Biomedical subjects

A Hasilik

Publications and source records attributed to A Hasilik.

At least 37 records · Page 2Linked to original sources

PubMed1996

Purification of lysosomal membrane proteins from human placenta.

A major portion of the intracellular hydrolytic reactions are confined to lysosomes, whose membrane proteins take care of the proper lumenal milieu and the release of the degradation products. To obtain material for structural characterization of the transport proteins, we elaborated a procedure for isolation of lysosomal membranes and separation of their proteins in a two-dimensional electrophoresis. In a first step dense lysosomes were isolated from human placenta using a Percoll gradient. Subsequently, lysosomal membranes were purified by immunoadsorption. The procedure yielded mg-amounts of lysosomal membranes. Proteins associated with lysosomal membranes, acid beta-glucosidase, acetyl-coenzyme A:alpha-glucosaminide N-acetyltransferase, CD63/LIMP I, and h-lamp-2 were enriched approximately 300-fold as compared to the initial homogenate. Separation of the membrane proteins was achieved in a two-dimensional electrophoresis. The procedure is expected to yield material for structural studies on lysosomal membrane proteins with suspected defects in lysosomal storage diseases.

Cells, Cultured↗

PubMed1996

Isolation and cellular localization of the decorin endocytosis receptor.

The small dermatan sulfate proteoglycans decorin and biglycan are efficiently internalized by a variety of cells of mesenchymal origin. This process is modulated, at least under tissue culture conditions, by cell surface-associated heparan sulfate proteoglycans. Receptor proteins of 51 and 26 kDa, respectively, bind to leucine-rich repeat structures of the core proteins of the small proteoglycans but also to highly sulfated domains of heparan sulfate. The 51 kDa protein was purified from rat brain tissue by subcellular fractionation, heparin affinity chromatography and subsequent SDS-PAGE, and was used for raising a polyclonal antiserum. Affinity-purified antibodies also recognize the 26 kDa protein and a few other low molecular weight proteins, suggesting that these proteins represent proteolytic degradation products of the 51 kDa receptor. By confocal laser microscopy, it could be demonstrated that the affinity-purified antibody reacted at 0 degree C with a protein that became internalized and was transported to a perinuclear compartment during 15 min of incubation at 37 degrees C. These findings provide direct evidence that the receptor protein(s) are internalized together with the ligand and reach an endosomal compartment where further sorting can occur.

Animals↗

PubMed1995

Immunocytochemical localisation of some lysosomal hydrolases, their presence in luminal fluid and their directional secretion by human epididymal cells in culture.

The way in which the human epididymis modifies spermatozoa during their sojourn in this structure might be clarified by knowledge of the nature of its secretions. We have examined the presence of several lysosomal hydrolases in human epididymal tissue and fluids, and their synthesis and secretion by monolayer cultures. Tissues were obtained from men undergoing orchidectomy for prostatic carcinoma. The enzymes cathepsin D and acid alpha-glucosidase were localised in the lysosomes of epithelial cells from the corpus epididymidis, by an immunocytochemical technique. Cathepsin D was also found in epithelial cells of the efferent ducts within lysosomes, apical vesicles and multivesicular bodies. No immunolocalisation of acid glucosidase in the efferent ducts or on the microvilli of the corpus was demonstrable. Cathepsin D, beta-hexosaminidase (N-acetylglucosaminidase) and alpha-glucosidase were measurable in the luminal fluid from the human corpus epididymidis; beta-hexosaminidase was secreted into the culture medium by confluent monolayers of epididymal and efferent duct cells. Immunoprecipitation of cell extracts and culture medium of these cultures incubated with 35S-methionine revealed that the precursors of cathepsin D and beta-hexosaminidase were synthesized and secreted by such monolayers. Thus, active lytic enzymes are secreted by the human epididymis and could modify sperm membranes.

Adult↗

PubMed1995

Elevated levels of activities of beta-hexosaminidase and alpha-mannosidase in human immunodeficiency virus-infected patients.

