BR 96 is an internalizing antibody that binds to Lewis Y (Le(y)), a carbohydrate determinant expressed at high levels on many human carcinomas (Hellstrom, I., H. J. Garrigues, U. Garrigues, and K. E. Hellstrom. 1990. Cancer Res. 50:2183-2190). Breast carcinoma cell lines grown to confluence bind less BR96 than subconfluent cultures (Garrigues, J., U. Garrigues, I. Hellstrom, and K. E. Hellstrom. 1993. Am. J. Path. 142:607-622). However, when the confluent cells are induced to migrate by scratch wounding, they again bind BR96 suggesting that antigens bearing the Le(y) determinant may promote cell migration. In the present study, BR96 was found to be highly enriched on microspikes and ruffled membranes, cell surface structures involved in cell migration. In addition, BR96 was a potent inhibitor of cell migration in vitro. When stationary BR96 treated cells were exposed to fresh culture media, membrane ruffles and microspikes developed at the cell margin and migration resumed. Immunogold microscopy showed that BR96 antigens were enriched on these membrane protrusions. BR96 cell surface immunoprecipitation analysis of 3H-glucosamine labeled breast carcinoma cells identified antigens with approximate molecular weights of 135 kd (upper antigen) and 85 kd (lower antigen). A short amino terminal sequence (8 residues) of the upper antigen matched that of human lysosomal membrane glycoprotein 1 (LAMP-1). In addition, the upper antigen was detected on immunoblots probed with anti-LAMP-1, and within the intracellular compartment BR96 was found predominantly in endosomes and lysosomes. A soluble LAMP-1/immunoglobulin fusion protein (LAMP-1/Ig) was transiently expressed in both BR96 binding and nonbinding cell lines. Immunoblot analysis of LAMP-1/Ig's from the various cell lines showed that (a) acquisition of the BR96 epitope is probably controlled at the level of polylactosamine modification (e.g., fucosylation) rather than LAMP-1 gene expression; (b) alternate forms of LAMP-1/Ig comigrate with the lower BR96 antigen raising the possibility that it may be a degradation product of the upper antigen; and (c) LAMP-1/Ig expressed in 3396 breast carcinoma cells has approximately 30-fold more BR96 epitopes than LAMP-1/Ig from non- tumorigenic mammary epithelial cells. Together these data indicate that a major BR96 antigen, LAMP-1, is present on unique cell surface domains involved in cell locomotion as well as membranes of the endocytic compartment. Altered glycosylation of LAMP-1 expressed in transformed cells may contribute to their ability to disseminate.
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.
LAMP-2; LAMP-1; cholesterol; LDL; lysosome; late endosome; NPC2
An endogenous Madin-Darby canine kidney (MDCK) lysosomal membrane glycoprotein that exhibits a basolateral targeting pathway to the lysosome is shown here to exhibit significant N-terminal amino acid sequence identity to lysosomal associated membrane proteins (LAMP-2) of other species. During establishment of the MDCK monolayer after only 1 d of culture, this canine LAMP-2 has a larger molecular size (110 kDa) than following formation of a confluent monolayer after 3 d of culture (100 kDa) due to the increased presence of N-linked polylactosamine oligosaccharide chains. Neither polylactosamine glycosylation of LAMP-2 in MDCK cells nor truncation of N-linked oligosaccharide chains of LAMP-2 in a ricin-resistant MDCK-RCAR cell line influenced the basolateral polarity of its targeting. However, the rate of basolateral delivery of LAMP-2 in MDCK cells plated for 3 d was significantly faster (t1/2 = 28 min) than in 1-d cells (t1/2 = 40 min); in MDCK-RCAR cells the rate of basolateral delivery at both 1 and 3 d of plating was similar (t1/2 = 40 min). The rate differential in MDCK cells occurred after arrival of LAMP-2 to the Golgi apparatus because the rate of acquisition of endoglycosidase H resistance was the same (t1/2 = 25 min) at both days of plating. The rate of transit of LAMP-2 through the Golgi apparatus to the basolateral domain was therefore far more rapid (approximately 4-fold) in 3 d compared with 1-d MDCK cultures. The increased polylactosamine glycosylation of MDCK LAMP-2 at early times of plating during the establishment of a confluent epithelial monolayer may thus be related to its longer residence time in the Golgi apparatus.
