ABNORMAL GLYCOSYLATION OF PROCATHEPSIN-L DUE TO N-TERMINAL POINT MUTATIONS CORRELATES WITH FAILURE TO SORT TO LYSOSOMES

A single point mutation in the lysosomal proenzyme receptor-inhibiting sequence near the N terminus of mouse procathepsin L can result in gl ycosylation of a normally cryptic site near its C terminus. When alani ne replaced His(36), Arg(38), or Tyr(40), the nascent chain of the mut ant protein cotranslationally acquired a high mannose oligosaccharide chain at Asn(268). In contrast, when alanine replaced Ser(34), Arg(37) , or Leu(39), this second carbohydrate chain was not added, This alter nating pattern of abnormal glycosylation suggested that propeptide res idues 36-40 normally assume an extended conformation having the side c hains of residues 36, 38, and 40 facing in the same direction. When ty rosine conservatively replaced His(36) or lysine replaced Arg(38), Asn (268) was not glycosylated, But the procathepsin L mutant having pheny lalanine in place of Tyr(40) was carboxylated at Asn(268), which indic ates that the hydrogen bond between the hydroxyl group of Tyr(40) and the carboxylate group of Asp(82) is necessary for normal folding of th e nascent proenzyme chain. Mutation of the adjacent alpha 2p (ERININ) helix of the propeptide or addition of a C terminal epitope tag sequen ce to procathepsin L also induced misfolding of the proenzyme, as indi cated by addition of the second oligosaccharide chain. In contrast, th e propeptide mutation KAKK99-102AAAA had no effect on carbohydrate mod ification 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 pro cathepsin L for targeting to lysosomes.