To determine their value as markers of the clinical stage of human immunodeficiency virus (HIV) disease, plasma activities of lysosomal glycosidases were determined in the plasma of 97 HIV-infected patients: molecular forms of cathepsin D were characterized by Western blot to examine the mode of enzyme release. In patients with Centers for Disease Control and Prevention stage II and III of HIV disease, plasma activity of beta-hexosaminidase was significantly increased. In patients with stage III infection, alpha-mannosidase activity was also significantly increased and cathepsin D was elevated and present only in its premature form. Thus, determination of plasma activities of beta-hexosaminidase and alpha-mannosidase in HIV-positive persons may be useful for distinguishing the clinical stage of disease. The elevation of precursors of cathepsin D in patients with stage III HIV disease indicates that secretion of lysosomal enzymes rather than leakage of enzymes from damaged cells is markedly elevated.

Cathepsin D↗

PubMed1995

Carbohydrate-deficient glycoprotein syndrome (CDGS)--glycosylation, folding and intracellular transport of newly synthesized glycoproteins.

Carbohydrate-deficient glycoprotein syndrome (CDGS) is a hereditary glycosylation disorder of unknown origin. In this study we used skin fibroblasts from patients with CDGS to study the glycosylation of three well characterized glycoproteins using gel mobility analysis, endoglycosidase treatments and protein folding studies. We show that glycoprotein transport along the secretory pathway was delayed. Dilation of the rough endoplasmic reticulum indicated a retention phenomenon for selected glycoproteins. However, for all examined glycoproteins cotranslational glycosylation in CDGS fibroblasts was normal.

Abnormalities, Multiple↗

PubMed1994

Selective inactivity of TGF-beta/decorin complexes.

Previous studies had shown that binding of TGF-beta to the small proteoglycan decorin results in its inactivation. Indeed, in osteosarcoma cells the addition of decorin prevented the TGF-beta 1-mediated up-regulation of biglycan synthesis. However, the down-regulation of proteoglycan-100 remained unaltered. Even in the presence of a 100,000-fold molar excess of decorin, TGF-beta 1 was fully active in U937 monocytes with respect to the inhibition of cell proliferation. There was no inhibition of the TGF-beta-mediated stimulation of the retraction of fibroblast-populated collagen lattices. Thus, the formation of TGF-beta/decorin complexes leads to the neutralization of distinct effects only.

Biglycan↗

PubMed1994

Distinctive inhibition of the lysosomal targeting of lysozyme and cathepsin D by drugs affecting pH gradients and protein kinase C.

Morphological and biochemical evidence indicates that in several cell types, lysozyme is found in both lysosomes and the medium. Here we report that in calcitriol-treated human promonocytes U937, in which approx. two-thirds of the synthesized lysozyme is secreted, most of the intracellular lysozyme co-localizes with cathepsin D in lysosomal organelles. In the presence of NH4Cl the lysosomal targeting of procathepsin D, but not that of lysozyme, is inhibited. In the presence of 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA; 'TPA'), the lysosomal packaging of lysozyme is almost completely inhibited, while that of procathepsin D is only partially so. However, the inhibition of the lysosomal targeting of procathepsin D by NH4Cl and 4 beta-PMA is additive. The targeting of lysozyme is partially inhibited in the presence of R-59022, an inhibitor of diacylglycerol kinase, whereas it is not affected by 4 alpha-phorbol 12-myristate 13-acetate, an isomer of 4 beta-PMA that does not activate protein kinase C. It is concluded that in U937 cells both carbohydrate-dependent and -independent recognition contributes to the lysosomal targeting of soluble proteins. We suggest that the carbohydrate-independent traffic of proteins to lysosomal compartments is controlled by a signalling pathway involving protein kinase C.

Ammonium Chloride↗

PubMed1993

Synthesis of phosphorylated oligosaccharides in lysozyme is enhanced by fusion to cathepsin D.

