Biomedical subjects

R Willemsen

Publications and source records attributed to R Willemsen.

At least 91 records · Page 5Linked to original sources

PubMed1990

Expression and routeing of human lysosomal alpha-glucosidase in transiently transfected mammalian cells.

Previously isolated lysosomal alpha-glucosidase cDNA clones were ligated to full-length constructs for expression in vitro and in mammalian cells. One of these constructs (pSHAG1) did not code for functional enzyme, due to an arginine residue instead of a tryptophan residue at amino acid position 402. The mutation does not affect the rate of enzyme synthesis, but interferes with post-translational modification and intracellular transport of the acid alpha-glucosidase precursor. Using immunocytochemistry it is demonstrated that the mutant precursor traverses the endoplasmic reticulum and the Golgi complex, but does not reach the lysosomes. Pulse-chase experiments suggest premature degradation. The Trp-402-containing enzyme (encoded by construct pSHAG2) is processed properly, and has catalytic activity. A fraction of the enzyme is localized at the plasma membrane. It is hypothesized that membrane association of the acid alpha-glucosidase precursor, as demonstrated by Triton X-114 phase separation, is responsible for transport to this location. Transiently expressed acid alpha-glucosidase also enters the secretory pathway, since a catalytically active precursor is found in the culture medium. This precursor has the appropriate characteristics for use in enzyme replacement therapy. Efficient uptake via the mannose 6-phosphate receptor results in degradation of lysosomal glycogen in cultured fibroblasts and muscle cells from patients with glycogenosis type II.

Animals↗

PubMed1990

Rat heart perfusion as model system for enzyme replacement therapy in glycogenosis type II.

Cardiac failure and skeletal muscle weakness are the main clinical features of glycogenosis type II, a lysosomal storage disorder caused by acid alpha-glucosidase deficiency. In our study, we have investigated in a rat heart perfusion-recirculation system whether acid alpha-glucosidase can be taken up from the vascular system into cardiomyocytes. When rat hearts were perfused with mannose 6-phosphate-containing acid alpha-glucosidase purified from bovine testis, a 3- to 4-fold increase of enzyme activity was obtained. Perfusion with human placental acid alpha-glucosidase not containing the mannose 6-phosphate recognition marker did not have such an effect. The presence of bovine testis acid alpha-glucosidase in heart tissue was demonstrated by immunoblotting. Immunocytochemistry provides evidence for uptake of the exogenous enzyme in lysosomes of the cardiomyocytes. The relevance of these findings for enzyme therapy in glycogenosis type II is discussed.

Animals↗

PubMed1990

Plasmalemmal vesicles are involved in transendothelial transport of albumin, lysosomal enzymes and mannose 6-phosphate receptor fragments in capillary endothelium.

With the use of immunoelectron microscopy we have demonstrated the presence of lysosomal enzymes (acid alpha-glucosidase and glucocerebrosidase) and fragments of the 270 kDa receptor for mannose 6-phosphate and insulin-like growth factor II in blood plasma, plasmalemmal vesicles of endothelial cells and pericapillary spaces in human skeletal muscle tissue. At these locations, the three proteins colocalized with albumin known to be transported from the capillaries into the pericapillary spaces. Immunoblot analysis of plasma revealed the presence of relatively high molecular weight polypeptides in this material. These observations strongly suggest that high molecular weight species of lysosomal enzymes can pass the endothelial barrier in skeletal muscle tissue.

Albumins↗

PubMed1989

Alternative splicing of beta-galactosidase mRNA generates the classic lysosomal enzyme and a beta-galactosidase-related protein.

We have isolated two cDNAs encoding human lysosomal beta-galactosidase, the enzyme deficient in GM1-gangliosidosis and Morquio B syndrome, and a beta-galactosidase-related protein. In total RNA from normal fibroblasts a major mRNA of about 2.5 kilobases (kb) is recognized by cDNA probes. A minor transcript of about 2.0 kb is visible only in immunoselected polysomal RNA. A heterogeneous pattern of expression of the 2.5-kb beta-galactosidase transcript is observed in fibroblasts from different GM1-gangliosidosis patients. The nucleotide sequences of the two cDNAs are extensively colinear. However, the short cDNA misses two noncontiguous protein-encoding regions (1 and 2) present in the long cDNA. The exclusion of region 1 in the short molecule introduces a frameshift in its 3'-flanking sequence, which is restored by the exclusion of region 2. These findings imply the existence of two mRNA templates, which are read in a different frame only in the nucleotide stretch between regions 1 and 2. Sequence analysis of genomic exons of the beta-galactosidase gene shows that the short mRNA is generated by alternative splicing. The long and short cDNAs direct the synthesis in COS-1 cells of beta-galactosidase polypeptides of 85 and 68 kDa, respectively. Only the long protein is catalytically active under the assay conditions used, and it is capable of correcting beta-galactosidase activity after endocytosis by GM1-gangliosidosis fibroblasts. The subcellular localization of cDNA-encoded beta-galactosidase and beta-galactosidase-related proteins is different.

