Gamma-interferon-inducible lysosomal thiol reductase (GILT). Maturation, activity, and mechanism of action

We recently identified a gamma-interferon-inducible lysosomal thiol reductase (GILT), constitutively expressed in antigen-presenting cells, that catalyzes disulfide bond reduction both in vitro and in vivo and is optimally active at acidic pH. GILT is synthesized as a 35-kDa precursor, and following delivery to major histocompatibility complex (MHC) class II-containing compartments (MIICs), is processed to the mature 30-kDa form via cleavage of N- and C-terminal propeptides. The generation of MHC class II epitopes requires both protein denaturation and reduction of intra- and inter-chain disulfide bonds prior to proteolysis. GILT may be important in disulfide bond reduction of proteins delivered to MIICs and consequently in antigen processing. In this report we show that, like its mature form, precursor GILT reduces disulfide bonds with an acidic pH optimum, suggesting that it may also be involved in disulfide bond reduction in the endocytic pathway. We also show that processing of precursor GILT can be mediated by multiple lysosomal proteases and provide evidence that the mechanism of action of GILT resembles that of other thiol oxidoreductases.     

Targeting of lysosomal acid phosphatase with altered carbohydrate

Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes, where it is converted into a soluble protein by a limited proteolysis (Waheed et al., 1988, EMBO J. 7, 2351-2358). Transport of human lysosomal acid phosphatase in heterologous BHK-21 cells was examined under conditions that impair mannose-6-phosphate receptor-dependent transport, N-glycosylation or processing of N-linked oligosaccharides. Targeting of lysosomal acid phosphatase to lysosomes was neither affected by antibodies blocking the mannose-6-phosphate/IGF II receptor, nor by NH4Cl, which inhibited the mannose-6-phosphate receptor-dependent targeting of soluble lysosomal enzymes. 1-Deoxynojirimycin, 1-deoxymannojirimycin and swainsonine inhibited processing of N-linked oligosaccharides in lysosomal acid phosphatase without significantly affecting its transport. Tunicamycin inhibited N-glycosylation of lysosomal acid phosphatase. The non-glycosylated lysosomal acid phosphatase polypeptides accumulated within light membranes and were not transported to dense lysosomes. These results indicate that transport of lysosomal acid phosphatase is independent of mannose-6-phosphate receptors, does not involve an acid pH-dependent step and does not require processing of N-linked oligosaccharides. N-glycosylation appears to be necessary to achieve a transport competent form of lysosomal acid phosphatase.     

Lysosomal membrane proteins do not bind to mannose-6-phosphate-specific receptors

Lysosomal membrane proteins and soluble lysosomal material were isolated from pulse-chase labelled human skin fibroblasts and examined for incorporation of radioactivity and affinity to immobilized mannose-6-phosphate-specific receptors. Incorporation of radioactivity into lysosomal membrane proteins was delayed by about 2 h on average when compared to that of soluble lysosomal proteins. The lack of binding indicates that a mannose-6-phosphate-independent mechanism is responsible for targeting of lysosomal membrane proteins to lysosomes. In contrast to soluble lysosomal proteins, the membrane proteins did not bind to mannose-6-phosphate specific receptors. The delayed appearance of membrane proteins in lysosomes as compared to that of soluble lysosomal proteins suggested that different pathways are utilized by the two classes of lysosomal proteins.     

Glycosylation-independent Lysosomal Targeting of Acid α-Glucosidase Enhances Muscle Glycogen Clearance in Pompe Mice

We have used a peptide-based targeting system to improve lysosomal delivery of acid α-glucosidase (GAA), the enzyme deficient in patients with Pompe disease. Human GAA was fused to the glycosylation-independent lysosomal targeting (GILT) tag, which contains a portion of insulin-like growth factor II, to create an active, chimeric enzyme with high affinity for the cation-independent mannose 6-phosphate receptor. GILT-tagged GAA was taken up by L6 myoblasts about 25-fold more efficiently than was recombinant human GAA (rhGAA). Once delivered to the lysosome, the mature form of GILT-tagged GAA was indistinguishable from rhGAA and persisted with a half-life indistinguishable from rhGAA. GILT-tagged GAA was significantly more effective than rhGAA in clearing glycogen from numerous skeletal muscle tissues in the Pompe mouse model. The GILT-tagged GAA enzyme may provide an improved enzyme replacement therapy for Pompe disease patients.     

Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes in transfected baby hamster kidney cells.

BHK cells transfected with human lysosomal acid phosphatase (LAP) cDNA (CT29) expressed 70-fold higher enzyme activities of acid phosphatase than non-transfected BHK cells. The CT29-LAP was synthesized in BHK cells as a heterogeneously glycosylated precursor that was tightly membrane associated. Transfer to the trans-Golgi was associated with a small increase in size (approximately 7 kd) and partial processing of the oligosaccharides to complex type structures. CT29-LAP was transferred into lysosomes as shown by subcellular fractionation, immunofluorescence and immunoelectron microscopy. Lack of mannose-6-phosphate residues suggested that transport does not involve mannose-6-phosphate receptors. Part of the membrane-associated CT29-LAP was processed to a soluble form. The mechanism that converts CT29-LAP into a soluble form was sensitive to NH4Cl, and reduced the size of the polypeptide by 7 kd. In vitro translation of CT29-derived cRNA in the presence of microsomal membranes yielded a CT29-LAP precursor that is protected from proteinase K except for a small peptide of approximately 2 kd. In combination with the sequence data available for LAP, these observations suggest that CT29-LAP is synthesized and transported to lysosomes as a transmembrane protein. In the lysosomes, CT29-LAP is released from the membrane by proteolytic cleavage, which removes a C-terminal peptide including the transmembrane domain and the cytosolic tail of 18 amino acids.     

Functional requirements for the lysosomal thiol reductase GILT in MHC class II-restricted antigen processing

Ag processing and presentation via MHC class II is essential for activation of CD4(+) T lymphocytes. gamma-IFN-inducible lysosomal thiol reductase (GILT) is present in the MHC class II loading compartment and has been shown to facilitate class II Ag processing and recall responses to Ags containing disulfide bonds such as hen egg lysozyme (HEL). Reduction of proteins within the MHC class II loading compartment is hypothesized to expose residues for class II binding and protease trimming. In vitro analysis has shown that the active site of GILT involves Cys(46) and Cys(49), present in a CXXC motif that shares similarity with the thioredoxin family. To define the functional requirements for GILT in MHC class II Ag processing, a GILT-deficient murine B cell lymphoma line was generated and stably transduced with wild-type and cysteine mutants of GILT. Intracellular flow cytometric, immunoblotting, and immunofluorescence analyses demonstrated that wild-type and mutant GILT were expressed and maintained lysosomal localization. Transduction with wild-type GILT reconstituted MHC class II processing of a GILT-dependent HEL epitope. Mutation of either Cys(46) or Cys(49) abrogated MHC class II processing of a GILT-dependent HEL epitope. In addition, biochemical analysis of these mutants suggested that the active site facilitates processing of precursor GILT to the mature form. Precursor forms of GILT-bearing mutations in Cys(200) or Cys(211), previously found to display thiol reductase activity in vitro, could not mediate Ag processing. These studies demonstrate that the thiol reductase activity of GILT is its essential function in MHC class II-restricted Ag processing.     

