Adsorptive endocytosis of lysosomal enzymes by human fibroblasts: presence of two different functional systems that deliver an acid hydrolase to lysosomes

Endocytosis of human spleen beta-glucuronidase by human fibroblasts can be completely impaired by the competitive inhibitor mannose 6-phosphate or by pretreatment with acid phosphatase or endoglycosidases H or F. However, endocytosis of bovine spleen and liver beta-glucuronidase is partially impaired by the same treatments, suggesting that the bovine enzyme contains two endocytosis recognition markers located in separate enzyme domains. The mannose 6-phosphate recognition marker seems to be responsible for approximately 23% of the bovine enzyme endocytosis. The existence of two lysosomal endocytosis systems in human fibroblasts is supported by the following facts: (a) the rate of endocytosis of mannose 6-phosphate-containing human beta-glucuronidase was not affected by the presence of high levels of the bovine enzyme (which has only the other marker). (b) Anti-215K mannose 6-phosphate receptor antibodies selectively impair the endocytosis of the beta-glucuronidase containing mannose 6-phosphate. (c) Weak bases exert a differential effect on human and bovine endocytosis. beta-Glucuronidase internalized by either system is targeted to secondary lysosomes of human beta-glucuronidase-deficient fibroblasts, where it is able to degrade accumulated glycosaminoglycans. These results suggest that human fibroblasts have two different and independent endocytic systems for targeting of acid hydrolases to lysosomes.     

Secretion of the lysosomal acid triacylglycerol hydrolase precursor by J774 macrophages

J774, thioglycollate-elicited mouse peritoneal and BCG-induced rabbit alveolar macrophages all contain high levels of a triacylglycerol hydrolase (EC 3.1.1.3) (TGase) with optimal activity at pH 6.5. The J774 macrophages, a cell line deficient in the calcium-independent mannose 6-phosphate receptor, were found to secrete large quantities of the TGase into the culture medium. In contrast, mouse peritoneal and rabbit alveolar macrophages, which are both mannose 6-phosphate receptor-competent cell types, secreted much lower amounts of neutral TGase. The enzyme was localized in the lysosomes of rabbit alveolar macrophages. Addition of 25 mM NH4Cl induced a 6-fold increase in TGase secretion by alveolar macrophages, while 50 mM NH4Cl induced a 12-fold increase in TGase secretion. NH4Cl had no effect on TGase secretion by J774 macrophages. The TGase secreted by J774 macrophages was internalized by I-cell disease fibroblasts, increasing the cellular content of TGase 10-fold after 8 h. Internalization was inhibited 70% by the addition of 2 mM mannose 6-phosphate to the culture medium, but was not affected by 2 mM mannose or glucose 6-phosphate. After internalization, the neutral TGase was converted to a TGase with a pH optimum of 5.1. These data are consistent with the spontaneous release of a lysosomal enzyme precursor from a calcium-independent mannose 6-phosphate receptor-deficient cell line, indicating that the neutral TGase previously reported in several types of macrophages may be the precursor of the lysosomal acid TGase.     

Mannose 6-phosphate/insulin-like growth factor II receptor in I-cell disease fibroblasts: increased synthesis and defective regulation of cell surface expression.

The amount of mannose 6-phosphate/IGF II receptors in fibroblasts from five I-cell patients was about 2-fold higher than in control fibroblasts. The elevated receptor concentration, which led to a higher binding and uptake of mannose 6-phosphate containing ligands and to a higher binding of IGF II resulted from an increased rate of synthesis, while the stability of the receptor was comparable to that in control fibroblasts. Control fibroblasts respond to mannose 6-phosphate, IGF I, IGF II and tumor promoting phorbol esters with a rapid redistribution of mannose 6-phosphate/IGF II receptors from internal membranes to the cell surface. In I-cell fibroblasts only a moderate increase in cell surface receptors was seen after exposure to these effectors. In contrast to control fibroblasts the treatment of I-cell fibroblasts with lysosomotropic amines failed to affect the mannose 6-phosphate containing ligand binding to the receptor. These data provide evidence for multiple potential regulatory sites in intracellular mannose 6-phosphate/IGF II receptor pathway which differ in control and I-cell fibroblasts.     

