A. T. Hoogeveen

Fmr1 knockout mice: A model to study fragile X mental retardation

Male patients with fragile X syndrome lack FMR1 protein due to silencing of the FMR1 gene by amplification of a CGG repeat and subsequent methylation of the promoter region. The absence of FMR1 protein leads to mental retardation, aberrant behavior, and macroorchidism. Hardly anything is known about the physiological function of FMR1 and the pathological mechanisms leading to these symptoms. Therefore, we designed a knockout model for the fragile X syndrome in mice. The knockout mice lack normal Fmr1 protein and show macroorchidism, learning deficits, and hyperactivity. Consequently, this knockout mouse may serve as a valuable tool in the elucidation of the physiological role of FMR1 and the mechanisms involved in macroorchidism, abnormal behavior, and mental retardation.

Genetic heterogeneity in GM1-gangliosidosis.

GM1-GANGLIOSIDOSIS is an inherited lysosomal storage disease which is due to a deficiency of the acid hydrolase GM1-β-galactosidase1. During the past few years several clinical variants have been described2–6 that differ in time of onset of symptoms, involvement of visceral organs or skeletal tissue and in the degree of neuronal and mental deterioration. Some of these variants have been related to different properties of the deficient β-galactosidases5,7 but the significance of the experimental data8 has been questioned. Several investigators have speculated on the genetic background of the different variants2,8,9 but no experimental evidence has been provided to support the hypotheses.

A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease type A (MPS IIIA)

Summary4-Methylumbelliferyl-α-N-acetylglucosamine 6-sulphate was synthesized and shown to be a substrate for the lysosomalN-acetylglucosamine-6-sulphate sulphatase (GlcNAc-6S sulphatase). Fibroblasts and leukocytes from 3 different Sanfilippo D patients showed <1% of mean normal GlcNAc-6S sulphatase activity. The enzymatic liberation of the fluorochrome from 4-methyl-umbelliferyl-α-N-acetylglucosamine 6-sulphate requires the sequential action of the GlcNAc-6S sulphatase and α-N-acetylglucosaminidase. A normal level of α-N-acetylglucosaminidase activity was insufficient to complete the hydrolysis of the reaction intermediate 4-methylumbelliferyl-α-N-acetylgluco-saminide formed by the GlcNAc-6S sulphatase. A second incubation in the presence of excess α-N-acetyglucosaminidase is needed to avoid underestimation of the GlcNAc-6S sulphatase activity.

Somatic cell hybridisation studies showing different gene mutations in Niemann-Pick variants

SummaryCultured skin fibroblasts from patients with different clinical types of Niemann-Pick disease were hybridized and sphingomyelinase activities were measured in the heterokaryon cell population. Both the natural substrate (3H-choline) sphingomyelin and the chromogenic analogue hexadecanoylamino-4-nitrophenylphosphorylcholine were used in the complementation analysis. In fusions between cells from type C Niemann-Pick disease with those from type A or B a clear restoration of sphingomyelinase activity occurred, whereas no complementation was found in other fusion combinations. The results indicate that at least two different genes are involved in the mutations leading to the different Niemann-Pick variants.

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

SummaryImmunocytochemistry 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.

A comparative study of the accumulated sialic acid-containing oligosaccharides from cultured human galactosialidosis and sialidosis fibroblasts

Sialic acid-containing storage material was isolated from cultured human galactosialidosis fibroblasts, by a combination of gel filtration and anion-exchange chromatography on Mono Q. The obtained sialyloligosaccharides were analyzed by 500-MHz 1H-NMR spectroscopy in combination with sugar analysis and analytical HPLC. The storage material consisted of a series of completely sialylated N-acetyl-lactosamine type of structures having Man beta 1-4GlcNAc at the reducing terminus in common, similar to those recently reported for human sialidosis fibroblasts. Comparison of the storage material from both sources revealed only differences in their relative amounts. In control fibroblasts these compounds could not be detected. The nature of the accumulated compounds is in accordance with the alpha-neuraminidase deficiency in both genetic diseases. The additional deficiency of beta-galactosidase in case of galactosialidosis is not reflected in the storage material.

