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F W Verheijen

Publications and source records attributed to F W Verheijen.

32 records · Page 2Linked to original sources
PubMed1989

Characterization of a proton-driven carrier for sialic acid in the lysosomal membrane. Evidence for a group-specific transport system for acidic monosaccharides.

Highly purified lysosomal membrane vesicles, obtained from rat liver lysosomes, were used to study characteristics of NeuAc transport across the lysosomal membrane. Uptake of [14C]NeuAc was found to be strongly influenced by a pH gradient across the membrane. When a proton gradient (pHin greater than pHout) was generated by impermeable buffers, NeuAc uptake above equilibrium level (overshoot) was observed. The influence of membrane diffusion potentials was ruled out by experiments where K+ and valinomycin were present. The overshoot appeared to be specifically produced by protons, since gradients of other cations (Na+ and K+) did not give stimulation. Proton-driven uptake was saturable (Kt = 0.24 mM) and mediated by a single system, as shown by linearity of the Scatchard plot. Stimulation of transport was also obtained by preincubation of vesicles with MgATP and the effect was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by the protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Monocarboxylic sugars like glycuronic acids were competitive inhibitors of sialic acid transport. Transstimulation of [14C] NeuAc uptake was observed when vesicles were preloaded either with unlabeled NeuAc or with glucuronic acid. The data demonstrate that lysosomal membrane vesicles from rat liver are a suitable system for kinetic studies of solute transport events. The presence of a proton-driven carrier in the lysosomal membrane specific for sialic acid and other acidic sugars, including glucuronic acid, is shown. The possible physiological significance of these findings for the human lysosomal carrier and the patients with a sialic acid transport defect is discussed.

Amino Acids, Diamino↗
PubMed1989

Identification and in vitro reconstitution of lysosomal neuraminidase from human placenta.

Lysosomal neuraminidase from human placenta has been obtained in its active form by association of an inactive neuraminidase polypeptide with beta-galactosidase and the protective protein. Using a specific antiserum, we have now identified a 66-kDa protein as the inactive neuraminidase polypeptide. It is specifically recognized on immunoblots only in its nonreduced state, and it coprecipitates with neuraminidase activity. The 66-kDa polypeptide is substantially glycosylated (38-kDa protein core with 7-14 N-linked oligosaccharide chains), a feature characteristic of lysosomal integral membrane proteins. Specific removal of the 66-kDa neuraminidase polypeptide from glycoprotein preparations prevents the generation of neuraminidase activity. Removal of beta-galactosidase or destruction of the protective protein also hinders the formation of active neuraminidase. Reconstitution of neuraminidase activity is observed after mixing glycoprotein preparations, depleted in different components of the beta-galactosidase-neuraminidase-protective protein complex, indicating that all three components of the complex are required for neuraminidase activity. Association of the neuraminidase polypeptide and the protective protein generates unstable neuraminidase activity, whereas association with beta-galactosidase is required for stability.

Chromatography, Affinity↗
PubMed1988

Expression of cDNA encoding the human "protective protein" associated with lysosomal beta-galactosidase and neuraminidase: homology to yeast proteases.

The "protective protein" is a glycoprotein that associates with lysosomal beta-galactosidase and neuraminidase and is deficient in the autosomal recessive disorder galactosialidosis. We have isolated the cDNA encoding human "protective protein". The clone recognizes a 2 kb mRNA in normal cells that is not evident in fibroblasts of an early infantile galactosialidosis patient. The cDNA directs the synthesis of a 452 amino acid precursor molecule that is processed in vivo to yield mature "protective protein," a heterodimer of 32 kd and 20 kd polypeptides held together by disulfide bridges. This mature form is also biologically functional since it restores beta-galactosidase and neuraminidase activities in galactosialidosis cells. The predicted amino acid sequence of the "protective protein" bears homology to yeast carboxypeptidase Y and the KEX1 gene product. This suggests a protease activity for the "protective protein."

Amino Acid Sequence↗
PubMed1988

Isolation and structural characterization of sialic acid-containing storage material from mucolipidosis I (sialidosis) fibroblasts.

Sialic acid-containing storage material was isolated from cultured human mucolipidosis I (sialidosis) fibroblasts by gel permeation chromatography on Bio-Gel P-6 followed by medium-pressure anion-exchange chromatography on Mono Q. The structure determination of the isolated sialyloligosaccharides was carried out by 500-MHz 1H-NMR spectroscopy in conjunction with sugar analysis and analytical HPLC. The storage material showed completely sialylated mono-, di- and triantennary N-glycosidic N-acetyllactosamine oligosaccharides having the Man beta 1----4GlcNAc sequence at the reducing end in common. Heterogeneity occurred with respect to the linkages between terminal sialic acid and the penultimate galactose residues (alpha 2----3/alpha 2----6). It turned out that all the identified carbohydrate chains are consistent with the neuraminidase deficiency.