The Lysosomal Associated Membrane Protein type-2 (LAMP-2) is an abundant lysosomal membrane protein with an important role in immunity, macroautophagy (MA) and chaperone-mediated autophagy (CMA). Mutations within the Lamp2 gene cause Danon disease, an X-linked lysosomal storage disorder characterized by (cardio)myopathy and intellectual dysfunction. The pathological hallmark of this disease is an accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscle that, along with the myopathy, is also present in LAMP-2-deficient mice. Intellectual dysfunction observed in the human disease suggests a pivotal role of LAMP-2 within brain. LAMP-2A, one specific LAMP-2 isoform, was proposed to be important for the lysosomal degradation of selective proteins involved in neurodegenerative diseases such as Huntington’s and Parkinson’s disease.
To elucidate the neuronal function of LAMP-2 we analyzed knockout mice for neuropathological changes, MA and steady-state levels of CMA substrates. The absence of LAMP-2 in murine brain led to inflammation and abnormal behavior, including motor deficits and impaired learning. The latter abnormality points to hippocampal dysfunction caused by altered lysosomal activity, distinct accumulation of p62-positive aggregates, autophagic vacuoles and lipid storage within hippocampal neurons and their presynaptic terminals. The absence of LAMP-2 did not apparently affect MA or steady-state levels of selected CMA substrates in brain or neuroblastoma cells under physiological and prolonged starvation conditions.
Our data contribute to the understanding of intellectual dysfunction observed in Danon disease patients and highlight the role of LAMP-2 within the central nervous system, particularly the hippocampus.
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The online version of this article (doi:10.1186/s40478-014-0182-y) contains supplementary material, which is available to authorized users.
LAMP-2; Danon disease; Mouse model; Lysosome; Chaperone-mediated autophagy; Huntingtin; α-synuclein
Human lysosome membrane glycoprotein h-lamp-1 is a highly N- glycosylated protein found predominantly in lysosomes, with low levels present at the cell surface. The signal required for delivery of h-lamp- 1 to lysosomes was investigated by analyzing the intracellular distribution of h-lamp-1 with altered amino acid sequences expressed from mutated cDNA clones. A cytoplasmic tail tyrosine residue found conserved in chicken, rodent, and human deduced amino acid sequences was discovered to be necessary for efficient lysosomal transport of h- lamp-1 in COS-1 cells. In addition, the position of the tyrosine residue relative to the membrane and carboxyl terminus also determined lysosomal expression. Supplanting the wild-type h-lamp-1 cytoplasmic tail onto a cell surface reporter glycoprotein was sufficient to cause redistribution of the chimera to lysosomes. A similar chimeric protein replacing the cytoplasmic tyrosine residue with an alanine was not expressed in lysosomes. Altered proteins that were not transported to lysosomes were found to accumulate at the cell surface, and unlike wild- type lysosomal membrane glycoproteins, were unable to undergo endocytosis. These data indicate that lysosomal membrane glycoproteins are sorted to lysosomes by a cytoplasmic signal containing tyrosine in a specific position, and the sorting signal may be recognized both in the trans-Golgi network and at the cell surface.
We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.
The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb) that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7.
Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432) have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y), endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4) and not with late endosomal/lysosomal markers (LAMP-1, Rab7). Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71–75: KKGRR) disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6 and ClC-7 when cotransfected in COS-1 cells.
We conclude that human ClC-6 is an endosomal glycoprotein that partitions in detergent resistant lipid domains. The differential sorting of endogenous (late endosomal) versus overexpressed (early and recycling endosomal) ClC-6 is reminiscent of that of other late endosomal/lysosomal membrane proteins (e.g. LIMP II), and is consistent with a rate-limiting sorting step for ClC-6 between early endosomes and its final destination in late endosomes.
