Transport of human lysosomal neuraminidase to mature lysosomes requires protective protein/cathepsin A
1998; Springer Nature; Volume: 17; Issue: 6 Linguagem: Inglês
10.1093/emboj/17.6.1588
ISSN1460-2075
Autores Tópico(s)Infant Nutrition and Health
ResumoArticle16 March 1998free access Transport of human lysosomal neuraminidase to mature lysosomes requires protective protein/cathepsin A Aarnoud van der Spoel Aarnoud van der Spoel Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Erik Bonten Erik Bonten Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Alessandra d'Azzo Corresponding Author Alessandra d'Azzo Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Aarnoud van der Spoel Aarnoud van der Spoel Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Erik Bonten Erik Bonten Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Alessandra d'Azzo Corresponding Author Alessandra d'Azzo Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Author Information Aarnoud van der Spoel1, Erik Bonten1 and Alessandra d'Azzo 1 1Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:1588-1597https://doi.org/10.1093/emboj/17.6.1588 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Human lysosomal N-acetyl-α-neuraminidase is deficient in two lysosomal storage disorders, sialidosis, caused by structural mutations in the neuraminidase gene, and galactosialidosis, in which a primary defect of protective protein/cathepsin A (PPCA) leads to a combined deficiency of neuraminidase and β-D-galactosidase. These three glycoproteins can be isolated in a high molecular weight multi-enzyme complex, and the enzymatic activity of neuraminidase is contingent on its interaction with PPCA. To explain the unusual need of neuraminidase for an auxiliary protein, we examined, in transfected COS-1 cells, the effect of PPCA expression on post-translational modification, turnover and intracellular localization of neuraminidase. In pulse–chase studies, we show that the enzyme is synthesized as a 46 kDa glycoprotein, which is poorly phosphorylated, does not undergo major proteolytic processing and is secreted. Importantly, its half-life is not altered by the presence of PPCA. However, neuraminidase associates with the PPCA precursor shortly after synthesis, since the latter protein co-precipitates with neuraminidase using anti-neuraminidase antibodies. We further demonstrate by subcellular fractionation of transfected cells that neuraminidase segregates to mature lysosomes only when accompanied by wild-type PPCA, but not by transport-impaired PPCA mutants. These data suggest a novel role for PPCA in the activation of lysosomal neuraminidase, that of an intracellular transport protein. Introduction Neuraminidases (sialidases) are exoglycosidases that catalyze the removal of terminal sialic acid residues, α-ketosidically linked to mono- or oligosaccharide chains of glycoconjugates. In mammals, three distinct neuraminidases have been identified in the cytoplasm, plasma membrane and lysosomes. These enzymes differ in their pH optimum, interaction with detergents, and stability (reviewed in Saito and Yu, 1995; Schauer et al., 1995). Lysosomal neuraminidase preferentially cleaves terminal α(2,3)- and α(2,6)-linked sialic acid residues and has an acidic pH optimum. In man, deficiency of this enzyme is associated with two distinct genetic disorders of metabolism: sialidosis, caused by structural lesions in the neuraminidase gene (Thomas and Beaudet, 1995), and galactosialidosis (GS), in which neuraminidase deficiency is secondary to a primary defect in the serine carboxypeptidase protective protein/cathepsin A (PPCA) (d'Azzo et al., 1982). For both diseases, early onset forms with severe CNS pathology and systemic organ involvement, as well as milder late onset variants, have been identified. The lack of PPCA has also been shown to hamper neuraminidase activity severely in the mouse model of GS (Zhou et al., 1995). The cDNA for human lysosomal neuraminidase was isolated recently (Bonten et al., 1996; Milner et al., 1997; Pshezhetsky et al., 1997). It encodes a protein of ∼45 kDa, with three potential N-linked glycosylation sites, and 