Journal: The Journal of Experimental Medicine

Article Title: Identification of α-galactosylceramide as an endogenous mammalian antigen for iNKT cells

doi: 10.1084/jem.20240728

Figure Lengend Snippet: Serum contains antigens for iNKT cells. (A) DN32.D3 cells were co-cultured with 5.5 × 10 2 , 1.66 × 10 3 , 5.0 × 10 3 and 1.5 × 10 4 parental or CD1d-transduced MC38, LLC1, B16F10, 2B4, EL4, DN32.D3, HEK293T, HeLa, A549, MDA-MB-231, and PANC-1 cells for 16 h and analyzed for CD69 expression. (B) 5 × 10 5 CD1d −/− or CD1d-transduced B16F10 cells were injected subcutaneously into the right flank of C57BL/6J mice ( n = 8). Tumor volume was measured every 3–4 days. (C) DN32.D3 cells were co-cultured with the indicated cell number of WT or Ugcg −/− Ugt8a −/− CD1d-transduced B16F10 cells for 16 h and analyzed as in A (left). Concentrations of IL-2 in the supernatants were measured (right). (D) CD1d-transduced B16F10 cells were cultured in RPMI 1640 supplemented with 10% FCS or in RPMI 1640 with 0.6% FCS and 9.4% animal component-free cell culture supplement for 7 days. DN32.D3 cells were then co-cultured with those B16F10 cells for 16 h and analyzed as in A. (E) DN32.D3 cells were co-cultured with CD1d-transduced B16F10 cells that were cultured in RPMI 1640 supplemented with 0.6% FCS and 9.4% animal component-free cell culture supplement for 7 days as in D in the absence or presence of α-GalCer (t18:0/26:0) (KRN7000) for 16 h and analyzed as in A. (F) Lipids extracted from serum were separated into seven fractions by open column chromatography and analyzed by HPTLC using C:M:W (65:25:4; vol/vol/vol) followed by staining with copper acetate reagent. Commercial β-GlcCer was used as a reference (right lanes). Open and closed arrowheads denote the origin and solvent front, respectively. (G) CD1d −/− or CD1d-transduced DN32.D3 cells were stimulated with each fraction separated from serum lipids in F for 16 h and analyzed as in A. α-GalCer (t18:0/26:0) was used as a positive control. (H) CD1d-transduced DN32.D3 cells were stimulated with the C:M = 19:1 fraction of serum lipids with or without hydrolysis treatment for 16 h and analyzed as in A. α-GalCer (t18:0/26:0) was used as a positive control. (I) CD1d-transduced DN32.D3 cells were stimulated with the C:M = 19:1 fraction of serum lipids treated with Gba (left) or Galc (right) for 16 h and analyzed as in A. α-GalCer (t18:0/26:0) was used as a positive control. Data are presented as mean ± SD (A–E and G–I) and are representative of three independent experiments (A–I). Statistical significance was determined by Student’s t test. *, P < 0.05. Source data are available for this figure: .

Article Snippet: N-stearoyl-D-erythro-sphingosine was from Katayama Chemical Industries Co. Recombinant human glucosylceramidase and recombinant human Galc were from R&D Systems.

Techniques: Cell Culture, Expressing, Injection, Column Chromatography, High Performance Thin Layer Chromatography, Staining, Solvent, Positive Control

Journal: The Journal of Experimental Medicine

Article Title: Identification of α-galactosylceramide as an endogenous mammalian antigen for iNKT cells

