Human GM1-gangliosidosis is caused by a genetic deficiency of lysosomal acid beta-galactosidase (beta-gal). The disease manifests itself either as an infantile, juvenile or adult form and is primarily a neurological disorder with progressive brain dysfunction. A mouse model lacking a functional beta-gal gene has been generated by homologous recombination and embryonic stem cell technology. Tissues from affected mice are devoid of beta-gal mRNA and totally deficient in GM1-ganglioside-hydrolyzing capacity. Storage material was already conspicuous in the brain at 3 weeks. By 5 weeks, extensive storage of periodic acid Schiff-positive material was observed in neurons throughout the brain and spinal cord. Consistent with the neuropathology, abnormal accumulation of GM1-ganglioside in the brain progressed from twice to almost five times the normal amount during the period from 3 weeks to 3.5 months. Despite the accumulation of brain GM1-ganglioside at the level equal to or exceeding that seen in gravely ill human patients, these mice show no overt clinical phenotype up to 4-5 months. However, tremor, ataxia and abnormal gait become apparent in older mice. Thus, the beta-gal-deficient mice appear to mimic closely the pathological, biochemical and clinical abnormalities of the human disease.

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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.

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.

The Gr,,r activator protein is a glycolipid-binding protein required for the lysosomal degradation of ganglioside Gi,,r. A human fibroblast cDNA library was screened with mixtures of oligonucleotide probes corresponding to four different areas of the amino acid sequence. A putative clone (821 bp) which gave positive signals to all four probe mixtures was purified and sequenced. The sequence was colinear with the sequence of 160 amino acids of the mature GMZ activator protein. Availability of the cDNA clone should facilitate investigation into function of the GM* activator protein and also into genetic abnormalities underlying GM2 gangliosidosis AB variant.GM2 activator protein; GM2 gangliosidosis; cDNA sequence

A 6-month-old female emu (Dromaius novaehollandiae) died following acute central nervous system signs. Hematoxylin-and-eosin-stained sections revealed that neurons of the brain were distended with nonstaining 1-to-2-microns vacuoles. Ultrastructural examination of the affected neurons revealed numerous membranous cytoplasmic bodies (MCBs) similar in appearance to the MCBs seen in mammalian gangliosidoses. A full sibling of this emu was donated for study. This 7-month-old female emu was stunted compared with hatchmates. Neurologic examination revealed hypermetric gait, persistent head tremor, and mild ataxia. No gross lesions were evident at postmortem. Histopathologic and electron microscopic findings were similar to those in the index case in that swollen, pale neurons were present in the cerebrum, pons, medulla, cerebellum, spinal cord, spinal ganglia, autonomic ganglia, myenteric plexus, and ganglion cell layer of the retina. Analysis of brain gangliosides of the affected 7-month-old emu revealed 14- and 25-fold increases of GM1 and GM3 gangliosides, respectively, compared with control emus. The total brain ganglioside sialic acids were, on a wet weight basis, 519 micrograms/g (control A), 658 micrograms/g (control B), and 1800 micrograms/g (affected emu). The familial association seen with this condition suggests that emus are affected by an inherited disorder similar to mammalian gangliosidoses.

Full-length cDNAs coding for the human GMZ-activator protein has been isolated and characterized. and its genomic structure studied in two overlapping clones in ;1-EMBL-4 isolated from a human brain genomic library. Two different cDNAs were found that were identical to the 5'.terminus to nt 13 I I (counted from the A of the initiation codon. ATG) including the entire Protein coding scqueuce. 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 Y-portion of the other cDNA clone is not clear at this time.GMZ-activator protein: Genomic structure; Glycolipid-binding protein

Ciliary neurotrophic factor (CNTF) promotes survival in vitro and in vivo of several neuronal cell types including sensory and motor neurons. The primary structure of CNTF suggests it to be a cytosolic protein with strong similarity to the alpha-helical cytokine family which is characterized by a bundle of four anti-parallel helices. CNTF exerts its activity via complexation with CNTF receptor (CNTF-R). This complex consists of a CNTF-binding protein (CNTF-R) and two proteins important for signal transduction [gp130 and leukaemia inhibitory factor receptor (LIF-R)]. We have shortened the cDNA coding for CNTF at both the 5' and the 3' end and expressed the truncated proteins in bacteria. Biological activities of the protein preparations were determined by their ability to induce proliferation of BAF/3 cells that were stably transfected with CNTF-R, gp130 and LIF-R cDNAs. CNTF proteins with 14 amino acid residues removed from the N-terminus were biologically active whereas the removal of 23 amino acids resulted in an inactive protein. In addition, 18 amino acid residues could be removed from the C-terminus of the CNTF protein without apparent loss of bioactivity, but further truncation at the C-terminus yielded biologically inactive proteins. The introduction of two point mutations into the CNTF protein at a site that presumably interacts with one of the two signal-transducing proteins resulted in a CNTF mutant with no measurable bioactivity. In addition, a model of the three-dimensional structure of human CNTF was constructed using the recently established structural co-ordinates of the related cytokine, granulocyte colony-stimulating factor. CD spectra of CNTF together with our mutational analysis and our three-dimensional model fully support the view that CNTF belongs to the family of alpha-helical cytokines. It is expected that our results will facilitate the rational design of CNTF mutants with agonistic or antagonistic properties.