32–38% sequence homology to several bacterial sialidases as well as to the cytosolic mammalian enzyme. These homologous sequences include the characteristic FRIP sequence, three conserved copies of an 'Asp box' [consensus sequence Ser/Thr-X-Asp-(X)-Gly-X-Thr-Trp/Phe (Roggentin et al., 1993)] and two degenerated Asp boxes. Electroporation of the neuraminidase cDNA into sialidosis fibroblasts restores enzymatic activity (Bonten et al., 1996). Furthermore, analysis of the neuraminidase cDNA from different sialidosis patients has identified six independent mutations in the gene (Bonten et al., 1996; Pshezhetsky et al., 1997), two of which were shown to render the protein non-functional (Bonten et al., 1996). These studies have thus defined the molecular basis of sialidosis. Mammalian lysosomal neuraminidase is unique among other sialidases in that it requires the serine carboxypeptidase PPCA for enzymatic activity (d'Azzo et al., 1995; Thomas and Beaudet, 1995). Neuraminidase shares this feature with a third lysosomal enzyme, β-D-galactosidase (d'Azzo et al., 1995; Suzuki et al., 1995). The dependence of these glycosidases on the carboxypeptidase is evident in GS, where malfunctioning or absence of PPCA leads to the combined deficiency of neuraminidase and β-galactosidase (d'Azzo et al., 1995). These three lysosomal enzymes can be co-purified in a high molecular weight complex with either β-galactosidase or PPCA affinity matrices (Verheijen et al., 1985; Yamamoto and Nishimura, 1987; Pshezhetsky and Potier, 1994, 1996). Both neuraminidase and β-galactosidase activities in cultured GS fibroblasts are restored by the addition of exogenous PPCA precursor (54 kDa), which is internalized via the mannose-6-phosphate (M6P) receptor, routed to the lysosome and processed into its mature 32/20 kDa two-chain form (Galjart et al., 1988; Zhou et al., 1996). How PPCA influences the generation and maintenance of neuraminidase and β-galactosidase activities is not yet clear. It is known that the half-life of mature β-galactosidase is severely reduced in GS fibroblasts, and that treatment with the protease inhibitor leupeptin increases the amount and activity of β-galactosidase (d'Azzo et al., 1982; van Diggelen et al., 1982; Pshezhetsky and Potier, 1996). This implies that PPCA protects β-galactosidase against rapid proteolytic degradation. In contrast, the neuraminidase activity of GS cells is hardly affected by leupeptin treatment (d'Azzo et al., 1982; Pshezhetsky and Potier, 1996), suggesting that this enzyme is influenced by PPCA in a different way. In this study, we have investigated whether neuraminidase requires the presence of PPCA for protection against intralysosomal degradation, for specific post-translational modifications like proteolytic processing and phosphorylation, or for its intracellular transport. We also compared the effect of transport-deficient PPCA variants on the intracellular behavior of neuraminidase. Our results offer a first explanation for the PPCA dependence of lysosomal neuraminidase activity. Results Neuraminidase associates with PPCA and β-galactosidase precursors and has a short half-life To determine whether the PPCA-dependent activation of neuraminidase is accompanied by specific structural modifications of the enzyme en route to the lysosome, we transfected COS-1 cells with the neuraminidase cDNA either alone or in combination with the cDNA for PPCA and/or β-galactosidase. Transfected cells were pulse-labeled for 1 h and then chased for different time periods in medium containing cold leucine. The cells were then lysed in buffer at pH 7.4 and immunoprecipitated with anti-neuraminidase (anti-Neur) antibodies. As shown in Figure 1, neuraminidase was recovered from single and co-transfected cells in multiple forms, migrating on SDS–polyacrylamide gels either as a broad band or as discrete bands with molecular weights of ∼44–46 kDa. These multiple forms represent different glycosylation states of the enzyme (Bonten et al., 1996; Milner et al., 1997). In both single and co-transfections, the size of the newly synthesized neuraminidase did not change during the chase periods, suggesting