Amino Acid Sequence↗

PubMed1988

Ultrastructural localization of steroid sulphatase in cultured human fibroblasts by immunocytochemistry: a comparative study with lysosomal enzymes and the mannose 6-phosphate receptor.

Immunocytochemistry was used to study the subcellular localization of steroid sulphatase in cultured human fibroblasts. Ultra-thin cryosections were incubated with antibodies raised against steroid sulphatase purified from human placenta and immune complexes were visualized with gold probes as electron dense markers. Steroid sulphatase was found in rough endoplasmic reticulum, Golgi cisternae and in the trans-Golgi reticulum, where it co-distributes with lysosomal enzymes and the mannose 6-phosphate receptor. The enzyme was not detected in lysosomes. Steroid sulphatase was also found at the plasma membrane and in the endocytic pathway (i.e. coated pits, endosomes and multivesicular endosomes). These may be the sites where sulphated oestrogen precursors are hydrolysed. Also here, it co-localizes with lysosomal enzymes and the mannose 6-phosphate receptor. It is concluded that microsomal steroid sulphatase and lysosomal enzymes share several cellular compartments.

Carrier Proteins↗

PubMed1988

alpha-N-acetylgalactosaminidase deficiency, a new lysosomal storage disorder.

A new lysosomal storage disease with autosomal recessive inheritance is described in two male siblings of 5 1/2 and 4 years of age. Clinical manifestations started after 9 months of age with neurological symptoms, followed by progressive psychomotor deterioration. Urinary oligosaccharide excretion was abnormal and showed a characteristic pattern on chromatography. Enzyme assays showed a profound deficiency of lysosomal alpha-N-acetylgalactosaminidase in cultured fibroblasts, leukocytes and plasma from the patients and reduced activity in material from the parents. The deficiency was demonstrated both with an artificial substrate and a natural one, the blood group A trisaccharide. Excessive intra-lysosomal storage of alpha-N-acetylgalactosamine-containing material was demonstrated in cultured fibroblasts from the patients, using the lectin from Helix pomatia which is specific for terminal alpha-N-acetylgalactosamine residues.

Acetylgalactosamine↗

PubMed1987

Ultrastructural localization of glucocerebrosidase in cultured Gaucher's disease fibroblasts by immunocytochemistry.

The subcellular localization of glucocerebrosidase was studied in cultured skin fibroblasts from eight patients with Gaucher's disease. The enzyme, in situ, was visualized under the electron microscope by incubating ultrathin frozen sections of fibroblasts with antibodies against glucocerebrosidase, followed by a second incubation with goat anti- (rabbit IgG) coupled to colloidal gold. In control cells, most of the gold label was found in lysosomes, associated with the membrane. Labelling of the rough endoplasmic reticulum (RER) and Golgi complex was also observed. In fibroblasts from three Gaucher's disease patients without neurological symptoms (type 1 disease), a near normal amount of cross-reactive material (CRM) was detected in lysosomes, but in a fourth such patient, the lysosomal CRM was reduced. Little lysosomal glucocerebrosidase was detected in cells from patients with the acute neuronopathic form (type 2) or the subacute neuronopathic form (type 3) of Gaucher's disease. CRM in lysosomes correlates with amount of mature, 59 kDA glucocerebrosidase which is undetectable in type 2 and type 3 Gaucher's disease cell lines. These findings demonstrate that different mutations in the gene for glucocerebrosidase result in mutant proteins that have different intracellular localizations. They also suggest that there is a relationship between the amount of cross-reactive material in the lysosomes and the phenotypic expression of the disease.

Cell Line↗

PubMed1987

Molecular heterogeneity in human beta-galactosidase and neuraminidase deficiency.