Two murine lysosome-associated membrane proteins, LAMP-1 of 105,000- 115,000 D and LAMP-2 of 100,000-110,000 D, have been identified by monoclonal antibodies that bind specifically to lysosomal membranes. Both glycoproteins were distinguished as integral membrane components solubilized by detergent solutions but not by various chaotropic agents. The lysosome localization was demonstrated by indirect immunofluorescent staining, co-localization of the antigen to sites of acridine orange uptake, and immunoelectron microscopy. Antibody binding was predominantly located at the limiting lysosomal membrane, distinctly separated from colloidal gold-labeled alpha-2-macroglobulin accumulated in the lumen during prolonged incubation. LAMP-1 and LAMP-2 also appeared to be present in low concentrations on Golgi trans- elements but were not detected in receptosomes marked by the presence of newly endocytosed alpha-2-macroglobulin, or in other cellular structures. LAMP-1 and LAMP-2 were distinguished as different molecules by two-dimensional gel analysis, 125I-tryptic peptide mapping, and sequential immunoprecipitations of 125I-labeled cell extracts. Both glycoproteins were synthesized as a precursor protein of approximately 90,000 D, and showed a marked heterogeneity of apparent molecular weight expression in different cell lines. LAMP-2 was closely related or identical to the macrophage antigen, MAC-3, as indicated by antibody adsorption and tryptic peptide mapping. It is postulated that these glycoproteins, as major protein constituents of the lysosomal membrane, have important roles in lysosomal structure and function.
In neurodegenerative diseases, it remains unclear why certain brain regions are selectively vulnerable to protein aggregation. In transgenic mice expressing human A53T α-synuclein, the brainstem and spinal cord develop the most prominent α-synuclein inclusions which correlate with age-dependent motor dysfunction. Herein we present the novel finding that this selective aggregation is in part dependent on the inability of chaperone-mediated autophagy (CMA) to effectively degrade α-synuclein in these brain regions. Lysosomal assays revealed that CMA activity was significantly decreased in aggregation-prone regions compared to the remainder of the brain. Previously, CMA activity has been shown to be proportional to levels of the CMA receptor Lamp-2a. Using antibodies, brain tissue from Lamp-2a null mice, enzymatic deglycosylation, and mass spectrometry, we identified Lamp2a as a novel 72 kDa glycoprotein in the mouse brain. Examination of Lamp-2a levels revealed differences in expression across brain regions. The brainstem and the spinal cord had a more than three-fold greater levels of Lamp-2a as compared to regions less vulnerable to aggregation and exhibited a selective upregulation of Lamp-2a during development of α-synuclein inclusions. Despite this dynamic response of Lamp-2a, the levels of substrates bound to the brain lysosomes as well as the rates of substrate uptake and degradation were not proportional to the levels of Lamp-2a. These regional differences in CMA activity and Lamp-2a expression were found in both non-transgenic mice as well as A53T α-syn mice. Therefore, these are inherent variations and not a transgene-specific effect. However, differences in CMA activity may render select brain regions vulnerable to homeostatic dysfunction in the presence of stressors such as overexpression of human A53T α-syn. Collectively, the data provide a potential mechanism to explain the dichotomy of vulnerability or resistance that underlies brain regions during aggregate formation in neurodegenerative disease.
Parkinson’s Disease; protein aggregation; Lamp-2a; lysosomes
The extensively glycosylated lysosome-associated membrane proteins (LAMP)-2a, b, and c are derived from a single gene by alternative splicing that produces proteins with differences in the transmembrane and cytosolic domains. The lysosomal targeting signals reside in the cytosolic domain of these proteins. LAMPs are not restricted to lysosomes but can also be found in endosomes and at the cell surface. We investigated the subcellular distribution of chimeras comprised of the lumenal domain of avian LAMP-1 and the alternatively spliced domains of avian LAMP-2. Chimeras with the LAMP-2c cytosolic domain showed predominantly lysosomal distribution, while higher levels of chimeras with the LAMP-2a or b cytosolic domain were present at the cell surface. The increase in cell surface expression was due to differences in the recognition of the targeting signals and not saturation of intracellular trafficking machinery. Site-directed mutagenesis defined the COOH-terminal residue of the cytosolic tail as critical in governing the distributions of LAMP-2a, b, and c between intracellular compartments and the cell surface.
Danon disease is an X-linked dominant disorder characterized by the clinical triad of hypertrophic cardiomyopathy (HCM), skeletal myopathy and variable mental retardation. Pathologically, autophagic vacuoles are noted in both skeletal and cardiac muscle. It exhibits an X-linked dominant mode of inheritance and males are severely affected, while females develop milder and later-onset cardiac symptoms. Danon disease has been associated with mutations in the LAMP2 gene located at Xq24, typically resulting in splicing defects or protein truncation affecting the lysosome-associated membrane glycoprotein 2 (LAMP2). Because of its rarity, the full spectrum of genetic mutation resulting in Danon disease has not been elucidated.
