scholarly journals Four monoclonal antibodies inhibit the recognition of arylsulphatase A by the lysosomal enzyme phosphotransferase

1994 ◽  
Vol 297 (1) ◽  
pp. 123-130 ◽  
Author(s):  
H J Sommerlade ◽  
A Hille-Rehfeld ◽  
K von Figura ◽  
V Gieselmann
Keyword(s):  
Monoclonal Antibodies ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Enzymic Activity ◽  
Chimeric Mouse ◽  
Amino Acid Residues ◽  
Lysosomal Protease ◽  
Arylsulphatase A ◽  
Ph Dependent

The critical step in the sorting of lysosomal enzymes is their recognition by a phosphotransferase in the Golgi apparatus. The topogenic sequences responsible for the recognition by this enzyme have so far only been defined for the lysosomal protease cathepsin D. We have generated four monoclonal antibodies directed against lysosomal arylsulphatase A (ASA). These antibodies inhibit the recognition of ASA by the phosphotransferase in vitro and thus define a region of topogenic sequences in the ASA polypeptide. The antibodies do not interfere with the enzymic activity nor with pH-dependent dimerization of ASA. The epitopes recognized by the antibodies have been located in the second quarter of the ASA polypeptide using chimeric mouse-human ASA molecules. Three of the monoclonal antibodies bind to identical or closely adjacent epitopes, which are formed by the interaction of amino acid residues 165-184 and 202-240. The fourth antibody recognizes a different epitope within amino acids 256-265.

scholarly journals Characterization of Chimpanzee/Human Monoclonal Antibodies to Vaccinia Virus A33 Glycoprotein and Its Variola Virus Homolog In Vitro and in a Vaccinia Virus Mouse Protection Model

2007 ◽  
Vol 81 (17) ◽  
pp. 8989-8995 ◽  
Author(s):  
Zhaochun Chen ◽  
Patricia Earl ◽  
Jeffrey Americo ◽  
Inger Damon ◽  
Scott K. Smith ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Vaccinia Virus ◽  
Protective Efficacy ◽  
Variola Virus ◽  
Binding Affinities ◽  
Amino Acid Residues ◽  
C Terminus ◽  
20 Nm

ABSTRACT Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (Kd of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.

Intralysosomal pH and release of lysosomal enzymes in the rat liver after exhaustive exercise

1993 ◽  
Vol 74 (4) ◽  
pp. 1628-1634 ◽  
Author(s):  
M. Tsuboi ◽  
K. Harasawa ◽  
T. Izawa ◽  
T. Komabayashi ◽  
H. Fujinami ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Rat Liver ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Osmotic Fragility ◽  
Exhaustive Exercise ◽  
Ammonia Accumulation ◽  
Exercise Induced ◽  
Beta Glucosidase

The mechanism underlying exhaustive exercise-induced release of lysosomal enzymes was studied in the rat liver. Exhaustive exercise resulted in the release of beta-glucuronidase and cathepsin D, but not beta-glucosidase and acid phosphatase, into the blood and cytosol, suggesting that the release of lysosomal enzymes is not due to disruption of lysosomal membranes. The intralysosomal pH of the liver, which was approximately 5.5 at the resting level, rose significantly after exhaustive exercise to pH 6.3. In vitro, beta-glucuronidase and cathepsin D were released at an intralysosomal pH exceeding 6.2. In contrast, beta-glucosidase and acid phosphatase were not released. The elevation of intralysosomal pH reduced the aggregation of beta-glucuronidase and cathepsin D. The rate of ammonia accumulation increased markedly in the lysosome-enriched subcellular fraction after exercise. There was a positive relationship between the rate of ammonia accumulation and the elevation of intralysosomal pH in vitro. Lysosomes isolated after exhaustive exercise showed significantly increased osmotic fragility. Our findings suggest that, during exhaustive exercise, the accumulation of ammonia in lysosomes leads to the elevation of intralysosomal pH, resulting in the reduced aggregation of certain lysosomal enzymes. Thus, less aggregated lysosomal enzymes may be released into the cytosol through the lysosomal membrane, the permeability of which has been increased.