Human lysosomal beta-galactosidase and neuraminidase exist in a complex together with a 32-kilodalton (kd) glycoprotein. The latter protein was found to have a dual function: it is required for the aggregation of monomeric 64-kd beta-galactosidase into high molecular weight (600-700 kd) multimers and it is an essential subunit of neuraminidase together with a 76-kd polypeptide. The severe neurological disorder galactosialidosis, characterized by a coexistent deficiency of beta-galactosidase and neuraminidase, was found to be due to a genetic defect of the 32-kd protective protein. The molecular background of the clinical heterogeneity within this syndrome is described and will undoubtedly be further elucidated since we have recently isolated the gene coding for the protective protein. The sequence of normal and mutant (enzyme) proteins will also provide better insight into the characteristics of the beta-galactosidase-neuraminidase-protective protein complex. Another interesting model for the study of posttranslational processing is the defective phosphorylation of beta-galactosidase in cells from patients with GM1-gangliosidosis.

Animals↗

PubMed1987

Clinical diversity in glycogenosis type II. Biosynthesis and in situ localization of acid alpha-glucosidase in mutant fibroblasts.

The molecular basis of clinical diversity in glycogenosis type II (Pompe's disease) was investigated by comparing the nature of acid alpha-glucosidase deficiency in cultured fibroblasts from 30 patients. Biosynthetic forms of acid alpha-glucosidase with different molecular mass were separated electrophoretically and identified by immunoblotting. Immuno-electron microscopy was employed to determine the intracellular localization of mutant enzyme. Our studies illustrate that maturation of acid alpha-glucosidase is associated with transport to the lysosomes. Deficiency of catalytically active mature enzyme in lysosomes is common to all clinical phenotypes but, in the majority of cases, is more profound in early onset than in late onset forms of the disease. Thus, the results suggest that the clinical course of glycogenosis type II is primarily determined by the amount of functional acid alpha-glucosidase. The role of secondary factors can, however, not be excluded because three adult patients were identified with very low activity and little enzyme in the lysosomes.

Adult↗

PubMed1987

Immunocytochemical localization of lysosomal acid phosphatase in normal and "I-cell" fibroblasts.

This study represents the first example of immunological localization of lysosomal acid phosphatase. The intracellular localization of lysosomal acid phosphatase was investigated with immunocytochemical methods at the light and electron microscopical level in cultured fibroblasts obtained from normal subjects and from a patient with I-cell disease. Double-labeling studies using fluorescence microscopy showed that acid phosphatase is present in the same organelles as other hydrolases. At the electron microscopic level in control fibroblasts acid phosphatase was found in the rough endoplasmic reticulum, lysosomes, at the plasma membrane, in vesicles just below the plasma membrane and in multivesicular bodies. This localization was comparable with that of other lysosomal enzymes tested (acid alpha-glucosidase, N-acetyl-beta-hexosaminidase, beta-galactosidase). Acid phosphatase labeling was mainly found in association with the lysosomal membrane and with membranous material present within the lysosome. In I-cell fibroblasts the label was present in the same subcellular organelles but always associated with membranous structures. We suggest that the association of acid phosphatase with membranes might explain the normal enzyme activity found in I-cell fibroblasts.

Acid Phosphatase↗

PubMed1986

Immunoelectron microscopical localization of lysosomal beta-galactosidase and its precursor forms in normal and mutant human fibroblasts.

Immunoelectron microscopy was performed to study the biosynthesis of lysosomal beta-galactosidase (beta-gal) in normal and mutant human fibroblasts. Using polyclonal and monoclonal antibodies we show in normal cells precursor forms of beta-gal in the rough endoplasmic reticulum (RER) and in the Golgi apparatus throughout the stack of cisternae. In the lysosomes virtually all beta-gal exists as a high molecular weight multimer of mature enzyme. In the autosomal recessive disease GM1-gangliosidosis caused by a beta-gal deficiency and in galactosialidosis, associated with a combined deficiency of lysosomal neuraminidase and beta-gal, precursor forms of the latter enzyme are found in RER, Golgi and some labeling is present at the cell surface. The lysosomes remain unlabeled, indicative for the absence of enzyme molecules in this organelle. In galactosialidosis fibroblasts also no mature beta-gal is found in the lysosomes but in these cells the presence of the monomeric form can be increased by leupeptin (inhibition of proteolysis) whereas addition of a partly purified 32 kDa "protective protein" results in the restoration of high molecular weight beta-gal multimers in the lysosomes.

Antibodies↗

PubMed1985

Sucrase-isomaltase and cystic fibrosis.

