Jos Jansen

3 75 TMEM199 Deficiency is characterized by elevated liverenzymes, hypercholesterolemia and abnormal glycosylation We performed ultrastructural analysis of liver tissue from individual F2-II2, directly fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer. Hepatocytes were enlarged with cytoplasmic degeneration. Severe vacuolization was observed, as well as the presence of large lipid vacuoles, in agreement with optical microscopy analysis (Figure 2D). A marked dilation and vesiculation of the Golgi and/or ER was seen (Figure 2E). In addition, mitochondria featured fragmented cristae, an altered inner matrix appearing as a granulo-filamentous material, and sometimes included electron-dense crystal-like structures (Figure 2F). All individuals were identified during routine metabolic diagnostics with abnormal glycosylation of serum Tf in CDG screening. Tf IEF showed increased disialo- and trisialotransferrin (see Figure 3A and Table S1 for quantification and ranges), indicating abnormal N-glycosylation. In addition, IEF of serum apolipoprotein C-III (ApoC-III) for analysis of mucin-type O-glycosylation showed an abnormal ApoC-III-1 profile for families F1 and F3 (see Figure 3B and Table S2 for quantification and ranges). To gain further insight into the aberrant N-glycan structures, we performed nanochip-C8 QTOF mass spectrometry of the intact Tf protein, which showed an accumulation of incompletely synthesized glycans lacking galactose and sialic acid for individual F1-II2, in comparison to the spectrum of a healthy individual (Figure 3C, peaks 2–6). Mass spectrometry of total plasma N-glycans showed a similar pattern, with an increase of glycans lacking galactose and sialic acid (Figure S1). Individuals with ATP6V0A2 deficiency show very similar glycosylation abnormalities of Tf, apoC-III, and total plasma N-glycans (Figures 3A and 3B).(11) Next, we labeled sialic acids with alkyne-tagged synthetic sugar analogs, as reported before.(21) These sialic acid precursors (alkyne-tagged mannosamine [ManNAl]) are incorporated into nascent glycoproteins by the cell, allowing specific detection with fluorescently labeled azides. The immunofluorescent intensity of the azides is a marker for Golgi glycosylation efficiency. As in previous studies, the main pool of labeled glycoproteins located to the Golgi apparatus in healthy control fibroblasts (Figure 3E).(21) Quantification of the immunofluorescence signal with TISGolgi, an ImageJ plugin, showed a clear reduction of absolute Golgi fluorescence intensity in individuals F1-II2, F1-II3, and F2-II2 (Figures 3D and 3E). Subsequently, fibroblasts from individual F1-II2 were complemented by transduction with either a pLenti6.2-GFP-V5 (mock) or a pLenti6.2-TMEM199-V5 (wild-type TMEM199 ) vector. .