Biomedical subjects

M Pasmanik-Chor

Publications and source records attributed to M Pasmanik-Chor.

7 recordsLinked to original sources

PubMed1999

EHD1--an EH-domain-containing protein with a specific expression pattern.

A cDNA that is a member of the eps15 homology (EH)-domain-containing family and is expressed differentially in testis was isolated from mouse and human. The corresponding genes map to the centromeric region of mouse chromosome 19 and to the region of conserved synteny on human chromosome 11q13. Northern analysis revealed two RNA species in mouse. In addition to the high levels in testis, expression was noted in kidney, heart, intestine, and brain. In human, three RNA species were evident. The smaller one was predominant in testis, while the largest species was evident in other tissues as well. The predicted protein sequence has an EH domain at its C-terminus, including an EF, a Ca2+ binding motif, and a central coiled-coil structure, as well as a nucleotide binding consensus site at its N-terminus. As such, it is a member of the EH-domain-containing protein family and was designated EHD1 (EH domain-containing 1). In cells in tissue culture, we localized EHD1 as a green fluorescent protein fusion protein, in transferrin-containing, endocytic vesicles. Immunostaining of different adult mouse organs revealed major expression of EHD1 in germ cells in meiosis, in the testes, in adipocytes, and in specific retinal layers. Results of in situ hybridization to whole embryos and immunohistochemical analyses indicated that EHD1 expression was already noted at day 9.5 in the limb buds and pharyngeal arches and at day 10.5 in sclerotomes, at various elements of the branchial apparatus (mandible and hyoid), and in the occipital region. At day 15.5 EHD1 expression peaked in cartilage, preceding hypertrophy and ossification, and at day 17.5 there was no expression in the bones. The EHD1 gene is highly conserved between nematode, Drosophila, mouse, and human. Its predicted protein structure and cellular localization point to the possibility that EHD1 participates in ligand-induced endocytosis.

Amino Acid Sequence↗

PubMed1999

Importance of splicing for prosaposin sorting.

The prosaposin gene encodes a 70 kDa protein. This protein might either reach the lysosomes and get processed there to four peptides, which are activators of known lysosomal enzymes, or be secreted by cells as a 70 kDa protein, recently anticipated to have several biological activities. The human prosaposin gene has a 9 bp exon (exon 8) that is alternatively spliced, thus encoding three prosaposin forms: one with an extra three amino acid residues, one with an extra two residues and a third form with no extra residues. With the aim of testing whether there is an association between the alternative splicing and the differential sorting of prosaposins, we cloned two human prosaposin cDNA forms in a T7/EMC/vaccinia virus-derived vector and expressed them in human cells. The results indicated that the prosaposin containing the three extra residues accumulated faster and in greater amounts in the medium, whereas the prosaposin with no extra residues was mainly destined for lysosomes. Point mutations created by mutagenesis in vitro in the 9 bp stretch had a diverse effect on prosaposin secretion. When supplied to cells in the medium, both prosaposins were endocytosed and reached the lysosomes, where they were processed to active saposin B and saposin C. The activities of the saposins were monitored qualitatively and quantitatively. Quantitatively, lipids were extracted from the cells, separated on TLC and measured fluorimetrically. Qualitatively, cells were detected by fluorescence microscopy.

Alternative Splicing↗

PubMed1999

Intracellular degradation of fluorescent glycolipids by lysosomal enzymes and their activators.

Fluorescent glycolipids were utilized for detection of the intracellular, activator-dependent, activities of beta-glucocerebrosidase and arylsulphatase A. Activities were measured in primary skin fibroblasts from normal individuals, from patients with Gaucher disease who had mutations within the beta-glucocerebrosidase gene, and from a prosaposin-deficient patient. Fluorescent microscopy demonstrated that glucosylceramide or sulphatide labelled with a fluorescent probe (lissamine-rhodamine) were endocytosed and reached the lysosomes. There, in the presence of active enzyme and the corresponding saposin, they were hydrolysed to fluorescent ceramide, which changed its intracellular localization. When these substrates were labelled with pH-sensitive lissamine-rhodamine, which loses its fluorescence at neutral or alkaline pH, the transport of the product, i.e. fluorescent ceramide, from the lysosomes resulted in disappearance of the cellular fluorescence. In cells of patients having mutations within the genes encoding the glucocerebrosidase or the prosaposin, there was a considerable reduction in the intracellular rate of substrate hydrolysis that could be followed by fluorescence microscopy or measured quantitatively in cell extracts.

Cerebroside-Sulfatase↗

PubMed1998

Prevalence of glucocerebrosidase mutations in the Israeli Ashkenazi Jewish population.

Gaucher disease is the most prevalent inherited disease among Ashkenazi Jews. It is very heterogeneous due to a large number of mutations within the glucocerebrosidase gene, whose impaired activity is the cause for this disease. Aiming at determining Gaucher carrier frequency among the Ashkenazi Jewish population in Israel, 1,208 individuals were molecularly diagnosed for six mutations known to occur among Ashkenazi Jewish Gaucher patients, using the newly developed Pronto Gaucher kit. The following mutations were tested: N370S, 84GG, IVS2+1, D409H, L444P, and V394L. Molecular testing of these mutations also allows identification of the recTL allele. The results indicated that Gaucher carrier frequency is 1:17 within the tested population. The prevalence of N370S carriers is 1:17.5. This implies that approximately 1:1225 Ashkenazi Jews will be homozygous for the N370S mutation. Actually, in our study of 1,208 individuals one was found to be homozygous for the N370S mutation. The actual number of known Ashkenazi Jewish Gaucher patients with this genotype is much lower than that expected according to the frequency of the N370S mutation, suggesting a low penetrance of this mutation. Results of loading experiments in cells homozygous for the N370S mutation, as well as cells homozygous for the L444P and the D409H mutations, exemplified this phenomenon.

