anti pmm2  (Proteintech)

Journal: Frontiers in Immunology

Article Title: Immunopathology in PMM2-CDG: Defective glycosylation impact in the TNFα -TNFR1 signalling pathway

doi: 10.3389/fimmu.2025.1655354

Figure Lengend Snippet: TNFR cell phenotyping in stimulated and non-stimulated PMM2-CDG and control fibroblasts. Fibroblasts were stimulated with 10 ng/ml of TNF-α for 24h and analysed using different techniques. (A) Western blot analysis of the PMM2 protein in PMM2-CDG and WT skin fibroblasts. Immunoblotting of cell lysates from three PMM2-CDG patients and three controls fibroblasts was performed using anti-PMM2 (Proteintech) at 1:1000. Mouse monoclonal anti-α-tubulin staining was performed as loading control; (B) Flow cytometry was performed for TNFR1 cell surface staining and (C) Western blot of cell lysates was performed for TNFR1 immunoblotting to access the overall expression of TNFR1. Staining with α-tubulin was used as loading control. The patient and control samples shown correspond to P1 and C1, respectively, as listed in Table 1 . (D) Ratio of TNFRI shedding (soluble form) to membrane bound TNFR1 in non-stimulated and stimulated conditions in fibroblasts from PMM2 patients and healthy controls. Each data point represents an individual cell line ( Table 1 ). p1: patient 1, p2: patient 2, p3: patient 3, c1: control 1, c2: control 2, c3: control 3. Data were confirmed for a normal distribution using a Normal Q-Q plot and Shapiro- Wilk test. Statistical comparisons were performed using an unpaired t-test; no statistically significant differences were found. Bars represent the mean ± standard deviation.

Article Snippet: Membranes were blocked with 5% non-fat dried milk and immunoblotting was carried out with antibodies against PMM2 (1:1000, 10666-1-AP, Proteintech), p38 (1:1000, sc-728, Santa Cruz Biotechnology), p-p38 (1:1000, #9211, Cell Signalling), ERK1/2 (1:1000, #9102, Cell Signalling), p-ERK1/2 (1:1000, #9101, Cell Signalling), IκBα (1:600, sc-371, Santa Cruz Biotechnology) and JNK2 (1:1000, #9258, Cell Signalling) overnight, followed by 1h incubation with the Peroxidase AffiniPure donkey anti-rabbit IgG (H+L) (1:10000, #711-035-152, Jackson Laboratories) secondary antibody.

Techniques: Control, Western Blot, Staining, Flow Cytometry, Expressing, Membrane, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Immunopathology in PMM2-CDG: Defective glycosylation impact in the TNFα -TNFR1 signalling pathway

doi: 10.3389/fimmu.2025.1655354

Figure Lengend Snippet: N-glycoprofiling in stimulated and non-stimulated PMM2-CDG and control fibroblasts. PMM2-CDG and control fibroblasts were stimulated (S) or non-stimulated (NS) with TNF-α (10 ng/ml) for 24h and analysed using different techniques. (A) Lectin staining was performed resorting to the ConA and GNL lectins and analysed by flow cytometry. Data are represented as the mean ± SD of normalized mean fluorescence intensity (MFI) values to the WT non-stimulated group (n = 3); (B) N-glycosylation biosynthetic pathways with emphasis on the N-glycan structures identified in this study. N-glycan synthesis begins in the cytosolic side of the endoplasmic reticulum (ER) with the attachment of N-acetylglucosamine (GlcNAc) to dolichol monophosphate. This is followed by the addition of one GlcNAc and five mannose residues, using a nucleotide-activated sugar, guanosine diphosphate-mannose (GDP-Man), as the donor. The PMM2 enzyme plays a critical role in the in GDP-Man biosynthesis, by converting mannose-1-phosphate in mannose-6-phosphate. The lipid precursor is then translocated by a flippase to the lumen of the ER, where it undergoes further elongation. Once the oligosaccharide (Glc3Man9GlcNAc2) is completed, it is transferred to an asparagine residue of the nascent protein. The processing of N-glycans is initiated in the ER and continues in the Golgi apparatus, resulting in the formation of various N-glycans forms, such as high mannose, galactosylated, early terminated, fucosylated agalactosylated bi-antennary and hybrid N-glycans. Image created using BioRender ( www.biorender.com ); (C) Semi-quantitative representation of four main N-glycan groups based on their type (high mannose; galactosylated and early terminated; hybrid; and other glycans) determined by the sum of relative intensities of individual structures by MALDI-TOF mass spectrometry; (D) relative distribution of individual high mannose N-glycans (Man3GlcNAc2-Man10GlcNAc2) determined by MALDI-TOF mass spectrometry; and (E) relative distribution of individual galactosylated and early terminated N-glycans determined by MALDI-TOF mass spectrometry. Data from mass spectrometry analysis is based on four technical replicates, except stimulated PMM2-CDG fibroblasts that were measured in triplicate due to limited sample amount. p < 0.05 (*), p < 0.01 (**). green circle – mannose, yellow circle – galactose, blue square – N-acetylglucosamine, red triangle – fucose, purple diamond – sialic acid.