Methods and Results
We analyzed three males with clinical and pathological findings consistent with Danon disease. Comprehensive mutational analysis failed to yield detectable products for selected LAMP2 exons and genomic DNA deletion was suspected. Genomic junction fragment PCR analysis in Case-1 identified a novel Alu-mediated 34kb microdeletion encompassing the entire 5’UTR and exon-1 of LAMP2. In Case-2 and -3, junctional PCR and Southern Blot analyses mapped the breakpoint to a MIRb and (TA)n simple repeats present in intron-3, which determined a 64kb and a 58Kb deletion, respectively, thereby ablating exons-4–10. Western blot analysis confirmed the absence of LAMP2 in protein extract from lymphocytes of index Case-2.
This is the first report of Danon disease caused by microdeletions at Xq24, which functionally ablate LAMP2. The microdeletion mechanism appears to involve one Alu-mediated unequal recombination and two chromosomal breakage points involving TA-rich repeat sequences.
LAMP2; HCM; Danon; Alu; (TA)n simple repeat; MIRb; MER21B; LIMA4A; Xq24; lysosome; vacuoles
We have used endocytic and phagocytic tracers in an EM immunocytochemical study to define the compartments of the phagocytic and endocytic pathways in mouse peritoneal macrophages. Endocytosed BSA- gold appeared successively in early endosomes, spherical endosomal vesicles, a late endosomal tubuloreticular compartment (TC), and terminal lysosomes. The TC appeared as an elaborate structure enriched for the lysosomal membrane glycoproteins Lamp 1 and Lamp 2, and expressing significant levels of rab7, a late endosome-specific GTP- binding protein. The cation-independent mannose-6-phosphate receptor was restricted to specialized regions of the TC that were predominantly adjacent to the Golgi complex. Both the early endosome and the TC had coated bud structures whose composition and function are presently unknown. Phagolysosomes containing latex beads expressed the same membrane antigens and received endocytic tracers simultaneously with the TC. Since the membrane surrounding both organelles was also in direct continuity, we assume that both structures form one functional compartment. Macrosialin, an antigen confined to macrophages and dendritic cells, was heavily expressed in TC and phagolysosomal membranes with low levels being detected in other endosomal compartments and on the cell surface. Treatment of cells with wheat germ agglutinin drastically altered the morphology of the TC, giving rise to sheets of tightly adherent membrane and greatly expanded vesicles, in which cell-associated wheat germ agglutinin was concentrated. The spherical endosomal carrier vesicles loaded with internalized gold tracers clustered nearby, often making contact without fusing. Since the delivery of endocytic tracer to the TC was significantly delayed these experiments suggest that the lectin is somehow preventing the endosome vesicles from fusing with the TC. Collectively, our data argue first that the PLC is equivalent to the "tubular lysosomes" commonly described in macrophages, and second that the meeting of the phagocytic and endocytic pathway occurs in this compartment.
The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin—and to a lesser extent the other AP complexes—are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.
The ability to control the movement of nanoparticles remotely and with high precision would have far-reaching implications in many areas of nanotechnology. We have designed a unique dynamic magnetic field (DMF) generator that can induce rotational movements of superparamagnetic iron oxide nanoparticles (SPIONs). We examined whether the rotational nanoparticle movement could be used for remote induction of cell death by injuring lysosomal membrane structures. We further hypothesized that the shear forces created by the generation of oscillatory torques (incomplete rotation) of SPIONs bound to lysosomal membranes would cause membrane permeabilization, lead to extravasation of lysosomal contents into the cytoplasm, and induce apoptosis. To this end, we covalently conjugated SPIONs with antibodies targeting the lysosomal protein marker LAMP1 (LAMP1-SPION). Remote activation of slow rotation of LAMP1-SPIONs significantly improved the efficacy of cellular internalization of the nanoparticles. LAMP1-SPIONs then preferentially accumulated along the membrane in lysosomes in both rat insulinoma tumor cells and human pancreatic beta cells due to binding of LAMP1-SPIONs to endogenous LAMP1. Further activation of torques by the LAMP1-SPIONs bound to lysosomes resulted in rapid decrease in size and number of lysosomes, attributable to tearing of the lysosomal membrane by the shear force of the rotationally activated LAMP1-SPIONs. This remote activation resulted in an increased expression of early and late apoptotic markers and impaired cell growth. Our findings suggest that DMF treatment of lysosome-targeted nanoparticles offers a noninvasive tool to induce apoptosis remotely and could serve as an important platform technology for a wide range of biomedical applications.