Chinese hamster ovary cells transfected with human lysozyme cDNA encoding Asn instead of Gly22 synthesize a mutant lysozyme, [Asn22]lysozyme, with about 60% of the molecules bearing carbohydrate. This carbohydrate is predominantly of the complex type and contains a varied number of lactosamine repeats. In this study we show that the glycosylation of [Asn22] lysozyme fused to human cathepsin D is altered relative to [Asn22]lysozyme alone. The fusion protein is synthesized as a 66-kDa precursor that is cleaved to enzymatically active and antigenically positive cathepsin D and lysozyme. As compared with [Asn22]lysozyme the lysozyme moiety of the fusion protein shows an increased N-glycosylation and a decreased synthesis of lactosamine repeats. Cleavage of the precursor with cathepsin L has revealed that the lysozyme portion of the secreted fusion protein bears a complex type carbohydrate. The intracellularly released lysozyme portion of the fusion protein contains trimmed oligosaccharides. In the presence of NH4Cl the lysosomal targeting of the fusion protein is inhibited. The secreted protein is then enriched in molecules bearing phosphorylated high mannose oligosaccharides in their lysozyme moiety. Our results indicate that carbohydrate processing in [Asn22]lysozyme, including the synthesis of mannose 6-phosphate residues and of lactosamine repeats, is altered by the attached cathepsin D. The phosphorylation of the carbohydrate on the lysozyme portion results in a very efficient lysosomal targeting of the concerned fusion protein molecules.

Animals↗

PubMed1993

A mild form of mucolipidosis type III in four Baluch siblings.

Four Baluch siblings with mucolipidosis type III (pseudo-Hurler polydystrophy) are described. The patients had features commonly found in mucolipidosis III, including claw hands, joint stiffness, aortic valve involvement and radiological dysostosis multiplex. However, intelligence was normal, there were no eye abnormalities on slit-lamp examination and skin elasticity was normal. Many lysosomal enzymes were elevated in serum and diminished in cultured fibroblasts, although the findings for beta-galactosidase were atypical. Assays for the two enzymes involved in formation of the phosphomannose recognition marker revealed normal activity of the phosphotransferase with alpha-methylmannoside as an acceptor, and normal activity of the phosphodiester glycosidase. Metabolic labelling of fibroblasts with 32P followed by immunoprecipitation of cathepsin D, electrophoresis and fluorography showed that this enzyme was not labelled in the patients' cells, although some label was detected in the secreted precursor polypeptide. The data are consistent with the assumption that activity of the phosphotransferase is low towards lysosomal enzymes as substrates, and that the patients belong to complementation group C.

Arylsulfatases↗

PubMed1992

The early and late processing of lysosomal enzymes: proteolysis and compartmentation.

Lysosomal enzymes are subjected to a number of modifications including carbohydrate restructuring and proteolytic maturation. Some of these reactions support lysosomal targeting, others are necessary for activation or keeping the enzyme inactive before being segregated, while still others may be adventitious. The non-segregated fraction of the enzyme is secreted and can be isolated from the medium. It is considered that the secreted lysosomal enzymes fulfill certain physiological and pathophysiological roles. By comparing the secreted and the intracellular enzymes it is possible to distinguish between the reactions that occur before and after the segregation. In this review the reactions that may influence the segregation are referred to as the early processing and those characteristic for the enzymes isolated from lysosomal compartments as the late processing. The early processing is characterized mainly by modifications of carbohydrate side chains. In the late processing, proteolytic fragmentation represents the most conspicuous changes. The review focuses on the compartmentation of the reactions and the proteolytic fragmentation of lysosomal enzyme precursors. While a plethora of proteolytic reactions are involved, our knowledge of the proteinases responsible for the particular maturation reactions remains very limited. The review points also to work with cells from patients affected with lysosomal storage disorders, which contributed to our understanding of the lysosomal apparatus.

Animals↗

PubMed1992

Human cathepsin D precursor is associated with a 60 kDa glycosylated polypeptide.

Human cathepsin D is synthesized as a 53 kDa precursor. Most of it is segregated into lysosomal compartments and subjected to a proteolytic fragmentation. Using a cross-linking reagent we show that a large proportion of the precursor is associated with a distinct protein which--under denaturing and reducing conditions--is characterized as a 60 kDa glycopeptide. Studies on cells cultured in the presence of drugs known to affect the intracellular transport (deoxynojirimycin, brefeldin A and NH4Cl) indicated that the association with cathepsin D precursor occurs early after the synthesis and is at least partially maintained after secretion.

1-Deoxynojirimycin↗

PubMed1992

Stimulation of the biosynthesis of lactosamine repeats in glycoproteins in differentiating U937 cells and its suppression in the presence of NH4Cl.

