Doris Schnabel

Mutation in the sphingolipid activator protein 2 in a patient with a variant of Gaucher disease

The lysosomal degradation of glucosylceramide requires the hydrolase, glucosylceramide‐β‐glucosidase and a sphingolipid activator protein (Gaucher factor, SAP‐2, saposin C). Genetic defects in either of these lysosomal proteins cause phenotypically similar disorders in man, the Gaucher disease. SAP‐2 originates from a gene which generates a mRNA that codes for four homologous proteins. In a patient with an immunologically proven SAP‐2 deficiency a G1154 → T transversion (counted from A of the initiation codon ATG) was found in the mRNA of the SAP‐2 precursor which results in the substitution of Phe for Cys385 in the mature SAP‐2. The rest of the coding sequence remained entirely normal.

Osteoblast-Derived Factors Induce Androgen-Independent Proliferation and Expression of Prostate-Specific Antigen in Human Prostate Cancer Cells

Purpose: Prostate cancer metastasizes to the skeleton to form osteoblastic lesions. Androgen ablation is the current treatment for metastatic prostate cancer. This therapy is palliative, and the disease will return in an androgen-independent form that is preceded by a rising titer of prostate-specific antigen (PSA). Here, we investigated the possibility that human osteoblasts might secrete factors that contribute to the emergence of androgen-independent prostate cancer. Experimental Design: Primary cultures of human osteoblasts were used as a source of conditioned medium (OCM). Proliferation, expression of androgen-regulated genes, and transactivation of the androgen receptor (AR) were monitored in LNCaP human prostate cancer cells in response to OCM using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Northern blot analysis, and reporter gene constructs. Levels of interleukin-6 (IL-6) present in OCM were measured, and its contribution to proliferation and expression of PSA were investigated by neutralization studies with anti IL-6 antibodies. Results: OCM increased the proliferation and expression of PSA at both the protein and RNA levels in LNCaP cells. Synergistic increases in the activities of PSA (6.1 kb)- and pARR3-tk-luciferase reporters were measured in cells cotreated with both OCM and androgen. OCM targeted the NH2-terminal domain of the AR. The effect of OCM on transcriptional activity of the AR was inhibited by an antiandrogen. Neutralizing antibodies to IL-6 blocked proliferation and expression of PSA by OCM. Conclusion: Osteoblasts secrete factors, such as IL-6, that cause androgen-independent induction of PSA gene expression and proliferation of prostate cancer cells by a mechanism that partially relies on the AR. Identifying such molecular mechanisms may lead to improved clinical management of metastatic prostate cancer.

A mutation in the gene of a glycolipid-binding protein (GM2 activator) that causes GM2-gangliosidosis variant AB

GM2‐gangliosidoses are neurological disorders caused by a genetic deficiency of either the β‐hexosaminidase A or the GM2 activator, a glycolipid binding protein. In a patient with an immunologically proven GM2 activator protein deficiency, A T412 → C transition (counted from A of the initiation codon) was found in the coding sequence, which results in the substitution of Arg for the normal Cys107 in the mature GM2 activator protein. The remainder of the coding sequence remained entirely normal.

Molecular genetics of GM2-gangliosidosis AB variant: a novel mutation and expression in BHK cells

The GM2 activator is a hexosaminidase A-specific glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. Genetic deficiency of GM2 activator leads to a neurological disorder, an atypical form of Tay-Sachs disease (GM2 gangliosidosis variant AB). Here, we describe a G506 to C transversion (Arg169 to Pro) in the mRNA of an infantile patient suffering from GM2-gangliosidosis variant AB. Using the polymerase chain reaction amplification and direct-sequencing technique, we found the patient to be homozygous for the mutation, whereas the parents were, as expected, heterozygous. BHK cells transfected with a construct of mutant cDNA gave no GM2 activator protein detectable by the Western blotting technique, whereas those transfected by a wild-type cDNA construct showed a significant level of human GM2 activator protein. The substitution of proline for the normal Arg169 therefore appears to result in premature degradation of the mutant GM2 activator, either during the post-translational processing steps or after reaching the lysosome. The basis for the phenotype of GM2 gangliosidosis variant AB may therefore be either inactivation of the physiological activator function by the point mutation or instability of the mutant protein.

Characterization of full-length cDNAs and the gene coding for the human GM2 activator protein

Full‐length cDNAs coding for the human GM2‐activator protein has been isolated and characterized, and its genomic structure studied in two overlapping clones in λ‐EMBL‐4 isolated from a human brain genomic library. Two different cDNAs were found that were identical to the 5′‐terminus to nt 1311 (counted from the A of the initiation codon, ATG) including the entire protein coding sequence. However, they were entirely dissimilar in the 3′‐non‐coding sequences. The genomic clones covered 94% of the full‐length cDNA sequence. Three introns were found. The last exon spans contiguously the carboxyl terminus of the protein and the entire 3′‐untranslated region of one of the two cDNAs with different 3′‐ends. The origin of the 3′‐portion of the other cDNA clone is not clear at this time.

The organization of the gene for the human cerebroside sulfate activator protein

The organization of 14 exons covering 97% of the cDNA sequence of human cerebroside sulfate activator protein precursor has been determined from two overlapping EMBL‐4 human genomic clones extending over 17kb. All exons and exon/intron splice junctions and five introns were sequenced. Exon 8 consists of only 9 bp and is involved in alternative splicing which generates three different mRNAs of cerebroside sulfate activator precursor.