Biosynthesis and turnover of the phosphomannosyl receptor in human fibroblasts.

The phosphomannosyl receptor mediates intracellular targeting of newly synthesized acid hydrolases to lysosomes, and is also expressed as a pinocytosis receptor on the cell surface of fibroblasts. We have purified the phosphomannosyl receptor from bovine liver and produced rabbit antibodies to the bovine receptor. The antibodies partially blocked pinocytosis of human spleen beta-glucuronidase by fibroblasts, a process mediated by the phosphomannosyl receptor. Affinity-purified antibodies to the phosphomannosyl receptor were used to study the biosynthesis and turnover of the receptor in human fibroblasts. Phosphomannosyl receptor immunoprecipitated after a 15 min pulse-labelling of fibroblasts with [35S]methionine exhibited an identical mobility on sodium dodecyl sulphate/polyacrylamide gels as purified bovine liver phosphomannosyl receptor. Pulse-chase experiments for up to 3 days provided no evidence for changes in molecular weight attributable to post-translational processing of the phosphomannosyl receptor. Turnover studies determined that the half-life of the phosphomannosyl receptor in normal human fibroblasts was 24-29 h. The half-life of the receptor was slightly longer (32 h) in I-cell disease fibroblasts and normal fibroblasts exposed to leupeptin (32 h), slightly shorter in fibroblasts exposed to NH4Cl (23 h) and saturating amounts of ligand (21 h) and unaffected in cells exposed to mannose 6-phosphate (24 h). These studies show that the turnover of the phosphomannosyl receptor in fibroblasts is very slow, in contrast with its rate of internalization in endocytosis, and that its rate of degradation is not greatly altered by a variety of agents that affect lysosomal protein turnover and/or receptor-mediated endocytosis. These results suggest that the degradative activities of the lysosomes do not play an important role in phosphomannosyl receptor turnover in cultured fibroblasts.     

Identification of novel lysosomal matrix proteins by proteome analysis

The lysosomal matrix is estimated to contain about 50 different proteins. Most of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive proteome analysis of MPR-binding proteins from mouse. Mouse embryonic fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to secrete the lysosomal matrix proteins. Secretions of these cells were affinity purified using an affinity matrix derivatized with MPR46 and MPR300. In the protein fraction bound to the affinity matrix and eluted with mannose 6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the lysosomal matrix and 4 non-lysosomal contaminants were identified by mass spectrometry after separation by two-dimensional gel electrophoresis or by multidimensional protein identification technology. For 3 of the candidate proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could verify that C-terminally tagged forms bound in an M6P-dependent manner to an MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix proteins.     

Intracellular transport of acid alpha-glucosidase in human fibroblasts: evidence for involvement of phosphomannosyl receptor-independent system

Intracellular transport of two lysosomal enzymes, acid alpha-glucosidase and beta-hexosaminidase, was analyzed in human fibroblasts. The precursors of beta-hexosaminidase in normal fibroblasts were released from the membrane fraction by treatment with mannose 6-phosphate, but the precursor of alpha-glucosidase was not. Percoll density gradient centrifugation revealed a normal amount of acid alpha-glucosidase activity in heavy lysosomes in I-cell disease fibroblasts despite impaired maturation and defective phosphorylation, and beta-hexosaminidase activity was markedly reduced in lysosomes. It was concluded that the membrane-bound precursor of acid alpha-glucosidase is transported to lysosomes by a phosphomannosyl receptor-independent system although the enzyme lacks the recognition marker for the phosphomannosyl receptor and processing of an intermediate form to mature forms does not occur in this disease.     

