The N370S (Asn370-->Ser) mutation affects the capacity of glucosylceramidase to interact with anionic phospholipid-containing membranes and saposin C

Abstract

The properties of the endolysosomal enzyme GCase (glucosylceramidase), carrying the most prevalent mutation observed in Gaucher patients, namely substitution of an asparagine residue with a serine at amino acid position 370 [N370S (Asn370-->Ser) GCase], were investigated in the present study. We previously demonstrated that Sap (saposin) C, the physiological GCase activator, promotes the association of GCase with anionic phospholipid-containing membranes, reconstituting in this way the enzyme activity. In the present study, we show that, in the presence of Sap C and membranes containing high levels of anionic phospholipids, both normal and N370S GCases are able to associate with the lipid surface and to express their activity. Conversely, when the amount of anionic phospholipids in the membrane is reduced (approximately 20% of total lipids), Sap C is still able to promote binding and activation of the normal enzyme, but not of N370S GCase. The altered interaction of the mutated enzyme with anionic phospholipid-containing membranes and Sap C was further demonstrated in Gaucher fibroblasts by confocal microscopy, which revealed poor co-localization of N370S GCase with Sap C and lysobisphosphatidic acid, the most abundant anionic phospholipid in endolysosomes. Moreover, we found that N370S Gaucher fibroblasts accumulate endolysosomal free cholesterol, a lipid that might further interfere with the interaction of the enzyme with Sap C and lysobisphosphatidic acid-containing membranes. In summary, our results show that the N370S mutation primarily affects the interaction of GCase with its physiological activators, namely Sap C and anionic phospholipid-containing membranes. We thus propose that the poor contact between N370S GCase and its activators may be responsible for the low activity of the mutant enzyme in vivo.

Figure 1. Effect of different activators on the GCase activity in control and N370S Gaucher fibroblast extracts

The GCase activity in normal and N370S Gaucher fibroblast extracts (cell lines 370a, 370b, 370c, and 370d) was determined in at least three separate preparations for each cell line utilizing assay mixtures containing either detergents (Triton X-100 and taurocholate) or Sap C and LUVs as activators (see the Experimental section). No enzyme activity was detected in both the normal and N370S Gaucher fibroblasts in the absence of activators. An identical amount of protein (10 μg) was used in each assay. The concentration of Sap C was 5 μM. LUVs were composed of one among PS/Chol, 75:25 (75% PS); PS/PC/Chol, 60:15:25 (60% PS); and PS/PC/Chol, 20:55:25 (20% PS). Data represent the means±S.D.

Figure 2. Level and molecular mass forms of N370S GCase in N370S Gaucher fibroblasts

The level and the pattern of molecular mass forms of GCase were determined by Western-blot analysis of GCase antigen from control and N370S Gaucher fibroblast homogenates (cell lines 370a, 370b, 370c and 370d). Identical amounts of protein (5 μg) were loaded in each lane. The number on the left refers to the molecular mass (kDa) of albumin standard. The blots were reprobed for β-actin as a control of protein content. Each experiment was repeated more than three times and gave similar results.

Figure 3. Level and processing of Sap C in N370S Gaucher fibroblasts

(A) Sap C level was determined by Western-blot analysis of control and N370S Gaucher fibroblast homogenates (cell lines 370a, 370b, 370c and 370d). Identical amounts of protein (5 μg) were loaded in each lane. The blots were reprobed for β-actin as a control of protein content. (B) Control and N370S Gaucher fibroblasts were pulsed for 1 h and then chased as indicated. Immunoprecipitation, SDS/PAGE and fluorography were performed as described in the Experimental section. The numbers on the left refer to the molecular-mass standards (kDa). Each experiment was repeated more than two times and gave similar results.

Figure 4. Immunoblots of normal and N370S GCases purified from fibroblast homogenates

Aliquots of the purified enzyme preparations containing predetermined enzyme units were analysed by Western blotting. Lanes were loaded with 0.5, 2 and 3 units (U) of control enzyme or with 0.1, 0.2 and 0.3 unit of N370S GCase as indicated. Each experiment was repeated more than two times and gave similar results.

Figure 5. Effect of anionic phospholipids and Sap C on the activation of control and N370S GCases

The activities of similar amounts of purified control (A) and N370S (B) GCases, corresponding to 1 unit of control and 0.1 unit of N370S GCase respectively were measured in the presence of LUVs and increasing amounts of Sap C as described in the Experimental section. LUVs were composed of one among PS/Chol, 75:25 (75% PS, ○); PS/PC/Chol, 60:15:25 (60% PS, ▲); PS/PC/Chol, 20:55:25 (20% PS, □); LBPA/PC/Chol, 20:55:25 (20% LBPA, ●); and PC/Chol, 75:25 (75% PC, ■). Each point represents the mean of at least three replicates. The deviation was within ±5% of each data point.

Figure 6. Comparison of the binding of control and N370S GCases to membranes containing anionic phospholipids and Sap C

For binding experiments, similar amounts of purified control and N370S GCases were incubated with Sap C and LUVs as reported in the Experimental section. The vesicles were composed of one among LBPA/PC/Chol, 20:55:25 (20% LBPA); PS/PC/Chol, 20:55:25 (20% PS); and PS/PC/Chol, 60:15:25 (60% PS). After centrifugation, the free and liposome-bound enzymes were evaluated by SDS/PAGE and Western blotting. Lanes 1, free enzyme; lanes 2, membrane-bound enzyme. Each experiment was repeated at least three times and gave similar results.

Figure 7. Chol accumulates in endosomal/lysosomal vesicles in N370S Gaucher fibroblasts

(A) Control and Gaucher fibroblasts (N370S a, b and c) were cytochemically stained with filipin for Chol as reported in the Experimental section. Scale bars, 10 μm. (B) The Gaucher cell line N370S d was cytochemically stained with filipin for Chol (left panel) and immunostained for LBPA, an endosomal/lysosomal marker (middle panel). The right panels show the merged image. Scale bars, 10 μm.

Figure 8. N370S GCase poorly co-localizes with Sap C and LBPA

(A) Normal fibroblasts were double-immunostained for GCase and Sap C (top panels) or GCase and LBPA (bottom panels) as reported in the Experimental section. Note that the enzyme was visualized with the monoclonal antibody 8E4 (green, top-left panel) or with a polyclonal anti-GCase antibody (red, bottom-left panel). The right panels show an enlargement of the regions outlined by the boxes in the merged panels. Scale bars, 10 μm. (B) N370S GCase fibroblasts were double-immunostained for the mutated enzyme and Sap C (top panels) or for the enzyme and LBPA (bottom panels). The right panels show an enlargement of the regions outlined by the boxes in the merged panels. Scale bars, 10 μm. A comparison of the merged images in (A) and (B) shows that, in normal fibroblasts, all the intracellular vesicles are yellow, indicating that each vesicle contains GCase, Sap C and LBPA, while in Gaucher cells, most of the vesicles are either green or red, indicating that the enzyme often resides in vesicles devoid of Sap C and LBPA.

Figure 9. N370S GCase resides in less-acidic organelles than Sap C

N370S GCase fibroblasts were incubated with LysoTracker Red as reported in the Experimental section. The cells were then immunostained for the enzyme (green, upper-middle panel) or Sap C (green, bottom-middle panel). A comparison of the merged images shows that the vesicles containing Sap C also contain LysoTracker Red (all the vesicles are yellow), while several vesicles containing the enzyme are green indicating that they are devoid of LysoTracker Red. Scale bars, 10 μm.