A stretch of 17 amino acids in the prosaposin C terminus is critical for its binding to sortilin and targeting to lysosomes

Abstract

Prosaposin, the precursor of four lysosomal cofactors required for the hydrolysis of sphingolipids, is transported to the lysosomes via the alternative receptor, sortilin. In this study, we identified a specific domain of 17 amino acids within the C terminus of prosaposin involved in binding to this sorting receptor. We generated six prosaposin deletion constructs and examined the effect of truncation by coimmunoprecipitation and confocal microscopy. The experiments revealed that the first half of the prosaposin C terminus (aa 524-540), containing a saposin-like motif, was required and necessary to bind sortilin and to transport it to the lysosomes. Based on this result, we introduced twelve site-directed point mutations within the first half of the C terminus. Although the interaction of prosaposin with sortilin was pH dependent, the mutation of hydrophilic amino acids that usually modulate pH-dependent protein interactions did not affect the binding of prosaposin to sortilin. Conversely, a tryptophan (W530) and two cysteines (C528 and C536) were essential for its interaction with sortilin and for its transport to the lysosomes. In conclusion, our investigation demonstrates that a saposin-like motif within the first half of the prosaposin C terminus contains the sortilin recognition site.

Figure 1

Effect of pH on the interaction of prosaposin and sortilin. (A) Schematic representation of wild-type prosaposin and sortilin. Both of the prosaposin and sortilin constructs are linked to myc tags. (B) Upper panel shows coimmunoprecipitation of sortilin with prosaposin at different pHs. The 70-kDa bands correspond to prosaposin pulled down by anti-prosaposin antibody. The 100-kDa bands represent sortilin coimmunoprecipitated with prosaposin at pH 6.0, 6.5, and 7.0. The sortilin band at pH 5.5 was very weak and no band was observed at pH 5.0. Lower panel shows a Western blot of prosaposin and sortilin in the crude cell lysate.

Figure 2

Effects of sequential truncations of prosaposin C terminus. (A) Schematic representation of truncated prosaposin constructs. PSAP-WT is a wild-type prosaposin; P-75 lacks 25% of the A-type domain; P-50 lacks 50% of the A-type domain. P-L50 lacks the first half of the A-type domain but contains the second half. P-25 lacks 75% of the A-type domain; P-0 contains only linker region; P-ΔC lacks the whole C terminus. (B) Coimmunoprecipitation with anti-sortilin antibody was conducted to identify the binding of truncated prosaposin to sortilin. While P-75 and P-50 were precipitated by sortilin, P-0 and P-25 were not. PSAP-WT was used as positive control and P-ΔC as negative control. NC was another negative control, in which the cells were only transfected with the sortilin construct. (C) Contrary to PSAP-WT, P-L50 was not pulled-down by sortilin. WB = Western blot, as 2% input of sortilin and prosaposin proteins in the crude lysate.

Figure 3

Lysosomal targeting of sequential truncations of prosaposin. The COS-7 cells transfected with the truncated constructs were examined by confocal microscopy. Truncated prosaposin was stained green with chicken anti-myc antibody. Lysosomes were stained red with anti-LAMP-1 antibody. Nuclei appear in blue. Anti-myc staining of cells transfected with PSAP-WT, P-75, and P-50 constructs labeled the perinuclear region and cytoplasmic vesicular structures and overlaid with anti-LAMP1 staining (A–C). P-L50, P-25, P-0, and P-ΔC labeled only the perinuclear region and did not overlay with anti-LAMP1 staining (D–G). Bar = 5 μm and applies to all micrographs.

Figure 4

Lysosomal transport of prosaposin truncation/mutation constructs. The percentage of LAMP-1 vesicles overlaid with each prosaposin truncation/mutation construct was calculated. (A) Among the truncated constructs, P-ΔC, P-0, P-25, and P-L50 showed significant decreases in lysosomal transport. P-50 and P-75 had no significant differences from PSAP-WT. (B) All of the point mutations on hydrophilic residues did not show a significant decrease compared with that of PSAP-WT. (C) C528D, W530P, and C536D demonstrated significant decrease in lysosomal transportation. Error bars indicate ± SE, and double asterisks represent p<0.01, compared with PSAP-WT.

Figure 5

Effects of point mutations of hydrophilic residues in the interaction of prosaposin and sortilin. (A) Schematic representation of the mutated prosaposin constructs. All of the hydrophilic amino acids in the first half of the A-type domain and the linker region were substituted with the hydrophobic amino acids phenylalanine or tryptophan. (B) Coimmunoprecipitation with anti-sortilin antibody was conducted to identify the binding of mutated prosaposin to sortilin. All of the mutations, K520F, E526F, K527F, Q537F, N538W, and E540F, were precipitated by sortilin. PSAP-WT was used as a positive control while the negative control (NC) consisted of cells transfected with only sortilin construct. WB indicates Western blot results, showing a 1.5% input of sortilin and prosaposin in the crude lysate.

Figure 6

Lysosomal targeting of prosaposin point mutations on hydrophilic residues. The COS-7 cells transfected with the mutation constructs K520F, E526F, K527F, Q537F, N538W, and E540F were examined by confocal microscopy. Prosaposin mutation constructs were stained green, lysosomes stained red, and nuclei stained blue. Anti-myc staining of cells transfected with K520F, E526F, K527F, Q537F, N538W, and E540F constructs labeled the perinuclear Golgi region and cytoplasmic vesicular structures and overlaid with anti-LAMP1 staining (A–F). Bar = 5 μm and applies to all micrographs.

Figure 7

Effects of point mutations on hydrophobic residues in the first half of prosaposin C terminus. (A) Schematic representation of mutated prosaposin constructs. The hydrophobic amino acid residues in the first half of the A-type domain, C528, W530, P532, W535, and C536, were replaced with the disfavored amino acids. (B–D) C528D, W530P, and C536D were stained with anti-myc antibody at the perinuclear region and did not overlay with vesicular structures stained with anti-LAMP-1. Bar = 5 μm and applies to all micrographs. (E) The cells transfected with P532W, P532H, or W535P were treated with or without MG-132. Before MG-132 treatment, these constructs were not detected by Western blot. However, the bands of all the mutated constructs appeared as bands at 70 kDa after MG-132 treatment.