Article Snippet: Membranes were blocked with 5% non-fat dried milk and immunoblotting was carried out with antibodies against PMM2 (1:1000, 10666-1-AP, Proteintech), p38 (1:1000, sc-728, Santa Cruz Biotechnology), p-p38 (1:1000, #9211, Cell Signalling), ERK1/2 (1:1000, #9102, Cell Signalling), p-ERK1/2 (1:1000, #9101, Cell Signalling), IκBα (1:600, sc-371, Santa Cruz Biotechnology) and JNK2 (1:1000, #9258, Cell Signalling) overnight, followed by 1h incubation with the Peroxidase AffiniPure donkey anti-rabbit IgG (H+L) (1:10000, #711-035-152, Jackson Laboratories) secondary antibody.

Techniques: Control, Staining, Flow Cytometry, Fluorescence, Glycoproteomics, Residue, Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: Immunopathology in PMM2-CDG: Defective glycosylation impact in the TNFα -TNFR1 signalling pathway

doi: 10.3389/fimmu.2025.1655354

Figure Lengend Snippet: Differential gene expression and functional annotation analysis of TNF-α stimulated and non-stimulated samples of control and PMM2-CDG samples. (A) Venn diagram of control vs PMM2-CDG DEGs upon TNF-α stimulus. A total of 305 (239 upregulated and 66 downregulated) and 222 DEGs (188 upregulated and 34 downregulated) were identified in the control and PMM2-CDG sample groups, respectively. These DEGs can be classified into three distinct groups: Control-exclusive, Common, and PMM2-CDG-exclusive. There is a total of 122 DEGs in the control-exclusive group, of which 80 are upregulated and 42 are downregulated. The Common group contains 183 DEGs, 159 upregulated and 24 downregulated. The PMM2-CDG-exclusive group comprises 39 DEGs, of which 29 are upregulated and 10 are downregulated. (B) Fold enrichment plot from the upregulated control and PMM2-CDG sample groups of the GO terms within the Biological Process domain. Blue dots represent the GO terms which are statistically significant, while the red dots comprise the GO terms which are not statistically significant. The size of the dots comprehends the number of DEGs which are inserted in the pathway of the GO term. (C) Heatmap of the log2FC of signalling, adaptor and regulatory proteins involved in TNFR1 signalling. (D) Log2 Fold-Change (FC) of differently expressed receptors and ligands upon TNF-α stimulus in PMM2-CDG and control skin fibroblasts, annotated with a prediction of cell–cell interactions with immune cells. These Log2FC are statistically significant, with False Discovery Rate (FDR) < 0.05 (*), FDR < 0.01 (**) or FDR < 0.001 (***). The receptor and ligands represented include common DEGs between PMM2-CDG and control samples (significant in both conditions), control-exclusive (only significant in control) and PMM2-CDG DEGs (only significant in PMM2) upon TNF-α stimulation. Different colours were used to distinguish between immune cell types.

Article Snippet: Membranes were blocked with 5% non-fat dried milk and immunoblotting was carried out with antibodies against PMM2 (1:1000, 10666-1-AP, Proteintech), p38 (1:1000, sc-728, Santa Cruz Biotechnology), p-p38 (1:1000, #9211, Cell Signalling), ERK1/2 (1:1000, #9102, Cell Signalling), p-ERK1/2 (1:1000, #9101, Cell Signalling), IκBα (1:600, sc-371, Santa Cruz Biotechnology) and JNK2 (1:1000, #9258, Cell Signalling) overnight, followed by 1h incubation with the Peroxidase AffiniPure donkey anti-rabbit IgG (H+L) (1:10000, #711-035-152, Jackson Laboratories) secondary antibody.