Cells, Cultured↗

PubMed1997

Expression of mutated glucocerebrosidase alleles in human cells.

Gaucher disease is a heterogeneous disease characterized by impaired activity of the lysosomal enzyme glucocerebrosidase. This heterogeneity is attributed to a large number of mutations in the corresponding gene. In order to test the biochemical properties of some mutations prevalent among Israeli populations, the normal human glucocerebrosidase cDNA and cDNAs carrying mutations N370S, L444P, D409H, recTL, recNcil, P415R and 84GG were coupled to the T7 RNA polymerase promoter in a vaccinia virus-derived expression vector (pTM-1). Recombinant viruses were produced and used to infect human tissue culture cells. RNA and protein stability, recognition by anti-glucocerebrosidase monoclonal antibodies and intracellular enzymatic activity were measured. The results demonstrated that the D409H allele directed synthesis of cytoplasmic RNA with decreased stability compared with its normal counterpart or other mutated forms. The D409H and L444P mutated proteins had lower stability than that of their normal counterpart, while the recNcil-mutated protein was more stable. Only glucocerebrosidase forms harboring leucine at position 444 were recognized by the anti-glucocerebrosidase monoclonal antibodies used (8E4 and 2C7). Measurements of enzymatic activity of the recombinant proteins in cells loaded with a fluorescent glucosylceramide demonstrated that the N370S mutated enzyme had activity similar to that of the normal enzyme. The other mutated enzymes exhibited varying degrees of activities, generally corresponding to the phenotypes with which they are associated. The results presented demonstrate the use of the vaccinia virus-derived expression system and of loading living cells with fluorescent substrate as efficient tools for studying mutants in Gaucher disease and in other lysosomal diseases.

Alleles↗

PubMed1996

Overexpression of human glucocerebrosidase containing different-sized leaders.

Gaucher disease results from impaired activity of the lysosomal enzyme glucocerebrosidase. Aiming at overexpressing the human glucocerebrosidase and testing the efficiency of the two in-frame ATGs of its gene in directing synthesis of an active enzyme, it was coupled to the T7 RNA polymerase promoter in a vaccinia virus-derived expression vector (pTM-1). cDNAs containing either one or both ATGs of the glucocerebrosidase mRNA were linked to the T7 polymerase promoter. Recombinant viruses were produced and used for infecting human cells in tissue culture. The results demonstrated that both ATGs directed translation of active glucocerebrosidase, resulting in a 10-fold increase in enzymic activity. Most of the protein remained sensitive to endoglycosidase H. The active enzyme represented a small fraction of the expressed glucocerebrosidase. The recombinant enzyme had the same Km and optimal pH towards the artificial substrate 4-methylumbelliferyl glucopyranoside as the authentic endogenous human enzyme. Measurements of intracellular enzymic activity directed by the cDNAs with either one or both ATGs in cells loaded with a fluorescent glucosylceramide demonstrated a 30% increase in activity directed by the cDNAs containing the first ATG over that containing the second ATG. This indicates that the protein synthesized from the first ATG, with a 38 amino acid leader, is translocated through the endoplasmic reticulum more readily than its counterpart directed by the second ATG, with a 19 amino acid leader. The elevation in glucocerebrosidase activity and the reproducibility of the data leads us to propose the use of the vaccinia virus-derived expression system as a tool for studying glucocerebrosidase mutants in Gaucher disease.

Bacteriophage T7↗

PubMed1996

The glucocerebrosidase D409H mutation in Gaucher disease.

Gaucher disease, resulting from the decreased activity of the lysosomal enzyme glucocerebrosidase, is the most prevalent sphingolipid storage disease. Due to considerable heterogeneity of phenotypic expression, it has been subdivided into the nonneurological type 1 disease, and types 2 and 3, the neurological types. We describe homozygosity for the D409H mutation within the glucocerebrosidase gene associated with a unique form of type 3 Gaucher disease. Twelve patients, originating from three Arab sibships, were found to be homozygous for the D409H mutation. They all presented with oculomotor apraxia and a progressive cardiac valve defect with minimal organomegaly. When expressed in human cells in tissue culture, using the T7/EMC/vaccinia virus hybrid expression system, we were able to demonstrate that the mRNA carrying the D409H mutation was less stable than the normal counterpart. Pulse-chase experiments demonstrated that the mutated protein exhibited lower stability than the normal counterpart. Its activity toward the artificial substrate 4-methyl umbelliferyl glucopyranoside was similar to that of the mutated enzymes carrying the N370S or the L444P mutations. However, in loading experiments using lissamine-rhodamine conjugated glucosyl ceramide as a substrate, the recombinant mutated protein carrying the D409H mutation exhibited 28.63 +/- 6.05% of the activity exhibited by the normal enzyme. L444P and N370S mutations exhibited 51.90 +/- 7.16 and 115.75 +/- 12.64% of normal enzyme activity, respectively. Loading of cells homozygous for the D409H mutation demonstrated 10. 05% of the activity shown by normal cells. L444P and N370S homozygous cells demonstrated 25.3 and 98.5% of foreskin fibroblast glucocerebrosidase activity, respectively. We demonstrate that homozygosity for the D409H mutation is a unique case of a peculiar phenotype associated with a specific intracellular glucocerebrosidase activity.

DNA, Complementary↗