that post-translational processing of the enzyme was completed within 1 h and was not influenced by the presence of PPCA and/or β-galactosidase (Figure 1A–D). Furthermore, in all transfected cells, the neuraminidase levels began to decrease after 3 h of chase, and the enzyme was largely degraded 24 h after synthesis. Therefore, co-expression of PPCA or β-galactosidase did not grossly alter the half-life of neuraminidase. After immunoprecipitation with anti-Neur antibodies, lysates from double- or triple-transfected cells were subjected to a second round of immunoprecipitation with anti-PPCA and anti-β-galactosidase antisera (Figure 1E and F). PPCA and β-galactosidase precursors were converted slowly to their mature forms (32/20 and 64 kDa, respectively) that were stable for >24 h after synthesis (see also Morreau et al., 1992; Zhou et al., 1996). This clearly illustrates that the turnover of neuraminidase was more rapid than that of PPCA or β-galactosidase. From triple-transfected cells, both PPCA precursor and small amounts of its mature form were co-precipitated with β-galactosidase. Figure 1.Pulse–chase labeling. COS-1 cells were transfected with vector alone [denoted M in (G)] or with cDNAs encoding neuraminidase (N), β-galactosidase (B) or PPCA (P), in various combinations (A–F), labeled with [3H]leucine for 1 h and chased with cold leucine for the time periods indicated. Cell lysates and medium samples were immunoprecipitated with anti-Neur antiserum (A–D and G), and selected lysates were used for a secondary round of immunoprecipitation with anti-PPCA antiserum (E) or anti-β-galactosidase antisera (F). Exposure was for 20 days (B and D upper panels, and E and F), 1 month (B and D, lower panels), 3 days (A and C, upper panels), 4 days (A and C, lower panels) or 14 days (G). Download figure Download PowerPoint During the 1 h pulse labeling, both PPCA and β-galactosidase precursors were co-precipitated with neuraminidase from double- and triple-transfected cells (Figure 1B–D), indicating that association of these three proteins occurred shortly after their synthesis. The mature forms of PPCA and β-galactosidase did not co-precipitate with neuraminidase under the neutral immunoprecipitation conditions. However, by immunotitration with Staphylococcus aureus-bound anti-PPCA antibodies, up to 60% of neuraminidase activity was co-precipitated with cathepsin A activity from co-transfected cells lysed in buffer at pH 5.5. From these results, we could infer that the majority of enzymatically active neuraminidase remains associated with mature PPCA (Figure 2). Figure 2.Precipitation of neuraminidase activity from co-transfected COS-1 cells with anti-PPCA antibodies. Aliquots of lysates of COS-1 cells overexpressing both neuraminidase and PPCA were incubated under acidic conditions with increasing amounts of fixed S.aureus cells that had been pre-loaded with IgG from either pre-immune or anti-PPCA antiserum. After removal of the bacteria by centrifugation, neuraminidase and cathepsin A activities were assayed in the supernatants. The data are expressed as a percentage of the level measured in samples incubated with pre-immune IgG-S.aureus. Error bars indicate standard deviation for duplicate experiments. Download figure Download PowerPoint Neuraminidase is secreted into the extracellular space We found overexpressed neuraminidase in the medium of transfected cells, and the time course of its secretion was independent of PPCA and β-galactosidase co-expression. The level of secreted enzyme was maximal after 3–6 h and had diminished at the 24 h time point (Figure 1A–D, lower panels). To compare the relative levels of secretion, the amount of neuraminidase immunoprecipitated from medium samples was quantitated by densitometry scanning and expressed as a percentage of the total amount immunoprecipitated from pulse-labeled cell lysates (Figure 1A and B, lower panels). The secretion of neuraminidase, quantitated in this fashion, was clearly reduced when co-expressed with PPCA, irrespective of β-galactosidase expression (Figure 1C and D, lower panels). This variation was not the result of differences