Exposure of the promonocytic cell line THP-1 to Escherichia coli induces IFN-gamma-inducible lysosomal thiol reductase expression by inflammatory cytokines

IFN-gamma-inducible lysosomal thiol reductase (GILT), which plays a role in MHC class II-restricted processing and presentation of Ags containing disulfide bonds, can be induced in various cell types by the cytokine IFN-gamma. APCs, including circulating macrophages, constitutively express high levels of GILT, although the pathways regulating its expression in these cells have not been characterized. In this study, we used the promonocytic cell line THP-1, an established model for monocyte to macrophage differentiation, to investigate the induction of GILT upon exposure to bacteria. We show that contact with LPS or intact Escherichia coli causes THP-1 cells to undergo programmed differentiation, characterized by adhesion, cytokine secretion, and up-regulation of Ag processing and presentation components, including GILT. Unlike GILT induction in response to IFN-gamma treatment, induction by bacteria is dependent on new protein synthesis, NF-kappaB signaling, and secretion of the inflammatory cytokines TNF and IL-1beta. Furthermore, we show that both cytokines are sufficient for GILT induction in the absence of a microbial stimulus. The majority of GILT synthesized by differentiated THP-1 cells is secreted as the precursor form rather than being transported to, and maturing in, lysosomes, suggesting a novel role for GILT in cells of the macrophage lineage.     

Human and hamster procathepsin D, although equally tagged with mannose-6-phosphate, are differentially targeted to lysosomes in transfected BHK cells

BHK cells transfected with human cathepsin D (CD) cDNA normally segregate the autologous hamster cathepsin D while secreting a large proportion of the human proenzyme. In the present work, we have utilized these transfectants to examine to what extent the mannose-6-phosphate-dependent pathway for lysosomal enzyme segregation contributes to the differential sorting of human and hamster CD. We report that, in recipient control BHK cells, the rate of mannose-6-phosphate-dependent endocytosis of human procathepsin D secreted by transfected BHK cells is lower than that of hamster procathepsin D and much lower than that of human arylsulphatase A. The missorted human enzyme bears phosphorylated oligosaccharides and most of its phosphate residues are “uncovered”, like the autologous enzyme. Thus, despite both the Golgi-associated modifications of oligosaccharides, i.e. the phosphorylation of mannose and the uncovering of mannose-6-phosphate residues, which proceed on human and hamster procathepsin D with comparable efficiency, only the latter is accurately packaged into lysosomes. Ammonium chloride partially affects the lysosomal targeting of cathepsin D in control BHK cells, whereas in transfected cells, this drug strongly inhibits the maturation of human procathepsin D and slightly enhances its secretion. These data indicate that: (1) over-expression of a lysosomal protein does not saturate the Golgi-associated reactions leading to the synthesis of mannose-6-phosphate; (2) a portion of cathepsin D is targeted independently of mannose-6-phosphate receptors in the transfected BHK cells; and (3) whichever mechanism for lysosomal delivery of autologous procathepsin D is involved, this is not saturated by the high rate of expression of human cathepsin D.     

Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion

Human lysosomal alpha-galactosidase A (alpha-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme overexpressor, produced intracellular alpha-Gal A levels of 20,900 U/mg (approximately 100 micrograms of enzyme/10(7) cells) and secreted approximately 13,000 U (or 75 micrograms/10(7) cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 microns crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity-purified anti-human alpha-Gal A antibodies. Pulse-chase studies revealed that approximately 65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the alpha-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6-phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted alpha-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification, presumably due to the inaccessibility of the enzyme's tyrosine residues for the sulfotransferase in the TGN. Overexpression of human lysosomal alpha-N-acetylgalactosaminidase and acid sphingomyelinase in CHO cell lines also resulted in their respective selective secretion. In vitro studies revealed that purified secreted alpha-Gal A was precipitated as a function of enzyme concentration and pH, with 30% of the soluble enzyme being precipitated when 10 mg/ml of enzyme was incubated at pH 5.0. Thus, it is hypothesized that these overexpressed lysosomal enzymes are normally modified until they reach the TGN where the more acidic environment of this compartment causes the formation of soluble and particulate enzyme aggregates. A significant proportion of these enzyme aggregates are unable to bind the M6PR and are selectively secreted via the constitutive secretory pathway, while endogenous lysosomal enzymes bind the M6PRs and are transported to lysosomes.     

Purification of a lysosomal DNase from Drosophila melanogaster.