Techniques: Gene Expression, Functional Assay, Control

Journal: Frontiers in Immunology

Article Title: Immunopathology in PMM2-CDG: Defective glycosylation impact in the TNFα -TNFR1 signalling pathway

doi: 10.3389/fimmu.2025.1655354

Figure Lengend Snippet: Illustration of the proposed deregulated signalling, adaptor and regulatory proteins involved in TNFR1 signalling due to defective N-glycosylation and TNFR1 shedding. Downregulated expression is shown in red, upregulated expression is shown in green. The proteins whose expression does not differ between control and PMM2-CDG or where is no indication of their expression are highlighted in yellow. For the proteins where differences were identified or inferred (down or upregulated in PMM2-CDG), when the levels of expression are indicated by only gene expression levels, these are outlines by a dotted border; when the expression levels were identified by protein expression, these are outlined by a continuous border. Expression levels confirmed by both gene and protein levels are in bold. AP-1, Activator protein 1; ASK1, Apoptosis-stimulated kinase 1; CASP8, caspase 8; CCL, Chemokine C-C motif ligand; cFLIP, cellular FLICE inhibitory protein; cIAP, Cellular inhibitor of apoptosis protein; CXCL, C-X-C motif chemokine ligand; ERK, Extracellular signal-regulated kinase; FADD, FAS-associated death domain; FOS(L), Fos Proto-Oncogene (like); IKKα/β, Inhibitory kappa B kinases alpha/beta; IL, Interleukin; ITCH, ubiquitin E3 ligase ITCH, also named atrophin-1 interacting protein 4; IκBα/β, Nuclear factor kappa-light-chain-enhancer of activated B cells inhibitor alpha (/beta); JNK, c-Jun N-terminal kinase; MEK/MKK, Mitogen-activated protein kinase kinase; MLKL, Mixed lineage kinase domain-like protein; NEMO, Nuclear factor kappa-light-chain-enhancer of activated B cells essential modulator; RIPK, Receptor-interacting serine/threonine-protein kinase; TAB, TAK1-binding protein; TAK, Transforming growth factor-β (TGFβ)-activated kinase; TNF(R), Tumour necrosis factor (receptor); TRADD, TNF receptor type 1-associated death domain; TRAF, TNF receptor associated factor. Several abbreviations used in the figure are not described in the text and are shown in grey. Image created using BioRender ( www.biorender.com ).

Article Snippet: Membranes were blocked with 5% non-fat dried milk and immunoblotting was carried out with antibodies against PMM2 (1:1000, 10666-1-AP, Proteintech), p38 (1:1000, sc-728, Santa Cruz Biotechnology), p-p38 (1:1000, #9211, Cell Signalling), ERK1/2 (1:1000, #9102, Cell Signalling), p-ERK1/2 (1:1000, #9101, Cell Signalling), IκBα (1:600, sc-371, Santa Cruz Biotechnology) and JNK2 (1:1000, #9258, Cell Signalling) overnight, followed by 1h incubation with the Peroxidase AffiniPure donkey anti-rabbit IgG (H+L) (1:10000, #711-035-152, Jackson Laboratories) secondary antibody.

Techniques: Glycoproteomics, Expressing, Control, Gene Expression, Ubiquitin Proteomics, Binding Assay

Journal: Molecular genetics and metabolism

Article Title: HepG2 PMM2-CDG knockout model: A versatile platform for variant and therapeutic evaluation.

