ISOLATION OF PNEUMOCYSTIS-CARINII GP120 BY FIBRONECTIN AFFINITY - EVIDENCE FOR MANGANESE DEPENDENCE

Pneumocystis carinii is a major opportunistic lung pathogen and a lead ing cause of death among patients with the human immunodeficiency viru s. Adherence of P. carinii to type I alveolar epithelial cells is esse ntial for growth and replication and has been shown to be mediated in part by fibronectin (Fn). To better understand the mechanisms underlyi ng this attachment, P. carinii-Fn interaction was characterized with r espect to divalent and monovalent ion concentration and pH using an I- 125-Fn binding assay to Il carinii. The results suggest that P. carini i has a receptor for Fn that was partially dependent on Ca2+, enhanced by Mn2+, and diminished somewhat by Mg2+. Additional data demonstrate d that P carinii-Fn interaction was sensitive to ionic strength. The p H profile revealed that P. carinii-Fn interaction increased with decre asing pH. The results from the binding assay provided the rationale fo r a simple isolation of the Fn receptor from P. carinii using a Fn-aff inity column involving nondenaturing conditions. The isolated receptor appeared highly purified by SDS-PAGE analysis, with apparent molecula r weights of 114 to 118 kD and 66 kD. Western blot analysis indicated that this receptor was gp120, a major surface glycoprotein of P. carin ii. Furthermore, the isolated receptor inhibited Fn binding to P. cari nii. Finally, a monoclonal antibody raised against the affinity-purifi ed gp120 blocked Fn binding to P. carinii.