An acid DNase was purified from Drosophila melanogaster till apparent homogeneity by six consecutive chromatographic steps. The enzyme is a lysosomal DNase, because it is glycosylated and carries 1.8-2.4 mol of mannose-6-phosphate/mol of enzyme. The enzyme is fully active without any divalent cation and introduces single stranded nicks into a supercoiled DNA.     

A Quantitative Mannose 6-Phosphate Receptor-Basedin VitroAssay for Recombinant HumanN-Acetylgalactosamine-4-sulfatase

An assay was developed, using two similar formats, to simultaneously measure both the lysosomal targeting receptor binding and enzyme activity of the recombinant human enzymeN-acetylgalactosamine-4-sulfatase. This assay also has potential application for all phosphorylated lysosomal enzymes that contain mannose-6-phosphate residues. The receptor was either purified from fetal bovine sera then adsorbed, or producedin situby growing and fixing diploid human fibroblast-like cells, to a solid phase. The enzyme substrate was 4-methylumbelliferyl sulfate which fluoresces after cleavage of the sulfate moiety. Both the precursor and mature forms of the recombinant enzyme were used to demonstrate the specificity and usefulness of the assay. The assay is rapid and sensitive and has a wide dynamic range. Association between the receptor and the mannose-6-phosphate residues was abrogated in the presence of a competitive inhibitor, mannose 6-phosphate. However, partial activity was still measured when the mature enzyme was incubated in the presence of mannose 6-phosphate when using the fixed fibroblast format. This would indicate that the recombinant enzymes contain at least one terminal sugar moiety other than mannose 6-phosphate which can recognize receptors on the surface of human fibroblast-like cells. Other possible applications of this assay are also discussed.     

Structural requirements for efficient processing and activation of recombinant human UDP-N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase

Mannose 6-phosphate-modified N-glycans are the determinant for intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme responsible for the initial step in the synthesis of mannose 6-phosphate is UDP-N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosmine-1-phosphotransferase(GlcNAc-phosphotransferase). GlcNAc-phosphotransferase is a multisubunit enzyme with an alpha2beta2gamma2 arrangement that requires a detergent for solubilization. Recent cloning of cDNAs and genes encoding these subunits revealed that the alpha- and beta-subunits are encoded by a single gene as a precursor, whereas the gamma-subunit is encoded by a second gene. The hydropathy plots of the deduced amino acid sequences suggested that the alpha- and beta-subunits but not the gamma-subunit contain transmembrane domains. Access to these cDNAs allowed us to express a soluble form of human recombinant GlcNAc-phosphotransferase by removing the putative transmembrane and cytoplasmic domains from the alpha- and beta-subunits. Because this modification prevented precursor processing to mature alpha- and beta-subunits, the native cleavage sequence was replaced by a cleavage site for furin. When the modified alpha/beta-subunits (alpha'/beta'-subunits) precursor and wild type gamma-subunit cDNAs were co-expressed in 293T or CHO-K1 cells, a furin-like protease activity in these cells cleaved the precursor and produced an active and processed soluble GlcNAc-phosphotransferase with an alpha'2beta'2gamma2-subunits arrangement. Recombinant soluble GlcNAc-phosphotransferase exhibited specific activity and substrate preferences similar to the wild type bovine GlcNAc-phosphotransferase and was able to phosphorylate a lysosomal hydrolase, acid alpha-glucosidase in vitro.     

Inhibition by lysosomal enzymes and mannose-6-phosphate of the phosphorylation of the lysosomal enzyme binding receptor protein from monkey brain

The lysosomal enzymes beta-glucuronidase and alpha-L-fucosidase and mannose-6-phosphate inhibited the phosphorylation of the lysosomal enzyme binding receptor protein prepared from monkey brain. Inhibition of both serine and tyrosine phosphorylation was observed. A non-lysosomal glycoprotein enzyme butyrylcholinesterase, mannose or glucose did not inhibit phosphorylation. Tyrosine phosphorylation of histone by the receptor protein was also inhibited by the lysosomal enzymes and mannose-6-phosphate.     