dynamic magnetic field; nanoparticle rotation; iron oxide; magnetic nanoparticles; lysosomes; antibody; LAMP1; permeabilization; apoptosis
Chaperone-mediated autophagy (CMA) is a selective type of autophagy by which specific cytosolic proteins are sent to lysosomes for degradation. Substrate proteins bind to the lysosomal membrane through the lysosome-associated membrane protein type 2A (LAMP-2A), one of the three splice variants of the lamp2 gene, and this binding is limiting for their degradation via CMA. However, the mechanisms of substrate binding and uptake remain unknown. We report here that LAMP-2A organizes at the lysosomal membrane into protein complexes of different sizes. The assembly and disassembly of these complexes are a very dynamic process directly related to CMA activity. Substrate proteins only bind to monomeric LAMP-2A, while the efficient translocation of substrates requires the formation of a particular high-molecular-weight LAMP-2A complex. The two major chaperones related to CMA, hsc70 and hsp90, play critical roles in the functional dynamics of the LAMP-2A complexes at the lysosomal membrane. Thus, we have identified a novel function for hsc70 in the disassembly of LAMP-2A from these complexes, whereas the presence of lysosome-associated hsp90 is essential to preserve the stability of LAMP-2A at the lysosomal membrane.
Time for primary review: 15 days
Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation–contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown.
Methods and results
To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA.
These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels.
Ryanodine receptor; Chaperone-mediated autophagy; Geldanamycin; Protein degradation; Cardiomyocyte
Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec188.8.131.52 cells. Using preparative cell sorting, stable, high-expressing GFP ‘master’ cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).
Members of our group reported recently that neisseria infection of human epithelial cells results in accelerated degradation of the major lysosomal integral membrane protein LAMP1 and that this is due to hydrolysis of this glycoprotein at its immunoglobulin A1 (IgA1)-like hinge by the neisseria type 2 IgA1 protease (L. Lin et al., Mol. Microbiol. 24:1083–1094, 1997). We also reported that the IgA1 protease plays a major role in the ability of the pathogenic neisseriae to survive within epithelial cells and hypothesized that this is due to alteration of lysosomes as a result of protease-mediated LAMP1 degradation. In this study, we tested the hypothesis that neisseria infection leads to multiple changes in lysosomes. Here, we report that neisseria infection also reduces the levels of three other lysosomal markers: LAMP2, lysosomal acid phosphatase (LAP), and CD63. In contrast, neither the epidermal growth factor receptor level nor the β-tubulin level is affected. A detailed examination of LAMP2 indicated that the reduced LAMP2 levels are not the result of an altered biosynthetic rate or of cleavage by the IgA1 protease. Nevertheless, the protease plays a role in reducing LAMP2 and LAP activity levels, as these are partially restored in cells infected with an iga mutant. We conclude that neisseria infection results in multiple changes to the lysosomes of infected epithelial cells and that these changes are likely an indirect result of IgA1 protease-mediated cleavage of LAMP1.
Mutations of the intrinsic lysosomal membrane protein SCARB2 cause action myoclonus-renal failure syndrome (AMRF syndrome), a rare disease characterized by renal and neurological manifestations. In this study, examination of Cos7 cells transfected with SCARB2 cDNA derived from two patients with AMRF syndrome showed that the resultant protein was truncated and was not incorporated into vesicular structures, as occurred with full-length SCARB2 cDNA. Mutant SCARB2 protein failed to colocalize with lysosomes and was found in the endoplasmic reticulum or the cytosol indicating a loss of function. Cultured skin fibroblast and Epstein–Barr virus–transformed lymphoblastoid B cell lines (LCLs) were created from these two patients. Despite the loss of SCARB2 function, studies with lysosomal-associated membrane protein (LAMP) 1 and LAMP2 demonstrated normal lysosomal numbers in fibroblasts and LCLs. Immunofluorescence microscopy using anti-LAMP1 and anti-LAMP2 antibodies also showed normal lysosomal structures in fibroblasts. There was no change in the morphology of fibroblasts examined by electron microscopy compared with cells from unaffected individuals. By contrast, LCLs from individuals bearing SCARB2 mutations had large intracellular vesicles that resembled autophagosomes and contained heterogeneous cellular debris. Some of the autophagosomes were seen to be extruding cellular contents into the media. Furthermore, LCLs had elevated levels of microtubule-associated protein light chain 3-II, consistent with increased autophagy. These data demonstrate that SCARB2 mutations are associated with an inability to process autophagosomes in B lymphocytes, suggesting a novel function for SCARB2 in immune function.