scholarly journals IN VITRO INDUCTION OF LYSOSOMAL ENZYMES BY PHAGOCYTOSIS

1970 ◽  
Vol 131 (6) ◽  
pp. 1239-1260 ◽  
Author(s):  
Stanton G. Axline ◽  
Zanvil A. Cohn
Keyword(s):  
Amino Acids ◽  
Acid Phosphatase ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Gamma Globulin ◽  
Peritoneal Macrophages ◽  
Lysosomal Hydrolases ◽  
Mouse Peritoneal Macrophages ◽  
In Vitro Induction

The in vitro induction of lysosomal enzymes by phagocytosis was demonstrated in cultivated mouse peritoneal macrophages. The contribution of each of several steps in the endocytic process to enzyme induction was examined. The enzymatic response after the uptake of equal numbers of erythrocytes (RBC) and nondigestible particles were compared. Phagocytosis of RBC produced a marked increase in the levels of acid phosphatase, ß-glucuronidase, and cathepsin D. Puromycin (1 µg/ml) inhibited the enzyme response. In contrast, phagocytosis of polyvinyl toluene, polystyrene, and insoluble starch particles produced no increase in macrophage lysosomal enzymes, although fusion of phagosomes with preexisting lysosomes occurred normally. The endocytic stimulus to synthesis of inducible lysosomal enzymes, therefore, occurred at or beyond the stage of digestion. Purified protein (bovine gamma globulin) aggregates and homopolymer coacervates of poly-l-glutamic acid: poly-l-lysine were effective inducers of lysosomal acid phosphatase, ß-glucuronidase, and cathepsin D, whereas homopolymers of the same D-amino acids were ineffective as inducers. Both the quantity of phagocytized substrate and its rate of enzymatic hydrolysis appear to control the level and persistance of lysosomal hydrolases.

Oestrogen-induced pro-cathepsin D in breast cancer: from biology to clinical applications

1989 ◽  
Vol 95 ◽  
pp. 107-118
Author(s):  
Henri Rochefort ◽  
Patrick Augereau ◽  
Pierre Briozzo ◽  
François Capony ◽  
Vincent Cavailles ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cathepsin D ◽  
Breast Cancer Cells ◽  
Mammary Epithelial Cells ◽  
Tissue Type ◽  
Breast Cancer Cell Lines ◽  
Lysosomal Protease ◽  
Mannose 6 Phosphate

SynopsisIn addition to secreted growth factors, acting as autocrine or paracrine mitogens, breast cancer cells secrete other proteins whose function and significance in mammary carcinogenesis may be important. Among them, proteases are particularly interesting since it has been suggested that they play a role in metastatic process, and since at least two of them, the tissue type plasminogen activator and pro-cathepsin D, the precursor of a lysosomal protease, are induced by oestrogens and secreted in excess in some mammary cancer cells.In oestrogen-receptor-positive human breast cancer cell lines (MCF7, ZR75–1), oestrogens stimulate cell proliferation and specifically increase the secretion into the culture medium of a 52,000-dalton (52-kDa) glycoprotein identified as the secreted precursor of a cathepsin D bearing mannose-6-phosphate signals, which is routed to lysosomesviamannose-6-phosphate-IGF-II receptors. We have determined the structure of this procathepsin D by sequencing its complete cDNA sequence, its chromosomal localisation and its transcriptional regulation by oestrogens and other mitogens. In breast cancer cells, pro-cathepsin D production and secretion is much higher and its processing is altered compared to normal mammary epithelial cells in culture.In vitro, pro-cathepsin D acts as an autocrine mitogen on breast cancer cells and can be activated at acidic pH to degrade extracellular matrix, suggesting a role in mediating the effect of oestrogens on tumour growth and invasion. Retrospective clinical studies indicate a significant correlation between high 52-kDa cathepsin D concentrations in the cytosol of primary breast cancer and poor prognosis (Danish Breast Cancer Group, S. Thorpe, Copenhagen). We propose that among the proteases secreted by cancer cells, 52-kDa cathepsin D is important both as a tissue marker in breast cancer and as a potential factor involved in carcinogenesis.

