Biochemical characterization of pikachurin-dystroglycan interaction. A, schematic representation of recombinant pikachurin and α-DG. Pikachurin contains a signal sequence (ss), two fibronectin 3 (FN) domains, three laminin globular (LG) domains, and two calcium-binding EGF-like (EGF like) domains. Recombinant pikachurin LG domains (PikaLG) contain amino acid residues 391–1071 and a tandem myc-His tag at the C terminus. α-DG contains the signal sequence (ss), N-terminal, mucin-like, and C-terminal domains. Recombinant α-DG (DGFc) has an Fc tag at the C terminus. B, divalent cation is necessary for pikachurin-dystroglycan interaction. PikaLG binding to DGFc-protein A beads was tested in the presence of 2 mm EDTA (lane 1) and 2 mm each of Ca²⁺ (lane 2), Mg²⁺ (lane 3), or Mn²⁺ (lane 4). Bound PikaLG was detected by Western blotting with an anti-His tag antibody (upper panel, indicated by PikaLG). Comparable amounts of DGFc proteins on protein A beads were confirmed by staining with an anti-Fc antibody (lower panel, indicated by DGFc). C, quantitative solid-phase binding assays for divalent cation dependence. PikaLG binding to immobilized DGFc was tested in the presence of 2 mm EDTA and 2 mm each of Ca²⁺, Mg²⁺, or Mn²⁺. Binding in the presence of Ca²⁺ was set as 100%. Data shown are the average of three independent experiments with standard deviations. D, Ca²⁺-dependent binding of pikachurin to dystroglycan. PikaLG binding to DGFc was tested in various Ca²⁺ concentrations by solid-phase binding assays. The binding data were fit to the equation Y = B_max x/(K_d + x), where K_d is the concentration required to reach half-maximal binding, and B_max is maximal binding. Maximal binding was set as 100%. K_d = 78 ± 15 μm. Data shown are the average of four independent experiments with standard deviations. E and F, effects of NaCl (E) and heparin (F) on the pikachurin-dystroglycan interaction. PikaLG binding to DGFc was tested in various NaCl or heparin concentrations by solid-phase binding assays. Binding in the presence of 150 mm NaCl (E) or in the absence of heparin (F) was set as 100%. Data shown are the average of four (E) and six (F) independent experiments with standard deviations. *, p < 0.05. G, binding of pikachurin LG domains to heparin. Lysates from PikaLG-expressing cells were incubated with heparin affinity beads. Total lysate sample (total, lane 1), flow-through (void, lane 2), and bound (bound, lane 3) fractions were analyzed by Western blotting with an antibody to anti-Myc tag.

Dystroglycan functional domains for pikachurin binding. A, schematic representation of deletion mutants of DGFc proteins. ss, signal sequence. B and C, dissection of dystroglycan domains necessary for pikachurin binding. Each deletion construct was expressed in HEK293 cells and recovered from the culture media using protein A beads. The DG-wt construct was expressed without or with LARGE (B, lanes 4 and 5). Lysates from PikaLG-expressing cells were subjected to protein A beads that had captured each DGFc mutant protein. PikaLG binding was detected by Western blotting with an anti-Myc antibody (upper panel, PikaLG). Comparable amounts of DGFc mutant proteins on protein A beads were confirmed by Western blotting (WB) with an anti-Fc antibody (middle panel, Fc). The blot was also tested using a laminin-111 overlay assay (bottom panel, laminin O/L).

Disruption of pikachurin localization in dystroglycanopathy animals. A and C, immunofluorescence analysis of pikachurin in the outer plexiform layer (OPL). Retinal sections of POMGnT1-deficient (−/−) and Large^myd (myd/myd) mice, and their littermate heterozygous controls, were immunostained using antibodies to pikachurin (red, left panels) or β-DG (green, middle panels). Nuclei were stained with DAPI (blue). Merged images are shown in the right panels. Scale bar, 10 μm. ONL, outer nuclear layer; INL, inner nuclear layer. B and D, reduced pikachurin binding to α-DG in dystroglycanopathy models. α-DG was immunoprecipitated from eyes of POMGnT1-deficient (−/−) and Large^myd (myd/myd) mice, and their littermate heterozygous controls. PikaLG-containing cell lysates were incubated with the immunoprecipitated materials to examine PikaLG-DG binding. PikaLG binding was detected by Western blotting (WB) with anti-Myc antibody (upper panel, PikaLG). Comparable amounts οf α-DG were confirmed by Western blotting with anti-α-DG antibody (lower panel, α-DG core). Normal and hypoglycosylated (hypo.) sizes of α-DG are indicated on the left side of the blots.

Dissection of the dystroglycan binding region in pikachurin. A, schematic representation of pikachurin deletion mutant proteins. All constructs contain a tandem myc-His tag at the C terminus. ss, signal sequence. B, binding of pikachurin deletion constructs to dystroglycan. Each deletion construct was expressed in HEK293 cells, and cell lysates were subjected to the DGFc binding assay. PikaLG in the reaction mixture (left panel) and PikaLG bound to DGFc-protein G-beads (right panel) were analyzed by Western blotting with an anti-Myc tag antibody. C, solid-phase binding assays for pikachurin deletion constructs. Cell lysates containing comparable amounts of each deletion construct were tested for DGFc binding. Binding of full-length DGFc was set as 100%. Data shown are the average of four independent experiments with standard deviations. Inset, Western blot analysis to confirm the amount of each LG protein used in the binding assays. D, oligomer formation of pikachurin. Cell lysates containing comparable amounts of each construct were dissolved in SDS sample buffer containing 2-mercaptoethanol (2-ME) and then subjected to SDS-PAGE with (+2-ME, +heat denaturing) or without (+2-ME, –heat denaturing) heat denaturing (95 °C, 5 min). Constructs containing LG1 (LG1, LG1-2, and LG FL) showed several higher molecular weight bands (arrowheads), which might indicate oligomeric structure formation by pikachurin.

Reduced pikachurin binding to α-dystroglycan in dystroglycanopathy animals. α-DG was immunoprecipitated from the brains of POMGnT1-deficient (A) and Large^myd (B) mice. Littermates were used as controls. Lysates from PikaLG-expressing cells were incubated with the immunoprecipitated materials to examine PikaLG-DG binding. PikaLG binding was detected by Western blotting (WB) with an anti-Myc antibody (upper panel, PikaLG). Comparable amounts of α-DG were confirmed by Western blotting with anti-α-DG antibody (lower panel, α-DG core). Normal and hypoglycosylated (hypo.) sizes of α-DG are indicated on the left side of the blots. C, quantitative solid-phase binding assays for brain DG. Wheat germ agglutinin-enriched brain DG preparations from POMGnT1-deficient, Large^myd, and their littermates were immobilized and tested for PikaLG binding. Binding to DG preparations from littermate controls in the presence of Ca²⁺ was set as 100%. Data shown are the average of three individual preparations with standard deviations.