Effect of carbohydrate position on lysosomal transport of procathepsin L.
To study the role of carbohydrate in lysosomal protein transport, we engineered two novel glycosylation signals (Asn-X-Ser/Thr) into the cDNA of human procathepsin L, a lysosomal acid protease. We constructed six mutant cDNAs encoding glycosylation signals at mutant sites Asn-138, Asn-175, or both sites together, in the presence or absence of the wild-type Asn-204 site. We stably transfected wild-type and mutant cDNAs into NIH3T3 mouse fibroblasts and then used species-specific antibodies to determine the glycosylation status, phosphorylation, localization, and transport kinetics of recombinant human procathepsin L containing one, two, or three glycosylation sites. Both novel glycosylation sites were capable of being glycosylated, although Asn-175 was utilized only 30-50% of the time. Like the wild-type glycosylation at Asn-204, carbohydrates at Asn-138 and Asn-175 were completely sensitive to endoglycosidase H, and they were phosphorylated. Mutant proteins containing two carbohydrates were capable of being delivered to lysosomes, but there was not a consistent relationship between the efficiency of lysosomal delivery and carbohydrate content of the protein. Pulse-chase labeling revealed a unique biosynthetic pattern for proteins carrying the Asn-175 glycosylation sequence. Whereas wild-type procathepsin L and mutants bearing carbohydrate at Asn-138 appeared in lysosomes by about 60 min, proteins with carbohydrate at Asn-175 were processed to a lysosome-like polypeptide within 15 min. Temperature shift, brefeldin A, and NH4Cl experiments suggested that the rapid processing did not occur in the endoplasmic reticulum and that Asn-175 mutants could interact with the mannose 6-phosphate receptor. Taken together, our results are consistent with the interpretation that Asn-175 carbohydrate 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.
Lingeman
R G
RG
Department of Cell and Tumor Biology, City of Hope National Medical Center, Duarte, California, 91010, USA.
Joy
D S
DS
Sherman
M A
MA
Kane
S E
SE
D016475:3T3 Cells; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D050260:Carbohydrate Metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D008360:Mannosephosphates / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D017527:Receptor, IGF Type 2 / Q000378:metabolism
D004792:Enzyme Precursors; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; C027693:mannose-6-phosphate; D001216:Asparagine; D002403:Cathepsins; C063905:procathepsin L; D056668:Cathepsin L