Secretion by mononuclear phagocytes of lysosomal hydrolases bearing ligands for the mannose-6-phosphate receptor system of fibroblasts: Evidence for a second mechanism of spontaneous secretion?

β-Hexosaminidase secreted by peritoneal macrophages in response to stimulation by zymosan or NH₄Cl, or spontaneously by a macrophage-like cell line (P388D₁), is susceptible to receptor-mediated endocytosis by human fibroblasts. This endocytosis is almost completely blocked by exogenous mannose-6-phosphate and therefore seems to depend on a mannose-6-phosphate ligand on the enzyme. It is suggested that macrophage lysosomal enzyme packaging may involve mannose-6-phosphate recognition markers, and that a continuous hypersecretion mechanism may exist which does not depend on a defect in this ligand.     

Suppression of the 'uncovering' of mannose-6-phosphate residues in lysosomal enzymes in the presence of NH4Cl

The uncovering ratio of phosphate groups in lysosomal enzymes is defined as the percentage of phosphomonoester groups in the oligosaccharide side chains based on the sum of phosphomonoester and phosphodiester groups. Using a new procedure for the specific and complete hydrolysis of uncovered phosphomonoester groups in denatured immunoprecipitates of human cathepsin D, we show that the uncovering ratio varies between different forms of the enzyme and may be used as an indicator of the maturation of its carbohydrate side chains. The uncovering ratio in the total (cellular and secreted) cathepsin D from U937 promonocytes is greater than 95%. It is only slightly decreased in cells incubated in the presence of 1 alpha,25-dihydroxycholecalciferol, in which the rate of synthesis of cathepsin D is several times higher than in the control cells. In U937 cells and also in fibroblasts, the uncovering is nearly complete in intermediate and mature forms of the intracellular cathepsin D but less extensive in the intracellular and secreted precursor. In both cell types, incubation with 10 mM NH4Cl results in a decrease in the uncovering ratio of total cathepsin D. However, the activity of the uncovering enzyme, N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, as determined with UDP-N-acetylglucosamine is not affected with up to 60 mM NH4Cl. Our results suggest that NH4Cl, in addition to its known effects on the acidic-pH-dependent functions of lysosomal compartments and of mannose-6-phosphate receptors, impairs the processing or transport of lysosomal enzyme precursors at, or proximally to, the site of the uncovering of their mannose-6-phosphate residues.     

Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).

Sphingolipid activator proteins SAP-A, -B, -C and -D (also called saposins) are generated by proteolytic processing from a 73 kDa precursor and function as obligatory activators of lysosomal enzymes involved in glycosphingolipid metabolism. Although the SAP precursor can be recognized by the mannose-6-phosphate (M-6-P) receptor and shuttled directly from the secretory pathway to the lysosome, a substantial fraction of newly synthesized precursor is secreted from the cell where it may participate in sphingolipid transport and signaling events. Re-uptake of the secreted precursor is mediated by high-affinity cell surface receptors that are apparently distinct from the M-6-P receptor. We found that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic receptor that is expressed on most cells, can mediate cellular uptake and lysosomal delivery of SAP precursor. Additional in vivo experiments in mice revealed that the mannose receptor system on macrophages also participates in precursor internalization. We conclude that SAP precursor gains entry into cells by at least three independent receptor mechanisms including the M-6-P receptor, the mannose receptor and LRP.     

Five related Lebanese individuals with high plasma lysosomal hydrolases: a new defect in mannose-6-phosphate receptor recognition?

Five healthy related individuals in 3 generations of a Lebanese family have been found to have highly elevated plasma lysosomal enzyme levels inherited as a dominant Mendelian trait. The same enzymes in other extracellular fluids were within normal limits. While the pattern and extent of plasma enzyme elevation was similar to that found in mucolipidoses II and III, the physicochemical properties of the elevated enzymes were different from those of both control and I-cell disease plasma. Secretion of lysosomal hydrolases into cell media by fibroblasts from one of the individuals was increased two to seven times more than that from controls. The results suggest faulty recognition between lysosomal hydrolases and mannose-6-phosphate receptors. This could be caused by a defect either in the phosphodiesterase that normally uncovers mannose-6-phosphate hydrolase markers or in the mannose-6-phosphate receptor itself.     