in the rate of synthesis of neuraminidase, because the intracellular levels of neuraminidase were similar in all transfected cells (Figure 1A–D, upper panels). Thus, the intracellular routing of the enzyme was influenced by the concomitant overexpression of PPCA. Extracellular neuraminidase, on the other hand, did not associate with PPCA or β-galactosidase, since we could not co-precipitate the three proteins from the medium of triple-transfected cells (Figure 1D, lower panel); in turn, no neuraminidase activity was detected in concentrated medium samples from co-transfected cells, even though high activity was measured in the corresponding lysates. Moreover, secreted neuraminidase was not activated when we mixed concentrated media from PPCA- and neuraminidase-overexpressing cells, in an attempt to promote in vitro association of the two molecules (data not shown). PPCA controls the intracellular routing of neuraminidase By using immunofluorescence, we and others have reported that when neuraminidase is expressed in COS-1 cells it has either a punctate distribution or it is localized in the endoplasmic reticulum (ER)/Golgi region. Further, depending on the expression levels, the enzyme can also form small square crystals in the perinuclear area that stain strongly with anti-Neur antiserum (Bonten et al., 1996; Milner et al., 1997). Here, we analyzed whether the proportion of cell populations showing a different subcellular distribution of neuraminidase was contingent on the level of expressed PPCA. In cells transfected with the same amount of neuraminidase cDNA but increasing concentrations of PPCA cDNA, neuraminidase activity increased in parallel with the number of cells exhibiting punctate staining (Figure 3A and B). In contrast, cells transfected with different amounts of PPCA cDNA and no neuraminidase cDNA displayed slightly higher endogenous neuraminidase activity, which remained constant irrespective of the amount of PPCA cDNA added (Figure 3A). This result demonstrates that PPCA affects the subcellular localization of neuraminidase, which in turn may influence its activation. Figure 3.Titration of neuraminidase. COS-1 cells were transfected with vector alone (M), or with increasing amounts of PPCA (P) cDNA (0.2, 0.4, 0.8 or 1.2 μg per 35 mm dish), alone or in combination with a fixed amount of neuraminidase (N) cDNA (0.2 μg per 35 mm dish). Cells were (A) assayed for neuraminidase activity or (B) processed for immunofluorescence using affinity-purified anti-Neur antiserum. Enzyme activity is expressed in nmol/mg/h. At least 70 cells were examined microscopically and scored for neuraminidase staining. Download figure Download PowerPoint To analyze the subcellular distribution of neuraminidase further, we separated the organelles of transfected cells on self-generating Percoll density gradients (Figure 4). This procedure separates mature dense lysosomes from lighter membranes such as microsomes and early and late endosomes. β-Hexosaminidase and horseradish peroxidase (HRP) were used as lysosomal and endosomal markers, respectively. In cells transfected with the β-hexosaminidase cDNA (β-chain), enzyme activity peaked in both the dense bottom fractions (fractions 1 and 2) and in fraction 5 (Figure 4A). After a 4 min incubation of the cells with HRP, the internalized enzyme was detected only in fractions 4–6, confirming the presence of endosomal vesicles in this part of the gradient (Figure 4B). During the 3 h chase period that followed, however, the internalized enzyme shifted to the denser fractions (Figure 4B). In parallel experiments, we monitored the distribution and enzymatic activities of PPCA and neuraminidase. Neuraminidase expressed alone was detected on immunoblots with anti-Neur antibodies exclusively in the lighter fractions (Figure 4C). Its activity was marginal throughout the gradient (Figure 4C). In contrast, blots probed with anti-PPCA antibodies showed the PPCA precursor in both the light and dense fractions, while its mature two-chain form was confined to the bottom of the gradient (Figure 4D). In keeping with this finding, cathepsin A activity increased only in the