunoassay was developed using a rabbit antiserum raised to the partially purified calmodulin binding protein and (('125)I) calmodulin covalently crosslinked to the principal calmodulin binding protein in the preparation. The radioimmunoassay confirmed the unique cellular distribution of this protein suggesting that it may be a marker for certain stages of macrophage differentiation.;Monoclonal antibodies were prepared and one of these was used to further purify the protein by immunoaffinity chromatography. A protein of molecular weight 50,000 to 60,000 was isolated. It could be selectively adsorbed to wheat germ agglutinin agarose and subsequently eluted with N-acetyl glucosamine. This property plus its sensitivity to endoglycosidase F led to the conclusion that it is a glycoprotein. The cellular distribution, subcellular localization and evidence for glycosylation suggest that this protein may be a macrophage-specific receptor with a high affinity for calcium-calmodulin.