The intestinal microvillar enzyme complex sucrase-isomaltase has been studied in cystic fibrosis and control ileum. A number of biochemical parameters of the enzyme in ileum homogenates have been determined. Both solubilized as well as membrane-bound sucrase-isomaltase were analyzed with respect to their reaction with monoclonal antibodies against human sucrase-isomaltase. Finally the subcellular localization of sucrase-isomaltase was verified by immunoelectronmicroscopy or via the analysis of purified brush-border membrane preparations. At all levels no significant differences could be detected between sucrase-isomaltase of cystic fibrosis and control ileum. It is concluded that an abnormal subcellular localization and/or abnormal enzymatic activity of sucrase-isomaltase in cystic fibrosis intestine cannot explain the markedly decreased disaccharidase activities in amniotic fluids from pregnancies resulting in a child affected with cystic fibrosis.

Antibodies, Monoclonal↗

PubMed1985

The subcellular localization of soluble and membrane-bound lysosomal enzymes in I-cell fibroblasts: a comparative immunocytochemical study.

Using electron microscopic immunocytochemistry with gold probes, we have studied the localization of acid alpha-glucosidase, N-acetyl-beta-hexosaminidase and beta-glucocerebrosidase in cultured skin fibroblasts from control subjects and patients with mucolipidosis II (I-cell disease). In control fibroblasts, a random distribution of acid alpha-glucosidase and N-acetyl-beta-hexosaminidase within the lysosomes was observed, whereas beta-glucocerebrosidase was found to be localized on or near the lysosomal membrane. The observations confirm the soluble character of acid alpha-glucosidase and N-acetyl-beta-hexosaminidase and the membrane-bound character of beta-glucocerebrosidase. In I-cell fibroblasts an abnormal localization of the two soluble enzymes was found. Labeling in lysosomes was very weak, but instead, small 'presumptive' vesicles containing both enzymes were detected throughout the cytoplasm and close to the plasma membrane. These vesicles could be involved in the secretion of the two enzymes. In contrast, a normal membrane-bound lysosomal localization was observed for beta-glucocerebrosidase. It is concluded that the intracellular transport of beta-glucocerebrosidase to the lysosomes can occur even when the mannose-6-phosphate recognition system is defective. This explains the normal activity of beta-glucocerebrosidase in I-cells in contrast to the deficiency of most other lysosomal enzymes.

Cells, Cultured↗

PubMed1985

Effect of monensin on the metabolism, localization, and biosynthesis of N- and O-linked oligosaccharides of galactosyltransferase.

The effect of monensin on the metabolism and glycosylation of the trans-Golgi enzyme galactosyltransferase (GT) was studied in HeLa cells. The synthesis of GT was not affected by monensin; however, in the presence of the drug GT precursor molecules (Mr 54 000 and 47 000) were transported to the Golgi system at a slower rate than in control cells. the half-life of GT as an intrinsic membrane protein of the trans-Golgi system was prolonged in the presence of monensin. Mature GT (Mr 54 000) contained in addition to one N-linked oligosaccharide several O-linked oligosaccharides; the majority of them consisted of the disaccharide galactose-N-acetylgalactosamine containing one or two sialic acid residues. We conclude that GT resides at an intracellular location where significant sialylation occurs. The sialylation of O-linked oligosaccharides was not hampered by the presence of monensin. Two-dimensional gel electrophoresis combined with neuraminidase treatment revealed that GT consists of a number of species in a pI range between 4 and 7. Removal of sialic acid residues resulted in a less acidic pI range of GT but the charge heterogeneity persisted. Immunoelectron microscopy with antibodies against GT showed that monensin affects primarily the GT-containing cisternae.

Biological Transport, Active↗

PubMed1985

Immunocytochemical localization of acrosin and hyaluronidase in epididymal and ejaculated porcine spermatozoa.

Acrosin and hyaluronidase demonstrated different release patterns following treatment of living spermatozoa with the Ca2+-ionophore A 23187. One hour after the acrosomal reaction about 50% of the acrosin was still associated with the spermatozoal membranes, while hyaluronidase could no longer be detected in the spermatozoal remnants. Strong fixation conditions with acrolein and glutaraldehyde were used to prevent redistribution and leakage of these sperm proteins. Lost antigenicity was restored with sodium-borohydride and pronase E treatment. Immunofluorescent localization showed hyaluronidase to be confined to the anterior portion of the acrosome. Acrosin was localized throughout the entire acrosome including the equatorial segment. By immunoelectron microscopy, hyaluronidase was exclusively found in the acrosomal matrix. The equatorial segment was devoid of hyaluronidase. Acrosin was found in the acrosomal matrix as well as on the outer acrosomal membrane. Furthermore, labeling for acrosin in the equatorial segment was clearly demonstrated. Localization of hyaluronidase was the same for epididymal and ejaculated sperm cells but in approximately 70% of the epididymal spermatozoa acrosin could not be detected in the equatorial segment. In most of these epididymal cells acrosin was confined to the anterior part of the acrosome comparable with hyaluronidase. These observations support the motion that the appearance of acrosin in the equatorial segment is part of the maturation process during passage through the epididymis.