Biosynthesis of cathepsin B in cultured normal and I-cell fibroblasts

Biosynthesis and processing of cathepsin B in cultured human skin fibroblasts were investigated using immunological procedures. Upon metabolic labeling with [35S]methionine for 10 min, a precursor form with Mr 44,500 was identified. During an 80-min chase, about 50% of it was converted to an Mr 46,000 form. Further processing yielded mature forms with Mr 33,000 and 27,000, in a final quantitative ratio of about 3:1. Processing of cathepsin B was inhibited by leupeptin, which led to an accumulation of the Mr 33,000 polypeptide. The Mr 33,000 form appeared to be the most active form and showed a half-time of about 12 h. About 5% of newly synthesized enzyme was secreted as precursor, being detectable extracellularly already after 40 min. NH4Cl enhanced the secretion of the precursor about 20-fold. The precursor and the 33-kDa form contained phosphorylated N-linked oligosaccharides. Cleavage by peptide N-glycosidase F or biosynthesis in the presence of tunicamycin yielded a precursor with Mr 39,000. Evidence of a mannose 6-phosphate-dependent transport of cathepsin B in fibroblasts was obtained on the basis of the following results: (i) cathepsin B precursor from NH4Cl-stimulated secretions was internalized in a mannose 6-phosphate inhibitable manner, and (ii) I-cell fibroblasts secreted more than 95% of newly synthesized cathepsin B precursor. In conclusion, cathepsin B from human skin fibroblasts shows an analogous biosynthetic behavior as other lysosomal enzymes.     

Is there a mechanism for introducing acid hydrolases into liver lysosomes that is independent of mannose 6-phosphate recognition? evidence from I-cell disease

We have examined frozen liver tissue for N-acetylglucosamine-l-phosphotransferase, an enzyme required for the formation of the mannose 6-phosphate recognition marker of lysosomal enzymes. Using [β³²P]-UDPGlcNAc and placental β-hexosaminidase B as N-acetylglucosamine l-phosphate donor and acceptor, respectively, we were unable to find activity of the transferase in 100,000 × g membranes prepared from livers of patients with I-cell disease, whereas activity was readily observed in membranes from control livers stored under the same conditions. Yet the activity of several lysosomal enzymes (β-N-acetylglucosaminidase, β-glucuronidase, α-mannosidase and α-$\text{L}$-iduronidase) was comparable in liver tissue of I-cell patients and controls, and only β-galactosidase activity showed a marked reduction. These results suggest that in contrast to cultured skin fibroblasts, liver may be able to introduce into lysosomes acid hydrolases that lack the mannose 6-phosphate recognition marker.     

Biosynthesis and endocytosis of lysosomal enzymes in human colon carcinoma SW 1116 cells: impaired internalization of plasma membrane-associated cation-independent mannose 6-phosphate receptor.

The human colon adenocarcinoma cell lines SW 948, SW 1116, and SW 1222 were tested for their ability to sort and internalize lysosomal enzymes. The biosynthesis of the lysosomal enzymes cathepsin B, arylsulfatase A, and beta-hexosaminidase in these cell lines exhibits no significant differences to that in human fibroblasts. The intracellular targeting of newly synthesized hydrolases to the lysosomes relies in colon carcinoma cells on the mannose 6-phosphate receptor system. Both the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor are expressed in all colon carcinoma cell lines investigated. Endocytosis of lysosomal enzymes via mannose 6-phosphate receptors is reduced in colon carcinoma cells as compared with human fibroblasts. SW 1116 cells were shown to be deficient in receptor-mediated endocytosis of mannose 6-phosphate containing ligands. Ligands of other endocytic receptors as well as the fluid-phase marker horseradish peroxidase were internalized at normal rates. While antibodies against CI-MPR bind to the surface of SW 1116 cells, these antibodies cannot be internalized. These data suggest that the cycling of CI-MPR is specifically impaired in SW 1116 cells.     

Domain 5 of the cation-independent mannose 6-phosphate receptor preferentially binds phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester)

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46 kDa cation-dependent MPR (CD-MPR) are key components of the lysosomal enzyme targeting system that bind newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and divert them from the secretory pathway. Previous studies have mapped two high-affinity Man-6-P binding sites of the CI-MPR to domains 1-3 and 9 and one low-affinity site to domain 5 within its 15-domain extracytoplasmic region. A structure-based sequence alignment predicts that domain 5 contains the four conserved residues (Gln, Arg, Glu, Tyr) identified as essential for Man-6-P binding by the CD-MPR and domains 1-3 and 9 of the CI-MPR. Here we show by surface plasmon resonance (SPR) analyses of constructs containing single amino acid substitutions that these conserved residues (Gln-644, Arg-687, Glu-709, Tyr-714) are critical for carbohydrate recognition by domain 5. Furthermore, the N-glycosylation site at position 711 of domain 5, which is predicted to be located near the binding pocket, has no influence on the carbohydrate binding affinity. Endogenous ligands for the MPRs that contain solely phosphomonoesters (Man-6-P) or phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester, Man-P-GlcNAc) were generated by treating the lysosomal enzyme acid alpha-glucosidase (GAA) with recombinant GlcNAc-phosphotransferase and uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase). SPR analyses using these modified GAAs demonstrate that, unlike the CD-MPR or domain 9 of the CI-MPR, domain 5 exhibits a 14-18-fold higher affinity for Man-P-GlcNAc than Man-6-P, implicating this region of the receptor in targeting phosphodiester-containing lysosomal enzymes to the lysosome.     