biochemistry; cellular biology; genetics; physiology
OA1 (GPR143) is a pigment cell-specific intracellular glycoprotein consisting of 404 amino acid residues that is mutated in patients with Ocular Albinism Type 1, the most common form of ocular albinism. While its cellular localization is suggested to be endolysosomal and melanosomal, the physiological function of OA1 is currently unclear. Recent reports predicted that OA1 functions as a G protein coupled receptor (GPCR) based on its weak amino acid sequence similarity to known GPCRs, and on demonstration of GPCR activity in OA1 mislocalized to the plasma membrane. Because mislocalization of proteins is often caused by or induces defects in their proper folding/assembly, the significance of these studies remains unclear. A characteristic feature of GPCRs is a seven transmembrane domain structure. We analyzed the membrane topology of OA1 properly localized to intracellular lysosomal organelles in COS-1 cells. To accomplish this analysis, we established experimental conditions that allowed selective permeabilization of the plasma membrane while leaving endolysosomal membranes intact. Domains were mapped by the insertion of a hemagglutinin (HA) tag into the predicted cytosolic/luminal regions of OA1 molecule and the accessibility of tag to HA antibody was determined by immunofluorescence. HA-tagged lysosome associated membrane protein 1 (LAMP1), a type I membrane protein, was employed as a reporter for selective permeabilization of the plasma membrane. Our results show experimentally that the C-terminus of OA1 is directed to the cytoplasm and that the protein spans the intracellular membrane 7 times. Thus, OA1, properly localized intracellularly, is a 7 transmembrane domain integral membrane protein consistent with its putative role as an intracellular GPCR.
Membrane topology; Lysosome; Cellular localization; Ocular albinism type 1; Melanosome; G protein coupled receptor; Selective membrane permeabilization
Lysosome-associated membrane protein type 2A (LAMP2A) is a key protein in the chaperone-mediated autophagy (CMA) pathway. LAMP2A helps in lysosomal uptake of modified and oxidatively damaged proteins directly into the lumen of lysosomes for degradation and protein turnover. Elevated expression of LAMP2A was observed in breast tumor tissues of all patients under investigation, suggesting a survival mechanism via CMA and LAMP2A. Reduced expression of the CMA substrates, GAPDH and PKM, was observed in most of the breast tumor tissues when compared with the normal adjacent tissues. Reactive oxygen species (ROS) mediated oxidative stress damages regulatory cellular components such as DNA, proteins and/or lipids. Protein carbonyl content (PCC) is widely used as a measure of total protein oxidation in cells. Ectopic expression of LAMP2A reduces PCC and thereby promotes cell survival during oxidative stress. Furthermore, inhibition of LAMP2A stimulates accumulation of GAPDH, AKT1 phosphorylation, generation of ROS, and induction of cellular apoptosis in breast cancer cells. Doxorubicin, which is a chemotherapeutic drug, often becomes ineffective against tumor cells with time due to chemotherapeutic resistance. Breast cancer cells deficient of LAMP2A demonstrate increased sensitivity to the drug. Thus, inhibiting CMA activity in breast tumor cells can be exploited as a potential therapeutic application in the treatment of breast cancer.
PCC; TUNEL; autophagy; CMA; tumor; tissues; doxorubicin
Danon disease is a rare X-linked disorder comprising hypertrophic cardiomyopathy, skeletal myopathy, intellectual disability, and retinopathy; mutations of the lysosome-associated membrane protein gene LAMP2 are responsible. Most affected persons exhibit “private” point mutations; small locus rearrangements have recently been reported in four cases. Here, we describe the clinical, pathologic, and molecular features of a male proband and his affected mother with Danon disease and a small LAMP2 microduplication. The proband presented at age 12 years with exercise intolerance, hypertrophic cardiomyopathy, and increased creatine kinase. Endomyocardial biopsy findings were nonspecific, showing myocyte hypertrophy and reactive mitochondrial changes. Quadriceps muscle biopsy demonstrated the characteristic autophagic vacuoles with sarcolemma-like features. LAMP2 tissue immunostaining was absent; however, LAMP2 sequencing was normal. Deletion/duplication testing by multiplex ligation-dependent probe amplification (MLPA) assay revealed a 1.5kb microduplication containing LAMP2 exons 4 and 5. RT-PCR studies were consistent with the inclusion of these two duplicated exons in the final spliced transcript, resulting in a frameshift. The proband’s mother, who had died following cardiac transplantation due to suspected myocarditis at age 35, was reviewed and was shown to be affected upon immunostaining of banked myocardial tissue. This case constitutes the second report of a pathogenic microduplication in Danon disease, and illustrates a number of potential diagnostic pitfalls. Firstly, given the imperfect sensitivity of LAMP2 sequencing, tissue immunostaining and/or MLPA should be considered as a diagnostic adjunct in the workup for this disorder. Secondly, the pathological findings in myocardium may be falsely indicative of relatively common conditions such as myocarditis.
