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. 2016 May 27:7:11600.
doi: 10.1038/ncomms11600.

ATP6AP1 deficiency causes an immunodeficiency with hepatopathy, cognitive impairment and abnormal protein glycosylation

Eric J R Jansen  1 , Sharita Timal  2   3 , Margret Ryan  4 , Angel Ashikov  2   3 , Monique van Scherpenzeel  2   3 , Laurie A Graham  4 , Hanna Mandel  5 , Alexander Hoischen  6 , Theodore C Iancu  7 , Kimiyo Raymond  8 , Gerry Steenbergen  3 , Christian Gilissen  6 , Karin Huijben  3 , Nick H M van Bakel  1 , Yusuke Maeda  9 , Richard J Rodenburg  3   10 , Maciej Adamowicz  11 , Ellen Crushell  12 , Hans Koenen  13 , Darius Adams  14 , Julia Vodopiutz  15 , Susanne Greber-Platzer  15 , Thomas Müller  16 , Gregor Dueckers  17 , Eva Morava  18   19   20 , Jolanta Sykut-Cegielska  21 , Gerard J M Martens  1 , Ron A Wevers  3 , Tim Niehues  17 , Martijn A Huynen  22 , Joris A Veltman  6   23 , Tom H Stevens  4 , Dirk J Lefeber  2   3
Affiliations

ATP6AP1 deficiency causes an immunodeficiency with hepatopathy, cognitive impairment and abnormal protein glycosylation

Eric J R Jansen et al. Nat Commun. .

Abstract

The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1, encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed ∼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function.

