Lamp-1 is upregulated in human glioblastoma cell lines induced to undergo apoptosis

Lysosome-associated membrane protein (LAMP)-1, one of the major protein components of the lysosomal membrane, is upregulated in the human glioblastoma cell lines, U-373 MG and LN-Z308, which undergo cisplatin-induced apoptosis. These human brain tumor cell lines demonstrated apoptosis in response to cisplatin/nifedipine treatment. Both cell lines demonstrated an apoptotic response by more than one criterion. Apoptosis was demonstrated by DNA fragmentation techniques such as DNA laddering, ApopTag in situ labeling, and an ELISA-based method of detecting liberated oligosomes. These cells also had characteristic morphologic changes and upregulation of bax consistent with apoptosis. LAMP-1 expression at the protein and mRNA level was examined and found to increase with cisplatin/nifedipine treatment. LAMP-1 expression was examined using indirect immunofluorescent staining, Northern blot analysis and Western blot analysis. The finding of an augmentation of LAMP-1 in these cells induced to die is enigmatic. These findings raise the possibility of LAMP-1 involvement in the apoptotic process.     

At the acidic edge: emerging functions for lysosomal membrane proteins

It has recently become clear that lysosomes have more complex functions than simply being the end-point on a degradative pathway. Similarly, it is now emerging that there are interesting functions for the limiting membranes around these organelles and their associated proteins. Although it has been known for several decades that the lysosomal membrane contains several highly N-glycosylated proteins, including the lysosome-associated membrane proteins LAMP-1 and LAMP-2 and lysosomal integral membrane protein-2/lysosomal membrane glycoprotein-85 (LIMP-2/LGP85), specific functions of these proteins have only recently begun to be recognized. Although the normal functions of LAMP-1 can be substituted by the structurally related LAMP-2, LAMP-2 itself has more specific tasks. Knockout of LAMP-2 in mice has revealed roles for LAMP-2 in lysosomal enzyme targeting, autophagy and lysosomal biogenesis. LAMP-2 deficiency in humans leads to Danon disease, a fatal cardiomyopathy and myopathy. Furthermore, there is evidence that LAMP-2 functions in chaperone-mediated autophagy. LIMP-2/LGP85 also seems to have specific functions in maintaining endosomal transport and lysosomal biogenesis. The pivotal function of lysosomal membrane proteins is also highlighted by the recent identification of disease-causing mutations in cystine and sialic acid transporter proteins, leading to nephropathic cystinosis and Salla disease.     

Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy

The lysosomal membrane proteins LAMP-1 and LAMP-2 are estimated to contribute to about 50% of all proteins of the lysosome membrane. Surprisingly, mice deficient in either LAMP-1 or LAMP-2 are viable and fertile. However, mice deficient in both LAMP-1 and LAMP-2 have an embryonic lethal phenotype. These results show that these two major lysosomal membrane proteins share common functions in vivo. However, LAMP-2 seems to have more specific functions since LAMP-2 single deficiency has more severe consequences than LAMP-1 single deficiency. Mutations in LAMP-2 gene cause a lysosomal glycogen storage disease, Danon disease, in humans. LAMP-2 deficient mice replicate the symptoms found in Danon patients including accumulation of autophagic vacuoles in heart and skeletal muscle. In embryonic fibroblasts, mutual disruption of both LAMPs is associated with an increased accumulation of autophagic vacuoles and unesterified cholesterol, while protein degradation rates are not affected. These results clearly show that the LAMP proteins fulfil functions far beyond the initially suggested roles in maintaining the structural integrity of the lysosomal compartment.     

Cathepsin E deficiency induces a novel form of lysosomal storage disorder showing the accumulation of lysosomal membrane sialoglycoproteins and the elevation of lysosomal pH in macrophages

Cathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE(-/-)) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE(-/-) macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H(+)-ATPase activity in both cell types, the elevated lysosomal pH in CatE(-/-) macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder.     

Neuraminidase 1 is a negative regulator of lysosomal exocytosis

Lysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase NEU1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein LAMP-1. In macrophages from NEU1-deficient mice, a model of the disease sialidosis, and in patients' fibroblasts, oversialylated LAMP-1 enhances lysosomal exocytosis. Silencing of LAMP-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In neu1-/- mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases.     

Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/LAMP-2 double-deficient fibroblasts

Mice double deficient in LAMP-1 and -2 were generated. The embryos died between embryonic days 14.5 and 16.5. An accumulation of autophagic vacuoles was detected in many tissues including endothelial cells and Schwann cells. Fibroblast cell lines derived from the double-deficient embryos accumulated autophagic vacuoles and the autophagy protein LC3II after amino acid starvation. Lysosomal vesicles were larger and more peripherally distributed and showed a lower specific density in Percoll gradients in double deficient when compared with control cells. Lysosomal enzyme activities, cathepsin D processing and mannose-6-phosphate receptor expression levels were not affected by the deficiency of both LAMPs. Surprisingly, LAMP-1 and -2 deficiencies did not affect long-lived protein degradation rates, including proteolysis due to chaperone-mediated autophagy. The LAMP-1/2 double-deficient cells and, to a lesser extent, LAMP-2 single-deficient cells showed an accumulation of unesterified cholesterol in endo/lysosomal, rab7, and NPC1 positive compartments as well as reduced amounts of lipid droplets. The cholesterol accumulation in LAMP-1/2 double-deficient cells could be rescued by overexpression of murine LAMP-2a, but not by LAMP-1, highlighting the more prominent role of LAMP-2. Taken together these findings indicate partially overlapping functions for LAMP-1 and -2 in lysosome biogenesis, autophagy, and cholesterol homeostasis.     

Altered gene expression in cells from patients with lysosomal storage disorders suggests impairment of the ubiquitin pathway

By comparing mRNA profiles in cultured fibroblasts from patients affected with lysosomal storage diseases, we identified differentially expressed genes common to these conditions. These studies, confirmed by biochemical experiments, demonstrated that lysosomal storage is associated with downregulation of ubiquitin C-terminal hydrolase, UCH-L1 in the cells of eight different lysosomal disorders, as well as in the brain of a mouse model of Sandhoff disease. Induction of lysosomal storage by the cysteine protease inhibitor E-64 also reduced UCH-L1 mRNA, protein level and activity. All cells exhibiting lysosomal storage contained ubiquitinated protein aggregates and showed reduced levels of free ubiquitin and decreased proteasome activity. The caspase-mediated apoptosis in E-64-treated fibroblasts was reversed by transfection with a UCH-L1 plasmid, and increased after downregulation of UCH-L1 by siRNA, suggesting that UCH-L1 deficiency and impairment of the ubiquitin-dependent protein degradation pathway can contribute to the increased cell death observed in many lysosomal storage disorders.     

Normal lysosomal morphology and function in LAMP-1-deficient mice

Lysosomal membranes contain two highly glycosylated proteins, designated LAMP-1 and LAMP-2, as major components. LAMP-1 and LAMP-2 are structurally related. To investigate the physiological role of LAMP-1, we have generated mice deficient for this protein. LAMP-1-deficient mice are viable and fertile. In LAMP-1-deficient brain, a mild regional astrogliosis and altered immunoreactivity against cathepsin-D was observed. Histological and ultrastructural analyses of all other tissues did not reveal abnormalities. Lysosomal properties, such as enzyme activities, lysosomal pH, osmotic stability, density, shape, and subcellular distribution were not changed in comparison with controls. Western blot analyses of LAMP-1-deficient and heterozygote tissues revealed an up-regulation of the LAMP-2 protein pointing to a compensatory effect of LAMP-2 in response to the LAMP-1 deficiency. The increase of LAMP-2 was neither correlated with an increase in the level of lamp-2 mRNAs nor with increased half-life time of LAMP-2. This findings suggest a translational regulation of LAMP-2 expression.     

A kinetic analysis of biosynthesis and localization of a lysosome-associated membrane glycoprotein

The biosynthesis and subcellular distribution of a major lysosomal membrane glycoprotein of mouse embryo 3T3 cells, LAMP-1, have been examined by [35S]methionine pulse-labeling, sucrose density gradient fractionation, and oligosaccharide analysis. Mature LAMP-1, immunoprecipitated after labeling for 4 h, had a molecular mass of about 110,000 Da. It comigrated during sucrose density fractionation with lysosomal markers, consistent with previous electron microscopic evidence for its localization in lysosomal membranes. Precursor molecules, pulse-labeled for 5 min and extracted during the first 15 min of post-translational processing, were concentrated in the rough endoplasmic reticulum fraction as a species of 92,000 Da. Within 30 min after synthesis, LAMP-1 was found in fractions enriched in Golgi and lysosomal marker enzyme activities as the mature 110,000-Da glycoprotein. Oligosaccharide processing was complete by 1 h after synthesis, and the mature glycoprotein remained in a fraction bearing lysosomal markers. Treatment of the 92,000-Da precursor with endo-beta-N-acetyl-glucosaminidase H produced a core polypeptide of 43,000 Da. Pulse-labeling in the presence of tunicamycin yielded a 42,000-Da form of LAMP-1, which was converted within 30 min to a 43,000-Da molecule. Bio-Gel column chromatography and hexosamine/hexosaminitol analyses indicated that the mature 110,000-Da molecule contained both complex-type and high-mannose N-linked oligosaccharides.     