Acrosin↗

PubMed1983

Transport and topology of galactosyltransferase in endomembranes of HeLa cells.

HeLa cell membranes were studied for the distribution and orientation of the Golgi marker enzyme uridine diphosphate-galactose:beta-D-N-acetylglucosamine beta, 1-4 transferase (GT). Short pulse labeling in the presence of [35S]methionine resulted in two precursor species (Mr = 44,000 and 47,000), present in a microsomal fraction with a density of 1.18 g/ml in sucrose, presumably derived from the rough endoplasmic reticulum. Processing of the N-linked oligosaccharide(s) occurred only after the precursor molecules migrated to lighter density fractions, presumably derived from the Golgi complex. The mature GT molecules (Mr = 54,000) contain O-linked oligosaccharides as shown by beta-elimination of metabolically incorporated [3H]galactose. The O-glycosylation occurred mainly in the light density fractions. The topology of GT was studied on membrane fractions after labeling with [35S]methionine as well as immunocytochemically on ultrathin cryosections at the electron microscope level. Our results indicate that both the antigenic determinants of GT as well as polypeptide chain are present intramembraneously and at the luminal side of the membranes of the Golgi complex and rough endoplasmic reticulum.

Animals↗

PubMed1983

Vesicular stomatitis virus glycoprotein, albumin, and transferrin are transported to the cell surface via the same Golgi vesicles.

Human hepatoma cells, infected by vesicular stomatitis virus, offer a good system to study simultaneously the intracellular localization of a well defined transmembrane glycoprotein (VSV-G), a secretory glycoprotein (transferrin), and a nonglycosylated secretory protein (albumin). We used monospecific antibodies in combination with 5- and 8-nm colloidal gold particles complexed with protein A to immunolabel these proteins simultaneously in thin frozen sections of hepatoma cells. VSV-G, transferrin, and albumin are present in the same rough endoplasmic reticulum cisternae, the same Golgi compartments, and the same secretory vesicles. In the presence of the ionophore monensin intracellular transport is blocked at the trans cisternae of the Golgi complex, and VSV-G, transferrin, and albumin accumulate in dilated cisternae, which are apparently derived from the trans-Golgi elements. Glycoproteins, synthesized and secreted in the presence of monensin, are less acidic than those in control cultures. This is probably caused by a less efficient contact between the soluble secretory proteins and the membrane-bound glycosyltransferases that are present in the most monensin-affected (trans) Golgi cisternae.

Albumins↗

PubMedDate not supplied

A biochemical and ultrastructural evaluation of the type 2 Gaucher mouse.

Gaucher mice, created by targeted disruption of the glucocerebrosidase gene, are totally deficient in glucocerebrosidase and have a rapidly deteriorating clinical course analogous to the most severely affected type 2 human patients. An ultrastructural study of tissues from these mice revealed glucocerebroside accumulation in bone marrow, liver, spleen, and brain. This glycolipid had a characteristic elongated tubular structure and was contained in lysosomes, as demonstrated by colocalization with both ingested carbon particles and cathepsin D. In the central nervous system (CNS), glucocerebroside was diffusely stored in microglia cells and in brainstem and spinal cord neurons, but not in neurons of the cerebellum or cerebral cortex. This rostralcaudal pattern of neuronal lipid storage in these Gaucher mice replicates the pattern seen in type 2 human Gaucher patients and clearly demonstrates that glycosphingolipid catabolism and/or accumulation varies within different brain regions. Surprisingly, the cellular pathology of tissue from these Gaucher mice was relatively mild, and suggests that the early and rapid demise of both Gaucher mice and severely affected type 2 human neonates may be the result of both a neurotoxic metabolite, such as glucosylsphingosine, and other factors, such as skin water barrier dysfunction secondary to the absence of glucocerebrosidase activity.

Animals↗