Lysosomal integral membrane glycoproteins are expressed at high levels in the inclusion bodies of I-cell disease fibroblasts

The localization, expression, and transport of two lysosomal integral membrane glycoproteins of human cells, hLAMP-1 and hLAMP-2, have been studied in mucolipidosis II (I-cell disease) fibroblasts. These cells are deficient in N-acetylglucosaminylphosphotransferase, one of the enzymes required for addition of the mannose 6-phosphate recognition signal to newly synthesized lysosomal hydrolases and a prerequisite for the sorting and transport of the hydrolases to lysosomes. I-cells analyzed by immunofluorescence microscopy with monoclonal antibodies against hLAMP-1 and hLAMP-2 showed intense staining of the inclusion bodies covering most of the cytoplasm of the cells. Immunoelectron microscopy confirmed this localization and showed that the hLAMP-positive vesicles commonly contained membrane structures or electron-dense homogeneous material characteristic of secondary lysosomes. Studies of the biosynthesis of hLAMP-2 in I-cells pulse-labeled with [35S]methionine indicated that the molecule is glycosylated in the Golgi system, is transported to vesicles with the high density characteristic of lysosomes, and has chemical properties similar to those of the glycoprotein synthesized in normal cells. The concentration of the hLAMP-2 glycoprotein was three- to fourfold greater than that in normal fibroblasts, in sharp contrast to the reduced levels of lysosomal hydrolases seen in I-cells. These experiments demonstrate that the inclusion bodies in I-cells have properties of secondary lysosomes and that the transport and targeting of the lysosomal membrane glycoproteins to the inclusion bodies of these cells is not coupled to the mannose 6-phosphate system for transporting soluble acid hydrolases.     

Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins.

Lysosomal enzymes containing mannose 6-phosphate recognition markers are sorted to lysosomes by mannose 6-phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I-cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6-phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46-deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 -/- mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 -/- mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo.     

I-cell disease-like phenotype in mice deficient in mannose 6-phosphate receptors

Mannose 6-phosphate receptor deficient mice were generated by crossing mice carrying null alleles for Igf2 and the 300 kDa and 46 kDa mannose 6-phosphate receptors, Mpr300 and Mpr46. Pre- and perinatal lethality of mice nullizygous for Igf2, Mpr300 and Mpr46 was increased. Triple deficient mice surviving the first postnatal day had normal viability and developed a phenotype resembling human I-cell disease. The triple deficient mice were characterized by dwarfism, facial dysplasia, waddling gait, dysostosis multiplex, elevated lysosomal enzymes in serum and histological signs of lysosomal storage predominantly in fibroblasts, but also in parenchymal cells of brain and liver. A paternally inherited Mpr300 wild type allele that is normally inactive in mice due to imprinting was reactivated in some tissues of mice lacking IGF II and MPR 46 and carrying a maternal Mpr300 null allele. Inspite of the partial reactivation the phenotype of these mice was similar to that of triple deficient mice.     