fractions that contained the mature enzyme (Figure 4D). When neuraminidase cDNA was transfected into COS-1 cells together with PPCA cDNA, the two proteins co-distributed, and equal amounts of neuraminidase polypeptide were found in the light and dense fractions (Figure 4E and F). However, enzyme activity was detected primarily in the mature lysosomal fractions, with only a moderate increase in fraction 5 (Figure 4E and F). These results show that when the neuraminidase polypeptide is expressed alone it localizes in only light biosynthetic vesicles, but it reaches mature lysosomes when accompanied by PPCA. The generation of neuraminidase activity seems, therefore, to depend on the lysosomal localization of the enzyme. The interaction of neuraminidase with PPCA in an early biosynthetic compartment is sufficient merely for partial activation, which is fully unraveled in the lysosomal milieu. Figure 4.Subcellular fractionation of COS-1 cells on density gradients. (A) Density distribution of Percoll gradients, as determined with density marker beads (Pharmacia), and profile of β-hexosaminidase activity (nmol/h/ml) in COS-1 cells overexpressing β-hexosaminidase β-chain. (B) After pulse-labeling with HRP, COS-1 cells were either stored on ice (▾) or chased for 3 h in HRP-free medium (▪). Cells were then fractionated and the resulting gradient fractions assayed for HRP content (μg/ml). Alternatively, COS-1 cells, transfected with (C) neuraminidase cDNA, (D) PPCA cDNA or (E and F) co-transfected with both cDNAs were homogenized and loaded onto Percoll gradients. Fractions were analyzed for enzyme activities and used in Western blots, which were probed with anti-Neur (anti-N) or anti-PPCA (anti-P) antisera. Neuraminidase activity (♦) is expressed in nmol/h/ml and cathepsin A activity (●) in nmol/min/ml. Blots were developed with colorimetric substrates. Download figure Download PowerPoint Effect of PPCA mutants on neuraminidase compartmentalization We next investigated the effect of different PPCA variants on the intracellular transport and activation of neuraminidase. Four mutants were selected, each carrying a single amino acid substitution that impairs its lysosomal compartmentalization to a different degree: PPCA-Leu208Pro (LP) and PPCA-Val104Met (VM) were originally identified in a severe early infantile case of GS. These mutant proteins are not transported out of the ER and persist as immature precursors in this compartment (Zhou et al., 1996). In the patient with these mutations, neuraminidase activity is undetectable. In contrast, PPCA-Phe412Val (FV) and PPCA-Tyr221Asn (YN), which are associated with the mild, late-infantile phenotype, are partially transported to lysosomes, where PPCA-FV is degraded more rapidly than PPCA-YN (Zhou et al., 1991, 1996; Shimmoto et al., 1993). The presence of small amounts of mutant PPCA in lysosomes supports the finding of residual neuraminidase activity in patients with this phenotype. COS-1 cells were transfected with neuraminidase cDNA or co-transfected with neuraminidase cDNA and a cDNA encoding either wild-type PPCA or one of the PPCA mutants. Cells were then metabolically labeled for 16 h, lysed, and the cell lysates were immunoprecipitated sequentially with anti-Neur and anti-PPCA antisera. Figure 5 shows that all four mutant precursors co-precipitated with neuraminidase when treated with anti-Neur antiserum, demonstrating that the PPCA mutants associate with the enzyme as efficiently as the wild-type PPCA precursor does (lanes 5, 7, 9, 11 and 13). As observed previously (Zhou et al., 1991, 1996), all of the PPCA mutants accumulated in the precursor form, except for PPCA-YN which was partially converted to the mature form (Figure 5, lanes 6, 8, 10, 12 and 14). The amount of PPCA precursor associated with neuraminidase was proportional to the level of unbound precursor that was not brought down by the anti-Neur antibodies but was precipitated with anti-PPCA antibodies (Figure 5, compare adjacent lanes). Figure 5.Co-immunoprecipitation of PPCA mutants with neuraminidase. COS-1 cells, transfected with the neuraminidase cDNA alone, or in combination with a cDNA