Mannose 6-Phosphate-independent Targeting of Lysosomal Enzymes in I-CeU Disease

ease (ICD) maintain normal cellular levels of lysosomal enzymes despite a deficiency of the enzyme UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-l-phosphotransferase. We find that an ICD B lymphoblastoid cell line targets about 45 % of the lysosomal protease cathepsin D to dense lysosomes. This targeting occurs in the absence of detectable mannose 6-phosphate residues on the cathepsin D and is not observed in ICD fibroblasts. The secretory protein pepsinogen, which is closely related to cathepsin D in both amino acid sequence and threedimensional structure, is mostly excluded from dense lysosomes, indicating that the lymphoblast targeting pathway is specific. Carbohydrate residues are not required for lysosomal targeting, since a nonglycosylated mutant cathepsin D is sorted with comparable efficiency to the wild type protein. Analysis of a number of cathepsin D/pepsinogen chimeric proteins indicates that an extensive polypeptide determinant in the cathepsin D carboxyl lobe can confer efficient lysosomal sorting when introduced into the pepsinogen sequence. This determinant overlaps but is not identical to the recognition marker for phosphotransferase. These results indicate that a specific protein recognition event underlies Man-6-P-independent lysosomal sorting in ICD lymphoblasts. M OST lysosomal enzymes are soluble glycoproteins which, along with secretory proteins, are cotranslationaUy inserted into the lumen of the rough ER and glycosylated at selected asparagine residues by the en bloc transfer of a large preformed oligosaccharide (reviewed in Kornfeld and MeUman, 1989). In normal mammalian cells, the N-linked oligosaccharides of the lysosomal enzymes are subsequently modified by the concerted action of the enzymes UDP-GIcNAc: lysosomal enzyme N-acetylglucosamine-l-phosphotransferase (phosphotransferase) and N-acetylglucosamine-l-phosphate tx-N-acetylgluco saminidase to generate an exposed mannose 6-phosphate (Man-6-P)~ moiety. Lysosomal enzymes bearing phosphomannosyl residues bind to Man-6-P receptors present in the TGN and are then transported to an acidified prelysosomal compartment by means of clathrin-coated vesicles. Pbospbotransferase selects these acid hydrolases for modification by recognizing a conformation-dependent protein determinant present on lysosomal enzymes but not on structurally related secretory proteins (Baranski, 1990, 1991). I-cell disease (ICD) is an inherited human disorder in which an unknown mutation eliminates or severely reduces the activity of phosphotransferase (Reitman et al., 1981; 1. Abbreviations used in this paper: BLCL, B lymphoblastoid cell line; EBV, Epstein-Barr virus; endo H, endoglycosidase H; ICD, I-cell disease; Man-6-P, mannose 6-phosphate. Hasilik et al., 1981). As a result, newly synthesized lysosomal enzymes do not acquire Man-6-P residues and are quantitatively secreted by many cell types. This results in gross cellular deficiencies of most lysosomal enzymes and severe clinical manifestations in the first few years of life (Nolan and Sly, 1989). However, certain tissues and cell types isolated from ICD patients, such as liver and spleen (Waheed et al., 1982; Owada and Neufeld, 1982) and B lymphocytes immortalized by Epstein-Barr virus (EBV) (Little et al., 1987; Okada et al., 1988; Tsuji et al., 1988), have cellular lysosomal enzyme levels within the normal range for those cell types despite virtually undetectable phosphotransferase activity. In B lymphocytes, these enzymes have been localized to a dense membrane compartment likely representing lysosomes (Miller, A. L., M. Liddle, V. Norton, and D. Wright. 1988. J. Cell Biol. 107:341a), and a recent report indicates that newly synthesized lysosomal enzymes are retained intracellularly in the absence of phosphorylation (DiCioccio and Miller, 1991). The ability of these cells to retain intracellular lysosomal enzymes in the absence of phosphotransferase activity suggests that they may have a cell type-restricted, Man-6-P -independent mechanism of lysosomal enzyme targeting. We decided to investigate this possibility further by studying an established B lymphoblastoid cell line derived from an ICD patient (described in Little et al., 1987). We find that in these cells, both endogenous and transfected cathepsin D, © The Rockefeller University Press, 0021-9525/93/10/99/10 $2.00 The Journal of Cell Biology, Volume 123, Number 1, October 1993 99-108 99 on O cber 9, 2017 jcb.rress. org D ow nladed fom a lysosomal protease, are sorted directly to dense lysosomes without acquiring Man-6-P residues, consistent with previous reports on other lysosomal hydrolases. Furthermore, we find that transfected pepsinogen, the related nonlysosomal protease, is largely excluded from lysosomes, indicating that ICD lymphoblasts target lysosomal enzymes selectively. Finally, we present evidence that an extended protein determinant in the carboxyl lobe of cathepsin D directs its lysosomal sorting in these cells. Materials and Methods