Insights into post-translational processing of beta-galactosidase in an animal model resembling late infantile human G-gangliosidosis.

G(M1)-gangliosidosis is a lysosomal storage disorder caused by a deficiency of ss-galactosidase activity. Human GM1-gangliosidosis has been classified into three forms according to the age of clinical onset and specific biochemical parameters. In the present study, a canine model for type II late infantile human GM1-gangliosidosis was investigated 'in vitro' in detail. For a better understanding of the molecular pathogenesis underlying G(M1)-gangliosidosis the study focused on the analysis of the molecular events and subsequent intracellular protein trafficking of beta-galactosidase. In the canine model the genetic defect results in exclusion or inclusion of exon 15 in the mRNA transcripts and to translation of two mutant precursor proteins. Intracellular localization, processing and enzymatic activity of these mutant proteins were investigated. The obtained results suggested that the beta-galactosidase C-terminus encoded by exons 15 and 16 is necessary for correct C-terminal proteolytic processing and enzyme activity but does not affect the correct routing to the lysosomes. Both mutant protein precursors are enzymatically inactive, but are transported to the lysosomes clearly indicating that the amino acid sequences encoded by exons 15 and 16 are necessary for correct folding and association with protective protein/cathepsin A, whereas the routing to the lysosomes is not influenced. Thus, the investigated canine model is an appropriate animal model for the human late infantile form and represents a versatile system to test gene therapeutic approaches for human and canine G(M1)-gangliosidosis.

Confocal laser scanning analysis of an equine oral mast cell tumor with atypical expression of tyrosine kinase receptor C-KIT.

A 20-year-old female horse showed a nodular, firm, focal ulcerated mast cell tumor at the right dorsobuccal face of the tongue. Histologically, the nonencapsulated tumor consisted of dense, infiltrating aggregates of well-differentiated, Cresyl violet-positive mast cells accompanied by numerous eosinophils. Furthermore, they exhibited a strong, diffuse, intracytoplasmatic immunohistochemical signal for tryptase and a faint membrane-associated and perinuclear signal for tyrosine kinase receptor KIT. Confocal laser scanning microscopy confirmed an aberrant spatial colocalization of KIT in the Golgi apparatus, which may be the result of a defective protein processing within the tumor cells. The tumor was not associated with a poor prognosis.

Inhibition of transthyretin-met30 expression using Inosine(15.1)-Hammerhead ribozymes in cell culture.

Hereditary amyloidosis is primarily caused by mutations within the transthyretin gene. More than 75 mutations within transthyretin have been reported in causing amyloidosis. The most common mutation is the val30met mutation in the transthyretin protein (TTR-met30) caused by a mononucleic substitution from G to A (GUC to AUC) in the transthyretin gene resulting in the exchange for the amino acids valine to methionine in the corresponding protein sequence. The aim of this work is the development of a specific cleavage of TTR-met30 mRNA in the cell culture system using hammerhead ribozymes. We showed previously that chemically modified nuclease stable Inosine(15.1)-Hammerhead ribozymes are able to target the TTR-met30 mRNA with high specificity on the RNA level (Biochem. Biophys. Res. Commun. 260, 313-317, 1999). Now we present data confirming our observations on the cellular level. We used the wild-type human normal (hn) TTR expressing cell line HepG2 and the stable transfected cell line 293-TTR-met30 for TTR-met30 experiments. We cleaved the TTR-met30 and hnTTR mRNA with specific nuclease stable chemically modified Inosine(15.1)-Hammerhead ribozymes and analyzed the protein after immunoprecipitation and subsequent Western blotting. We were able to downregulate the TTR concentration by 54.5% (100% = 1.5 mg/l TTR) and also specifically to target the TTR-met30 expression in the cell culture system. The therapeutic effect was improved using cationic liposomes resulting in a total downregulation by 92.1 and 62.7% targeting hnTTR mRNA and TTR-met30 mRNA, respectively. The successful employment of Inosine(15.1)-Hammerhead ribozymes in cell culture is therefore a promising tool for the development of a gene therapeutic strategy for hereditary amyloidosis.

