Generation of specific deoxynojirimycin-type inhibitors of the non-lysosomal glucosylceramidase

The existence of a non-lysosomal glucosylceramidase in human cells has been documented (van Weely, S., Brandsma, M., Strijland, A., Tager, J. M., and Aerts, J. M. F. G. (1993) Biochim. Biophys. Acta 1181, 55-62). Hypothetically, the activity of this enzyme, which is localized near the cell surface, may influence ceramide-mediated signaling processes. To obtain insight in the physiological importance of the non-lysosomal glucosylceramidase, the availability of specific inhibitors would be helpful. Here we report on the generation of hydrophobic deoxynojirimycin (DNM) derivatives that potently inhibit the enzyme. The inhibitors were designed on the basis of the known features of the non-lysosomal glucosylceramidase and consist of a DNM moiety, an N-alkyl spacer, and a large hydrophobic group that promotes insertion in membranes. In particular, N-(5-adamantane-1-yl-methoxy)pentyl)-DNM is a very powerful inhibitor of the non-lysosomal glucosylceramidase at nanomolar concentrations. At such concentrations, the lysosomal glucocerebrosidase and alpha-glucosidase, the glucosylceramide synthase, and the N-linked glycan-trimming alpha-glucosidases of the endoplasmic reticulum are not affecte

Ortho-Carborane-modified N-substituted Deoxynojirimycins

Medical Biochemistr

Elevated globotriaosylsphingosine is a hallmark of Fabry disease

FWN – Publicaties zonder aanstelling Universiteit Leide

Drinking on masculinity: Alcohol and gender in Andalusia

FWN – Publicaties zonder aanstelling Universiteit Leide

Accumulation of weak bases in relation to intralysosomal pH in cultured human skin fibroblasts

The volume of the lysosomal compartment in cultured human skin fibroblasts was estimated from the distribution between the cells and the medium of tracer amounts of labelled methylamine and chloroquine, which accumulate in the lysosomes, 2,2-dimethyloxazolidine-2,4-dione, which accumulates in the soluble cytoplasmic compartment relative to the lysosomes, and sucrose, which is excluded by the cells. In a foetal fibroblast line, the fractional volume of the lysosomal compartment was 0.044 +/- 0.007 (n = 8). In fibroblasts from a patient with the I-cell disease, the fractional volume was 0.15. The fractional volume of the lysosomal compartment was used to calculate the intralysosomal pH from the accumulation of the weak bases in the cells. The mean value obtained was 5.29 +/- 0.04 (n = 8). In fibroblasts incubated with various concentrations of chloroquine, the fractional volume of the lysosomal compartment and the accumulation of chloroquine in the cells were used to calculate the concentration of chloroquine in the lysosomes. The intralysosomal concentration increased from 3 to 114 mM as the extracellular concentration increased from 1 to 100 microM. Concomitantly, the intralysosomal pH increased from 5.3 in the absence of chloroquine to 5.9 in the presence of 100 microM chloroquine. A similar increase in intralysosomal pH could be calculated in fibroblasts incubated with different concentrations of ammoni

Recombinant enzyme therapy for Fabry disease: absence of editing of human alpha-galactosidase A mRNA

For more than a decade, protein-replacement therapy has been employed successfully for the treatment of Gaucher disease. Recently, a comparable therapy has become available for the related lipid-storage disorder Fabry disease. Two differently produced recombinant α-galactosidase A (α-gal A) preparations are used independently for this purpose. Agalsidase α is obtained from human fibroblasts that have been modified by gene activation; agalsidase β is obtained from Chinese hamster ovary cells that are transduced with human α-gal A cDNA. It has previously been claimed that α-gal A mRNA undergoes editing, which may result in coproduction of an edited protein (Phe 396 Tyr) that might have a relevant physiological function. We therefore analyzed the occurrence of α-gal A editing, as well as the precise nature, in this respect, of the therapeutic enzymes. No indications were obtained for the existence of editing at the protein or RNA level. Both recombinant enzymes used in therapy are unedited and are capable of functionally correcting cultured fibroblasts from Fabry patients in their excessive globotriaosylceramide accumulation. Although RNA editing is apparently not relevant in the case of α-gal A, a thorough analysis of the potential occurrence of editing of transcripts is nevertheless advisable in connection with newly developed protein-replacement therapies

Demonstration of the existence of a second, non-lysosomal glucocerebrosidase that is not deficient in Gaucher disease

In addition to the lysosomal glucocerebrosidase, a distinct beta-glucosidase that is also active towards glucosylceramide could be demonstrated in various human tissues and cell types. Subcellular fractionation analysis revealed that the hitherto undescribed glucocerebrosidase is not located in lysosomes but in compartments with a considerably lower density. The non-lysosomal glucocerebrosidase differed in several respects from lysosomal glucocerebrosidase. The non-lysosomal isoenzyme proved to be tightly membrane-bound, whereas lysosomal glucocerebrosidase is weakly membrane-associated. The pH optimum of the non-lysosomal isoenzyme is less acidic than that of lysosomal glucocerebrosidase. Non-lysosomal glucocerebrosidase, in contrast to the lysosomal isoenzyme, was not inhibited by low concentrations of conduritol B-epoxide, was markedly inhibited by taurocholate, was not stimulated in activity by the lysosomal activator protein saposin C, and was not deficient in patients with Gaucher disease. Non-lysosomal glucocerebrosidase proved to be less sensitive to inhibition by castanospermine or deoxynojirimycin but more sensitive to inhibition by D-gluconolactone than the lysosomal glucocerebrosidase. The physiological function of this second, non-lysosomal, glucocerebrosidase is as yet unknow