scholarly journals Importance of splicing for prosaposin sorting

1999 ◽  
Vol 337 (3) ◽  
pp. 433-443 ◽  
Author(s):  
Liora MADAR-SHAPIRO ◽  
Metsada PASMANIK-CHOR ◽  
Anna Maria VACCARO ◽  
Tama DINUR ◽  
Arie DAGAN ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Amino Acid ◽  
Lysosomal Enzymes ◽  
Biological Activities ◽  
Point Mutations ◽  
Amino Acid Residues ◽  
Saposin C ◽  
Alternatively Spliced ◽  
Saposin B

The prosaposin gene encodes a 70 kDa protein. This protein might either reach the lysosomes and get processed there to four peptides, which are activators of known lysosomal enzymes, or be secreted by cells as a 70 kDa protein, recently anticipated to have several biological activities. The human prosaposin gene has a 9 bp exon (exon 8) that is alternatively spliced, thus encoding three prosaposin forms: one with an extra three amino acid residues, one with an extra two residues and a third form with no extra residues. With the aim of testing whether there is an association between the alternative splicing and the differential sorting of prosaposins, we cloned two human prosaposin cDNA forms in a T7/EMC/vaccinia virus-derived vector and expressed them in human cells. The results indicated that the prosaposin containing the three extra residues accumulated faster and in greater amounts in the medium, whereas the prosaposin with no extra residues was mainly destined for lysosomes. Point mutations created by mutagenesis in vitro in the 9 bp stretch had a diverse effect on prosaposin secretion. When supplied to cells in the medium, both prosaposins were endocytosed and reached the lysosomes, where they were processed to active saposin B and saposin C. The activities of the saposins were monitored qualitatively and quantitatively. Quantitatively, lipids were extracted from the cells, separated on TLC and measured fluorimetrically. Qualitatively, cells were detected by fluorescence microscopy.

Alternative mechanisms for trafficking of lysosomal enzymes in mannose 6-phosphate receptor-deficient mice are cell type-specific

1999 ◽  
Vol 112 (10) ◽  
pp. 1591-1597 ◽  
Author(s):  
F. Dittmer ◽  
E.J. Ulbrich ◽  
A. Hafner ◽  
W. Schmahl ◽  
T. Meister ◽  
...  
Keyword(s):  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Lysosomal Storage ◽  
Cell Type ◽  
Genes Encoding ◽  
Cell Type Specific ◽  
Mannose 6 Phosphate ◽  
Underlying Mechanisms

Viable mice nullizygous in genes encoding the 300 kDa and the 46 kDa mannose 6-phosphate receptors (MPR 300 and MPR 46) and the insulin like growth factor II (IGF II) were generated to study the trafficking of lysosomal enzymes in the absence of MPRs. The mice have an I-cell disease-like phenotype, with increase of lysosomal enzymes in serum and normal activities in tissues. Surprisingly, the ability of MPR-deficient cells to transport newly synthesized lysosomal enzymes to lysosomes and the underlying mechanisms were found to depend on the cell type. MPR-deficient thymocytes target newly synthesized cathepsin D to lysosomes via an intracellular route. In contrast, hepatocytes and fibroblasts secrete newly synthesized cathepsin D. In fibroblasts recapture of secreted lysosomal enzymes, including that of cathepsin D, is limited and results in lysosomal storage, both in vivo and in vitro, whereas recapture by hepatocytes is remarkably effective in vivo and can result in lysosomal enzyme levels even above normal.

scholarly journals Anti-Alpha-Hemolysin Monoclonal Antibodies Mediate Protection against Staphylococcus aureus Pneumonia