doi: 10.1084/jem.20240728

Figure Lengend Snippet: Serum contains antigens for iNKT cells. (A) Surface expression of CD1d on CD1d-transduced cell lines. Filled histogram, anti-mouse CD1d antibody; open histogram, isotype control antibody. (B) WT or TCRα −/− DN32.D3 cells were co-cultured with CD1d-transduced B16F10 cells for 16 h and analyzed for CD69 expression. (C) CD1d −/− or CD1d-transduced B16F10 cells were seeded onto 24-well plates. Growth curves were generated using cell counting by flow cytometer every 24 h. (D) 5 × 10 5 CD1d −/− or CD1d-transduced B16F10 cells were injected subcutaneously into the right flank of Jα18-deficient mice ( n = 7). Tumor volume was measured every 3–4 days. (E) The crude lipids extracted from WT, Ugcg −/− , Ugt8a −/− , and Ugcg −/− Ugt8a −/− B16F10 cells were analyzed by HPTLC using C:M:W (65:25:4; vol/vol/vol) and stained with copper acetate reagent. (F) Lipid extracts from B16F10 cells (5 × 10 6 ) were separated into 84 fractions in a 96-well plate by LC-FRC system and evaporated. DN32.D3 cells were stimulated in the 96-well plate for 16 h and analyzed for CD69 expression. Fractionation was performed in triplicate. (G) The C:M = 19:1 fraction of serum lipids before and after hydrolysis treatment was analyzed by HPTLC as in E. (H) Commercial α- and β-GalCer (d18:1/16:0) (left) and α- and β-GalCer (d18:1/24:1) (right) were treated with Galc and analyzed by HPTLC as in E. (I) The C:M = 19:1 fraction of serum lipids and commercial β-GlcCer or β-GalCer were treated with Gba (left) or Galc (right) and analyzed by HPTLC as in E. (J) Screening of columns to separate three diastereomers of synthesized HexCer (d18:1/16:0). MRM chromatograms of SFC/MRM analysis using the columns in are shown. The MRM transition was set to 700.57 > 264.27 (precursor ions selected as [M+H] + ). The SFC analysis conditions for 1-AA, 2-PC, BEH 2-EP, BEH, DEA, Diol, P4VP (PEEK), and PTZ (PEEK) (left) were as follows: column temperature, 50°C; mobile phase A, supercritical carbon dioxide; mobile phase B, M:W (95:5, vol/vol) with 0.1% (wt/vol) ammonium acetate; flow rate of mobile phase, 1.0 ml min −1 ; flow rate of make-up pump, 0.1 ml min −1 ; back-pressure regulator, 10 MPa. The gradient conditions were as follows: 1% B, 0–1 min; 1–75% B, 1–24 min; 75% B, 24–26 min; and 1% B, 26–30 min. The SFC analytical conditions for other columns (center and right) were as described above with modification as follows: column temperature, 40°C; gradient conditions, 1% B, 0–1 min; 1–50% B, 1–17 min; 50% B, 17–26 min; and 1% B, 26–30 min. The MRM operating conditions were identical to those of the SFC/MRM analysis method. The colored shadows indicate the peaks coincident with the RT of synthesized α-GalCer (red), α-GlcCer (blue), β-GlcCer (green), and β-GalCer (yellow), respectively. Open and close arrowheads denote the origin and solvent front, respectively (E and G–I). Data are presented as mean ± SD (B–D and F) and are representative of three independent experiments (B–E and G–J). Statistical significance was determined by Student’s t test (C and D). NS, not significant. Source data are available for this figure: .

Article Snippet: N-stearoyl-D-erythro-sphingosine was from Katayama Chemical Industries Co. Recombinant human glucosylceramidase and recombinant human Galc were from R&D Systems.

Techniques: Expressing, Control, Cell Culture, Generated, Cell Counting, Flow Cytometry, Injection, High Performance Thin Layer Chromatography, Staining, Fractionation, Synthesized, Modification, Solvent

Journal: bioRxiv

Article Title: Expression study of Krabbe Disease GALC missense variants – Insights from quantification profiles of residual enzyme activity, secretion and psychosine levels

doi: 10.1101/2024.10.17.618938

Figure Lengend Snippet: The schematic diagram shows that distribution of clinically-relevant missense mutation variants (MMVs), common polymorphic variants (green) and substrate binding site residues (red) on the GALC protein. The main structural domains of GALC are indicated: signal peptide (SP), TIM barrel domain, β-sandwich domain and lectin-binding domain. MMVs predicted to affect substrate binding are labeled purple color. Potential N-glycosylation sites are marked with asterisks.

Article Snippet: Standard curve samples were prepared by diluting recombinant human GALC protein (R&D Systems) in culture supernatant from KO cells to 0.8, 1.6, 3.2, 6.3, 12.5, 25 and 50 ng/ml.

Techniques: Mutagenesis, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Expression study of Krabbe Disease GALC missense variants – Insights from quantification profiles of residual enzyme activity, secretion and psychosine levels

doi: 10.1101/2024.10.17.618938

Figure Lengend Snippet: (A) Human GALC gene sequences (red) adjacent to PAM sites (bolded) were targeted for Cas9-mediated cleavage by the sgRNA vectors. A downstream nonsense mutation (asterisk) is introduced upon successful targeted deletion (highlighted yellow). (B) Confirmation of targeted deletion of the 85 bp region in the KO cell line by PCR amplification, compared to control WT cells. (C) Western blot analysis of GALC and GAPDH proteins in native MO3.13 cells (WT), GALC-KO cells (KO) and GALC-overexpressing cells (OE). (D) GALC activity and (E) psychosine levels in WT and KO cells. Statistical significance is determined using an unpaired t-test (n=4, 95% confidence interval, ** P < 0.01, *** P < 0.001).

Article Snippet: Standard curve samples were prepared by diluting recombinant human GALC protein (R&D Systems) in culture supernatant from KO cells to 0.8, 1.6, 3.2, 6.3, 12.5, 25 and 50 ng/ml.

Techniques: Mutagenesis, Amplification, Control, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: Expression study of Krabbe Disease GALC missense variants – Insights from quantification profiles of residual enzyme activity, secretion and psychosine levels

doi: 10.1101/2024.10.17.618938

Figure Lengend Snippet: Correlations among (A) GALC activity, (B) sec-GALC levels and (C) pre-GALC protein levels in the GALC MMV expressing cells are analyzed using the Pearson correlation method. (A) GALC activity is significantly correlated with sec-GALC levels in the cell models (Pearson r = 0.5, P < 0.01, n = 37). No significant correlations are found between (B) GALC activity and pre-GALC levels, and between (C) sec-GALC and pre-GALC levels.

Article Snippet: Standard curve samples were prepared by diluting recombinant human GALC protein (R&D Systems) in culture supernatant from KO cells to 0.8, 1.6, 3.2, 6.3, 12.5, 25 and 50 ng/ml.

Techniques: Activity Assay, Expressing