Complementation studies with enucleated fibroblasts from different variants of β-galactosidase deficiency

Abstract Somatic cell hybridization of β-galactosidase fibroblasts derived from patients with the infantile type 1 and the adult type 4 G M1 -gangliosidosis results in restoration of the β-galactosidase activity. The kinetic properties of the enzyme activity in heterokaryons were found to be similar as in controls. Genetic complementation did not occur after inhibition of protein synthesis by cycloheximide indicating the necessity of de novo protein synthesis. When fibroblasts of the adult type 4 patient were enucleated and fused with cells from an infantile type 1 variant no restoration of β-galactosidase in the hybrids was observed. Fusion of enucleated type 1 cells with nucleated type 4 cells, however, did result in genetic complementation. The results obtained in this study and observations by others fit with the hypothesis of intergenic complementation. In heterokaryons the adult type 4 genome codes for normal β-galactosidase monomers and the infantile type 1 cells or cytoplasts provide a protein factor necessary for the hydrolytic activity and aggregation of the molecule.

The presence of a reduced amount of 32-kd “protective” protein is a distinct biochemical finding in late infantile galactosialidosis

SummaryThe biochemical defect underlying the late infantile form of galactosialidosis has been investigated in fibroblasts from two patients presenting with this phenotype. Immunoprecipitation experiments demonstrated that a reduced amount of 32-kd “protective” protein and a normal amount of its precursor are present in late infantile galactosialidosis fibroblasts, while neither of the two polypeptides are detectable in early infantile and juvenile/adult fibroblasts. Leupeptin treatment led to a slight increase in the amount of 54-kd and 32-kd polypeptides in both late-infantile galactosialidosis cell lines. Uptake studies in one of the two cell lines confirmed the hypothesis that a block in the maturation of the protective protein is responsible for the late infantile type of galactosialidosis. This mutation seems to be a distinct finding in all patients affected by this form of the disease.

A two-year-old patient with an atypical expression of GM1-β-galactosidase deficiency: Biochemical, immunological, and cell genetic studies

SummaryCultured skin fibroblasts from a 2-year-old boy with an atypical form of β-galactosidase deficiency have been studied. With the artificial substrate 4-methylumbelliferyl-β-D-galactopyranoside, 5–15% residual activity was found in fibroblasts from this patient. Most of this activity was in the monomeric A form of the enzyme, very little in the multimeric B form. Km value, pH profile, and heat lability of the mutant enzyme were similar to those of β-galactosidase from control fibroblasts. Immunological studies showed that the mutant enzyme cross-reacted with an antiserum raised against human liver β-galactosidase, but the catalytic activity per unit antigenic activity was lower than normal. It was demonstrated by somatic cell hybridization that the gene mutation in this patient is different from that in patients with type 1 or type 2 GM1-gangliosidosis. No genetic complementation was found after fusion of fibroblasts from this patient with those from two other clinical variants of GM1-gangliosidosis formerly designated type 3 and adult type 4.

A quantitative immunoelectronmicroscopic study on soluble, membrane-associated and membrane-bound lysosomal enzymes in human intestinal epithelial cells

SummaryWe have used quantitative immunoelectronmicroscopy to compare thein situ localization of acid α-glucosidase, lysosomal acid phosphatase, β-hexosaminidase and glucocerebrosidase in intestinal epithelial cells of the human duodenum. Differences between these four lysosomal enzymes were observed with respect to their presence at the apical cell surface. Transport to the apical membrane seems to be a more important intracellular route for lysosomal acid phosphatase and acid α-glucosidase than it is for β-hexosaminidase. The membrane associated lysosomal enzyme glucocerebrosidase is not transported to the microvilli. The studies emphasize that lysosomal enzyme transport pathways are enzyme and cell type specific.