Gaucher disease is a lysosomal storage disorder caused by deficient glucocerebrosidase activity. We have previously shown that the cellular activity of the most common Gaucher disease-associated glucocerebrosidase variant, N370S, is increased when patient-derived cells are cultured with the chemical chaperone N-nonyl-deoxynojirimycin. Chemical chaperones stabilize proteins against misfolding, enabling their trafficking from the endoplasmic reticulum. Herein, the generality of this therapeutic strategy is evaluated with other glucocerebrosidase variants and with additional candidate chemical chaperones. Improved chemical chaperones are identified for N370S glucocerebrosidase. Moreover, we demonstrate that G202R, a glucocerebrosidase variant that is known to be retained in the endoplasmic reticulum, is also amenable to chemical chaperoning. The L444P variant is not chaperoned by any of the active site-directed molecules tested, likely because this mutation destabilizes a domain distinct from the catalytic domain.

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Point mutations in the lysosomal hydrolase, glucocerebrosidase (GC), can cause Gaucher disease, a common lysosomal storage disease. Several clinically important GC mutations impede folding in the endoplasmic reticulum (ER) and target these enzymes for ER-associated degradation (ERAD). The removal of these misfolded proteins decreases the lysosomal concentration of GC, which results in glucosylceramide accumulation. The most common GC variant, N370S, and other clinically relevant variants, G202R and L444P, exhibit different cellular localization patterns in patient-derived fibroblasts. We show that these distributions can be altered by manipulation of the ER folding environment, either by chemical chaperones or by temperature shifts. N370S, L444P, and G202R GC are destabilized in the neutral pH environment of the ER, rendering them prone to ERAD. Fibroblasts harboring the G202R and L444P GC mutations grown at 30 degrees C localize the mutant proteins to the lysosome, and this increases total GC activity. Both of these temperature-sensitive mutants appear to be stable at 37 degrees C once they are trafficked to the low pH environment of the lysosome. Chemical chaperones correct the ER instability and significant ER retention of G202R GC. N370S is also destabilized under ER simulating conditions, a deficiency that is corrected by chemical chaperone binding. These data clearly show manipulating the ER environment with chemical chaperones increases the lysosomal concentration of partially active GC variants and suggest that small molecules could be used to treat Gaucher disease.

The plasma membrane of polarised epithelial cells is characterised by two structurally and functionally different domains, the apical and basolateral domains. These domains contain distinct protein and lipid constituents that are sorted by specific signals to the correct surface domain [1]. The best characterised apical sorting signal is that of glycophosphatidylinositol (GPI) membrane anchors [2], although N-linked glycans on some secreted proteins [3] and O-linked glycans [4] also function as apical sorting signals. In the latter cases, however, the underlying sorting mechanisms remain obscure. Here, we have analysed the role of O-glycosylation in the apical sorting of sucrase-isomaltase (SI), a highly polarised N- and O-glycosylated intestinal enzyme, and the mechanisms underlying this process. Inhibition of O-glycosylation by benzyl-N-acetyl-alpha-D-galactosaminide (benzyl-GalNAc) was accompanied by a dramatic shift in the sorting of SI from the apical membrane to both membranes. The sorting mechanism of SI involves its association with sphingolipid- and cholesterol-rich membrane rafts because this association was eliminated when O-glycosylation was inhibited by benzyl-GaINAc. The results demonstrate for the first time that O-linked glycans mediate apical sorting through association with lipid rafts.

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.

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Background: Celiac disease (CD) is induced by wheat gliadins and related cereal proteins. Anti-gliadin antibodies (AGAs) are present in the serum of CD patients, but these antibodies have lower diagnostic specificity and sensitivity than autoantibodies [anti-endomysium antibodies (AEmAs) and anti-tissue transglutaminase antibodies (AtTGAs)]. Recently, AGAs from CD patients were found to recognize deamidated gliadin peptides, probably formed by the action of tissue transglutaminase. Methods: We synthesized several gliadin peptides and their glutamine-glutamic acid-substituted counterparts on cellulose membranes and tested their recognition by IgA in sera of 52 AEmA-positive CD patients and 76 AEmA-negative controls in a luminescence assay. For comparison, we assayed IgA concentrations of AGAs, AtTGAs, and AEmAs. For measurement of AtTGAs, we used the human recombinant antigen. Results: We identified several nonapeptides that were detected with high specificity by IgA in CD patients. Diagnostic accuracy of the peptide antibody assay was highest when peptide PLQPEQPFP was used in combination with peptide PEQLPQFEE within one assay. AGAs were above the cutoff in 14 of the controls, but only 5 of the controls were positive for peptide antibod-

Caveolin-1, a putative mediator of intracellular cholesterol transport, is generally assumed to be integrated into the cytoplasmic leaflets of all cellular membranes. Lipid droplets form by budding at the endoplasmic reticulum (ER), and caveolin-1 is thought to be transferred to the droplet surface along with the cytoplasmic leaflet of ER membranes and not to enter the droplet core. We explored how caveolin-1 accesses lipid droplets from the ER by localizing caveolin-1 in ER membranes and in lipid droplets in cultured smooth muscle cells using freeze-fracture immunocytochemistry. We detected caveolin-1 in endoplasmic leaflets of ER membranes but never in cytoplasmic leaflets. Caveolin-1 was also present in lipid droplet cores. These findings are incompatible with the current hypothesis of lipid droplet biogenesis. We suggest that the inherent high affinity of caveolin-1 for neutral lipids causes caveolin-1 molecules to be extracted from the endoplasmic leaflets of ER membranes and to be transferred into the droplet core by inundating lipids during droplet formation.