2009 ◽  
Vol 77 (7) ◽  
pp. 2712-2718 ◽  
Author(s):  
Brook E. Ragle ◽  
Juliane Bubeck Wardenburg
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibodies ◽  
Cell Injury ◽  
Active Immunization ◽  
Amino Acid Residues ◽  
Alpha Hemolysin ◽  
Immunization Strategies ◽  
Staphylococcal Pneumonia ◽  
Staphylococcus Aureus Pneumonia

ABSTRACT Staphylococcus aureus pneumonia is one of the most common invasive diseases caused by this human pathogen. S. aureus alpha-hemolysin, a pore-forming cytotoxin, is an essential virulence factor in the pathogenesis of pneumonia. Vaccine-based targeting of this toxin provides protection against lethal staphylococcal pneumonia in a murine model system, suggesting that a monoclonal antibody-based therapy may likewise prove to be efficacious for prevention and treatment of this disease. We report the generation of two distinct anti-alpha-hemolysin monoclonal antibodies that antagonize toxin activity, preventing human lung cell injury in vitro and protecting experimental animals against lethal S. aureus pneumonia. Each of these two monoclonal antibodies recognized an epitope within the first 50 amino acid residues of the mature toxin and blocked the formation of a stable alpha-hemolysin oligomer on the target cell surface. Active immunization with the first 50 amino acids of the toxin also conferred protection against S. aureus pneumonia. Together, these data reveal passive and active immunization strategies for prevention or therapy of staphylococcal pneumonia and highlight the potential role that a critical epitope may play in defining human susceptibility to this deadly disease.

scholarly journals Physicochemical characteristics of the glycosaminoglycan-lysosomal enzyme interaction in vitro. A model of control of leucocytic lysosomal activity

1976 ◽  
Vol 160 (2) ◽  
pp. 129-136 ◽  
Author(s):  
J L Avila ◽  
J Convit
Keyword(s):  
Phospholipase A2 ◽  
Intermolecular Interaction ◽  
Lysosomal Enzymes ◽  
Chondroitin Sulphate ◽  
Physicochemical Characteristic ◽  
Phagocytic Process ◽  
Ph Values ◽  
Ph Dependent ◽  
Sulphated Glycosaminoglycans

1. The activities of 30 different lysosomal enzymes were determined in vitro in the presence of the sulphated glycosaminoglycans, heparin and chondroitin sulphate, all the enzymes being measured on a density-gradient-purified lysosomal fraction. 2. Each enzyme was studied as a function of the pH of the incubation medium. In general the presence of sulphated glycosaminoglycans induced a strong pH-dependent inhibition of lysosomal enzymes at pH values lower than 5.0, with full activity at higher pH values. However, in the particular case of lysozyme and phospholipase A2 the heparin-induced inhibition was maintained in the pH range 4.0-7.0. 3. For certain enzymes, such as acid β-glycerophosphatase, α-galactosidase, acid lipase, lysozyme and phospholipase A2, the pH-dependent behaviour obtained in the presence of heparin was quite different to that obtained with chondroitin sulphate, suggesting the existence of physicochemical characteristic factors playing a role in the intermolecular interaction for each of the sulphated glycosaminoglycans studied. 4. Except in the particular case of peroxidase activity, in all other lysosomal enzymes measured the glycosaminoglycan-enzyme complex formation was a temperature-and time-independent phenomenon. 5. The effects of the ionic strength and pH on this intermolecular interaction reinforce the concept of an electrostatic reversible interaction between anionic groups of the glycosaminoglycans and cationic groups on the enzyme molecule. 6. As leucocytic primary lysosomes have a very acid intragranular pH and large amounts of chondroitin sulphate, we propose that this glycosaminoglycan might act as molecular regulator of leucocytic activity, by inhibiting lysosomal enzymes when the intragranular pH is below the pI of lysosomal enzymes. This fact, plus the intravacuolar pH changes described during the phagocytic process, might explain the unresponsiveness of lysosomal enzymes against each other existing in primary lysosomes as well as its full activation at pH values occurring in secondary lysosomes during the phagocytic process.