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Figures

Figure 1
Figure 1. Overview of identified ATP6AP1 mutations.
(a) Pedigree of index family 1. Black arrow (↑) indicates the index patient P1.1 with the mutation c.1284G>A (p.Met428Ile). (b) ATP6AP1 gene, located on chromosome Xq28, and its gene structure consisting of 10 exons. All mutations identified in the six families are indicated in boxes. Domain structure of Ac45 as published on uniprot.org for human Ac45 (http://www.uniprot.org/uniprot/Q15904; Entry version 146, 07 January 2015). CS, furin proteolytic cleavage site; SP, signal peptide; TM, transmembrane region. Stars (★) indicate the location of the mutations at the protein level. See also Supplementary Fig. 1.
Figure 2
Figure 2. Ultrastructural studies of a liver biopsy of patient 3.1.
(a) A hepatocyte is surrounded by fat globules of variable size having the typical aspect of triglycerides, × 4,000. (b) Hepatocyte showing relative translucency due to proliferated SER. Arrows point to lipofuscin bodies (lysosomes), × 6,000. (c) Higher magnification showing an atypical lipofuscin body with a central reticulate content (black arrow). White arrow: section through a Golgi apparatus, × 10,000. (d) A mitochondrion is engulfed in an autophagosome (arrow). BC, Bile canaliculus, × 12,000.
Figure 3
Figure 3. Glycosylation studies.
(a) Routine screening for N-glycosylation by isofocusing of serum transferrin and for mucin type O-glycosylation by isofocusing of apolipoprotein CIII. The numbers on the y axis mark the number of sialic acids, each column shows the profile of the respective control (C) or patient. (b) Analysis of total serum protein N-glycans by MALDI mass spectrometry. (c) Analysis of intact serum transferrin by nanoLC-chip-QTOF mass spectrometry. Glycans are synthesized from individual monosaccharide building blocks: a purple diamond represents one sialic acid, a yellow circle one galactose, a green circle one mannose, a blue square one N-acetylglucosamine and a red triangle one fucose. See also Supplementary Fig. 3 and Supplementary Tables 2 and 3.
Figure 4
Figure 4. Differential expression of the Ac45 protein in human brain, liver and B cells.
(a) Schematic representation of the human Ac45 protein. CS, furin proteolytic cleavage site; SP, signal peptide; TM, transmembrane domain; formula image represent predicted N-glycan structures, whereas the structures shown in black (formula image) are the experimentally confirmed glycans. (b) Western blot analysis of Ac45 in mouse cortex and in human brain and liver. Asterisk (*) indicates the deglycosylated form of cleaved-Ac45. Hash tags (#) indicate non-specific antibody reaction with PNGaseF present in the samples. (c) Western blot analysis of Ac45 in primary B cells from healthy controls in comparison with human liver. One of the two representative analyses is shown. (d) Western blot of Ac45 in liver tissue homogenates of control and patient 4.2. GapdH was used as loading control. (e) Analysis of newly synthesized Ac45 in immortalized human hepatocytes (IHH). Cells were transfected with Ac45 construct, pulsed for a 30-min period with 35S, and Ac45 was immunoprecipitated and analysed by SDS–PAGE. Cells were treated with or without tunicamycin during the 30-min pulse (left panel). Immunoprecipitated Ac45 protein was treated with or without Endo H or PNGaseF (right panel). Note during the 30-min pulse period, the presence of a minor portion of newly synthesized pre-intact-Ac45 protein is still in its unglycosylated proform and containing the signal peptide for translocation over the ER membrane. (f) IHH cells were stained with anti-Ac45 antibody (green) and antibodies against various organelle markers (magenta). Nuclear staining is shown in blue (DAPI). Co-localization is indicated by a white colour in the merged channel. The graph shows the fluorescent intensity profile along the cross-section indicated. Scale bar represents 10 μm. Staining for Sec31 is shown as example, other organelle markers are shown in Supplementary Fig. 5.
Figure 5
Figure 5. Identification of Voa1 as the yeast ortholog of human Ac45.
(a) Overview of the regions of Ac45 that are homologous to the yeast proteins Voa1 and Big1 and an alignment of Ac45's and Voa1's C-terminal transmembrane helices (in blue, based on TMHMM63) and their flanking amino acids. Ac45 and Voa1 are separated by a sequence logo representation of this region among all the homologs that could be detected using JACKHMMER. A pattern in which the level of sequence conservation in the transmembrane helix peaks every 3–4 amino acids is indicated with arrows. (b) Schematic of Voa1 and Ac45 proteins expressed from centromere plasmids in voa1::H vma21QQ yeast. Ac45 proteins are either full length (intact-Ac45) or processed (cleaved-Ac45), with (shown) or without KKNN appended to the natural C terminus. Numbers indicate amino-acid residues. Residues mutated in Ac45 are shown. (c) Cleaved-Ac45 can substitute for Voa1 when a C-terminal dilysine motif is present. The voa1::H vma21QQ strain was transformed with plasmids coding for the indicated proteins (HA-tagged, diagrammed in (b)), Voa1QQ denotes Voa1 with K262Q and K263Q mutations. (d) The Y313C or E346K mutation in cleaved-Ac45-KKNN reduces V-ATPase function while protein levels are unaffected. Serial dilution growth test of voa1::H vma21QQ yeast expressing the indicated proteins tagged with HA. Restrictive growth is on rich medium adjusted to pH 7.5 and supplemented with 60 mM CaCl2. Membrane proteins prepared from the same cultures used in the growth test were analysed by western blot using anti-HA antibody to detect Voa1, cleaved-Ac45-KKNN and its mutant forms (band locations marked on the right, molecular mass (kDa) is indicated on the left. (e) Voa1 and cleaved-Ac45 require a C-terminal dilysine motif for ER localization. Fluorescent microscopy of live yeast cells showing DAPI stained DNA, GFP, the merged image of both, and cells viewed by differential interference contrast (DIC) to locate the vacuole as apparent indentation. The indicated proteins are N-terminally tagged with HA-GFP and expressed in voa1::H vma21QQ yeast cells. Exposure times for GFP images of cleaved-Ac45 were 10 × longer than for Voa1 or Voa1QQ. Perinuclear GFP fluorescence indicates ER localization. Mutated and non-mutated cleaved-Ac45-KKNN show the same localization. See also Supplementary Fig. 7.
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