André T. Hoogeveen

OLIGOMERIZATION PROPERTIES OF FRAGILE-X MENTAL-RETARDATION PROTEIN (FMRP) AND THE FRAGILE-X-RELATED PROTEINS FXR1P AND FXR2P

The absence of fragile-X mental-retardation protein (FMRP) results in fragile-X syndrome. Two other fragile-X-related (FXR) proteins have been described, FXR1P and FXR2P, which are both very similar in amino acid sequence to FMRP. Interaction between the three proteins as well as with themselves has been demonstrated. The FXR proteins are believed to play a role in RNA metabolism. To characterize a possible functional role of the interacting proteins the complex formation of the FXR proteins was studied in mammalian cells. Double immunofluorescence analysis in COS cells over-expressing either FMRP ISO12/FXR1P or FMRP ISO12/FXR2P confirmed heterotypic interactions. However, Western-blotting studies on cellular homogenates containing physiological amounts of the three proteins gave different indications. Gel-filtration experiments under physiological as well as EDTA conditions showed that the FXR proteins were in complexes of >600 kDa, as parts of messenger ribonuclear protein (mRNP) particles associated with polyribosomes. Salt treatment shifted FMRP, FXR1P and FXR2P into distinct intermediate complexes, with molecular masses between 200 and 300 kDa. Immunoprecipitations of FMRP as well as FXR1P from the dissociated complexes revealed that the vast majority of the FXR proteins do not form heteromeric complexes. Further analysis by [(35)S]methionine labelling in vivo followed by immunoprecipitation indicated that no proteins other than the FXR proteins were present in these complexes. These results suggest that the FXR proteins form homo-multimers preferentially under physiological conditions in mammalian cells, and might participate in mRNP particles with separate functions.

Tay-Sachs and Sandhoff's disease: Intergenic complementation after somatic cell hybridization

Abstract Cultivated skin fibroblasts from patients with Tay-Sachs and Sandhoffs disease were fused and the isoenzymes of N-acetyl-β- d -hexosaminidase were investigated after 2 and 7 days of subsequent cultivation. Enzyme analyses after heat inactivation showed a clear increase in the thermolabile component of hexosaminidase when compared with assays on fusion of each of the parental cell strains. Electrophoretic studies revealed that in cell homogenates prepared at various time intervals after cell fusion of Tay-Sachs with Sandhoffs fibroblasts, all three hexosaminidase isoenzymes were present, including hexosaminidase A which lacks in both parental cell strains. These results show that genetic complementation has occurred, which indicates that two different gene mutations are involved in these variants of GM2-gangliosidosis. The relevance of the data obtained for the elucidation of the molecular properties of the (iso)enzymes involved is discussed.

Immortalization of nasal polyp epithelial cells from cystic fibrosis patients

We have developed immortalized epithelial cystic fibrosis (CF) cell lines by infecting cultured nasal polyp cells with a SV40/Adenol2 hybrid virus. The cell lines obtained are epithelial in nature as shown by cytokeratin production and morphology, although cytokeratins 4 and 13 typical of primary nasal polyp cells are produced at a much reduced rate. Ussing chamber experiments showed that the precrisis CF cell line NCF3 was able to perform trans-cellular chloride transport when activated by agents which elevate intracellular calcium. cAMP agonists had no effect on chloride flux in NCF3 as expected for CF cells. The apical chloride channels found with the patch clamp technique in NCF3 and in the postcrisis cell line NCF3A have a conductance similar to that of chloride channels found earlier in normal and CF epithelial cells. The channels show a delay in the onset of activity in off-cell patches and are not activated by increased cAMP levels in the cell. This indicates that immortalized CF epithelial cells will provide a useful model for the study of cystic fibrosis.

