Binding of purified ETFB to GCDH.

<p>ETFB-His₆ expressed and purified from <i>E. coli</i> was immobilized on Ni-NTA agarose and incubated with extracts from BHK cells overexpressing GCDH-Myc (ETFB+GCDH) for 2 h. Cell extracts overexpressing LC3-GFP were used as negative control (ETFB+LC3). Aliquots of cell extract (input, I: 10% of total), the unbound protein supernatant after precipitation of ETFB-Ni-NTA beads (S: 10%), last wash (W, 25%) and the eluted fraction (E, 100%), containing the bound proteins were separated by SDS-PAGE (10% acrylamide) and analyzed by anti-GCDH and anti-LC3 immunoblotting. The expression of ETFB used for pull-down was analyzed by anti-ETFB western blotting. The image shows representative blots of n = 5 independent experiments.</p

Co-Precipitation of DLST with GCDH.

<p>Extracts from HeLa cells overexpressing the DLST-His₆ alone (DLST) or together with GCDH-Myc (DLST+GCDH) were incubated with Ni-NTA agarose for 4 h. Aliquots of the cell extract (input, I: 10% of total), the unbound protein supernatant after precipitation of Ni-NTA beads (S, 10%), the last wash (W, 25%) and the eluted fraction (E, 100%) representing bound proteins, were analyzed by successively exposing the blot to anti-GCDH and, after stripping, to anti-DLST antibodies. Extracts of HeLa cells overexpressing DLST-His₆ and LC3-GFP (DLST+LC3) were used as negative control and analyzed by anti-LC3 western blotting. The expression of DLST was analyzed by anti-DLST western blotting. The position of the 40 kDa molecular mass marker protein is indicated. The figure shows a representative blot of n = 3 independent experiments.</p

Interaction of GCDH with ETFB <i>in vivo</i>.

<p>(<b>A</b>) Schematic representation of GCDH and ETFB fusion proteins with YFP1 (dark blue) and YFP2 (red). The 10-amino acid linker (GGGGS)₂ is indicated in green. The calculated molecular masses of the respective fusions proteins are shown in brackets. (<b>B</b>) The expression of the various fusion proteins used in this study were analyzed by western blotting. The 43 kDa band (*) reactive with the anti-ETFB antibody is unspecific. (<b>C</b>) Fluorescence microscopy of fixed BHK cells co-expressing GCDH and ETFB fusion proteins showed a strong YFP fluorescence. Nuclei were visualized using DAPI (blue). Scale bars = 40 µM. Representative images of n = 3 independent transfection experiments are shown.</p

Ni-NTA purification of GCDH His₆-fusion protein.

<p>(<b>A</b>) The expression of recombinant GCDH-His₆ in <i>E. coli</i> was induced by the addition of IPTG (lane 1: before induction, lane 2: after induction). After 4 h <i>E. coli</i> cells were lysed by sonication (lane 3) and centrifuged (lane 4: pellet with insoluble proteins; lane 5: supernatant with soluble proteins). The supernatant with GCDH-His₆ was incubated with Ni-NTA agarose and loaded on a column. Unbounded proteins (lane 6) were removed and the column was washed with increasing imidazole concentrations (lane 7: 10 mM imidazole; lane 8: 50 mM imidazole). Finally the GCDH-His₆ protein was eluted in four steps with increasing imidazole concentrations (lane 9–11: 150 mM imidazole; lane 12: 250 mM imidazole). Samples were separated by SDS-PAGE (10% acrylamide) and proteins were visualized by Coomassie Blue staining. The positions of molecular mass marker proteins (in kDa) are indicated. (<b>B</b>) Validation of the purified GCDH-His₆-fusion protein. Different amounts of purified GCDH-His₆ protein were separated by SDS-PAGE (10% acrylamide) and analyzed by anti-GCDH western blotting. Representative pictures of n = 10 independent preparations are shown.</p

Mitochondrial proteins binding to GCDH.

<p>GCDH-His₆ was immobilized on beads and incubated with isolated mitochondrial matrix proteins from pig liver. The identity of specifically co-purifying proteins was determined by LC-MS/MS.</p><p>M: mitochondrial matrix; IM: inner mitochondrial membrane.</p

GCDH affinity chromatography of mitochondrial matrix extracts.

<p>Purified GCDH was covalently coupled to Affi-Gel 10 matrix and incubated with isolated mitochondrial matrix proteins. Aliquots of the loaded extract (input, I: 10% of total protein), the last wash fraction (W, 25%) and the high salt elution fraction (E, 100%) were separated by SDS-PAGE (10% acrylamide) and tested by anti-DLST (<b>A</b>) and anti-ETFB (<b>B</b>) western blotting. Non-coupled Affi-gel 10 beads were used as a control for unspecific binding. The positions of the molecular mass marker proteins (in kDa) are given. A representative blot of n = 3 independent preparations is shown.</p

YFP fragment complementation assay demonstrates the interaction of GCDH with DLST <i>in vivo</i>.

<p>(<b>A</b>) Schematic composition of C-terminal YFP1 (dark blue) and YFP2 (red) fusion proteins of DLST, GCDH and MCFD2 used in this study. The 10-amino acid linker (GGGGS)₂ is indicated in green. The calculated molecular masses of the fusion proteins are shown in brackets. The ERGIC marker protein MCFD2-YFP2 was used as negative control. (<b>B</b>) Expression analysis in BHK cells of all fusion proteins visualized by western blotting, using anti-DLST, anti-GCDH and anti-GFP antibodies. (<b>C</b>) Fluorescence microscopy of the indicated single or co-expressed fusion proteins. Strong YFP fluorescence was observed in cells co-expressing either GCDH-YFP1 with DLST-YFP2 or GCDH-YFP2 with DLST-YFP1. Nuclei were visualized using DAPI (blue). Scale bars = 40 µM. Representative images of n = 3 independent transfection experiments are shown.</p

Additional file 2: of Growth charts for patients with Sanfilippo syndrome (Mucopolysaccharidosis type III)

Figure RD2. Raw data of weight (kg), showing the construction of the charts with individual data points. (DOCX 145 kb

Additional file 8: of Growth charts for patients with Sanfilippo syndrome (Mucopolysaccharidosis type III)

Figure C3. Reconstructed charts for BMI (kg/m2) for MPS III patients, A: for boys (blue); B: for girls (orange). (DOCX 79 kb

Additional file 1: Figure S1. of Musculoskeletal manifestations in mucopolysaccharidosis type I (Hurler syndrome) following hematopoietic stem cell transplantation

(a-d): Radiographs of dysostosis multiplex. (a) Anterior-posterior spine x-ray of patient 14 at 9.6 years of age: scoliosis (40°) and paddle shaped ribs. (b) Lateral spine x-ray of patient 12 at 4.6 years of age: thoracolumbar gibbus (62°), flattened and beaked vertebrae. (c) X-ray of the left hand of patient 14 at 7.8 years of age: hypoplastic and irregular carpal bones, proximal pointing of metacarpals, shortened phalanges and metacarpals and V-shaped hypoplastic distal ulna and radius, decalcification of the bones of the hand. (d) Anterior-posterior x-ray of patient 4 at 3.4 years of age: broad iliac wings, hip dysplasia. (PNG 804 kb