Academic literature on the topic 'Lysosome-associated membrane glycoproteins'

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.

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.

Abstract During granulocyte differentiation in the bone marrow (BM), neutrophilic leukocyte precursors synthesize large amounts of lysosomal enzymes. These enzymes are sequestered into azurophilic storage granules until used days later for digestion of phagocytized microorganisms after leukocyte emigration to inflamed tissues. This azurophil granule population has previously been defined as a primary lysosome, ie, a membrane-bound organelle containing acid hydrolases that have not entered into a digestive event. In this study, azurophil granules were purified and shown to contain large amounts of mannose 6-phosphate-containing glycoproteins (Man 6-P GP) but little lysosome-associated membrane proteins (LAMP). In addition, the fine structural localization of Man 6-P GP and LAMP was investigated at various stages of maturation in human BM and blood. Man 6-P GP were present within the azurophilic granules at all stages of maturation and in typical multivesicular bodies (MVB) as well as in multilaminar compartments (MLC), identified by their content of concentric arrays of internal membranes. LAMP was absent in all identified granule populations, but was consistently found in the membranes of vesicles, MVB, and MLC. The latter compartment has not been previously described in this cell type. In conclusion, the azurophilic granules, which contain an abundance of lysosomal enzymes and Man 6-P GP, lack the LAMP glycoproteins. By current criteria, they therefore cannot be classified as lysosomes, but rather may have the functional characteristics of a regulated secretory granule. Rather, the true lysosomes of the resting neutrophil are probably the MVB and MLC. Finally, the typical “dense bodies” or mature lysosomes described in other cells are not present in resting neutrophils.

During granulocyte differentiation in the bone marrow (BM), neutrophilic leukocyte precursors synthesize large amounts of lysosomal enzymes. These enzymes are sequestered into azurophilic storage granules until used days later for digestion of phagocytized microorganisms after leukocyte emigration to inflamed tissues. This azurophil granule population has previously been defined as a primary lysosome, ie, a membrane-bound organelle containing acid hydrolases that have not entered into a digestive event. In this study, azurophil granules were purified and shown to contain large amounts of mannose 6-phosphate-containing glycoproteins (Man 6-P GP) but little lysosome-associated membrane proteins (LAMP). In addition, the fine structural localization of Man 6-P GP and LAMP was investigated at various stages of maturation in human BM and blood. Man 6-P GP were present within the azurophilic granules at all stages of maturation and in typical multivesicular bodies (MVB) as well as in multilaminar compartments (MLC), identified by their content of concentric arrays of internal membranes. LAMP was absent in all identified granule populations, but was consistently found in the membranes of vesicles, MVB, and MLC. The latter compartment has not been previously described in this cell type. In conclusion, the azurophilic granules, which contain an abundance of lysosomal enzymes and Man 6-P GP, lack the LAMP glycoproteins. By current criteria, they therefore cannot be classified as lysosomes, but rather may have the functional characteristics of a regulated secretory granule. Rather, the true lysosomes of the resting neutrophil are probably the MVB and MLC. Finally, the typical “dense bodies” or mature lysosomes described in other cells are not present in resting neutrophils.

The subcellular localization of two members of a highly glycosylated protein group present in lysosomal membranes in most cells, the lysosome-associated membrane proteins 1 and 2 (Lamp-1 and Lamp-2), was examined in human neutrophil granulocytes. Antibodies that were raised against purified Lamp-1 adn Lamp-2 gave a distinct granular staining of the cytoplasm upon immunostaining of neutrophils. Subcellular fractionation was used to separate the azurophil and specific granules from a light-membrane fraction containing plasma membranes and secretory vesicles, and Western blotting was used to determine the presence of the Lamps in these fractions. The results show that Lamp-1 and Lamp-2 are present in the specific-granule-enriched fraction and in the light-membrane fraction, but not in the azurophil granules. Separation of secretory vesicles from plasma membranes disclosed that the light-membrane Lamps were present primarily in the secretory-vesicle-enriched fraction. During phagocytosis both Lamp-1 and Lamp-2 became markedly concentrated around the ingested particle and they both appear on the cell surface when the secretory organelles are mobilized.

