Journal: Life Science Alliance

Article Title: A biallelic variant in GORASP1 causes a novel Golgipathy with glycosylation and mitotic defects

doi: 10.26508/lsa.202403065

Figure Lengend Snippet: (A, B) Unenhanced T1/T2-weighted and FLAIR transverse images through the bodies of the lateral ventricles on initial MRI at age 5 showing severe, bilateral, and relatively symmetrical confluent hyperintensities in the periventricular white matter with preservation of the U-fibers in subcortical areas (A) and two tiny bilateral periventricular cysts unchanged during follow-up (B). (C) Unenhanced T1/T2-weighted and FLAIR transverse images through the lateral ventricle bodies at age 14, showing progressive reduction of bilateral periventricular hyperintensities and white matter. (D) Sagittal T2-weighted image showing normal appearance of the posterior fossa and cerebellum structures. (E) Family genetic tree and sequencing chromatograms showing segregation of the variant c.1170_1171del in the heterozygous state in healthy individuals and in the homozygous state in the patient. The red asterisk in the patient’s chromatogram indicates the biallelic CA deletion. (F) Structure of the human GRASP65 showing the N-terminal half composed of the GRASP domain (AA15–199) with PDZ1 and PDZ2 subdomains, the GM130 binding domain (AA190–202), and the C-terminal half containing the serine/proline-rich domain (SPR, AA202–440), as well as the location of the identified variant (in red). myr: myristoylated glycine in the N terminus.

Article Snippet: Primary antibodies used were as follows: GRASP65 (NP2-02665, 1:1,000; Novus, and MA5-34658, 1:1,000; Invitrogen), GRASP55 (GORASP2, 10598-1-AP, 1:2,000; Proteintech), GM130 (610823, 1:1,000; BD Biosciences), LAMP-1 (9091S, 1:1,000; Cell Signaling), LAMP-2 (ab25631, 1:500; Abcam), TGN46 (APH500G, 1:1,000; Bio-Rad), GAPDH (60004-1-1g, 1:10,000; Proteintech).

Techniques: Preserving, Sequencing, Variant Assay, Binding Assay

Journal: Life Science Alliance

Article Title: A biallelic variant in GORASP1 causes a novel Golgipathy with glycosylation and mitotic defects

doi: 10.26508/lsa.202403065

Figure Lengend Snippet: (A) Schematic representation of the human GORASP1 gene and genomic DNA chromatograms of the mutations identified in patient’s fibroblasts (b) and in CRISPR/Cas9-generated mutants in RPE cells (c, d) relative to the normal sequence (a). (B) Relative expression of GORASP1 mRNA normalized against GAPDH from two pairs of primers distributed across the transcript (exons 3/4 and exons 6/7), showing that the level of gene expression is not significantly different either between patient and control fibroblasts, or between mutant and WT RPE cells.

Article Snippet: Primary antibodies used were as follows: GRASP65 (NP2-02665, 1:1,000; Novus, and MA5-34658, 1:1,000; Invitrogen), GRASP55 (GORASP2, 10598-1-AP, 1:2,000; Proteintech), GM130 (610823, 1:1,000; BD Biosciences), LAMP-1 (9091S, 1:1,000; Cell Signaling), LAMP-2 (ab25631, 1:500; Abcam), TGN46 (APH500G, 1:1,000; Bio-Rad), GAPDH (60004-1-1g, 1:10,000; Proteintech).

