Journal: Human Mutation

Article Title: A Novel Missense Variant in Ultrarare SLC35A1-CDG Alters Cellular Glycosylation, Lipid, and Energy Metabolism Without Affecting CDG Serum Markers

doi: 10.1155/humu/6290620

Figure Lengend Snippet: Effect of p.Thr45Ala on SLC35A1 expression and transporter activity. (a) Illustration of the cellular route of Neu5Ac. CMP-Neu5Ac is generated by the bifunctional UDP- N -acetylglucosamine 2-epimerase/ N -acetylmannosamine kinase (GNE) converting UDP-GlcNAc to ManNAc and then to ManNAc-6-phosphate. Next, sialic acid synthase (NANS) and N -acetylneuraminate-9-phosphatase (NANP) convert ManNAc-6-phosphate to Neu5Ac via Neu5Ac-9-phosphate. The final step of CMP-Neu5Ac synthesis occurs in the nucleus, where Neu5Ac is linked to the nucleotide CMP by cytidine monophosphate N -acetylneuraminic acid synthetase (CMAS). CMP-Neu5Ac can also be generated through an alternative synthesis pathway, where sialic acid molecules that have been cleaved from glycoconjugates by the lysosomal neuraminidases NEU1-4 are recycled and subsequently reactivated with CMP in the nucleus. (b) Top: variant c.133A>G of Patient 6 localizes to Exon 2 of the SLC35A1 gene. Bottom: electropherogram (Sanger sequencing) of Exon 2 showing homozygosity for variant c.133A>G; p.Thr45Ala in fibroblasts of Patient 6. (c) Transcript and protein expression determined by qPCR and SLC35A1-ELISA revealed a normal mRNA level but significantly reduced SLC35A1 protein level in the patient's cells. (d) Measurement of the import activity of radioactively labelled sugar substrates into Golgi vesicles isolated from control and patient fibroblasts and normalisation of the CMP-[ 14 C]-Neu5Ac import to the import of control sugars GDP-[ 14 C]-fucose and UDP-[ 14 C]-galactose. (e) Protein structure analysis. Left: visualization was performed by PROTTER (Version 1.0; http://wlab.ethz.ch/protter/start/ ). Highlighted in different colours are variants of the six known SLC35A1-CDG patients and of amino acid residues which are important for substrate binding and activity. Threonine 45 (blue circle) of Patient 6 is predicted to be located at the border of the first luminal loop to the second transmembrane domain. Right: due to the p.Thr45Ala variant (Patient 6), an interhelical H-bond is predicted to get lost. Simulation based on the murine Slc35a1 structure generated by DynaMut (PDB: 6XBO). (f) Cycloheximide (CHX) treatment of SLC35A1 K.o. HEK293 cells expressing the wild-type (WT) or the mutated (p.Thr45Ala) SLC35A1-HA protein revealed a shorter half-life of the mutated SLC35A1. The relative amount of SLC35A1-HA was determined by quantification of the Western blot signals where the start of treatment was set to 100%. The regression lines were used to calculate the protein half-life ( y = 50%).

Article Snippet: Primary antibodies for detection of SLC35A1 (Proteintech, BIOZOL, MyBioSource), ADAMTS13, ATP5H, GP130 (all Thermo Fisher), GAPDH, histone 2B (H2B), H2B-GlcNAc (all Abcam), ICAM1 (Sigma), OGA, OGT, TGN46 (all Proteintech), O-GlcNAcylation (Santa Cruz), and HA-Tag (Sigma) were used in 1:1000 dilution, β -ACTIN (Sigma) in 1:10,000 dilution, and incubated overnight at 4°C under constant movement.

Techniques: Expressing, Activity Assay, Generated, Variant Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Isolation, Control, Binding Assay, Western Blot

Journal: Human Mutation

Article Title: A Novel Missense Variant in Ultrarare SLC35A1-CDG Alters Cellular Glycosylation, Lipid, and Energy Metabolism Without Affecting CDG Serum Markers

doi: 10.1155/humu/6290620

Figure Lengend Snippet: Effect of SLC35A1 deficiency on glycosylation in fibroblasts. (a) Lectin binding studies showed a significant increase of nonsialylated structures (RCAI) and a significant decrease in α -2,3 sialylated glycans (MAL-I) in the patient, whereas the amount of α -2,6 sialylated residues (SNA) in SLC35A1-CDG was comparable to controls. (b) Expression of ICAM1 and GP130 is significantly reduced in the patient's cells (Pat.) compared to controls (Ctrl.). For quantification, the fully glycosylated protein forms (glyco., black arrows) were compared to the hypoglycosylated forms (nonglyco, grey arrows). (c) LC-MS analysis of N-glycans in fibroblasts revealed a deviating amount of individual N-glycans in the patient's cells (indicated by black boxes and arrows).

Article Snippet: Primary antibodies for detection of SLC35A1 (Proteintech, BIOZOL, MyBioSource), ADAMTS13, ATP5H, GP130 (all Thermo Fisher), GAPDH, histone 2B (H2B), H2B-GlcNAc (all Abcam), ICAM1 (Sigma), OGA, OGT, TGN46 (all Proteintech), O-GlcNAcylation (Santa Cruz), and HA-Tag (Sigma) were used in 1:1000 dilution, β -ACTIN (Sigma) in 1:10,000 dilution, and incubated overnight at 4°C under constant movement.

Techniques: Glycoproteomics, Binding Assay, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: Human Mutation

Article Title: A Novel Missense Variant in Ultrarare SLC35A1-CDG Alters Cellular Glycosylation, Lipid, and Energy Metabolism Without Affecting CDG Serum Markers

doi: 10.1155/humu/6290620

Figure Lengend Snippet: Metabolic measurements and GlcNAc supplementation assay in fibroblasts. (a) Measurement of very long-chain fatty acids (VLCFAs) in the lysate of patient fibroblasts normalized to the mean values of the control cell lysate revealed partially increased levels. (b) Staining of lipids in control and patient fibroblasts with Oil Red O presented with an increase in pronounced coloration in the case of the patient's cells indicating an accumulation of neutral lipids. (c) Nanoelectrospray ionization tandem mass spectrometry of cellular lipids revealed significant alterations concerning ceramide (Cer), phosphatidylethanolamine plasmalogen (PE-P), and triacylglycerol (TAG) within the main lipid classes. Abbreviations: CE, cholesteryl ester; Chol, cholesterol; DAG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; Hex2Cer, dihexosylceramide; HexCer, hexosylceramide; LPC, lysophosphatidylcholine; PA, phosphatidic acid; PG, phosphatidylglycerol. (d) Reduced expression of ATP5H combined with a decreased ATP level (e) hint to a general mitochondrial impairment in SLC35A1-CDG. (f) Reduced proliferation due to decreased growth rate and increased doubling time in the patient's cells can be improved by 10 mM GlcNAc as a cell culture supplement.

Article Snippet: Primary antibodies for detection of SLC35A1 (Proteintech, BIOZOL, MyBioSource), ADAMTS13, ATP5H, GP130 (all Thermo Fisher), GAPDH, histone 2B (H2B), H2B-GlcNAc (all Abcam), ICAM1 (Sigma), OGA, OGT, TGN46 (all Proteintech), O-GlcNAcylation (Santa Cruz), and HA-Tag (Sigma) were used in 1:1000 dilution, β -ACTIN (Sigma) in 1:10,000 dilution, and incubated overnight at 4°C under constant movement.

Techniques: Control, Staining, Mass Spectrometry, Expressing, Cell Culture