ABSTRACT INTRODUCTION: YKL-40 is a glycoprotein believed potentially to be a marker of various pathological processes. High levels of YKL-40 have been found in cancer and chronic infl ammatory diseases. The function of the glycoprotein is not completely known yet. A possible involvement in angiogenesis and tumor aggressiveness is supposed. Lysosome-associated membrane glycoproteins (LAMP) 1 and 2 are highly conserved proteins with still undefi ned biological functions. There is evidence that they are implicated in autophagy, angiogenesis and tissue remodeling. AIM: The aim of the present study was to investigate the potential relationship between the tissue expression of YKL-40, LAMP-1 and LAMP-2 in glial tumors. MATERIAL AND METHODS: LAMPs and YKL-40 expression was determined by immunohistochemistry in 36 glial tumors. A morphometric analysis of the intensity of tissue expression was performed with the Quick-photo Micro 2.3. system. Area (μm), perimeter (μm), and expression level (%) of the three glycoproteins were calculated. RESULTS: LAMPs were found on cell membranes of glial and endothelial cells, while YKL-40 was detected in the cytoplasm of these cells. Intensive immunohistochemical reaction was present in tumor cells. LAMP-2 showed a more intensive staining compared to LAMP-1. CONCLUSION: We present the fi rst comparative study of YKL-40 and LAMPs in astroglial tumors. The relationship between the expression of the three glycoconjugates indicates a possible participation in the processes of angiogenesis and tissue remodeling during tumor development

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.

We have identified the bovine renal homologue of Lamp-1 (lysosomal-associated membrane glycoprotein 1). It has very similar physical characteristics to other Lamp-1 proteins from a wide variety of tissues and species. Partial sequence analysis has shown it to be 61% identical with human Lamp-1 and about 50% identical with rat and mouse Lamp-1. The extent of glycosylation of bovine Lamp-1 alters in response to changes in the concentration of extracellular phosphate. Bovine renal epithelial cells (NBL-1) grown in normal or phosphate-starved medium contain Lamp-1 of 120 kDa. However, if cells are grown in medium containing 8-10 mM phosphate, they contain Lamp-1 of only 100 kDa. The core protein and mRNA levels have been shown to remain constant under both conditions. Therefore the only conclusion is that the extent of Lamp-1 glycosylation must be changing in response to the extracellular concentration of phosphate. Unlike Carlsson and Fukuda [(1990) J. Biol. Chem. 265, 20488-20495], who showed that the human Lamp-1 protein contained polylactosaminoglycan residues, we have been unable to demonstrate the partial deglycosylation of bovine Lamp-1 by endo-beta-galactosidase. This enzyme removes polylactosaminoglycan groups from glycoproteins, and therefore indicates that the carbohydrate structure of bovine Lamp-1 is probably different from that of other Lamp-1 proteins. At present the physiological importance of bovine renal Lamp-1 and the changes in its extent of glycosylation are unknown. In this paper we postulate that Lamp-1 may be involved in the cycling of plasma-membrane proteins to the lysosome. This is based on the finding that the only other known effect of high extracellular phosphate on NBL-1 cells is to cause a decrease in the Vmax. of plasma-membrane-associated Na(+)-dependent phosphate transport [Helps and McGivan (1991) Eur. J. Biochem. 200, 797-803].

ABSTRACT Following uptake, Francisella tularensis enters a phagosome that acquires limited amounts of lysosome-associated membrane glycoproteins and does not acquire cathepsin D or markers of secondary lysosomes. With additional time after uptake, F. tularensis disrupts its phagosomal membrane and escapes into the cytoplasm. To assess the role of phagosome acidification in phagosome escape, we followed acidification using the vital stain LysoTracker red and acquisition of the proton vacuolar ATPase (vATPase) using immunofluorescence within the first 3 h after uptake of live or killed F. tularensis subsp. holarctica live vaccine strain (LVS) by human macrophages. Whereas 90% of the phagosomes containing killed LVS stained intensely for the vATPase and were acidified, only 20 to 30% of phagosomes containing live LVS stained intensely for the vATPase and were acidified. To determine whether transient acidification might be required for phagosome escape, we assessed the impact on phagosome permeabilization of the proton pump inhibitor bafilomycin A. Using electron microscopy and an adenylate cyclase reporter system, we found that bafilomycin A did not prevent phagosomal permeabilization by F. tularensis LVS or virulent type A strains (F. tularensis subsp. tularensis strain Schu S4 and a recent clinical isolate) or by “F. tularensis subsp. novicida,” indicating that F. tularensis disrupts its phagosomal membrane by a mechanism that does not require acidification.