Lysosomal storage causes cellular dysfunction in mucolipidosis II skin fibroblasts

Mucolipidosis II (ML-II) is a fatal inherited metabolic disease caused by deficiency of GlcNAc-phosphotransferase, which plays a role in generating the mannose 6-phosphate recognition marker on lysosomal enzymes. In ML-II, many lysosomal acid hydrolases are mistargeted out of cells, and lysosomes become filled with undigested substrates, which explains inclusion cell disease as an alternative name for this disease. In this study, we revealed various cellular phenotypes in ML-II skin fibroblasts. We quantitated phospholipid and cholesterol within cells and showed ~2-fold accumulation in ML-II as compared with normal cells. Lysosomal pH of ML-II cells was higher than that of normal cells (5.29 ± 0.08 versus 4.79 ± 0.10, p < 0.001). The proliferated lysosomes in ML-II cells were accumulated ~3-fold in amount as compared with normal cells. Intracellular logistics including endocytosis and mannose 6-phosphate receptor recycling were impaired in ML-II cells. To confirm whether these ML-II cellular phenotypes derive from deficient lysosomal acid hydrolases within lysosomes, we performed supplementation of lysosomal enzymes using a partially purified total enzyme mixture, which was derived from the conditioned culture medium of normal skin fibroblasts after NH(4)Cl treatment. This supplementation corrected all of the previously described ML-II phenotypes. In addition, the autophagic and mitochondrial impairment that we have previously reported improved, and inclusion bodies disappeared on electron micrography following total lysosomal enzyme supplementation. Our results indicate that various cellular phenotypes in ML-II are caused by the deficiency of many lysosomal enzymes and massive accumulation of undigested substrates.     

Fibroblast chemotaxis and prolidase activity modulation by insulin-like growth factor II and mannose 6-phosphate

Chemotactic locomotion of fibroblasts requires extensive degradation of extracellular matrix components. The degradation is provided by a variety of proteases, including lysosomal enzymes. The process is regulated by cytokines. The present study shows that mannose 6-phosphate and insulin-like growth factor II (IGF-II) enhance fibroblast chemotaxis toward platelet-derived growth factor (PDGF). It is suggested that lysosomal enzymes (bearing mannose 6-phosphate molecules) are involved in chemotactic activity of the cells. The suggestion is supported by the observation that a-mannosidase and cathepsin D inhibitor - pepstatin are very potent inhibitors of fibroblast chemotaxis. Simultaneously, mannose 6-phosphate stimulates extracellular collagen degradation. The final step in collagen degradation is catalyzed by the cytosolic enzyme - prolidase. It has been found that mannose 6-phosphate stimulates also fibroblast prolidase activity with a concomitant increase in lysosomal enzymes activity. The present study demonstrates that the prolidase activity in fibroblasts may reflect the chemotactic activity of the cells and suggests that the mechanism of cell locomotion may involve lysosomal enzyme targeting, probably through IGF-II/mannose 6-phosphate receptor.     

Biosynthesis and processing of lysosomal acid phosphatase in cultured human cells

The biosynthesis of lysosomal acid phosphatase was studied in a normal human embryonic lung cell line, WI-38. Cells were labeled with radioactive leucine under a variety of conditions, the enzyme was immunoprecipitated using a monospecific antiserum raised against human liver lysosomal acid phosphatase, and the products were separated by electrophoresis and were visualized by fluorography. Lysosomal acid phosphatase constitutes 60% of the total tartrate-inhibitable acid phosphatase in WI-38. It is initially synthesized as a high-molecular-weight precursor polypeptide of 69 kDa. The precursor polypeptide is rapidly glycosylated and processed to a mature enzyme of 53-45 kDa via intermediates of 65 and 60 kDa in WI-38 cells. The 69-kDa precursor polypeptide is also converted to larger precursor polypeptides of 74 and 80 kDa. The multiplicity of precursor polypeptides is due at least in part to differences in the glycosylation and phosphorylation of the polypeptides. Sensitivity of phosphorylated oligosaccharide chains from precursor, mature and small polypeptides to endo-beta-hexosaminidase H-catalyzed cleavage suggests the presence of high-mannose phosphorylated oligosaccharide chains similar to those present on many other lysosomal enzymes. The effects of tunicamycin and ammonium chloride were also studied. In contrast to the effect of ammonium chloride on arylsulfatase A secretion, the lysosomal acid phosphatase in WI-38 cells was not secreted in the presence of NH4Cl. This is consistent with the existence of an alternate route for the transfer of lysosomal acid phosphatase into lysosomes. This alternate route may be the reason that I-cell fibroblasts contain a normal level of lysosomal acid phosphatase.     