encoding wild-type PPCA, PPCA-YN, PPCA-FV, PPCA-LP or PPCA-VM, were metabolically labeled with [3H]leucine and used for sequential immunoprecipitations with anti-Neur (n) and anti-PPCA antiserum (p). Exposure was for 1 day. Download figure Download PowerPoint Fractionation of the COS-1 cells co-expressing neuraminidase and PPCA-YN or PPCA-LP revealed that the mutant precursors localized in light organelles (Figure 6B and D). This method was not sensitive enough to detect the small amount of mature PPCA-YN that could be immunoprecipitated from radiolabeled cells (see Figure 5), which also explains why we saw no increase over background levels in cathepsin A activity throughout these gradients (data not shown). In both sets of co-transfected cells, most of the neuraminidase was found in light organelles (fractions 4, 5 and 6) (Figure 6A and C); however, a small portion of the enzyme was detected in the denser fractions (fractions 2 and 3) from cells co-expressing PPCA-YN (Figure 6A). Consequently, only these cells displayed a low level of neuraminidase activity in both dense and light organelles (Figure 6A). These data further support the concept that neuraminidase must interact with transport-competent PPCA for its lysosomal localization and activation. Figure 6.Density fractionation of COS-1 cells expressing neuraminidase and PPCA mutants. Following co-transfection of COS-1 cells with neuraminidase cDNA and either (A and B) PPCA-YN or (C and D) PPCA-LP cDNA, cellular organelles were separated on Percoll gradients. Fractions were used to assay for neuraminidase activity (♦), expressed in nmol/h/ml, and to prepare Western blots, which were probed with anti-PPCA antiserum (anti-P) or affinity-purified anti-Neur antiserum (anti-N). For increased sensitivity, blots probed with anti-Neur antiserum were developed with chemiluminescent substrates. Download figure Download PowerPoint Neuraminidase is poorly phosphorylated Soluble lysosomal enzymes are segregated to lysosomes via the interaction of their M6P recognition marker with the M6P receptor (Hille-Rehfeld, 1995; Sabatini and Adesnik, 1995). We therefore investigated whether neuraminidase acquires an M6P recognition marker when it interacts with PPCA. COS-1 cells, transfected with the neuraminidase cDNA, the PPCA cDNA or both cDNAs, were metabolically labeled with [32P]orthophosphate. Cell lysates and media were immunoprecipitated with either anti-Neur or anti-PPCA antibodies. Figure 7 shows that in all of the transfected cells the level of phosphorylation of neuraminidase (lanes 5, 7, 13 and 15) was considerably lower than that of PPCA (lanes 3 and 11), to the extent that the neuraminidase signal from cell lysates barely exceeded background levels even though both proteins were synthesized in comparable amounts in similarly transfected cells (see Figure 1). A clearer 44/46 kDa band could be resolved when the neuraminidase was immunoprecipitated from samples of medium because of the higher signal/noise ratio, which allowed a longer exposure time of this autoradiograph (Figure 7, lanes 13 and 15). We know that neuraminidase was phosphorylated on one or more of its N-linked oligosaccharide chains because the phosphate label was lost after the cells were cultured in the presence of tunicamycin and after treatment with N-glycosidase F (data not shown). The phosphorylation of neuraminidase was not influenced by PPCA. Figure 7.Phosphorylation of neuraminidase. COS-1 cells were transfected as outlined in the legend to Figure 1, and metabolically labeled with [32P]orthophosphate. Cell lysates and media were used for immunoprecipitation with anti-PPCA antiserum (lanes 3, 4, 11 and 12) or anti-Neur antiserum (all other lanes). The recovered proteins were divided into two aliquots and incubated with or without alkaline phosphatase (CIP). Signals are stronger in the medium samples because of a longer exposure time (4 days) compared with cell lysates (16 h). Download figure Download PowerPoint Addition of the M6P marker to lysosomal enzymes is a two-step process: first, an N-acetylglucosamine-1-phosphate residue is bound to the N-linked high-mannose oligosaccharide