Fig 2: Knockdown of Mgat5 expression in cultured human RPE cells attenuates binding of Gal-3 to RPE cells.(A) Western blot analysis of Mgat5 expression in RPE cells transfected with siRNA containing the same nucleotides as Mgat5 siRNA in random order (sc siRNA), and the same cell line transfected with 50 pmol and 100 pmol of double stranded siRNA complementary to Mgat5 (Mgat5 siRNA), respectively. Lysates containing approximately equal amounts of protein were separated by SDS-PAGE and blotted for immunochemical detection of Mgat5 content. Experiments were repeated at least three times. MW; molecular weight. (B) Quantification of Mgat5 gene silencing. Values are normalized to expression of tubulin. (C) Fluorescence micrographs of Gal-3 binding to the RPE cell surface. Ninety-six hours after transfection cells were treated with 60 µg/mL biotinylated Gal-3. Cells were then fixed and stained with a fluorescent streptavidin conjugate. Nuclei were counterstained with DAPI. Localization of Gal-3 binding was visualized by fluorescence microscopy at a 40 fold magnification. Scale bars represent 100 µm. Untreated cells exposed to streptavidin conjugate alone served as negative controls and exhibited no fluorescence signal (data not shown). (D-E) The target sequence derived from the genomic sequence of Mgat5 was inserted into a CRISPR-Cas9 nuclease expressing lentiviral vector and ARPE19 cells were transfected using Lipfectamine 2000 Plus reagent. (D) Western blot analysis of Mgat5 expression in ARPE19 cells transduced with guide RNA leading to specific knockdown of the Mgat5 (gMgat5), or cells tranduced with a CRISPR-Cas9 lentiviral vector encoding for an none-coding filler RNA (LV), or wild-type ARPE-19 cells. Lysates containing approximately equal amounts of protein were separated by SDS-PAGE and blotted for immunochemical detection of Mgat5 content. (E) Flow cytometric analysis of Gal-3 binding in Mgat5-knockout cells. CRISPR-Cas9-mediated Mgat5 knockdown of cultured RPE cells reduces cells surface binding of Gal-3, when compared to cells transfected with a non-coding control vector alone (LV). Histograms represent the number of counted cells versus relative fluorescence intensity. Transduction experiments have been repeated three times.

Fig 3: Secretion of other SPPL3 substrates from GnT-I-KO cells.a Proteins in the lysates of WT and SPPL3-deficient HEK293 cells and in the culture medium of these cells were blotted for GnT-V, CANT1, B4GALT1, or GAPDH. b Proteins in the lysates of WT and GnT-I-deficient (I-KO) HEK293 cells and in the culture medium of these cells were blotted for GnT-V, CANT1, B4GALT1, or GAPDH. The graphs show quantification of the signal intensities of GnT-V, CANT1, and B4GALT1 secreted from I-KO cells relative to secretion from WT cells. Error bars represent SD (n = 4). Statistical analysis was by Welch’s t-test. **p < 0.005; n.s. not significant.

Fig 4: Inhibition of SPPL3-dependent secretion of GnT-V in complex-type N-glycan-deficient cells.a Relative mRNA expression of MGAT5 (encoding GnT-V) in WT, GnT-I-deficient (I-KO), and GnT-I-rescued HEK293 cells. The levels of MGAT5 mRNA were normalized by those of rRNA. Error bars represent SD (n = 3). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. b Proteins from HEK293 cells treated with dimethylsulfoxide (DMSO), MG132, or chloroquine (CQ) were blotted for GnT-V, ß-catenin, p62, or GAPDH. The samples were treated with PNGase F (PNG-F) before SDS-PAGE. c The activity of GnT-V in DMSO-, MG132-, or CQ-treated HEK293 cells. Error bars represent SD (n = 3). Statistical analysis was by one-way ANOVA with post-hoc Dunnett test. d Proteins in the lysates of WT and GnT-I-deficient cells and in the culture media of these cells were blotted for GnT-V or GAPDH. e WT and SPPL3-deficient B16 cells were treated with or without kifunensine (kif). Proteins in the cell lysates and the culture media of these cells were blotted for GnT-V, APP, or GAPDH. The samples were treated with or without PNGase F before SDS-PAGE. APP was blotted as a positive control of secreted protein94. The proteins in the cell lysates were also blotted with Con A or PHA-L4. f The cellular activity of GnT-V in WT and SPPL3-KO B16 cells treated with or without kifunensine. Error bars represent SD (n = 3). Statistical analysis was by two-way ANOVA with post-hoc Sidak test. ***p < 0.0005; n.s. not significant.

Fig 5: Impact of loss of complex-type N-glycans on the expression, localization, and enzyme activity of SPPL3.a Relative mRNA expression of SPPL3 in WT, GnT-I-deficient (I-KO), and GnT-I-rescued HEK293 cells. The levels of SPPL3 mRNA were normalized by those of GAPDH. Error bars represent SD (n = 3). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. b SPPL3-3HA was expressed in HEK293 cells, and the lysates were treated with or without PNGase F (PNG-F). The proteins in the lysates were blotted for HA, N-cadherin, or GAPDH. c SPPL3-3HA was expressed in WT or GnT-I-deficient cells and the cell lysates were blotted for HA or GAPDH. d (Left) HeLa cells expressing SPPL3-3HA by pTK plasmid were treated with or without kifunensine and immunostained for HA and GM130, Golgin97, or Calnexin. (Right) Line plots of relative fluorescence intensity are shown. Arrows indicate the plotted regions. Scale bars: 10 or 2 µm. e (Left) B16 cells stably expressing GnT-V-Halo was transfected with SPPL3-3HA and were treated with or without kifunensine. SPPL3-3HA was immunostained with anti-HA antibody and GnT-V-Halo was visualized by TMR. (Right) Line plots of relative fluorescence intensity are shown. Arrows indicate the plotted regions. Scale bars: 10 or 2 µm. f Domain structure of human GnT-V. TM: Transmembrane domain. g GnT-V WT or its N-glycosylation site mutants were expressed in HEK293 cells. Proteins in the cell lysates and culture media of these cells were blotted for GnT-V, APP, or GAPDH. The samples were treated with or without PNGase F before SDS-PAGE. The graph shows quantification of the signal ratio of secreted GnT-V to total (cell + medium) GnT-V in PNGase F-treated blot. APP was blotted as a positive control of secreted protein. Error bars represent SD (n = 5). Statistical analysis was by one-way ANOVA with post-hoc Dunnett test. *p < 0.05; n.s. not significant.