scholarly journals Secretion of phosphomannosyl-deficient arylsulphatase A and cathepsin D from isolated human macrophages

2002 ◽  
Vol 368 (3) ◽  
pp. 845-853 ◽  
Author(s):  
Nicole MUSCHOL ◽  
Ulrich MATZNER ◽  
Stephan TIEDE ◽  
Volkmar GIESELMANN ◽  
Kurt ULLRICH ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Cell Line ◽  
Marrow Transplantation ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Brain Cells ◽  
Therapeutic Effects ◽  
Human Macrophages ◽  
Arylsulphatase A

The transfer of macrophage-secreted arylsulphatase A (ASA) to enzyme-deficient brain cells is part of the therapeutic concept of bone marrow transplantation in lysosomal storage diseases. Here we have investigated this transfer in vitro. The uptake of 125I-labelled recombinant human ASA purified from ASA-overexpressing mouse embryonic fibroblasts deficient for mannose 6-phosphate (M6P) receptors in a mouse ASA-deficient astroglial cell line was completely inhibited by M6P. In contrast, when ASA-deficient astroglial cells were incubated with secretions of [35S]methionine-labelled human macrophages or mouse microglia, containing various lysosomal enzymes, neither ASA nor cathepsin D (CTSD) were detected in acceptor cells. Co-culturing of metabolically labelled macrophages with ASA-deficient glial cells did not result in an M6P-dependent transfer of ASA or CTSD between these two cell types. In secretions of [33P]phosphate-labelled macrophages no or weakly phosphorylated ASA and CTSD precursor polypeptides were found, whereas both intracellular and secreted ASA from ASA-overexpressing baby hamster kidney cells displayed 33P-labelled M6P residues. Finally, the uptake of CTSD from secretions of [35S]methionine-labelled macrophages in rat hepatocytes was M6P-independent. These data indicated that lysosomal enzymes secreted by human macrophages or a mouse microglial cell line cannot be endocytosed by brain cells due to the failure to equip newly synthesized lysosomal enzymes with the M6P recognition marker efficiently. The data suggest that other mechanisms than the proposed M6P-dependent secretion/recapture of lysosomal enzymes might be responsible for therapeutic effects of bone marrow transplantation in the brain.

scholarly journals EpsinR: an ENTH Domain-containing Protein that Interacts with AP-1

2003 ◽  
Vol 14 (2) ◽  
pp. 625-641 ◽  
Author(s):  
Jennifer Hirst ◽  
Alison Motley ◽  
Kouki Harasaki ◽  
Sew Y. Peak Chew ◽  
Margaret S. Robinson
Keyword(s):  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Brefeldin A ◽  
Membrane Association ◽  
Mature Form ◽  
Enth Domain ◽  
Binding Partners ◽  
Cell Cytosol

We have used GST pulldowns from A431 cell cytosol to identify three new binding partners for the γ-adaptin appendage: Snx9, ARF GAP1, and a novel ENTH domain-containing protein, epsinR. EpsinR is a highly conserved protein that colocalizes with AP-1 and is enriched in purified clathrin-coated vesicles. However, it does not require AP-1 to get onto membranes and remains membrane-associated in AP-1–deficient cells. Moreover, although epsinR binds AP-1 via its COOH-terminal domain, its NH2-terminal ENTH domain can be independently recruited onto membranes, both in vivo and in vitro. Brefeldin A causes epsinR to redistribute into the cytosol, and recruitment of the ENTH domain requires GTPγS, indicating that membrane association is ARF dependent. In protein-lipid overlay assays, the epsinR ENTH domain binds to PtdIns(4)P, suggesting a possible mechanism for ARF-dependent recruitment onto TGN membranes. When epsinR is depleted from cells by RNAi, cathepsin D is still correctly processed intracellularly to the mature form. This indicates that although epsinR is likely to be an important component of the AP-1 network, it is not necessary for the sorting of lysosomal enzymes.

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