CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation

Abstract

Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal glycosylation in plasma.

Keywords: Golgi homeostasis; V-ATPase assembly; Vma22p; alkaline phosphatase; glycosylation; hepatosplenomegaly.

Figure 1

Pedigrees and Overview of the Structure, Variants, and Conservation of CCDC115 (A) Pedigrees and chromatograms of families F1 to F5 are shown. Partial chromatograms show autosomal-recessive segregation for all families. For family F3, DNA for parents and the healthy sibling was not available. For affected individual F4-II1, DNA was unavailable. The asterisk indicates the respective nucleotide change. (B) Schematic representation of the intron-exon structure and homology of CCDC115. The red lines indicate the positions of the missense mutations and substitutions within the families. The green regions indicate the two predicted coiled-coil domains (CC1 and CC2).

Figure 2

CCDC115-Deficient Individuals Have Abnormal Golgi Glycosylation (A) IEF of serum Tf (left) and serum ApoC-III (right). For individual F2-II1, HPLC was used to assess Tf glycosylation status. Reference ranges and quantifications are shown in Tables S5 and S6. (B) MALDI-LTQ mass spectrometry profiles of total serum N-glycans of a representative healthy control individual and of individual F1-II1. An increase in hypoglycosylated glycans with loss of sialic acid (purple diamond) and galactose (yellow dot) can be seen for individual F1-II1. (C) For individual F1-II1 and his unaffected mother, nanochip-C8 QTOF mass spectra are shown for the intact Tf protein (including two attached glycans) at 79,555 amu (peak 1). Any subsequent loss of sialic acid and/or galactose can be calculated on the basis of mass difference with the main peak. Individual F1-II1 shows a reduction in sialic acid and galactose residues (peaks 2–8, see Table S7 for glycan structures). m/z, mass-to-charge ratio; amu, atomic mass units.

Figure 3

Metabolic Labeling of Sialic Acids Shows Decreased Glycosylation in CCDC115-Deficient Fibroblasts (A and B) Metabolic labeling of fibroblasts with alkynyl-tagged sialic acid precursor ManNAl for 8 hr. Fibroblasts from three healthy controls were used, and the experiment was performed twice. A reduced absolute Golgi fluorescence signal was observed for siblings F1-II1 and F1-II2. Scale bars indicate 75 μm. (C and D) Fibroblasts of F1-II4, transfected with empty vector or wild-type CCDC115, were incubated with ManNAl for 6 hr, followed by fluorescent staining. The graphs indicate the absolute Golgi fluorescence intensity in a.u. Scale bars indicate 50 μm. (E) Healthy control fibroblasts and fibroblasts from CCDC115-deficient individual F2-II1 were stained with anti-calnexin antibody. The graph shows the percentage of cells with a dilated ER. Approximately 50 cells were counted, and the experiment was performed twice. The graph shows the percentage of cells (mean ± SEM) with a dilated ER. Scale bars indicate 10 μm. N.D., not detectable.

Figure 4

CCDC115-V5 Is Located in the ER-to-Golgi Region HeLa cells were transiently transfected with a V5-tagged CCDC115 construct and then fixed and stained with immunofluorescently labeled antibodies against V5 (green in merge) and different organelle markers (magenta in merge). Shown are representative cells stained for CCDC115-V5, organelle markers, and a merge with DAPI stain (blue), including a 3-fold magnification. Co-localization is indicated by white color in the merged channel. The graphs show the fluorescence intensity profiles along the cross-sections indicated. Scale bars represent 5 μm.