Techniques: CRISPR, Generated, Sequencing, Expressing, Gene Expression, Control, Mutagenesis

Journal: Life Science Alliance

Article Title: A biallelic variant in GORASP1 causes a novel Golgipathy with glycosylation and mitotic defects

doi: 10.26508/lsa.202403065

Figure Lengend Snippet: (A, B, C) Immunocytochemical analysis of GRASP65 (A, C) and GRASP55 (B) in control and patient’s fibroblasts labeled with GM130 (A, B) or giantin (C). Scale bars: 30 μm. (D) Western blot analysis of GRASPs and GM130 normalized on GAPDH in control and patient’s fibroblasts. Note that GRASP65 C-ter is an antibody specific to the C terminus of the protein. GRASP65 FL corresponds to an antibody directed against the whole protein (full length). (E) Analysis of the overall morphology of cis-Golgi based on the area defined by GM130. Each point represents the surface area of the Golgi for a cell analyzed (n = 80–125). Data are presented as the mean ± SEM. * P ≤ 0.05, t test.

Article Snippet: Primary antibodies used were as follows: GRASP65 (NP2-02665, 1:1,000; Novus, and MA5-34658, 1:1,000; Invitrogen), GRASP55 (GORASP2, 10598-1-AP, 1:2,000; Proteintech), GM130 (610823, 1:1,000; BD Biosciences), LAMP-1 (9091S, 1:1,000; Cell Signaling), LAMP-2 (ab25631, 1:500; Abcam), TGN46 (APH500G, 1:1,000; Bio-Rad), GAPDH (60004-1-1g, 1:10,000; Proteintech).

Techniques: Control, Labeling, Western Blot

Journal: Life Science Alliance

Article Title: A biallelic variant in GORASP1 causes a novel Golgipathy with glycosylation and mitotic defects

doi: 10.26508/lsa.202403065

Figure Lengend Snippet: (A) Surface staining of fluorophore-conjugated lectin WGA on non-permeabilized primary fibroblasts showing the reduced glycosylation of surface membranes in patient’s cells, which is rescued upon the re-expression of GRASP65-GFP. Scale bar: 50 μm. Quantification is presented as a percentage of the mean integrated signal density of control values ± SEM. * P ≤ 0.05, one-way ANOVA. (B) Surface staining of fluorophore-conjugated lectin MAAII on non-permeabilized primary fibroblasts showing the reduced glycosylation of surface membranes in patient’s cells, which is rescued upon the re-expression of GRASP65-GFP. Scale bar: 50 μm. Quantification is presented as a percentage of the mean integrated signal density of control values ± SEM. * P ≤ 0.05, one-way ANOVA. (C) Western blot analysis of LAMP-1, LAMP-2, and TGN46 normalized on GAPDH in control and patient’s fibroblasts showing a shift of the bands in patient’s fibroblasts. (D) Two-dimensional electrophoresis of the mucin core1 O-glycosylated apolipoprotein C-III (apoC-III) showing a reduced ratio of sialylation in the patient’s sample. Yellow square = N-acetylgalactosamine; yellow circle = galactose; purple diamond = sialic acid.

Article Snippet: Primary antibodies used were as follows: GRASP65 (NP2-02665, 1:1,000; Novus, and MA5-34658, 1:1,000; Invitrogen), GRASP55 (GORASP2, 10598-1-AP, 1:2,000; Proteintech), GM130 (610823, 1:1,000; BD Biosciences), LAMP-1 (9091S, 1:1,000; Cell Signaling), LAMP-2 (ab25631, 1:500; Abcam), TGN46 (APH500G, 1:1,000; Bio-Rad), GAPDH (60004-1-1g, 1:10,000; Proteintech).

Techniques: Staining, Glycoproteomics, Expressing, Control, Western Blot, Electrophoresis

