Napsin is an aspartic proteinase which has two essential asparagine residues in the active peptide (Tatnell et al., 1997). Napsin may participate in the processing of pulmonary surfactant protein B (Weaver et al., 1992; Chuman et al., 1999). in addition, changes in napsin expression have been linked to primary adenocarcinoma as well as kidney dysfunction (Hirano et al., 2000; Schauer-Vukasinovic et al., 2000). Localization studies by light microscope immunocytochemistry and in situ hybridization detected napsin in the lungs as well as in the kidney (Tatnell et al., 1997; Chuman et al, 1999; Schauer- Vukasinovic et al., 1999). However, these studies did not resolve the ultrastructure localization of napsin. in this study, EM cryoimmunogold labeling was used to characterize the subcellular distribution of napsin in lung and kidney cells.
A truncated murine napsin propeptide construct (1212 b.p.) without the signal peptide sequence (47 b.p.) was cloned into pProEX™HTb plasmid vector for the generation of recombinant protein. Polyclonal antibodies were generated against purified recombinant protein. The specificity of polyclonal antibodies was determined by immunoprecipitation and Western blotting using isolated mouse alveolar type 2 epithelial cells and kidney homogenates. The napsin antibodies detected a single protein with Mr= 43 kD.
Localization of napsin by EM cryoimmunogold labeling was performed using protein A gold probes as previously described (Na et al, 2000). in the lungs, napsin was localized in both alveolar type 2 epithelial cells and in alveolar macrophages. in alveolar type 2 epithelial cells, napsin was detected in multivesicular bodies and in lamellar bodies; internalized BSA gold colocalized with napsin in the lysosomes of alveolar macrophages (Figure 1A and 1B), consistent with the observation that napsin is active at low pH (Schauer-Vukasinovic et al., 1999). Although Schauer-Vukasinovic et al. (1999) reported that napsin was expressed by some bronchiolar cells, we did not observe any significant napsin labeling in Clara cells or in ciliated epithelial cells.