doi: 10.1016/j.ymgme.2024.108538

Figure Lengend Snippet: Fig. 1. Generation of the PMM2-CDG knockout model in HepG2 cells. (A) Nucleotide sequence of PMM2 exon 5 and sequences of the two guide RNA specifically designed for the knockout of PMM2, gRNA1 and gRNA2. The PAM sequences are highlighted in green, and the codons that codify crucial aminoacids for PMM2 function (F119, R123, R134 and R141) are indicated in red. (B) Immunodetection of PMM2 in the selected cell populations, from 1 to 15. The number between brackets indicate the gRNA used for their edition, gRNA1 (1) or gRNA2 (2). Same amounts of total protein from the soluble extracts were loaded onto SDS-PAGE gels. GAPDH was used as loading control. (C) Targeted deep sequencing reads of cell population 4 in the Integrative Genomics Viewer (IGV) showing the nucleotide sequence of PMM2 exon 5, the coverage, and the reads with the 16-bp and 22-bp deletions. (D) Relative PMM enzymatic activity of cell populations 4, 9 and 11 expressed as a percentage (%) considering that the enzymatic activity of HepG2 wildtype cells (WT) is the 100%. Data represents the mean ± SD of at least three independent experiments (*** p < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Immunodetections were performed using PMM2 antibody at 1:1000 (10666–1-AP, Proteintech, USA), ICAM-1 antibody at 1:1000 (sc-7891; Santa Cruz Biotechnology, USA), LAMP1 antibody at 1:1000 (3243, Cell signaling, USA), A1AT antibody at 1:500 (sc-59438; Santa Cruz), GAPDH antibody at 1:5000 (ab8245, Abcam, UK) and β-actin antibody at 1:5000 (TA811000S; Origene, USA).

Techniques: Knock-Out, Sequencing, Immunodetection, SDS Page, Control, Activity Assay

Journal: Molecular genetics and metabolism

Article Title: HepG2 PMM2-CDG knockout model: A versatile platform for variant and therapeutic evaluation.

doi: 10.1016/j.ymgme.2024.108538

Figure Lengend Snippet: Fig. 2. Characterization of the HepG2 PMM2 KO. (A) PMM2 mRNA expression levels in the knockout model (KO) relative to the PMM2 mRNA levels in the HepG2 wildtype cells (WT), considered´1′. (B) Representative western blot of ICAM-1, LAMP1, A1AT and PMM2. β-actin (β-ACT) was used as loading control. (C) Repre- sentative western blot of the PNGaseF treatment of the cell extract of treated (+) and untreated (−) HepG2 wildtype (WT) and HepG2-KO (KO) cells. β-actin (β-ACT) was used as loading control. G: glycosylated; PG: partially glycosylated; NG: non-glycosylated; ICAM-1: intercellular adhesion molecule 1; LAMP1: lysosomal associated membrane protein 1; A1AT: alpha-1 antitrypsin. (D) Representative western blot of ICAM-1, LAMP1 and PMM2 in HepG2 WT, KO, in the HepG2-KO cells transduced with the lentiviral vector carrying the PMM2 wildtype cDNA fused with GFP (LV-WT) and in the HepG2-KO cells transduced with the empty GFP lentiviral vector (LV-ᴓ). GAPDH was used as loading control. Same amounts of total protein from the soluble extracts were loaded onto SDS-PAGE gels. (E) Relative PMM activity measured in the cellular extract of HepG2 WT, KO and LV-WT, and expressed as percentage (%) considering that the activity of the LV-WT is 100%. Data represents the mean ± SD of at least three independent experiments (*** p < 0.001). (F) Proliferation of HepG2 WT (solid line, circles), KO (dashed line, squares) and LV-WT (dotted line, triangles) cells measured at 0, 24, 48 and 72 h with the CCK8 assay and expressed as absorbance at 460 nm. Data represents the mean ± SD of at least three independent experiments (* p < 0.05). (G) Number of viable HepG2 WT (solid line, circles), KO (dashed line, squares) and LV-WT (dotted line, triangles) cells at 0, 24, 48 and 72 h in culture. Data represents the mean ± SD of three technical replicates in one experiment.

Article Snippet: Immunodetections were performed using PMM2 antibody at 1:1000 (10666–1-AP, Proteintech, USA), ICAM-1 antibody at 1:1000 (sc-7891; Santa Cruz Biotechnology, USA), LAMP1 antibody at 1:1000 (3243, Cell signaling, USA), A1AT antibody at 1:500 (sc-59438; Santa Cruz), GAPDH antibody at 1:5000 (ab8245, Abcam, UK) and β-actin antibody at 1:5000 (TA811000S; Origene, USA).