Journal: Life Science Alliance

Article Title: A biallelic variant in GORASP1 causes a novel Golgipathy with glycosylation and mitotic defects

doi: 10.26508/lsa.202403065

Figure Lengend Snippet: (A) Immunocytochemical analysis of GRASP65 in WT versus mutant RPE cells labeled with giantin. Scale bars: 30 μm. (B) Western blot analysis of GRASP65 normalized on GAPDH in WT versus mutant RPE cells. (C) Analysis of the overall morphology of cis-Golgi based on the area defined by giantin. Each point represents the surface area of the Golgi for a cell analyzed. Data are presented as the mean ± SEM. * P ≤ 0.05, t test. (D) Kinetics of Golgi reconstitution after brefeldin A (BFA) treatment and washout (WO) at different time points. Golgi structure reconstitution was quantified by evaluating the number of objects present in each cell based on the giantin signal. Scale bar: 20 μm. Data are presented as the mean ± SEM. * P ≤ 0.05, two-way ANOVA. (E) Immunocytochemical analysis of metaphases in WT versus mutant RPE cells labeled with pericentrin and α-tubulin showing the overrepresentation of misaligned and polar spindles in the patient’s cells, as well as the increased ratio of prophases, prometaphases, and metaphases. Scale bar: 10 μm.

Article Snippet: Primary antibodies used were as follows: GRASP65 (NP2-02665, 1:1,000; Novus, and MA5-34658, 1:1,000; Invitrogen), GRASP55 (GORASP2, 10598-1-AP, 1:2,000; Proteintech), GM130 (610823, 1:1,000; BD Biosciences), LAMP-1 (9091S, 1:1,000; Cell Signaling), LAMP-2 (ab25631, 1:500; Abcam), TGN46 (APH500G, 1:1,000; Bio-Rad), GAPDH (60004-1-1g, 1:10,000; Proteintech).

Techniques: Mutagenesis, Labeling, Western Blot

Journal: Life Science Alliance

Article Title: A biallelic variant in GORASP1 causes a novel Golgipathy with glycosylation and mitotic defects

doi: 10.26508/lsa.202403065

Figure Lengend Snippet: (A) Immunocytochemical analysis of GRASP65 in WT versus mutant RPE cells labeled with either giantin or GM130. Note that mutant 2 results in a Golgi-localized protein truncated at its C terminus as only the GRASP65 FL antibody can detect it. Scale bars: 30 μm. (B) Western blot analysis of GRASP65 normalized on GAPDH in WT versus CRISPR/Cas9-generated mutants in RPE cells. GRASP65 C-ter is an antibody specific to the C terminus of the protein. GRASP65 FL corresponds to an antibody directed against the whole protein (full length). Note that mutant 2 results in a protein truncated at its C terminus. (C) Immunocytochemical analysis of metaphases in WT versus CRISPR/Cas9-generated mutants in RPE cells labeled with pericentrin and α-tubulin showing the multipolar spindles observed in mutant 2. Scale bar: 10 μm. (D) Proliferation index (Ki67/DAPI) and mitotic index (PH3/DAPI) in WT and mutant RPE cells. Data are presented as the mean ± SEM. * P ≤ 0.05, one-way ANOVA. (E) Growth curves of WT versus CRISPR/Cas9-generated mutants in RPE cells, showing the reduced growth capacity of mutant 2 over time as compared to WT and mutant 1 cells. (F) Surface staining of fluorophore-conjugated lectins WGA, MAAII, and SNA on non-permeabilized RPE cells showing the reduced glycosylation of surface membranes in mutant 1 but not in mutant 2. Scale bar: 50 μm. Quantification is presented as a percentage of the mean integrated signal density of control values ± SEM. * P ≤ 0.05, one-way ANOVA.

Article Snippet: Primary antibodies used were as follows: GRASP65 (NP2-02665, 1:1,000; Novus, and MA5-34658, 1:1,000; Invitrogen), GRASP55 (GORASP2, 10598-1-AP, 1:2,000; Proteintech), GM130 (610823, 1:1,000; BD Biosciences), LAMP-1 (9091S, 1:1,000; Cell Signaling), LAMP-2 (ab25631, 1:500; Abcam), TGN46 (APH500G, 1:1,000; Bio-Rad), GAPDH (60004-1-1g, 1:10,000; Proteintech).

Techniques: Mutagenesis, Labeling, Western Blot, CRISPR, Generated, Staining, Glycoproteomics, Control