through the formation of a phosphodiester bond; second, the terminal N-acetylglucosamine is removed by N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase, leaving the M6P exposed (reviewed in von Figura and Hasilik, 1986; Sabatini and Adesnik, 1995). Because the efficiency of lysosomal transport depends on how accessible the M6P marker is to its receptor, we examined the extent to which the mannose-bound phosphate of neuraminidase is exposed. We incubated immunoprecipitated neuraminidase with calf intestinal alkaline phosphatase (CIP), which removes only terminal, monoester-bound phosphate from N-linked oligosaccharides (Isidoro et al., 1991). After CIP treatment, the PPCA was almost completely dephosphorylated (Figure 7, lanes 4 and 12), whereas the neuraminidase retained a substantial proportion of its phosphate label (Figure 7, lanes 14 and 16). Furthermore, we found that neuraminidase from the culture medium of transfected COS-1 cells could not be internalized by deficient sialidosis fibroblasts (data not shown). Discussion Human lysosomal neuraminidase is the only member of the sialidase superfamily that needs another protein, PPCA, to be catalytically active, although influenza virus neuraminidase and some of the bacterial sialidases depend on Ca2+ for optimal activity (reviewed in Saito and Yu, 1995). To explain this unusual requirement of lysosomal neuraminidase, we have studied the various biochemical properties of this enzyme in relation to PPCA. We show that most of the neuraminidase activity, generated through co-expression of neuraminidase and PPCA, is measured in mature dense lysosomes, with only low levels detected in light organelles, although equal amounts of the neuraminidase polypeptide are present in these two compartments. This apparent discrepancy indicates that it is the subcellular location of neuraminidase that determines, to a large extent, its enzymatic activity. Moreover, in the absence of PPCA, neuraminidase is found only in light organelles and is completely inactive. These data suggest that PPCA activates neuraminidase first by interacting with it in a pre-lysosomal compartment, and then by mediating its transport to dense lysosomes. Various mechanisms govern intracellular transport of lysosomal proteins, which depends on their primary structure and solubility. Integral membrane proteins have a hydrophobic transmembrane domain and a specific lysosomal targeting motif in their cytoplasmic tails, containing the characteristic G-Y-X-X-hydrophobic sequence (reviewed in Hunziker and Geuze, 1996). If the cytoplasmic tails of lamp-1, lamp-2, limp-1 and acid phosphatase, for example, are introduced into integral plasma membrane proteins, the latter are re-routed to lysosomes. Lysosomal enzymes without a transmembrane domain are tagged in the Golgi with the M6P recognition marker (Hille-Rehfeld, 1995). Such proteins are dependent on their phosphomannosyl residues for intracellular transport, with the exception of the GM2 activator protein, cathepsin D, the sphingolipid activator protein (SAP) precursor and aspartylglucosaminidase that apparently can reach the lysosome even in the non-glycosylated state (Rijnboutt et al., 1991b; Tikkanen et al., 1995; Vielhaber et al., 1996; Glombitza et al., 1997). For cathepsin D and the SAP precursor, transient membrane association is thought to play a role in lysosomal transport (Rijnboutt et al., 1991a). Hydropathy analysis of neuraminidase did not demonstrate a prominent hydrophobic domain besides the signal sequence (data not shown). Furthermore, it is unlikely that phosphorylation mediates routing of neuraminidase, since the enzyme is poorly phosphorylated, even in the presence of PPCA. Our data suggest that the oligosaccharide-linked phosphates on neuraminidase are only partially unmasked; in addition, those that are unblocked may not be sufficiently multivalent, a requirement for high-affinity binding to M6P receptors (reviewed in Hille-Rehfeld, 1995). This may explain why the M6P marker on neuraminidase is not functional in receptor-mediated endocytosis of thi
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