Carbohydrate Conformation↗
PubMed1987

Purification and partial characterization of lysosomal neuraminidase from human placenta.

Lysosomal neuraminidase and beta-galactosidase are present in a complex together with a 32-kDa protective protein. This complex has been purified and the different components have been dissociated using potassium isothiocyanate (KSCN) treatment. beta-Galactosidase remains catalytically active, but neuraminidase loses its activity upon dissociation. The inactive dissociated neuraminidase was purified by removing the remaining non-dissociated beta-galactosidase/protective protein complex using beta-galactosidase-specific affinity chromatography. The dissociated neuraminidase material shows two major polypeptides on SDS-PAGE with an apparent molecular mass of 76 kDa and 66 kDa. Subsequently the 32-kDa protective protein was dissociated from the beta-galactosidase/protective protein complex, and purified. Antibodies raised against the dissociated inactive neuraminidase preparation specifically immunoprecipitate the active neuraminidase present in the complex with beta-galactosidase and protective protein. By immunoblotting evidence is provided that the 76-kDa protein is a subunit of neuraminidase which, in association with the 32-kDa protective protein, is essential for neuraminidase activity.

Antibody Specificity↗
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↗
PubMed1986

Free N-acetylneuraminic acid (NANA) storage disorders: evidence for defective NANA transport across the lysosomal membrane.

To study the biochemical defect underlying N-acetylneuraminic acid (NANA) storage disorders (NSD), a tritium-labeled NANA-methylester was prepared and its metabolism was studied in normal and mutant human fibroblasts. The uptake of methylester, its conversion into free NANA, and the release of free NANA was studied in lysosome-enriched fractions. In three clinically different types of NSD accumulation of free NANA was observed and the half-life of this compound was significantly increased. Our observations indicate the existence of a transport system for NANA across the lysosomal membrane, which is deficient in all variants of NSD.

Biological Transport↗
PubMed1986

Galactosialidosis: molecular heterogeneity among distinct clinical phenotypes.

The lysosomal storage disorder galactosialidosis has been recognized as a distinct genetic and biochemical entity, associated with a combined beta-galactosidase and neuraminidase deficiency that is due to the lack of a 32-kilodalton (kDa) glycoprotein. The molecular basis of different clinical variants of galactosialidosis has been investigated. In the early-infantile form, the synthesis of the 52-kDa precursor of the 32-kDa "protective protein" is markedly reduced and the absence of the latter protein explains the severe neuraminidase deficiency. In the juvenile-adult form, there is relatively more 52-kDa precursor but no 32-kDa protein can be detected. Cells from the late-infantile form have in comparison with controls, besides a small amount of the 32-kDa glycoprotein, an accumulation of the 52-kDa precursor. Apparently, this protein is genetically altered in such a way that its further processing is impaired. Furthermore, in this mutant, the residual neuraminidase activity is stimulated four- to sixfold upon leupeptin treatment together with an increase of the 32-kDa glycoprotein.

Adult↗
PubMed1985

Human placental neuraminidase. Activation, stabilization and association with beta-galactosidase and its protective protein.

Supernatant of homogenized human placenta hardly contains lysosomal neuraminidase activity. It is, however, possible to generate remarkably high activity by concentration of a partially purified glycoprotein fraction. This activity is labile to dilution, but can be stabilized by incubation at 37 degrees C and acid pH. Using beta-galactosidase specific affinity chromatography and immunotitration, we show that the activated and stabilized human lysosomal neuraminidase exists in a complex with beta-galactosidase. Sucrose density gradient centrifugation experiments demonstrate that the neuraminidase activity is exclusively present in a high density multimeric form of beta-galactosidase. The formation of multimeric forms of beta-galactosidase is known to require a 32000-Mr 'protective' protein. Monospecific antibodies against this 'protective' protein were purified from a conventional antiserum containing a mixture of antibodies against the 64000-Mr beta-galactosidase protein and against the 32000-Mr 'protective' protein, using a nitrocellulose blot immunoaffinity purification procedure. Immunotitration experiments with these antibodies show that the 32000-Mr 'protective' protein is present both in association with the beta-galactosidase multimer and with the high-density multimeric form together with neuraminidase. Our data further suggest that association of the 32000-Mr 'protective' protein and another yet unidentified subunit is essential for the catalytic activity of lysosomal neuraminidase. These results explain the absence of neuraminidase activity in the autosomal recessive human lysosomal storage disorder galactosialidosis, where the 32000-Mr 'protective' protein is known to be absent.