Prenatal diagnosis of sialidosis with combined neuraminidase and ø-galactosidase deficiency

Recently a combined deficiency of neuraminidase and P-galactosidase was reported in two patients (Wenger et al. 1978, O’Brien 1978), who had earlier been described as having variant types of Gq-gangliosidosis (Justice et al. 1977, Koster et al. 1976). We have found similar deficiencies in the second of two children who both died at birth with severe hydrops fetalis (Niermeijer et al. unpublished). The parents are consanguineous, of Turkish origin, and living in The Netherlands. Cultured skin fibroblasts and liver, obtained at autopsy, showed a deficiency of p-galactosidase with a residual activity of 5-10 % of normal. In addition, we found an almost complete deficiency of neuraminidase activity in the fibroblasts and an increased level of sialic acid in both fibroblasts and liver (3 and 23 times the normal levels, respectively). We now wish to report the results of the prenatal diagnosis which was offered to the parents during the third pregnancy. Amniocentesis was done in the 17th week (Dr. R. Vosters, University Hospital, Rotterdam). After 18 days of growth, the amniotic fluid cells were harvested in their first passage for enzyme assays. P-galactosidase and neuraminidase activities were determined using the fluorogenic 4-methyl-umbelliferyl substrates and micromethods, as described earlier for p-galactosidase in the prenatal analysis for GM1-gangliosidosis (Kleijer et al. 1976). The results are shown in Table 1. Both B-galactosidase and neuraminidase activity are strongly reduced in the amniotic fluid cells from the pregnancy at risk, as was the case in fibroblasts of the index patient. These results indicated that the fetus was affected with the variant type of sialidosis. Further evidence was obtained 1 week later by the demonstration of a 2-fold elevated sialic acid content of the amniotic fluid cells (44.3 nmol/mg protein, control: 22.1 nmoYmg). The parents decided on termination of the pregnancy, which was done by prostaglandin induction in the 21st week of pregnancy. The prenatal diagnosis was confirmed by the demonstration of reduced activities in the freshly obtained fetal liver and brain for P-galactosidase (5-10 % of normal activity) and neuraminidase (undetectable). The sialic acid content was 20-fold

Enhanced proteolytic degradation of normal β-galactosidase in the lysosomal storage disease with combined β-galactosidase and neuraminidase deficiency

Abstract The endocytosis of β-galactosidase purified from bovine and monkey testis was studied in cultured fibroblasts from patients with G M1 -gangliosidosis, mucolipidosis I and combined β-galactosidase and neuraminidase deficiency (β-gal − /neur − ). The uptake of 3 H-labelled β-galactosidase was of the same order of magnitude in all cell strains. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate of cell free extracts after endocytosis indicated that in β-gal − /neur − cells the majority of ingested enzyme was degraded within 4 h, whereas the molecular weight remained unchanged in the other cell strains for at least 12 h. The intracellular accumulation of β-galactosidase activity in β-gal − /neur − cells levels off rapidly and the ultimate activity remains very low. In contrast, the β-galactosidase activity ingested by other cell strains increased almost linearly in time, and eventually exceeded the endogenous β-galactosidase activity in normal cells. Chase experiments indicated that the ingested β-galactosidase activity was short-lived in β-gal − /neur − cells and long-lived in the other cell strains. The enhanced degradation of ingested normal β-galactosidase in β-gal − /neur − fibroblasts could be prevented by addition of leupeptin, which inhibits the action of thiol-cathepsins. We conclude from these various experiments that the basic defect in combined β-galactosidase and neuraminidase deficiency results in an inadequate protection of both endogenous and exogenous β-galactosidase towards intralysosomal proteolytic degradation. The relationship to neuraminidase deficiency is discussed.

The transcriptional corepressor MTG16a contains a novel nucleolar targeting sequence deranged in t (16; 21)-positive myeloid malignancies.

The MTG (Myeloid Translocation Gene) proteins are a family of novel transcriptional corepressors. We report that MTG16a, a protein isoform encoded by the MTG16 gene deranged by the t (16; 21) in myeloid malignancies, is targeted to the nucleolus. The amino acid sequence necessary for nucleolar localization was mapped to the MTG16a N-terminal region. MTG16a, like MTG8, the nuclear corepressor deranged by the t (8; 21), is capable to interact with specific histone deacetylases (HDACs) suggesting that the protein may mediate silencing of nucleolar gene transcription. In addition, MTG16a is capable to form oligomers with other MTG proteins. As a consequence of the t (16; 21) the AML1 DNA-binding domain replaces the MTG16a N-terminal region. The AML1-MTG16 fusion protein is targeted to the nucleoplasm where it is capable to oligomerize with MTG16a and interact with HDAC1 and HDAC3. The deficiency of HDAC-containing complexes at nucleolar sites and the accumulation of HDAC-containing complexes at AML1-sites may be critical in the pathogenesis of t (16; 21) myeloid malignancies.

