Functional studies of human intestinal alkaline sphingomyelinase by deglycosylation and mutagenesis

Abstract

Intestinal alk-SMase (alkaline sphingomyelinase) is an ectoenzyme related to the NPP (nucleotide phosphodiesterase) family. It has five potential N-glycosylation sites and predicated transmembrane domains at both the N- and C-termini. The amino acid residues forming the two metal-binding sites in NPP are conserved, and those of the active core are modified. We examined the functional changes of the enzyme induced by deglycosylation and mutagenesis. Treating alk-SMase cDNA-transfected COS-7 cells with tunicamycin rendered the expressed enzyme completely inactive. Mutations of the five potential N-glycosylation sites individually and in combination showed that these sites were all glycosylated and deficient glycosylation decreased the enzyme activity. Immunogold labelling showed that the wild-type enzyme was mainly located in the plasma membrane, whereas the C-terminal domain-truncated enzyme was released into the medium. Deglycosylation blocked the release of the enzyme that accumulated in endosome-like structures. The enzyme activity was also decreased by mutations of the residues forming the putative metal-binding sites and the active core. Substitution of the active core sequence with that of NPP or mutation of T75 in the core abolished the enzyme activity against sphingomyelin but failed to render the enzyme NPP active. Our results indicate that alk-SMase activity is severely affected by defective N-glycosylation and structural alterations of the putative metal-binding sites and the predicted active core.

Figure 1. Schematic representation of the putative structure of human alk-SMase

Transmembrane domains at both the N- and C-terminal, the potential glycosylation sites, the modified activity core and the conserved amino acids forming the metal-binding sites in NPP are indicated.

Figure 2. Effect of tunicamycin on the activity of alk-SMase expressed in COS-7 cells

COS-7 cells were transfected with wild-type alk-SMase cDNA. Tunicamycin (Tu) was added to the medium 6 h after transfection, to a final concentration of 10 μg/ml, followed by incubation for 42 h. The control (Ctl) cells were cultured in the absence of tunicamycin. Alk-SMase activities in cell lysates from three separate experiments were determined (upper panel) and the results are presented as means±S.E.M. The expressed protein was examined by Western blotting (lower panel).

Figure 3. Alk-SMase activity after mutation of the potential N-glycosylation sites

The potential glycosylation sites were mutated individually and the enzyme was expressed in COS-7 cells. The changes in alk-SMase activity in cell lysate were measured (top panel) and the proteins expressed were examined by Western blotting (second panel). The predicted fully glycosylated alk-SMase form is indicated by an arrow. In the third panel, the mutation was performed in an additive manner as indicated in the Figure. The alk-SMase activities in the expressed enzyme were determined. The changes in the molecular mass were examined by Western blotting. The predicted fully glycosylated and deglycosylated forms are indicated by an arrow on the left and right respectively (bottom panel). Results are expressed as the means±S.E.M. for three independent experiments. **P<0.01 or less. The statistical significance was not analysed for samples 3, 4, 5 and tunicamycin (TU) in the bottom panel, owing to absence of the activity.

Figure 4. Release of C-terminal-truncated alk-SMase into the culture medium

Wild-type (WT) alk-SMase or C-terminal-truncated enzyme was expressed in COS-7 cells. After 48 h, the enzyme activity in the culture medium was determined (top panel). R440, the C-terminal anchor from P441 to A458 was truncated; T415, the C-terminal from E416 to A458 encoded by exon 4 was truncated. Results are expressed as the means±S.E.M. for three independent experiments. **P<0.01 compared with WT. No statistical significance was identified between R440 and T415. In the middle panel, 10 μl of the medium from each group was subjected to Western blotting. The bottom panel shows the effect of deglycosylation on the release of alk-SMase into the medium. Both the C-terminal anchor-truncated alk-SMase and the enzyme with glycosylation sites further mutated were expressed in COS-7 cells. Either 10 μl of the medium or 50 μg of the cell lysate was subjected to Western blotting. Lane 1, medium, C-terminal-truncated; lane 2, medium, C-terminal-truncated, with mutant glycosylation sites; lane 3, lysate of COS-7 cells expressing C-terminal-truncated alk-SMase; lane 4, lysate of COS-7 cells expressing C-terminal-truncated enzyme with mutant glycosylation sites.

Figure 5. Immunogold labelling of alk-SMase in COS-7 cells transfected with wild-type alk-SMase (A, B), C-terminal-truncated alk-SMase (C, D) and C-terminal-truncated alk-SMase with five glycosylation sites mutated (E, F)

(A, B) The enzyme is mainly found in the plasma membrane (PM) and only in low concentration in endosome (EN). (C, D) The truncated enzyme is absent from the PM and found in vesicular structures (C) and EN. (E, F) The truncated alk-SMase with deglycosylation is absent from the plasma membrane and found in high concentration in the endosomes. Scale bars, 200 nm.

Figure 6. Changes of alk-SMase activity by mutation of the predicted activity core

The amino acid residues in the predicted active core were mutated individually to the corresponding ones in most NPPs. The expressed proteins were examined by Western blotting (upper panel) and alk-SMase activity in the cell lysate was assayed (lower panel). Results were obtained from three independent experiments and are presented as means±S.E.M.

Figure 7. Effects of mutation of the putative metal-binding sites on alk-SMase activity

COS-7 cells were transfected with either wild-type alk-SMase or alk-SMase in which one of the residues forming the two triad metal-binding sites was mutated. Alk-SMase activity in the cell lysate was determined (upper panel) and the expressed proteins were examined by Western blotting (lower panel). Results were obtained from three separate experiments and are presented as means±S.E.M.