Intracellular Distribution of Lysozyme in Rat Alveolar Type II Epithelial Cells

Abstract

This study investigated the intracellular distribution of lysozyme, a protein that is synthesized and secreted by rat alveolar type II epithelial (ATII) cells and alveolar macrophages, using a polyclonal antibody generated against purified rat lysozyme. Lysozyme was immunoprecipitated with this antibody from Triton X-100 lysates of ATII cells cultured on a basement membrane derived from Englebreth-Holme-Swarm mouse sarcoma (EHS) and radiolabeled with ³⁵S-methionine. ATII cells cultured on EHS basement membrane for several days were fixed and labeled with antibodies to surfactant apoprotein A (SP-A) and lgp-120 (a lysosomal glycoprotein), or lysozyme and lgp-120, and studied by confocal microscopy. Organelles were identified that stained positively for either anti-lysozyme or anti-lgp-120; a second population of organelles contained both markers. Similarly, two populations of SP-A-containing organelles were identified; one contained the lysosomal glycoprotein lgp-120. In addition, confocal images demonstrated that both SP-A and lysozyme were secreted by ATII cells, as evidenced by the accumulation of secretory products within the lumen of the cyst-like aggregates. When the subcellular localization of SP-A and lysozyme was studied by analytical cell fractionation, two populations of organelles were identified that contained SP-A or lysozyme. The lighter population accounted for approximately 32% of SP-A and 33% of total intracellular lysozyme and was recovered in the same region of the gradient as secretory lamellar bodies. The more dense population co-localized with lysosomes and accounted for approximately 67% of both SP-A and lysozyme recovered. Western blots of cell fractions revealed intact lysozyme in all the cell fractions. The results of these experiments suggest that lysozyme has a similar intracellular distribution as surfactant apoprotein A in ATII cells. Lysozyme is found in fractions containing lamellar bodies where it is packaged for secretion, and in lysosomal fractions where it may undergo degradation.