Carboxypeptidases↗
PubMed1983

Two genetically different MU-NANA neuraminidases in human leucocytes.

Human leucocytes contain two different MU-NANA neuraminidases, which can be distinguished by Concanavalin A binding. The Con A binding form is predominant in lymphocytes (more than 80%) and the non-binding form predominates in granulocytes. The pH optima of both these neuraminidases as well as their subcellular localization as determined by Percoll gradient centrifugation suggest that they are both lysosomal. Immunological studies indicate that the Con A binding form is present in a complex with beta-galactosidase whereas the non-binding form is not. Leucocytes from patients with sialidosis or galactosialidosis are deficient in the Con A binding neuraminidase, whereas the non-binding form is normal. In sialolipidosis both forms are normal. These results demonstrate that leucocytes contain at least two genetically different MU-NANA neuraminidases. Thus, the use of leucocytes should be avoided for the diagnosis of sialidosis and galactosialidosis, and isolated lymphocytes should be used to obtain reliable results.

Chromatography, Affinity↗
PubMed1983

The relation between human lysosomal beta-galactosidase and its protective protein.

Cultured skin fibroblasts from patients with the lysosomal storage disease galactosialidosis lack a 54-kDa protein which is a precursor of 32-kDa and 20-kDa proteins, which immunoprecipitate with human anti-beta-galactosidase antiserum. The lack of a 32-kDa "protective protein" results in a combined deficiency of beta-galactosidase and sialidase. The mechanism of protection of lysosomal beta-galactosidase against proteolytic degradation is elucidated by sucrose density gradient centrifugation and immunoprecipitation studies. In normal fibroblasts at the low intralysosomal pH, more than 85% of beta-galactosidase exists as a high molecular weight (600-700 kDa) multimer and about 10% as a monomer of 64-kDa. In mutant cells from galactosialidosis patients, the residual enzyme activity, about 10%, is present as a monomer and no multimer exists. After addition of the 54-kDa precursor form of the protective protein, the density pattern of beta-galactosidase in galactosialidosis cells is normalized. Immunoprecipitation studies after sucrose density gradient centrifugation on homogenate and on purified beta-galactosidase from normal fibroblasts show that the protective protein is associated only with the multimeric form of beta-galactosidase. We propose that intralysosomal protection against proteolysis of beta-galactosidase and sialidase is accomplished by aggregation into a high molecular weight complex consisting of multimeric beta-galactosidase, sialidase, and protective protein. The genetic deficiency of the latter, as in galactosialidosis, results in a rapid degradation of monomeric beta-galactosidase and a loss of sialidase activity.

Electrophoresis, Polyacrylamide Gel↗
PubMed1981

Complementation analysis of human sialidase deficiency using natural substrates.

Complementation analysis by somatic cell hybridization to produce heterokaryons has shown that at least three complementation groups exist within the disorders in which the enzyme sialidase is deficient. We have confirmed these results by electrophoretic analysis of two glycoprotein enzymes, adenosine deaminase and acid phosphatase, which show aberrant electrophoretic mobilities in these disorders. These abnormal forms, which have excess sialic acid bound, disappear on complementation and are replaced by normal mobility components. It is suggested that the sialidase produced on complementation uses the abnormal forms as natural substrates and that they may represent normal intermediates in the processing of glycoprotein enzymes.

Acid Phosphatase↗
PubMed1980

Genetic heterogeneity in human neuraminidase deficiency.

There is a deficiency of human alpha-N-acetylneuraminidase in several inherited diseases. In patients with mucolipidosis I (refs 1,2) and in adults with a variant form with out bony abnormalities and mental retardation, both also classified as sialidoses, it is the only deficient enzyme. In mucolipidosis II ('I-cell' disease) neuraminidase is one of many deficient lysosomal hydrolases and a third manifestation combines deficiency of neuraminidase and beta-galactosidase. We have investigated the genetic background of these various neuraminindase deficiencies by somatic cell hybridization and co-cultivation. The principal conclusions from work on mutant fibroblasts, reported here, are that at least three gene mutations are involved and that the combined beta-galactosidase/neuraminidase deficiency is likely to be due to defective post-translational modification of these enzymes.

Cell Communication↗