Inosine(15.1) hammerhead ribozymes for targeting the transthyretin-30 mutation.

The most common cause of hereditary amyloidosis (HA) is the val30met mutation in the transthyretin protein (TTR-met30). The mutation is caused by a mononucleic substitution from G to A (GUC to AUC) in the transthyretin gene resulting in the exchange for the amino acids valine to methionine in the corresponding protein sequence. The aim of our work was the development of a specific cleavage of TTR-30 mRNA using hammerhead ribozymes. We chemically modified nuclease stable hammerhead ribozymes to target the TTR-30 mRNA with high specificity. The exchange of adenosine(15.1) with inosine(15.1) in the catalytic core of the hammerhead ribozyme resulted in a change of the cleavable target sequence from N(16.2)U(16.1)H(17) to N(16. 2)C(16.1)H(17) without loss in ribozymal activity (Nucleic Acids Res. 26, 2279-2285, 1998). This modification allowed a specific cleavage of the TTR-30 mutation ("gCC Gug" to "gCC Aug"). In vitro experiments with TTR-30 mRNA demonstrated that the RNase stable inosine(15.1) hammerhead ribozyme cleaved the TTR-30 mRNA with 100% specificity and with a velocity of 0.23 min(-1), whereas no cleavage occured in the wildtype mRNA of TTR. In conclusion, the development of this NCH specific hammerhead ribozyme represents a promising tool for future in vivo therapeutic application for TTR-met30 induced hereditary amyloidosis.

Familial Amyloidotic Polyneuropathy: domino liver transplantation.

BACKGROUND/AIMS: The primary cause of Familial Amyloidotic Polyneuropathy is a variant transthyretin gene on chromosome 18. Progressive polyneuropathy followed by fatal cardiac and renal failure commonly manifest during middle age. Within 10 years after onset of clinical symptoms, affected individuals usually die due to malnutrition or heart failure. Currently, liver transplantation is the only available therapeutic option. METHODS: We performed liver transplantation in two patients with Familial Amyloidotic Polyneuropathy carrying the transthyretin-30 mutant. Two patients aged more than 50 years received the two explanted amyloidotic livers. This procedure is called Domino liver transplantation. We report the outcome in the studied subjects and analyze the metabolic consequences of this procedure. RESULTS: We determined the serum half-life of transthyretin-30 as 2.25 days using daily monitoring of transthyretin-30 levels. An affected amyloidotic patient had an increased serum concentration of lipoprotein(a) of 78 mg/dl before transplantation. The tumor patient, who received the organ from this affected patient, developed an almost identical serum concentration of lipoprotein(a) after liver transplantation, confirming the liver as the primary site of synthesis of this lipoprotein. CONCLUSION: Once Domino liver transplantation has been performed, the impact of the liver-dependent metabolism of specific proteins of interest can be studied.

Apolipoprotein A-I induced amyloidosis.

Amyloidosis is characterized by extracellular deposits of protein fibrils with a high content of beta-sheets in secondary structure. The protein forms together with proteoglycans amyloid fibrils causing organ damage and serious morbidity. Intact apolipoprotein A-I (apoA-I) is an important protein in lipid metabolism regulating the synthesis and catabolism of high density lipoproteins (HDL). Usually, apoA-I is not associated with amyloidosis. However, four naturally occurring mutant forms of apoA-I are known so far resulting in amyloidosis. The most important feature of all variants is the very similar formation of N-terminal fragments which were found in the amyloid deposits (residues 1-83 to 1-94). The new insights in the understanding of the association of apoA-I with HDL, its metabolism, and its hypothesized structural findings may explain a common mechanism for the genesis of apoA-I induced amyloidosis. Here we summarized the specific features of all known amyloidogenic variants of apoA-I and speculate about its metabolic pathway, which may have general implications for the metabolism of apoA-I.