Correction of combined β-galactosidase/neuraminidase deficiency in human fibroblasts

The combined deficiency of β-galactosidase and neuraminidase in human fibroblasts can be corrected to nearly normal values. This can be accomplished by addition of concentrated culture medium obtained after NH4Cl stimulation of different types of human fibroblasts, including those with an isolated β-galactosidase or neuraminidase deficiency. The corrective factor is a macromolecular glycoprotein, which is labile at 60°C. Its uptake by human fibroblasts is competitively inhibited by mannose-6-phosphate and its corrective action within β-gal−/neur− fibroblasts continues during a “chase” of 72 hours.

Immunocytochemical characterization of FMRP, FXR1P and FXR2P during embryonic development in the mouse

The absence of the FMR1 (fragile X mental retardation gene 1) gene product, protein FMRP (fragile X mental retardation protein) is causing the fragile X syndrome. FMRP, together with two homologues, called FXR1P and FXR2P, belongs to a small family of RNA-binding proteins (FXR proteins). The precise physiological function of the FXR proteins is unknown, but a role in mRNA transport has been suggested. In the present study, we have performed immunolocalization of these proteins during the embryonic development of the mouse to get more insight in their physiological function. All three proteins are expressed during mouse embryonic development, however, the pattern and intensity varies for each protein at the different developmental stages. During early development, the distribution of the Fxr proteins exhibits high similarities, however, during late development and in the neonate a more differential expression is observed especially in some non-neural tissues. The results of this descriptive study are discussed in relation to the pathogenesis of the fragile X syndrome.

Immunological localization of cystic fibrosis candidate gene products

The recent identification of the cystic fibrosis (CF) gene and its putative protein product, the CF transmembrane conductance regulator (CFTR), enabled us to synthesize oligopeptides corresponding with a predicted extracellular domain (position 103-117; peptide A) and a cytoplasmic domain (position 501-515; peptide B) constituting the phenylalanine deletion (F 508) observed in the majority of CF mutations. Immunobiochemical studies with antibodies directed against these peptides revealed the presence of two CFTR candidate proteins (155 and 195 kDa) in various types of epithelial cells. Immunolocalization studies performed on slices of human duodenum showed the strongest expression in the endoplasmic reticulum (RER) of the mucus-producing Goblet cells. Labeling is also demonstrated in the RER and apical membranes of villus and crypt cells, however, to a weaker extent.

FMR1 Gene Expression in Olfactory Neuroblasts From Two Males With Fragile X Syndrome

The fragile X mental retardation 1 gene (FMR1) mutation is strongly correlated with specific and marked neurobehavioral and neuroanatomical abnormalities. The protein product, FMRP, is highly expressed in neurons of the normal mammalian brain, and absent or in low levels in leukocytes from individuals with fragile X (FraX)-associated mental impairment. Inferences which arise from these findings are that FMRP has a critical role in the development and functioning of the brain, and that leukocyte-derived molecular assessments provide a good indicator of FMR1 expression in that organ. This latter conclusion appears true in most cases even though the typical FMR1 mutation is an unstable triplet repeat expansion which demonstrates somatic heterogeneity within and across tissues. Blood to brain correspondence in FraX patients has only rarely been confirmed by the direct study of human brain specimens and, to our knowledge, it has never been studied in living individuals with the FMR1 mutation. In this report, we describe the FMR1 patterns in olfactory neuroblasts (ON) from two living brothers with expansion mutations in their leukocytes who are mentally retarded and autistic. ON were chosen for study because they are accessible neurons closely linked to the brain. In both subjects, the ON genotype was highly, but not perfectly, consistent with that observed in leukocytes. Protein phenotypes across tissues were completely consistent showing the absence of FMRP-immunoreactivity (-ir). These results augment the limited amount of direct evidence which indicates that FMR1 mutation patterns in leukocytes are a good, albeit potentially fallible, reflection of such patterns in the brain. This report further demonstrates the feasibility of using ON samples to evaluate the FMR1 mutation in humans in vivo.