ABSTRACT Francisella tularensis, the agent of tularemia, is an intracellular pathogen, but little is known about the compartment in which it resides in human macrophages. We have examined the interaction of a recent virulent clinical isolate of F. tularensis subsp. tularensis and the live vaccine strain with human macrophages by immunoelectron and confocal immunofluorescence microscopy. We assessed the maturation of the F. tularensis phagosome by examining its acquisition of the lysosome-associated membrane glycoproteins (LAMPs) CD63 and LAMP1 and the acid hydrolase cathepsin D. Two to four hours after infection, vacuoles containing live F. tularensis cells acquired abundant staining for LAMPs but little or no staining for cathepsin D. However, after 4 h, the colocalization of LAMPs with live F. tularensis organisms declined dramatically. In contrast, vacuoles containing formalin-killed bacteria exhibited intense staining for all of these late endosomal/lysosomal markers at all time points examined (1 to 16 h). We examined the pH of the vacuoles 3 to 4 h after infection by quantitative immunogold staining and by fluorescence staining for lysosomotropic agents. Whereas phagosomes containing killed bacteria stained intensely for these agents, indicating a marked acidification of the phagosomes (pH 5.5), phagosomes containing live F. tularensis did not concentrate these markers and thus were not appreciably acidified (pH 6.7). An ultrastructural analysis of the F. tularensis compartment revealed that during the first 4 h after uptake, the majority of F. tularensis bacteria reside within phagosomes with identifiable membranes. The cytoplasmic side of the membranes of ∼50% of these phagosomes was coated with densely staining fibrils of ∼30 nm in length. In many cases, these coated phagosomal membranes appeared to bud, vesiculate, and fragment. By 8 h after infection, the majority of live F. tularensis bacteria lacked any ultrastructurally discernible membrane separating them from the host cell cytoplasm. These results indicate that F. tularensis initially enters a nonacidified phagosome with LAMPs but without cathepsin D and that the phagosomal membrane subsequently becomes morphologically disrupted, allowing the bacteria to gain direct access to the macrophagic cytoplasm. The capacity of F. tularensis to alter the maturation of its phagosome and to enter the cytoplasm is likely an important element of its capacity to parasitize macrophages and has major implications for vaccine development.

Cytotoxic T lymphocytes (CTL) kill virally infected and tumourigenic cells via the regulated secretion of specialised secretory lysosomes. These secretory lysosomes contain cytolytic effector molecules, such as perforin and granzymes, which are able to induce apoptosis in target cells. Secretion occurs at the contact point between the CTL and its target, in a highly structured region termed the immunological synapse (IS). Upon formation of the IS, CTL undergo polarisation of their microtubule cytoskeleton and movement of the microtubule organising centre (MTOC) to the IS. Secretory lysosomes are then able to polarise along microtubules, fuse with the plasma membrane and deliver their effector molecules to the IS. The Adaptor Protein 3 complex (AP-3) sorts transmembrane proteins to lysosomes and deficiency in AP-3 results in missorting of proteins from the lysosomal to plasma membrane. CTL from AP-3 deficient patients, who suffer from Hermansky-Pudlak Syndrome Type 2 (HPS2), show reduced killing of target cells. This thesis describes two new patients with HPS2, both with homozygous mutations in the AP3B1 gene, which codes for the β3A subunit of the AP-3 complex. CTL from the new HPS2 patients show reduced cytotoxicity, which is shown here to be due to impaired secretory lysosome polarisation towards the IS. This impairment is common to HPS2 CTL, but varies between patients. In order to determine differences between HPS2 and wild type CTL, the localisation of a range of lysosomal, cytolytic, transmembrane, inhibitory and activation marker proteins is examined. This shows that in HPS2 CTL, LAMP1, CD63 and CD9 are potential AP-3 cargos. In addition, a possible effect on the key lytic effector perforin is identified. Preliminary experiments to allow proteomic comparison of HPS2 and wild type CTL are also presented. Further investigation of these results will help to shed light on the mechanisms involved in secretory lysosome polarisation in CTL.