Identification and characterization of LAMP-1 as an activation-dependent platelet surface glycoprotein

Platelets normally circulate in a quiescent state. When activated, they undergo biochemical and morphological changes which greatly alter their function and contribute to their role in thrombosis and hemostasis. We have identified, cloned, and sequenced a cDNA from a human unbilical vein endothelial cell library that encodes a 110-kDa integral membrane protein. This protein is present on the surface of activated but not resting platelets and has previously been identified as lysosomal-associated membrane protein 1 (LAMP-1). Half-maximal surface expression of platelet LAMP-1 was induced by concentrations of thrombin that resulted in lysosome enzyme release, not alpha-, or dense granule release. Also consistent with lysosome enzyme studies, there was little surface expression of LAMP-1 in response to the weak agonists ADP and epinephrine. In addition, sucrose density gradient fractionation of platelet granules showed colocalization of LAMP-1 with the lysosomal enzyme, beta-galactosidase, and not with markers of alpha- or dense granules. While we found virtually no LAMP-1 on the resting platelet surface (0-90 molecules/cell), we estimated a mean of 1175 LAMP-1 molecules on the thrombin-activated platelet surface. The translocation of this heavily glycosylated protein to the platelet surface upon stimulation may play a role in the adhesive, prothrombic nature of these cells.     

Effects of lysosomal membrane protein depletion on the Salmonella-containing vacuole

Salmonella is an intracellular bacterial pathogen that replicates within a membrane-bound vacuole in host cells. The major lysosomal membrane proteins 1 and 2 (LAMP-1 and LAMP-2) are recruited to the Salmonella-containing vacuole as well as Salmonella- associated filaments (Sifs) that emerge from the vacuole. LAMP-1 is a dominant membrane marker for the vacuole and Sifs. Its colocalization with both is dependent on a major secreted bacterial virulence protein, SifA. Here, we show that SifA is required for the recruitment of LAMP-2 and can be used as a second independent marker for both the bacterial vacuolar membrane and Sifs. Further, RNAi studies revealed that in LAMP-1 depleted cells, the bacteria remain membrane bound as measured by their association with LAMP-2 protein. In contrast, LAMP-2 depletion increased the amount of LAMP-1 free bacteria. Together, the data suggests that despite its abundance, LAMP-1 is not essential, but LAMP-2 may be partially important for the Salmonella-containing vacuolar membrane.     

Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants

Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal enzymes tyrosinase-related protein 1 (Tyrp1) and tyrosinase follow an intracellular Golgi to melanosome pathway, whereas in the absence of glycosphingolipids, they are observed to pass over the cell surface. Unexpectedly, the lysosome-associated membrane protein 1 (LAMP-1) and 2 behaved exactly opposite: they were found to travel through the cell surface in control melanocytes but followed an intracellular pathway in the absence of glycosphingolipids. Chimeric proteins having the cytoplasmic tail of Tyrp1 or tyrosinase were transported like lysosomal proteins, whereas a LAMP-1 construct containing the lumenal domain of Tyrp1 localized to melanosomes. In conclusion, the lumenal domain contains sorting information that guides Tyrp1 and probably tyrosinase to melanosomes by an intracellular route that excludes lysosomal proteins and requires glucosylceramide.     

Immunochemical analysis of CD107a (LAMP-1)

CD107a, also known as the lysosome associated membrane protein-1 (LAMP-1), is expressed largely in the endosome-lysosome membranes of cells, but is also found on the plasma membrane (1-2% of total LAMP-1). LAMP-1 has been implicated in a variety of cellular functions, including cancer metastasis. It has been proposed as a therapeutic agent for some cancers, and is a marker for lysosomal storage disorders and different cell types such as cytotoxic T cells. In light of this diversity of applications, it is important to have well characterized immune-reagents for the detection and quantification of LAMP-1. We have compared a new monoclonal antibody 80280 against LAMP-1 to an existing monoclonal antibody BB6 and a rabbit polyclonal antibody. While all antibodies gave similar results by immunofluorescence, the monoclonal antibody 80280 showed no epitope reactivity to LAMP-1 peptides, suggesting the possibility of a carbohydrate epitope. Western blotting revealed a weaker activity of the monoclonal antibody 80280 relative to either the BB6 monoclonal or the polyclonal antibodies. The monoclonal antibody 80280 is distinct from BB6, providing an additional reagent for CD107a analysis.     