Targeted disruption of the mouse cation-dependent mannose 6-phosphate receptor results in partial missorting of multiple lysosomal enzymes.

In mammalian cells two mannose 6-phosphate receptors (MPRs) are involved in lysosomal enzyme transport. To understand the precise function of the cation-dependent mannose 6-phosphate receptor (CD-MPR), one allele of the corresponding gene has been disrupted in mouse embryonic stem cells and homozygous mice lacking this receptor have been generated. The homozygous mice appear normal, suggesting that other targeting mechanisms can partially compensate for the loss of the CD-MPR in vivo. However, homozygous receptor-deficient cells and animals clearly exhibit defects in targeting of multiple lysosomal enzymes when compared with wild-types. Increased levels of phosphorylated lysosomal enzymes were present in body fluids of homozygous animals. In thymocytes from homozygous mice or in primary cultures of fibroblasts from homozygous embryos, there is a marked increase in the amount of phosphorylated lysosomal enzymes that are secreted into the extracellular medium. The cultured fibroblasts have decreased intracellular levels of multiple lysosomal enzymes and accumulate macromolecules within their endosomal/lysosomal system. Taken together, these results clearly indicate that the CD-MPR is required for efficient intracellular targeting of multiple lysosomal enzymes.     

Processing and Secretion of Lysosomal Acid α-Glucosidase In Tetrahymena Wild Type and Secretion-Deficient Mutant Cells

ABSTRACT. The proteolytic processing and secretion of a lysosomal enzyme, acid α-glucosidase, was studied by pulse-chase labeling with [³⁵S]methionine in Tetrahymena thermophila CU-399 cells treated with ammonium chloride. This cell secreted a large amount of acid α-glucosidase into the cultured medium during starvation. the secretion was found to be repressed by addition of ammonium chloride (NH₄Cl). Acid α-glucosidase was produced as a precursor form (108 kDa) and then processed to a mature polypeptide (105 kDa) within 60 min. This mature enzyme was secreted into the media within 2-3 h after chase, whereas the precursor form was not secreted by either control cells or NH₄Cl-treated cells. NH₄Cl did not affect the processing of the precursor acid α-glucosidase. Processing profile of this enzyme was apparently indistinguishable from that of the mutant MS-1 defective in lysosomal enzyme secretion. Furthermore, the purified extracellular (CU-399) and intracellular (MS-1) acid a-glucosidases were the same in molecular mass (105 kDa) and enzymatic properties. They contained no mannose 6-phosphate residues in N-linked oligosaccharides. These results suggested that unlike mammalian cells, Tetrahymena acid α-glucosidase may be transferred to lysosomes by a mannose 6-phosphate receptor-independent mechanism, and also that low pH was not essential for the proteolytic processing of precursor polypeptide.     

Lysosomal acid alpha-glucosidase consists of four different peptides processed from a single chain precursor

Pompe's disease is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA is synthesized as a 110-kDa precursor containing N-linked carbohydrates modified with mannose 6-phosphate groups. Following trafficking to the lysosome, presumably via the mannose 6-phosphate receptor, the 110-kDa precursor undergoes a series of complex proteolytic and N-glycan processing events, yielding major species of 76 and 70 kDa. During a detailed characterization of human placental and recombinant human GAA, we found that the peptides released during proteolytic processing remained tightly associated with the major species. The 76-kDa form (amino acids (aa) 122-782) of GAA is associated with peptides of 3.9 kDa (aa 78-113) and 19.4 kDa (aa 792-952). The 70-kDa form (aa 204-782) contains the 3.9- and 19.4-kDa peptide species as well as a 10.3-kDa species (aa 122-199). A similar set of proteolytic fragments has been identified in hamster GAA, suggesting that the multicomponent character is a general phenomenon. Rabbit anti-peptide antibodies have been generated against sequences in the proteolytic fragments and used to demonstrate the time course of uptake and processing of the recombinant GAA precursor in Pompe's disease fibroblasts. The results indicate that the observed fragments are produced intracellularly in the lysosome and not as a result of nonspecific proteolysis during purification. These data demonstrate that the mature forms of GAA characterized by polypeptides of 76 or 70 kDa are in fact larger molecular mass multicomponent enzyme complexes.