Jos Jansen

3 77 TMEM199 Deficiency is characterized by elevated liverenzymes, hypercholesterolemia and abnormal glycosylation ◄ Figure 3. Glycosylation Abnormalities in TMEM199-Deficient Individuals (A) Glycosylation screening by IEF of serum Tf. The accompanying numbers represent the total number of sialic acids in the different protein isoforms. Representative N-glycosylation profiles from a healthy control individual, an ATP6V0A2-deficient individual, and TMEM199-deficient individuals are shown. (B) Glycosylation screening by IEF of serum ApoC-III. ApoC-III has one mucintype O-linked glycan with one or two sialic acids. For normal ranges and quantification of Tf-IEF and ApoC-III-IEF, see Tables S1 and S2. (C) The nanochip-C8 QTOF mass spectrum of a healthy control individual (left spectrum) and individual F1-II2 (right spectrum) are shown. Peak 1 (79,556 amu) represents the intact Tf protein with two attached complete glycans. Any subsequent loss of sialic acid and/or galactose can be calculated based on the mass difference with the main peak (i.e., loss of one sialic acid [purple diamond, peak 2]). For glycan structural annotation of peaks 1 to 6, see Table S3. (D–E) Fibroblasts from healthy control individuals and individuals F1-II2, F1-II3, and F2-II2 were incubated with ManNAl for metabolic labeling of sialic acids. The fluorescent signal indicates incorporation of sialic acids in glycoconjugates. Three healthy control fibroblasts were pooled from experiments performed twice. Scale bars indicate 75 μ m. (F) Fibroblasts of individual F1-II2 were transduced with an empty vector or TMEM199 wild-type construct and incubated with ManNAl, similarly to (D) and (E). Scale bars indicate 50 μ m. As shown in Figure 3F, the overall Golgi fluorescence intensity increased after transduction with wildtype TMEM199 but not with the mock vector. Together, these data confirm a generalized defect in Golgi processing of protein-linked glycans due to deficiency of TMEM199 Our study indicated TMEM199 as the human homolog of yeast V-ATPase assembly factor Vph2p. In yeast, V-ATPase assembly factors Vph2p, Vma21p, Pkr1p, and Vma22p are localized to the ER.(17) To define the subcellular location of TMEM199, a pLenti6.2-TMEM199-V5 plasmid for expression of C-terminally V5-tagged proteins was transiently overexpressed in HeLa cells. Imaging was done with a Leica SP8 (Leica Microsystems) confocal microscope with 603 water immersion 1.2 NA objective. Figure 4 shows clear co-localization of V5-tagged TMEM199 with ER-Golgi intermediate compartment (ERGIC) and coat protein complex I (COPI) vesicle markers. In addition, partial co-localization was seen with giantin, a Golgi marker, but not with markers for ER or COPII vesicles. On the basis of these results, we conclude that TMEM199 predominantly localizes to the ER-to-Golgi region, but not to the ER. In parallel with identification of TMEM199 mutations, we identified mutations in CCDC115 ina cohort of individualswithabnormal Golgi glycosylationanda storage disease-like clinical phenotype including hepatosplenomegaly, neurological symptoms, elevated ATs, alkaline phosphatase, and hypercholesterolemia (see the related paper in this issue of AJHG).(22) CCDC115 was predicted to be homologous to yeast V-ATPase assembly factor Vma22p. In yeast, Vph2p and Vma22p are localized to the ER and cooperatively stabilize the V0 domain during early assembly of the V-ATPase in the ER.(16,17) The human V-ATPase proton pump consists of 14 subunits divided between the membranebound V0 domain and a cytosolic V1 domain.(13) It participates in the acidification of the