Lysosomal membrane proteins: life between acid and neutral conditions

Whereas we have a profound understanding about the function and biogenesis of the protein constituents in the lumen of the lysosomal compartment, much less is known about the functions of proteins of the lysosomal membrane. Proteomic analyses of the lysosomal membrane suggest that, apart from the well-known lysosomal membrane proteins, additional and less abundant membrane proteins are present. The identification of disease-causing genes and the in-depth analysis of knockout mice leading to mutated or absent membrane proteins of the lysosomal membrane have demonstrated the essential role of these proteins in lysosomal acidification, transport of metabolites resulting from hydrolytic degradation and interaction and fusion with other cellular membrane systems. In addition, trafficking pathways of lysosomal membrane proteins are closely linked to the biogenesis of this compartment. This is exemplified by the recent finding that LIMP-2 (lysosomal integral membrane protein type-2) is responsible for the mannose 6-phosphate receptor-independent delivery of newly synthesized β-glucocerebrosidase to the lysosome. Similar to LIMP-2, which could also be linked to vesicular transport processes in certain polarized cell types, the major constituents of the lysosomal membrane, the glycoproteins LAMP (lysosome-associated membrane protein)-1 and LAMP-2 are essential for regulation of lysosomal motility and participating in control of membrane fusion events between autophagosomes or phagosomes with late endosomes/lysosomes. Our recent investigations into the role of these proteins have not only increased our understanding of the endolysosomal system, but also supported their major role in cell physiology and the development of different diseases.     

Lysosomal exocytosis: An important event during invasion of lamp deficient cells by extracellular amastigotes of Trypanosoma cruzi

Trypanosoma cruzi is an obligate intracellular organism in vertebrate hosts. Lysosomes are involved in parasite invasion. LAMP-1 and LAMP-2 are the most abundant glycoproteins of the lysosomal membrane. This study is the first report on the invasion of T. cruzi extracellular amastigotes (EA) in single LAMP-1 or LAMP-2 knockouts, respectively, or in two independent LAMP-1/2 double-knockout cell lines. When compared to their respective wild type clones, the EA show higher infectivity in LAMP-2 knockouts, but no difference was seen in LAMP-1 knockout cells. Similarly, EA invasion rate was higher for one of the double knockout clones but not for the other. Higher lysosomal exocytosis correlated with a higher invasion rate and early lysosomal marker acquisition. These findings suggest that lysosomal exocytosis is important to EA cell invasion. Also, phagolysosome maturation in knockout cell lines differed from previous results revealing that EA enter cells by a mechanism other than receptor-mediated phagocytosis.     

Role for LAMP‐2 in endosomal cholesterol transport

The mechanisms of endosomal and lysosomal cholesterol traffic are still poorly understood. We showed previously that unesterified cholesterol accumulates in the late endosomes and lysosomes of fibroblasts deficient in both lysosome associated membrane protein-2 (LAMP-2) and LAMP-1, two abundant membrane proteins of late endosomes and lysosomes. In this study we show that in cells deficient in both LAMP-1 and LAMP-2 (LAMP^−/−), low-density lipoprotein (LDL) receptor levels and LDL uptake are increased as compared to wild-type cells. However, there is a defect in esterification of both endogenous and LDL cholesterol. These results suggest that LAMP^−/− cells have a defect in cholesterol transport to the site of esterification in the endoplasmic reticulum, likely due to defective export of cholesterol out of late endosomes or lysosomes. We also show that cholesterol accumulates in LAMP-2 deficient liver and that overexpression of LAMP-2 retards the lysosomal cholesterol accumulation induced by U18666A. These results point to a critical role for LAMP-2 in endosomal/lysosomal cholesterol export. Moreover, the late endosomal/lysosomal cholesterol accumulation in LAMP^−/− cells was diminished by overexpression of any of the three isoforms of LAMP-2, but not by LAMP-1. The LAMP-2 luminal domain, the membrane-proximal half in particular, was necessary and sufficient for the rescue effect. Taken together, our results suggest that LAMP-2, its luminal domain in particular, plays a critical role in endosomal cholesterol transport and that this is distinct from the chaperone-mediated autophagy function of LAMP-2.