Electronic supplementary material
The online version of this chapter (doi:10.1007/8904_2013_277) contains supplementary material, which is available to authorized users.
Necrotizing and crescentic glomerulonephritis (NCGN) is frequently associated with circulating antineutrophil cytoplasmic autoantibodies (ANCA). It is established that ANCA are specific for soluble enzymes of granules of polymorphonuclear neutrophil granulocytes (PMN), such as myeloperoxidase (MPO) or protease 3 (PR3). The purpose of this study was to identify membrane proteins of PMNs, and/or glomerular cells, as additional autoantigenic ANCA targets. When membrane protein fractions were prepared from PMNs and isolated human glomeruli, and immunoblotted with ANCA sera of NCGN patients, two bands with apparent molecular masses of 170 and 80-110 kD (gp170/80-110) were labeled in PMNs, and a 130-kD glycoprotein (gp130) in glomeruli. Gp130 was purified, and monoclonal and rabbit antibodies (Abs) were produced which showed the same double specificity as the patient's ANCA. Using these probes, evidence was provided that gp170/80-110 is identical with human lysosomal-associated membrane protein 2 (h-lamp-2), because both proteins were immunologically cross-reactive and screening of a cDNA expression library from human promyelocytic leukemia cells with anti- gp130 Ab yielded a clone derived from h-lamp-2. Gp170/80-110 was localized primarily in granule membranes of resting PMNs, and was translocated to the cell surfaces by activation with FMLP. By contrast, gp130 was localized in the surface membranes of endothelial cells of human glomerular and renal interstitial capillaries, rather than in lysosomes, as found for h-lamp-2. Potential clinical relevance of autoantibodies to gp170/80-110 and gp130 was assessed in a preliminary trial, in which ANCA sera of patients (n = 16) with NCGN were probed with purified or recombinant antigens. Specific reactivity was detected in approximately 90% of cases with active phases of NCGN, and frequently also in combination with autoantibodies specific for PR3 or MPO. Collectively, these data provide evidence that h-lamp-2 in PMNs and a different, structurally related 130-kD membrane protein on the cell surface of renal microvascular endothelial cells are autoantigenic targets for ANCA in patients with active NCGN.
The class III sugar transport facilitator GLUT8 co-localizes with the lysosomal protein LAMP1 in heterologous expression systems. GLUT8 carries a [D/E]XXXL[L/I]-type dileucine sorting signal that has been postulated to retain the protein in an endosomal/lysosomal compartment via interactions with clathrin adaptor protein (AP) complexes. However, contradictory findings have been described regarding the subcellular localization of the endogenous GLUT8 and the adaptor proteins that interact with its dileucine motif. Here we demonstrate that endogenous GLUT8 is localized in a late endosomal/lysosomal compartment of spermatocytes and spermatids, and that the adaptor complexes AP1 and AP2, but not AP3 or AP4, interact with its N-terminal intracellular domain (NICD). In addition, fusion of the GLUT8 NICD to the tailless lumenal domain of the IL-2 receptor alpha chain (TAC) protein (interleukin-2 receptor α chain) targeted the protein to intracellular membranes, indicating that its N-terminal dileucine signal is sufficient for endosomal/lysosomal targeting of the transporter. The localization and targeting of GLUT8 show striking similarities to sorting mechanisms reported for lysosomal proteins. Therefore, we suggest a potential role for GLUT8 in the so far unexplored substrate transport across intracellular membranes.
