Abnormal Glycosylation of Procathepsin L Due to N-terminal Point Mutations Correlates with Failure to Sort to Lysosomes

• Published in: The Journal of biological chemistry Vol. 272; no. 13; pp. 8808 - 8816
• Main Authors: Chapman, Richard L., Kane, Susan E., Erickson, Ann H.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.03.1997; American Society for Biochemistry and Molecular Biology
• Subjects: 3T3 Cells; Animals; Aspartic Acid; Cathepsin L; Cathepsins - chemistry; Enzyme Precursors - chemistry; Glycosylation; HeLa Cells; Humans; Lysosomes - metabolism; Mannose - metabolism; Mice; Molecular Weight; Oligosaccharides - chemistry; Phosphorylation; Point Mutation; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.13.8808

A single point mutation in the lysosomal proenzyme receptor-inhibiting sequence near the N terminus of mouse procathepsin L can result in glycosylation of a normally cryptic site near its C terminus. When alanine replaced His36, Arg38, or Tyr40, the nascent chain of the mutant protein cotranslationally acquired a high mannose oligosaccharide chain at Asn268. In contrast, when alanine replaced Ser34, Arg37, or Leu39, this second carbohydrate chain was not added. This alternating pattern of abnormal glycosylation suggested that propeptide residues 36-40 normally assume an extended conformation having the side chains of residues 36, 38, and 40 facing in the same direction. When tyrosine conservatively replaced His36 or lysine replaced Arg38, Asn268 was not glycosylated. But the procathepsin L mutant having phenylalanine in place of Tyr40 was glycosylated at Asn268, which indicates that the hydrogen bond between the hydroxyl group of Tyr40 and the carboxylate group of Asp82 is necessary for normal folding of the nascent proenzyme chain. Mutation of the adjacent α2p (ERININ) helix of the propeptide or addition of a C-terminal epitope tag sequence to procathepsin L also induced misfolding of the proenzyme, as indicated by addition of the second oligosaccharide chain. In contrast, the propeptide mutation KAKK99-102AAAA had no effect on carbohydrate modification even though it reduced the positive charge of the proenzyme. Misfolded mutant mouse procathepsin L was not efficiently targeted to lysosomes on expression in human HeLa cells, even though it acquired phosphate on mannose residues. The majority of the mutant protein was secreted after undergoing modification with complex sugars. Similarly, epitope-tagged mouse procathepsin L was not targeted to lysosomes in homologous mouse cells but was efficiently secreted. Since production of mature endogenous protease was not reduced in cells expressing the tagged protein, the tagged protein did not compete with endogenous procathepsin L for targeting to lysosomes.