Techniques: Expressing, Knock-Out, Western Blot, Control, Membrane, Transduction, Plasmid Preparation, SDS Page, Activity Assay, CCK-8 Assay

Journal: Molecular genetics and metabolism

Article Title: HepG2 PMM2-CDG knockout model: A versatile platform for variant and therapeutic evaluation.

doi: 10.1016/j.ymgme.2024.108538

Figure Lengend Snippet: Fig. 4. Characterization of variants of uncertain significance (VUS) in the PMM2-CDG knockout model. (A) Representative western blot of ICAM-1, LAMP1, PMM2 and A1AT in the soluble extract of HepG2 wildtype (WT), HepG2-KO (KO), HepG2-KO cells transduced with the lentiviral vector carrying the PMM2 wildtype cDNA fused with FLAG (LV-WT), HepG2-KO cells transduced with the FLAG lentiviral vector carrying the nonsense variant p.Arg123* (LV-R123*) and in the HepG2-KO cells transduced with the FLAG lentiviral vector carrying different PMM2 VUS (p.Thr18Ser: LV-T18S; p.Asp65Gly: LV-D65G; p.Thr237Lys: LV-T237K). GAPDH was used as loading control. Same amounts of total protein from the soluble extracts were loaded onto SDS-PAGE gels. (B) Relative PMM activity measured in the cellular extract of HepG2-KO cells transduced with the FLAG lentiviral vector LV-R123* and in the soluble extract of HepG2-KO cells transduced different VUS (LV-T18S, LV- D65G, LV-T237K). The PMM activity is expressed as percentage (%) considering that the activity of the HepG2-KO cells with the lentiviral vector carrying the PMM2 wildtype cDNA is the 100%. Data represents the mean ± SD of at least three independent experiments (*** p < 0.001).

Article Snippet: Immunodetections were performed using PMM2 antibody at 1:1000 (10666–1-AP, Proteintech, USA), ICAM-1 antibody at 1:1000 (sc-7891; Santa Cruz Biotechnology, USA), LAMP1 antibody at 1:1000 (3243, Cell signaling, USA), A1AT antibody at 1:500 (sc-59438; Santa Cruz), GAPDH antibody at 1:5000 (ab8245, Abcam, UK) and β-actin antibody at 1:5000 (TA811000S; Origene, USA).

Techniques: Knock-Out, Western Blot, Transduction, Plasmid Preparation, Variant Assay, Control, SDS Page, Activity Assay

Journal: Molecular genetics and metabolism

Article Title: HepG2 PMM2-CDG knockout model: A versatile platform for variant and therapeutic evaluation.

doi: 10.1016/j.ymgme.2024.108538

Figure Lengend Snippet: Fig. 3. Characterization of clinical variants in the PMM2-CDG knockout model. (A) Relative PMM activity measured in the cellular extract of HepG2-KO cells transduced with the lentiviral vector carrying the PMM2 cDNA with different clinical variants fused with FLAG (LV-FLAG, clear grey) or GFP (LV-GFP, dark grey), and expressed as percentage (%) considering that the activity of the HepG2-KO cells transduced with the lentiviral vector carrying the PMM2 wildtype cDNA is 100%. Data represents the mean ± SD of at least three independent experiments (*** p < 0.001). (B) Representative western blot of ICAM-1, LAMP1, PMM2 and A1AT in the soluble extract of HepG2 WT, KO, KO cells transduced with the FLAG lentiviral vector carrying the PMM2 wildtype cDNA (LV-WT), HepG2-KO cells transduced with the lentiviral vector carrying the nonsense variant p.Arg123* (LV-R123*) and in the HepG2-KO cells transduced with the FLAG lentiviral vector carrying different PMM2 clinical variants (LV-V44 A, LV-D65Y, LV-P113L, LV-F119L, LV-R162W, LV-T237M). GAPDH was used as loading control. Same amounts of total protein from the soluble extracts were loaded onto SDS-PAGE gels.

Article Snippet: Immunodetections were performed using PMM2 antibody at 1:1000 (10666–1-AP, Proteintech, USA), ICAM-1 antibody at 1:1000 (sc-7891; Santa Cruz Biotechnology, USA), LAMP1 antibody at 1:1000 (3243, Cell signaling, USA), A1AT antibody at 1:500 (sc-59438; Santa Cruz), GAPDH antibody at 1:5000 (ab8245, Abcam, UK) and β-actin antibody at 1:5000 (TA811000S; Origene, USA).

Techniques: Knock-Out, Activity Assay, Transduction, Plasmid Preparation, Western Blot, Variant Assay, Control, SDS Page