We prepared a mouse monoclonal antibody, 2D5, which recognized a highly glycosylated human lysosomal membrane antigen. The apparent molecular mass of this antigen was cell type dependent and ranged between 100 kDa and 130 kDa. The difference was due to a variation in the carbohydrate moiety, since upon removal of the N-linked oligosaccharides the size of the glycoprotein was reduced to approximately 50 kDa in all cases. The high carbohydrate contents, subcellular localization and N-terminal sequence indicated a high similarity or identity of this antigen with the lamp-2 protein. In U937 cells several agents known to elicit differentiation induced synthesis of a larger form of the lamp antigen. Thus, treatment of cells with calcitriol resulted in a shift in its average molecular mass from 115 kDa to 130 kDa. The difference was due to an increase in the contents of lactosamine repeats. In subcellular membranes from calcitriol-treated cells the specific activity of the UDP-N-acetylglucosamine: N-acetyllactosamine N-acetylglucosaminyltransferase was enhanced 3-fold. The enhancement was accompanied with an elongation of lactosamine repeats in N-linked oligosaccharides in the 46 kDa mannose 6-phosphate receptor and the homing receptor, the leucocyte antigen CD44. In contrast, the apparent size of the leucocyte antigen CD43 which bears numerous O-linked oligosaccharides was not changed indicating a selectivity in the modulation of the formation of lactosamine repeats in N- and O-linked carbohydrates. It is shown further that the synthesis of lactosamine repeats in U937 cells is impeded in the presence of NH4Cl.

Amino Acid Sequence↗

PubMed1991

Biosynthesis of glycosylated human lysozyme mutants.

Complementary DNA encoding human lysozyme was subjected to oligonucleotide-directed mutagenesis. At one of three selected positions, amino acid residues 22, 68, or 118, the signal for N-linked glycosylation was created. The mutant DNAs were inserted into a eucaryotic vector and transfected into cultured hamster cells. The three mutant cDNAs directed synthesis of lysozyme mutants, which were named LI, LII, and LIII. The mutant lysozymes LI and LII comprised mixtures of glycosylated and nonglycosylated forms. The glycosylated and nonglycosylated forms of mutant LI were found to have an enzymatic activity similar to normal human milk lysozyme. The usage of the glycosylation sites in the mutants was similar in Chinese hamster ovary (CHO) and baby hamster kidney cells. Approximately two of every three molecules in mutant LI, approximately one of every eight molecules in mutant LII, and practically no molecules in mutant LIII became glycosylated. In CHO cells, the processing of the oligosaccharide side chains yielded several larger products than in baby hamster kidney cells. This size variability of glycosylated lysozyme from CHO cells may be explained by the presence of biantennary and triantennary endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides with N-acetyllactosamine repeats of variable length and by the presence of hybrid oligosaccharides, as suggested by affinity to several lectins and sensitivity to endo-beta-galactosidase. In both cell types, the majority of the glycosylated forms were secreted and thus behaved similarly to nonglycosylated lysozyme. A small proportion of mutant LI lysozyme remained associated with the cells. The retained lysozyme was recruited predominantly from the molecules bearing high mannose oligosaccharides. These molecules were targeted to lysosomes, and their carbohydrate was trimmed to an endo-beta-N-acetylglucosaminidase H-resistant form. Owing to the small size of mutant LI lysozyme, minor changes in the size of its carbohydrate moiety result in detectable changes in the electrophoretic mobility of the whole glycoprotein. We suggest that this novel glycoprotein could be used as a reporter in studies on processing and segregation of glycoproteins.

Animals↗

PubMed1991

UGA is translated as cysteine in pheromone 3 of Euplotes octocarinatus.

Pheromone 3 mRNA of the ciliate Euplotes octocarinatus contains three in-frame UGA codons that are translated as cysteines. This was revealed from cDNA sequencing and from plasma desorption mass spectrometry of cleaved pheromone 3 in connection with pyridylethylation of the fragments. N-terminal sequence analysis of carboxymethylated protein confirmed this conclusion for the first of the three UGA codons. Besides UGA the common cysteine codons UGU and UGC are also used to encode cysteine. UAA functions as a termination codon. No UAG codon was found. In connection with results reported for other ciliates, this suggests that the role of the classic termination codons had not yet been established when the ciliates started to diverge from other eukaryotes.

