Identification of the iduronate-2-sulfatase proteome in wild-type mouse brain

Abstract

Iduronate-2-sulfatase (IDS) is a lysosomal enzyme involved in the metabolism of the glycosaminoglycans heparan (HS) and dermatan (DS) sulfate. Mutations on IDS gene produce mucopolysaccharidosis II (MPS II), characterized by the lysosomal accumulation of HS and DS, leading to severe damage of the central nervous system (CNS) and other tissues. In this study, we used a neurochemistry and proteomic approaches to identify the brain distribution of IDS and its interacting proteins on wild-type mouse brain. IDS immunoreactivity showed a robust staining throughout the entire brain, suggesting an intracellular reactivity in nerve cells and astrocytes. By using affinity purification and mass spectrometry we identified 187 putative IDS partners-proteins, mainly hydrolases, cytoskeletal proteins, transporters, transferases, oxidoreductases, nucleic acid binding proteins, membrane traffic proteins, chaperons and enzyme modulators, among others. The interactions with some of these proteins were predicted by using bioinformatics tools and confirmed by co-immunoprecipitation analysis and Blue Native PAGE. In addition, we identified cytosolic IDS-complexes containing proteins from predicted highly connected nodes (hubs), with molecular functions including catalytic activity, redox balance, binding, transport, receptor activity and structural molecule activity. The proteins identified in this study would provide new insights about IDS physiological role into the CNS and its potential role in the brain-specific protein networks.

Keywords: Biochemistry; Bioinformatics; Biotechnology; Cell biology; Computational biology.

Fig. 1

Western blotting analysis. Cross-reactivity assessment for chicken polyclonal antibody. A) Anti-IDS_99-122 (anti-peptide against amino acids 99–122). B) Anti-IDS_262-286 (anti-peptide against amino acids 262–286). Equivalent amounts of protein extracts (35 μg) of whole tissue extracts liver (lane 1), brain (lane 2), leukocytes (lane 3) and 10 μg of non-purified IDS recombinant protein (lane 4) were loaded and run on 10 % sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes (the unedited version is provided in Supplementary Images).

Fig. 2

IDS immunohistochemistry in mouse brain. The immunoreactivity reveals robust staining in cerebellum and cortex (brown staining). A) cortex; B) magnified image indicating the presence of IDS in cerebral cortex layers (amplified image in C and detailed image inset after the H&E removal); D) detail of a positive cellular reactivity in the layer III; E) immunopositive nerve cell with different morphology; F) positive immunoreactivity for cerebellum (magnified image for a nerve cell from the molecular layer); (G and H) negative controls. Scale bar: 200 μm m, molecular layer; P, Purkinje cell layer; g, granular cell layer.

Fig. 3

Confocal microscopy. Double immunofluorescence for GFAP (red) and IDS (green) in cerebellum (A-C) and cortex (D-F). Scale bars: 20μm. The Pearson correlation value for C and I was calculated by Fiji Is Just (Image J) with 1.000 of R-total and 91,13 % of co-localization volume.

Fig. 4

Purification of recombinant IDS and proteome isolation. A) SDS of size-exclusion chromatography. The collected volumes were at maximum peak height corresponding to the protein IDS. The pooled fractions were analyzed by SDS-PAGE after molecular exclusion chromatography on Sephacryl-200. B) The presence of IDS was confirmed by Western blot using specific antibodies against human IDS and was visualized by chemiluminescence. Lanes 1–3 the collected fractions of the most abundant peak. C) The purity of recombinant protein was evaluated through silver staining before covalent coupling to the sepharose matrix pre-activated with cyanogen bromide. Under reducing conditions, the bands corresponding to the heavy chain (above 75 kDa) and the light chain (above 15 kDa) were observed. The IDS-binding proteins eluted with high astringency were precipitated, loaded for SDS-PAGE and, once stained, lanes were cut in four pieces (highlighted with segmented lines) for MS/MS analysis (the unedited version of this figures are provided in Supplementary Images).

Fig. 5

Bioinformatics analysis of IDS-interacting proteins. A) Interaction map constructed in Cytoscape. GeneMANIA analysis was performed selecting physical and predicted interaction networks and using query gene-based weighting. Known physical interactions were included (purple line) and novel interactions (orange lines) culled from mouse and orthologous protein databases. B) Magnified image of predicted primary IDS-network. C) DAVID GO annotation analysis for the 17 genes included in the primary IDS network.

Fig. 6

Protein Analysis Through evolutionary relationships (PANTHER). The proteins were classified according to the molecular function of the protein by itself or with directly interacting proteins at a biochemical level. Chart tooltips are read as: category name (accession): # genes; percent of gene hit against total # genes; percent of gene hit against total # protein class hits.

Fig. 7

Co-immunoprecipitation of IDS from mouse brain tissue extracts. Once the proteins are transferred to the nitrocellulose membrane, the light and heavy chains observed as contaminants around 50 and 25 kDa, were blocked with a secondary unconjugated antibody. Immunoblotting was performed using chicken anti-IDS_{262-286 .} Native IDS was immunopositive for Myelin PLP, 14-3-3 isoform zeta, 14-3-3 isoform gamma and aldolase C. IDS was not detected with anti synaptotagmin (Syn), heat shock protein 70 kDa (Hsp7C) and IgG negative control (NC) (the unedited version is provided in Supplementary Images).

Fig. 8

BN-PAGE and Western blot analysis. A) 60 μg of cytosolic (F1), membranes (F2) and nuclei (F3) protein fractions were subjected to BN-PAGE and stained with Coomassie G-250. B) Western Blot and immunodetection of cytosolic complexes. Proteins from brain and liver were separated and transferred to the nitrocellulose membrane under native conditions, and IDS-complex were immunodetected with specific chicken anti-IDS antibody (1:1000) in an approximate mass of 100 kDa and 300 kD. Segmented lines indicate the bands excised for MS/MS analysis (the unedited versions are provided in Supplementary Images).

Fig. 9

Venn diagram for native complexes. Venn diagram displays the overlap of proteins identified in each protein complex and the isolated proteome.

Fig. 10

ClusterONE analyses of high molecular complex for IDS. A) Images corresponding with the selected nodes from a PPI view in Cytoscape. Clusters are ordered according to the p-value. Nodes: number of nodes in the cluster, density: the sum of the edge weights within the cluster divided by the number of theoretically possible edges, quality: ratio (in-weight/(in-weight + out weight), P-value: p-values less than 0.05 are denoted by red colors. B) Functional annotation analysis using PANTHER database. The code corresponding to the protein class (PC) is shown in parentheses.

Fig. 11

Motif prediction with Scansite (3 Beta). The figure shows output detailing results for human IDS (A) and mouse IDS (B). A phosphoserine/threonine binding group (pST-bind) was identified at high stringency scoring in human sequence and two binding sites were identified on mouse sequence at low astringency scoring. Motif site, denoted by a vertical line, show an abbreviated name for the identified motif at the corresponding position. The plots of surface accessibility and the amino acid fractional surface probabilities are shown below each cartoon. The default scan condition reveals only motif scoring in the top 0.2858 percentile for human IDS and 2.28 and 2.66 percentile for mouse IDS.