Sialidose é uma doença neurossomática causada pela deficiência congênita da neuraminidase-1 (Neu1), enzima envolvida na regulação do catabolismo de sialoglicoconjugados nos lisossomos. Com o acúmulo de sialoglicoconjugados, ocorre comprometimento sistêmico e neurológico. Achados histológicos musculares incluem expansão da matriz extracelular (MEC) devido à proliferação anormal de fibroblastos, invasão das fibras musculares por componentes da MEC, fragmentação do citoplasma, formação vacuolar e atrofia das fibras musculares. Entretanto o mecanismo da atrofia muscular na deficiência de Neu1 não está completamente esclarecido, sendo o objetivo desse estudo. Desnervou-se o músculo gastrocnêmio direito de camundongos com deficiência de Neu1 (Neu1 -/-) e de controles Neu1 +/+. Os animais foram eutanasiados 0, 3, 7, 14 e 21 dias pós desnervação. Os músculos desnervados e contralaterais foram submetidos às seguintes análises: 1) histologia geral e medida da área transversa das fibras; 2) autofagia, através da avaliação da presença de vacúolos autofágicos por estudo ultraestrutural e da análise da expressão da proteína LC3; 3) ativação do sistema lisossomal, por reação de fosfatase ácida e análise da expressão proteica de catepsina L e lamp1; 4) deposição de colágeno e infiltração de tecido conjuntivo no tecido muscular; 5) níveis das proteínas Akt e GSK3b; 6) expressão dos atrogenes MuRF1 e Atrogina-1; 7) níveis da proteína MyoD, relacionada à diferenciação muscular; e 8) expressão dos genes Neu1, Neu2, Neu3 e Neu4. Os animais Neu1-/- apresentaram menor peso corporal e muscular compararando-se com animais Neu1 +/+. Houve redução progressiva da área das fibras dos músculos desnervados em relação aos músculos contralaterais. Os animais Neu1-/- apresentaram atrofia muscular basal, com aumento acentuado dos espaços endomisiais e perimisiais. Ocorreu formação de vacúolos autofágicos a partir de 14 dias de desnervação tanto em animais Neu1+/+ quanto em Neu1-/-. Os níveis de expressão proteica de catepsina L e de lamp1 aumentaram a partir de 14 dias de desnervação, mais notadamente em músculos desnervados de camundongos Neu1-/-. A expressão proteica de colágeno III mostrou-se aumentada em animais Neu1-/-, principalmente após desnervação. A expressão proteica da forma fosforilada do Akt (forma ativada) diminuiu após 21 dias de desnervação principalmente em músculos desnervados de animais Neu1+/+. Os níveis de PGSK3 b, forma inativa de GSK3b, diminuíram após a desnervação, em animais Neu1+/+ e animais Neu1-/-. Houve aumento na expressão gênica de Atrogina-1 e MuRF1 após 3 e 7 dias de desnervação, respectivamente; a expressão gênica de Atrogina-1 nos camundongos Neu1-/- teve um aumento atrasado, mostrando diferença significante após 7 dias de desnervação. Não houve diferença significativa entre níveis proteicos de MyoD. A expressão gênica de Neu1 mostrou-se elevada em músculos desnervados de animais Neu1+/+. Conclui-se, portanto, que a Neu1 parece atuar na regulação da massa muscular principalmente controlando o processo de ativação do sistema lisossomal, porém aparentemente sem afetar a autofagia
Sialidosis, a severe neurosomatic disease, results from congenital neuraminidase-1 (Neu1) deficiency. This enzyme regulates the catabolism of sialoglycoconjugates in the lysosomes. Systemic and neurologic manifestations occur due to the sialoglycoconjugates accumulation. In the mouse model for Neu1 deficiency, the muscle histologic findings include extracellular matrix (ECM) expansion, due to abnormal fibroblast proliferation, muscle fibers invasion by ECM components, cytoplasm fragmentation, vacuolar formation and muscle atrophy. Nevertheless the mechanisms of muscle atrophy in Neu1 deficiency are not completely known. This study was designed to investigate Neu1 involvement in muscle atrophy process. Denervation of gastrocnemius muscle was performed by sectioning sciatic nerve from Neu1 deficient mice (Neu1 -/-) and from normal control Neu1 +/+; the animals were euthanized 0, 3, 7, 14 and 21 days after denervation. Denervated and control muscles were collected and submitted to several analysis: 1) histological; 2) autophagic vacuoles formation, performed by ultrastructural analysis and LC3 protein expression; 3) acid phosphatase reaction, lamp1 and cathepsin L protein expression, to analyze lysosomal activation; 4) collagen deposition and fibrous formation; 5) proteins involved with muscle trophism, Akt and GSK3b; 6) MuRF1 and Atrogin-1 gene expression; 7) MyoD protein expression; 8) Neu1, Neu2, Neu3 and Neu4 genes expression. Neu1 -/- mice presented decreased body and muscle weight comparing to Neu1 +/+ animals. Muscle fiber cross-sectional area was reduced in denervated muscles comparing to contralateral muscles. Neu1 -/- mice muscles presented basal atrophy and increase of endomisial and perimisial spaces, which became more evident after denervation. After 14 days of denervation, autophagosome formation was noticed on Neu1 +/+ and Neu1-/- animals. Cathepsin L protein levels were increased after 14 and 21 days of denervation, especially in denervated muscles from Neu1 -/- mice. Lamp1 protein expression was increased in Neu1-/- animals. Type III collagen protein levels were increased in Neu1-/- animals. There were no significant differences between MyoD protein levels. P-Akt, active form of Akt protein levels, decreased after 21 days of denervation, especially in denervated muscles from control group animals, indicating that protein synthesis is decreased. P-GSK3b, inactive form of GSK3b decreased in denervated muscles from Neu1 -/- and Neu1 +/+ animals, which indicates that this protein remained activated during muscle atrophy process. There were significant differences in Atrogin-1 and MuRF1 gene expression levels after 3 and 7 days of denervation. Neu1 -/- animals muscles presented a delayed Atrogin-1 response. Neu1 gene expression was increased in denervated muscles from Neu1 +/+ mice. These findings suggest that Neu1 seems to act in the regulation of muscle mass mainly by controlling the process of lysosomal system activation, but apparently without affecting autophagy