Amino Acid Sequence↗

PubMed1991

Expression and maturation of human cathepsin D in baby-hamster kidney cells.

In medium and in homogenates from baby-hamster kidney cells (BHK) transfected with human cathepsin D cDNA, an elevated activity of cathepsin D was found as compared to non-transfected cells. The elevated activity was removed by titrating the homogenates with an anti-(human cathepsin D) antibody. Metabolic labelling and immunoprecipitation revealed that, in the transfected cells, human cathepsin D was synthesized as a 53-kDa precursor indistinguishable from that found in human cells. A portion of the precursor was secreted and the remainder was processed to intermediate and mature chains within a few hours of synthesis. The precursor that was released from the transfected cells had a slightly smaller apparent size than that from cultured human fibroblasts. This difference was abrogated when the precursors were treated with glycopeptidase F. In the intracellular small chain a difference was observed in the size of carbohydrate chains that were cleavable with endo-beta-N-acetylglucosaminidase H. Sequence analysis of the N-termini of mature intracellular cathepsin D indicated a N-terminal trimming in both large and small chains from both human and transfected hamster cells. The proteolytic maturation of human cathepsin D in BHK cells closely resembles that in human cells, whereas a portion of the carbohydrate side chains is processed differently. The trimming of the N-termini in mature cathepsin D is proposed to be a part of the maturation and aging of this protein.

Acetylglucosaminidase↗

PubMed1991

Differential segregation of human and hamster cathepsin D in transfected baby-hamster kidney cells.

The segregation of human cathepsin D, studied in baby-hamster kidney cells (BHK) transfected with human cathepsin D cDNA and compared with that of hamster cathepsin D in the same cells, showed that, in cells that expressed human cathepsin D at a low rate, most of the enzyme remained intracellular. In contrast, when the enzyme was expressed at a high rate, most was secreted. The segregation was examined with an anti-(human cathepsin D) antibody that reacted with the human enzyme exclusively and an anti-(rat cathepsin D) antibody that reacted with both enzymes. In one protocol the cells were metabolically labelled and the two antibodies were used in sequence to precipitate the enzymes from extracts of cells and medium. High expression of the human enzyme did not interfere with the segregation of hamster cathepsin D. In another protocol the activity of cathepsin D in cells and medium was measured before and after titration with anti-(human cathepsin D) antiserum. Human cathepsin D was found predominantly in the medium, and hamster cathepsin D mainly in the cells. In the presence of 10 mM-NH4Cl the intracellular segregation of hamster cathepsin D was strongly inhibited, while the segregation of human cathepsin D was only slightly diminished. In BHK cells, at least two systems participate in the sorting of the two cathepsins, one of them being rather insensitive to NH4Cl.

Ammonium Chloride↗

PubMed1991

Determination of the phosphorylation, uncovering of mannose 6-phosphate groups and targeting of lysosomal enzymes.

There are at least three stages in the targeting of soluble lysosomal enzymes: transfer of N-acetylglucosaminyl 1-phosphate to high-mannose oligosaccharide side chains, removal of N-acetylglucosamine and recognition of the "uncovered" mannose 6-phosphate residues. Defects in the transfer reaction cause mucolipidoses II and III. Those in the subsequent stages of the targeting may result in similar clinical disorders. To differentiate between possible defects of the targeting in cultured cells we have developed a procedure for a combined detection of the phosphorylation, uncovering of the transferred phosphate residues and the targeting of lysosomal enzymes. For this purpose cultured cells are metabolically labelled with [32P]phosphate and a lysosomal enzyme, such as cathepsin D, is isolated from the labelled cells and the medium by immunoprecipitation. The immunoprecipitates are dissolved with sodium dodecylsulphate and incubated in the presence and absence of calf intestine alkaline phosphatase. We show that the treatment of the denatured protein results in hydrolysis of phosphomonoester groups and that the phosphodiester and the peptide bonds remain intact. The initial and the residual radioactivity associated with the lysosomal enzyme which represent the total phosphate and the phosphodiester groups, respectively, are determined by gel-electrophoresis, fluorography and densitometry. This procedure extends one of the previously established methods for the diagnosis of mucolipidoses II and III.

Calcitriol↗

PubMed1990

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

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

Ammonium Chloride↗