Expression and localization of lysosomal-associated membrane protein 1- and perforin-based hybrid molecules in the murine cytotoxic T cell line CTLL-2.

Cytotoxic T lymphocytes (CTL) and natural killer cells harbor lytic granules as secretory lysosomes. Lytic granules contain perforin as a soluble protein that forms pores and is essential for target cell lysis. Lysosomal-associated membrane protein 1 (LAMP1) is a single transmembrane protein that is enriched in lysosomes and is also present in lytic granules. In this study, to investigate the feasibility of membrane-integral markers for lytic granules, LAMP1- and perforin-based hybrid molecules were constructed and their expression and localization were examined in the murine cytotoxic T-cell line CTLL-2. Stable CTLL-2 transfectants were established by nucleofection using linearized vectors encoding human LAMP1 (1-417) fused to enhanced green fluorescent protein (EGFP) and FLAG (LAMP1-EGFP-FLAG) and human perforin (1-555) fused to human LAMP1 (325-417), EGFP, and FLAG (Perforin-LAMP1-EGFP-FLAG). Confocal microscopy showed that LysoTracker Red colocalized with LAMP1-EGFP-FLAG and Perforin-LAMP1-EGFP-FLAG, and Perforin-LAMP1-EGFP-FLAG showed a broader cytoplasmic distribution than LAMP1-EGFP-FLAG. A Percoll density gradient centrifugation analysis revealed that LAMP1-EGFP-FLAG and Perforin-LAMP1-EGFP-FLAG were distributed in fractions associated with the endogenous expression of perforin and LAMP1. The effects of glycosylation inhibitors on the expression and intracellular transport of LAMP1-EGFP-FLAG were also investigated. Concanamycin A, an inhibitor of vacuolar-type H(+)-ATPase, was unable to induce the proteolytic degradation of Perforin-LAMP1-EGFP-FLAG, in contrast to perforin. The present results demonstrated that some LAMP1-EGFP-FLAG and Perforin-LAMP1-EGFP-FLAG localized to acidic granules or dense granules containing perforin and LAMP1 in CTLL-2 cells.