Figure 6.

Glycosylation defect of cubilin abrogated amnionless-dependent membrane expression of cubilin. (a) Schematic representations of N-glycosylation sites in mini-cubilin. The conservation across multiple species of consensus amino acid sequences for N-glycosylation sites is shown. Hs: Homo sapiens; Rn: Rattus norvegicus; Mm: Mus musculus. (b) A schematic diagram of SILAC labeling and proteome analysis. HEK293T cells were labeled with light (L-¹²C₆¹⁴N₄-Arg and L-¹²C₆-Lys) or heavy (L-¹³C₆¹⁵N₄-Arg and L-¹³C₆-Lys) isotope-labeled amino acids. Cells transiently transfected with wild-typed cubilin-Flag only or wild-type cubilin-Flag and amnionless-mycDDK were cultured in ‘Light’ or ‘Heavy’ medium, respectively. Equal amounts of cell lysate from ‘Light’ and ‘Heavy’ cells were mixed and immunoprecipitated with anti-Flag antibody. After trypsin/lysyl endopeptidase digestion, peptides were analysed by LC-MS/MS. (c) Representative spectral data of cubilin peptides including N-glycosylation site (P-3) or control not including N-glycosylation site using SILAC. (d) HEK293T cells transfected with both wild-type cubilin-flag and wild-type amnionless-mycGFP were incubated in absence or presence of tunicamycin (4 mg/mL) for 18 h at 6 h after transfection. Non-permeabilised cells were stained for membrane-targeted cubilin (red). (Scale bar: 10 µm.) (e) Expression levels of cubilin-flag in (d) were analysed by immunoblotting. Anti-flag immunoprecipitates were separated on a 7.5% SDS-PAGE gel, followed by western blotting. Full-length blots are presented in Supplementary Figure 13 (f) Non-permeabilised HEK293T cells transfected with amnionless-mycGFP and cubilin-Flag (wild-type or Asn to Asp (ND) mutants) were stained for membrane-targeted cubilin. Plasmid constructions of the ND mutant were created by replacement of Asn for Asp in the potential N-glycosylation sites as shown in Fig. 6a. Cubilin 7ND, replacing N857 with D857; Cubilin 456ND, replacing a combination of N711, N749, and N781 with a combination of D711, D749, and D781; Cubilin 4567ND, replacing N857 with D857 added to Cubilin 456ND. Membrane expression of cubilin and amnionless analysed by flow cytometry. (g) The ratio of cells with amnionless-dependent membrane-targeted cubilin to amnionless-expressing cells was analysed by flow cytometry in (f). Data represent means ± SEM. Statistical significance: *P < 0.01.