γ-Interferon-inducible Lysosomal Thiol Reductase (GILT) Maintains Phagosomal Proteolysis in Alternatively Activated Macrophages

Although it is known that lysosomal cysteine cathepsins require a reducing environment for optimal activity, it is not firmly established how these enzymes are maintained in their reduced-active state in the acidic and occasionally oxidative environment within phagosomes and lysosomes. γ-Interferon-inducible lysosomal thiol reductase (GILT) has been the only enzyme described in the endosomes, lysosomes, and phagosomes with the potential to catalyze the reduction of cysteine cathepsins. Our goal in the current study was to assess the effect of GILT on major phagosomal functions with an emphasis on proteolytic efficiency in murine bone marrow-derived macrophages. Assessment of phagosomal disulfide reduction upon internalization of IgG-opsonized experimental particles confirmed a major role for GILT in phagosomal disulfide reduction in both resting and interferon-γ-activated macrophages. Furthermore we observed a decrease in early phagosomal proteolytic efficiency in GILT-deficient macrophages, specifically in the absence of an NADPH oxidase-mediated respiratory burst. This deficiency was more prominent in IL-4-activated macrophages that inherently possess lower levels of NADPH oxidase activity. Finally, we provide evidence that GILT is required for optimal activity of the lysosomal cysteine protease, cathepsin S. In summary, our results suggest a role for GILT in maintaining cysteine cathepsin proteolytic efficiency in phagosomes, particularly in the absence of high NADPH oxidase activity, which is characteristic of alternatively activated macrophages.     

Role of acidic intracellular compartments in the biosynthesis of Dictyostelium lysosomal enzymes. The weak bases ammonium chloride and chloroquine differentially affect proteolytic processing and sorting

Radiolabel pulse-chase and subcellular fractionation procedures were used to analyze the transport, proteolytic processing, and sorting of two lysosomal enzymes in Dictyostelium discoideum cells treated with the weak bases ammonium chloride and chloroquine. Dictyostelium lacks detectable cation-independent mannose-6-phosphate receptors and represents an excellent system to investigate alternative mechanisms for lysosomal enzyme targeting. Exposure of growing cells to ammonium chloride, which increased the pH in intracellular vacuoles from 5.4 to 5.8-6.1, slowed but did not prevent the proteolytic processing and correct localization of pulse-radiolabeled precursors to the lysosomal enzymes alpha-mannosidase and beta-glucosidase. Additionally, ammonium chloride did not affect transport of the enzymes to the Golgi complex, as they acquired resistance to the enzyme endoglycosidase H at the same rate as in control cells. When the pH of lysosomal and endosomal organelles was raised to 6.4 with higher concentrations of ammonium chloride, the percentage of secreted (apparently mis-sorted) precursor polypeptides increased slightly, but proteolytic processing of intermediate forms of lysosomal enzymes to mature forms was greatly reduced. The intermediate and mature forms of alpha-mannosidase and beta-glucosidase did, however, accumulate intracellularly in vesicles similar in density to lysosomes. In contrast, in cells exposed to low concentrations of chloroquine the intravacuolar pH increased only slightly (to 5.7); however, enzymes were inefficiently processed and, instead, rapidly secreted as precursor molecules. Experiments involving the addition of chloroquine at various times during the chase of pulse-radiolabeled cells demonstrated that this weak base acted on a distal Golgi or prelysosomal compartment to prevent the normal sorting of lysosomal enzymes. These results suggest that although acidic endosomal/lysosomal compartments may be important for the complete proteolytic processing of lysosomal enzymes in Dictyostelium, low pH is not essential for the proper targeting of precursor polypeptides. Furthermore, certain amines may induce mis-sorting of these enzymes by pH-independent mechanisms.