Structured digital abstract
MINT-7035377: GLUT8 (uniprotkb:Q9JIF3) physically interacts (MI:0915) with AP2 (uniprotkb:P62944) by pull down (MI:0096)MINT-7035218: GLUT8 (uniprotkb:Q9JIF3) physically interacts (MI:0915) with AP1 (uniprotkb:O43747) by pull down (MI:0096)MINT-7035273: GLUT8 (uniprotkb:Q9JIF3) physically interacts (MI:0915) with AP1 (uniprotkb:P22892) by pull down (MI:0096)MINT-7035235: GLUT8 (uniprotkb:Q9JIF3) physically interacts (MI:0915) with AP1 (uniprotkb:Q8R525) by pull down (MI:0096)MINT-7035360: GLUT8 (uniprotkb:Q9JIF3) physically interacts (MI:0915) with AP2 (uniprotkb:Q9DBG3) by pull down (MI:0096)MINT-7035789, MINT-7035807: lamp1 (uniprotkb:P11438) and GLUT8 (uniprotkb:Q9JIF3) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7039929, MINT-7039945: lamp2 (uniprotkb:P17047) and GLUT8 (uniprotkb:Q9JIF3) colocalize (MI:0403) by fluorescence microscopy (MI:0416)
adaptor proteins; endocytosis; glucose transporter; GLUT8; lysosomes; targeting
Legionella pneumophila, the causative agent of Legionnaires' disease, evades phago-lysosome fusion in mammalian and protozoan hosts to create a suitable niche for intracellular replication. To modulate vesicle trafficking pathways, L. pneumophila translocates effector proteins into eukaryotic cells through a Type IVB macro-molecular transport system called the Icm-Dot system. In this study, we employed a fluorescence-based translocation assay to show that 33 previously identified Legionella eukaryotic-like genes (leg) encode substrates of the Icm-Dot secretion system. To assess which of these proteins may contribute to the disruption of vesicle trafficking, we expressed each gene in yeast and looked for phenotypes related to vacuolar protein sorting. We found that LegC3-GFP and LegC7/YlfA-GFP caused the mis-secretion of CPY-Invertase, a fusion protein normally restricted to the yeast vacuole. We also found that LegC7/YlfA-GFP and its paralog LegC2/YlfB-GFP formed large structures around the yeast vacuole while LegC3-GFP localized to the plasma membrane and a fragmented vacuole. In mammalian cells, LegC2/YlfB-GFP and LegC7/YlfA-GFP were found within large structures that co-localized with anti-KDEL antibodies but excluded the lysosomal marker LAMP-1, similar to what is observed in Legionella-containing vacuoles. LegC3-GFP, in contrast, was observed as smaller structures which had no obvious co-localization with KDEL or LAMP-1. Finally, LegC3-GFP caused the accumulation of many endosome-like structures containing undigested material when expressed in the protozoan host Dictyostelium discoideum. Our results demonstrate that multiple Leg proteins are Icm/Dot-dependent substrates and that LegC3, LegC7/YlfA, and LegC2/YlfB may contribute to the intracellular trafficking of L. pneumophila by interfering with highly conserved pathways that modulate vesicle maturation.
Legionella pneumophila is a Gram-negative bacterial species that causes a severe pneumonia known as Legionnaires' disease. Inhalation of L. pneumophila–contaminated aerosols results in the infection of lung macrophages. Following infection, the bacteria use a Type IVB secretion system to deliver multiple effector proteins into the macrophages to create a membrane-bound replicative compartment called the Legionella-containing vacuole, or LCV. The LCV is defined by its recruitment of early secretory vesicles and avoidance of the bactericidal lysosomes. We identified several effector proteins that contain eukaryotic domains and share significant homology with eukaryotic organelle trafficking proteins. We demonstrate that 33 Legionella eukaryotic-like genes (leg) encode proteins that are translocated into host cells. When artificially expressed in yeast, three Leg proteins (LegC2, LegC3, and LegC7) were able to disrupt normal vesicle trafficking and vacuole morphology. In addition, the Leg proteins induced the formation of, and were localized within, distinct structures when expressed in mammalian cells. In the protozoan host Dictyostelium discoideum, expression of LegC3 resulted in the accumulation of membranous compartments containing partially digested material. Thus, LegC3, LegC2, and LegC7 represent novel effector proteins that may contribute to the intracellular lifestyle of L. pneumophila by disrupting normal vacuolar trafficking pathways in host cells.