No abstract

A series of Moloney murine leukemia virus (Mo-MuLV) envelope gene constructs were analyzed for biological activity. Three classes of recombinant envelopes were examined: insertions, deletions, and chimeras. Insertion (4 to 5 amino acids) and deletion (31 to 62 amino acids) mutants spanned most of the SU (gp7O)-coding region and were all biologically inactive. Radioimmunoprecipitation demonstrated that the mutant envelope proteins were incorrectly processed. The Pr8r"" envelope precursor proteins failed to obtain the proper posttranslational modifications and were not cleaved into SU (gp70) and TM (pl5E), suggesting that disruption of Pr8O"V structure prevents intracellular transport and processing. To analyze the functional domains of the SU portion of the Env protein, we assembled several chimeric constructs. In these constructs, portions of the ecotropic Mo-MuLV envelope gene were replaced with corresponding sequences from the 4070A amphotropic MuLV envelope. Using a retroviral vector pseudotyping assay, 5 of 12 chimeric envelope proteins were shown to be biologically active. Host range was determined by retroviral vector transduction of the appropriate cell, by viral interference studies, and by the productive infection of Chinese hamster ovary cells expressing the murine ecotropic receptor. These results permit assignment of the amino acids responsible for host range determination. Ecotropic host range is determined by the first 88 amino acids of the Mo-MuLV SU, while the amphotropic host range-determining region spans the first 157 amino acids of the 4070A SU.

The endothelial dysfunction of Fabry disease results from α-galactosidase A deficiency leading to the accumulation of globotriaosylceramide. Vasculopathy in the α-galactosidase A null mouse is manifest as oxidant induced thrombosis, accelerated atherogenesis, and impaired arterial reactivity. To better understand the pathogenesis of Fabry disease in humans, we generated a human cell model by using RNA interference. Hybrid endothelial cells were transiently transfected with siRNA specifically directed against α-galactosidase A. Knockdown of α-galactosidase A was confirmed by immunoblotting and globotriaosylceramide accumulation. Endothelial nitric oxide synthase (eNOS) activity was correspondingly decreased by greater than sixty percent. Levels of 3-nitrotyrosine (3NT), a specific marker for reactive nitrogen species and quantified by mass spectrometry, increased by 40 to 120 fold without corresponding changes in other oxidized amino acids, consistent with eNOS derived reactive nitrogen species as the source of the reactive oxygen species. eNOS uncoupling was confirmed by the observed increase in free plasma and protein bound aortic 3NT levels in the α-galactosidase A knockout mice. Finally, 3NT levels, assayed in biobanked plasma samples from patients with classical Fabry disease, were over 6-fold elevated compared to age and gender matched controls. Thus, 3NT may serve as a biomarker for the vascular involvement in Fabry disease.

Fabry disease is a lysosomal storage disorder that results in an accumulation of globotriaosylceramide in vascular tissue secondary to a deficiency in ␣-galactosidase A. The glycolipid-associated vasculopathy results in strokes and cardiac disease, but the basis for these complications is poorly understood. Recent studies in the ␣-galactosidase A-knockout mouse suggested that a decrease in nitric oxide (NO) bioavailability may play a role in the abnormal thrombosis, atherogenesis, and vasorelaxation that are characteristic of these mice. To understand better the association between impaired NO bioavailability and glycolipid accumulation, we studied ␣-galactosidase A-knockout mice or primary cultures of their aortic endothelial cells. Treatment of knockout mice with a potent inhibitor of glucosylceramide synthase reversed accumulation of globotriaosylceramide but failed to normalize the defect in vasorelaxation. Basal and insulin-stimulated endothelial NO synthase (eNOS) activities in endothelial cells derived from knockout mice were lower than those observed from wild-type mice; normalization of glycolipid only partially reversed this reduction in eNOS activity. The loss of eNOS activity associated with a decrease in high molecular weight caveolin oligomers in endothelial cells and isolated caveolae, suggesting a role for glycolipids in caveolin assembly. Finally, concentrations of ortho-tyrosine and nitrotyrosine in knockout endothelial cells were markedly elevated compared with wild-type endothelial cells. These findings are consistent with a loss of NO bioavailability, associated with eNOS uncoupling, in the ␣-galactosidase A-knockout mouse.