Effect of XXXX position on lysosomal transport of procathepsin L.

@entity1 @entity8777 @entity19 @entity1735 @entity2734 @entity7788 @entity10613 @entity3363
To study the role of @entity1735 in lysosomal protein transport, we engineered two novel glycosylation signals ( @entity2734 -X-Ser/Thr) into the cDNA of @entity1 procathepsin L, a lysosomal acid protease. We constructed six mutant cDNAs encoding glycosylation signals at mutant sites @entity2734 -138, @entity2734 -175, or both sites together, in the presence or absence of the wild-type @entity2734 -204 site. We stably transfected wild-type and mutant cDNAs into NIH3T3 @entity19 fibroblasts and then used species-specific antibodies to determine the glycosylation status, phosphorylation, localization, and transport kinetics of recombinant @entity1 procathepsin L containing one, two, or three glycosylation sites. Both novel glycosylation sites were capable of being glycosylated, although @entity2734 -175 was utilized only 30-50% of the time. Like the wild-type glycosylation at @entity2734 -204, @entity3363 at @entity2734 -138 and @entity2734 -175 were completely sensitive to endoglycosidase H, and they were phosphorylated. Mutant proteins containing two @entity3363 were capable of being delivered to lysosomes, but there was not a consistent relationship between the efficiency of lysosomal delivery and @entity1735 content of the protein. Pulse-chase labeling revealed a unique biosynthetic pattern for proteins carrying the @entity2734 -175 glycosylation sequence. Whereas wild-type procathepsin L and mutants bearing @entity1735 at @entity2734 -138 appeared in lysosomes by about 60 min, proteins with @entity1735 at @entity2734 -175 were processed to a lysosome-like polypeptide within 15 min. Temperature shift, @entity10613 , and @entity8777 experiments suggested that the rapid processing did not occur in the endoplasmic reticulum and that @entity2734 -175 mutants could interact with the @entity7788 receptor. Taken together, our results are consistent with the interpretation that @entity2734 -175 @entity1735 confers rapid transport to lysosomes. We may have identified a recognition domain in procathepsin L that is important for its interactions with the cellular transport machinery.
