GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

Abstract

MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4(+) T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We sought to determine whether GILT's reductase activity regulates CatS expression and function. Confocal microscopy confirmed that GILT and CatS colocalized within lysosomes of B cells. GILT expression posttranscriptionally decreased the steady-state protein expression of CatS in primary B cells and B-cell lines. GILT did not substantially alter the expression of other lysosomal proteins, including H2-M, H2-O, or CatL. GILT's reductase active site was necessary for diminished CatS protein levels, and GILT expression decreased the half-life of CatS, suggesting that GILT-mediated reduction of protein disulfide bonds enhances CatS degradation. GILT expression decreased the proteolysis of a CatS selective substrate. This study illustrates a physiologic mechanism that regulates CatS and has implications for fine tuning MHC class II-restricted Ag processing and for the development of CatS inhibitors, which are under investigation for the treatment of autoimmune disease.

Figure 1

GILT and CatS colocalize in lysosomes of primary B cells. Confocal microscopy of WT and GILT^−/− primary B cells isolated by magnetic bead negative selection from murine splenocytes. Cells were fixed, permeabilized, and stained with (A) rabbit anti-GILT serum followed by Alexa Fluor 555-conjugated goat anti-rabbit (red, left), Alexa Fluor 488-conjugated anti-LAMP-1 (green, middle) and Hoechst (blue, right), or (B) rabbit anti-GILT serum followed by Alexa Fluor 488-conjugated goat anti-rabbit (green, left), goat anti-CatS followed by Alexa Fluor 555-conjugated donkey anti-goat (red, middle) and Hoechst (blue, right). Merged images (yellow) along with Hoechst are shown (right). Photographs were taken with 63× magnification under oil, and scale bar indicates 5 μm. Images are from a single 0.6 μm section. Data are representative of at least three independent experiments.

Figure 2

GILT acts posttranscriptionally to decrease the steady-state protein expression of CatS in primary B cells. Immunoblot analysis of (A) CatS, (D) H2-M, (E) H2-O, and (F and G) CatL in detergent lysates of WT and GILT^−/− primary B cells (A, D, E, and G) or thymus (F). Postnuclear supernatants of lysates (9, 15, 5, 22, and 15 μg/lane for A, D, E, F, and G, respectively) were resolved by 4–20% gradient SDS-PAGE under reducing conditions and probed with goat anti-CatS polyclonal Ab, mouse anti-H2-Mα mAb, rabbit anti-H2-Oβ Ab, or rabbit anti-CatL serum. GRP94 served as a loading control. (A) Immunoblot analysis and (B) densitometry of CatS expression are shown. (B) Band intensities were estimated using Quantity One software, and CatS expression was normalized to GRP94 expression. Data are shown as the mean ± SEM of three sets of lysates from three performed experiments. ^*p < 0.05, independent sample Student’s t-test. (C) Quantitative RT-PCR of CatS in primary B cells showed similar CatS mRNA transcript levels in WT and GILT^−/− B cells. CatS mRNA expression was normalized to GAPDH mRNA expression and expressed as fold change relative to expression in WT cells. Data are shown as the mean ± SD of three independent sets of WT and GILT^−/− B cells. (D–G) Immunoblot analysis and densitometry of H2-M, H2–O, and CatL are shown. Data are shown as the mean ± SEM of the signal intensities in WT and GILT^−/− B-cell lysates pooled from four independent experiments in (D), three independent experiments in (E and F) and six independent experiments in (G).

Figure 3

GILT’s reductase active site is necessary for diminished CatS steady-state protein levels. Immunoblot analysis of (A) GILT and (B) CatS protein expression in BμMyc.GKO.1 cells stably transduced with vector alone, WT GILT, or mutant C46S, C49S, or C46SC49S GILT. Post-nuclear supernatants of lysates (25 μg/lane) were resolved by 4–20% gradient SDS-PAGE under nonreducing (GILT) or reducing (CatS) conditions and probed with rabbit anti-GILT serum or goat anti-CatS Ab. GRP94 served as a loading control. (C) CatS expression was normalized to GRP94 and shown relative to cells transduced with vector alone. Data are shown as the mean ± SEM of the signal intensities from lysates of BμMyc.GKO.1 cells transduced with vector alone, WT, or mutant GILT from five independent experiments. ^*p < 0.05, ^**p < 0.01, ANOVA with a Bonferroni adjustment for multiple comparisons.

Figure 4

GILT decreases the half-life of CatS. (A and B) Immunoblot analysis of CatS protein expression in GILT-deficient BμMyc.GKO.1 cells stably transduced with vector alone (GILT -) or WT GILT (GILT +). Cells were treated with cycloheximide (CHX +) or DMSO (CHX -) for the indicated times. Postnuclear supernatants of lysates (40 μg/lane) were resolved by 4–20% gradient SDS-PAGE under reducing conditions and probed with goat anti-CatS Ab and rat anti-GPR94Ab, as a loading control. (C) CatS expression was quantified, normalized to GRP94, and shown relative to expression at 0 h. Data are shown as the mean ± SEM of vector alone or WT GILT samples pooled from three independent experiments. ^*p < 0.05, ^**p < 0.01, Student’s t-test.

Figure 5

GILT decreases the proteolysis of a CatS-selective substrate. CatS, CatB, and CatL activity was measured in lysates (1 × 10⁶ cell equivalents) from primary B cells isolated from WT and GILT^−/− murine splenocytes based upon the cleavage of a CatS, CatB, or CatL-selective substrate. Cathepsin inhibitor was included as indicated. Activity is expressed as the concentration of the released fluorophore (free AFC, μM). Data are shown as the mean ± SEM of triplicates from one experiment representative of two independent experiments. Lines represent the result of linear regression. The scale for CatB and CatL activity is smaller to highlight any differences between the activity of WT and GILT^−/− lysates. Comparison of the slopes by ANCOVA revealed a significant difference in CatS (p < 0.0001) and CatL (p < 0.01) activity between WT and GILT^−/− B-cell lysates.