Impact of Hypermannosylation on the Structure and Functionality of the ER and the Golgi Complex

Abstract

Proteins of the secretory pathway undergo glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. Altered protein glycosylation can manifest in serious, sometimes fatal malfunctions. We recently showed that mutations in GDP-mannose pyrophosphorylase A (GMPPA) can cause a syndrome characterized by alacrima, achalasia, mental retardation, and myopathic alterations (AAMR syndrome). GMPPA acts as a feedback inhibitor of GDP-mannose pyrophosphorylase B (GMPPB), which provides GDP-mannose as a substrate for protein glycosylation. Loss of GMPPA thus enhances the incorporation of mannose into glycochains of various proteins, including α-dystroglycan (α-DG), a protein that links the extracellular matrix with the cytoskeleton. Here, we further characterized the consequences of loss of GMPPA for the secretory pathway. This includes a fragmentation of the Golgi apparatus, which comes along with a regulation of the abundance of several ER- and Golgi-resident proteins. We further show that the activity of the Golgi-associated endoprotease furin is reduced. Moreover, the fraction of α-DG, which is retained in the ER, is increased. Notably, WT cells cultured at a high mannose concentration display similar changes with increased retention of α-DG, altered structure of the Golgi apparatus, and a decrease in furin activity. In summary, our data underline the importance of a balanced mannose homeostasis for the secretory pathway.

Keywords: Golgi network; endoplasmic reticulum; mannosylation.

Figure 1

Loss of GMPPA alters the morphology of the Golgi apparatus in skeletal muscle fibers and in neuronal cells. (A,B) Representative images of skeletal muscle fibers from 5-month-old mice stained for TGN38 and GM130 (scale bar: 10 µm). (A) WT fibers show a Golgi signal with partial overlap of TGN38 with GM130. (B) KO fibers do not show an obvious overlap of the cis-Golgi protein GM130 with the trans-Golgi protein TGN38. (C,D) Representative images of skeletal muscle fibers from 5-month-old mice stained for GLG1 for (C) WT and (D) KO (scale bar: 10 µm). White dashed lines indicate cell borders. White arrows indicate co-localized structures. Right panel: schematic presentation of a skeletal muscle fiber with Golgi and Golgi-network positive structures. (E,F) GLG1 and TGN38 stainings in fibers reveal more trans-Golgi fragments in KO fibers with ImageJ analysis (n = 3 mice per group, 5 images per animal). (E) Particles were counted in the ImageJ menu “analyze particles” after converting pictures to a binary format. (F) Particles were counted via the ImageJ plugin ComDet v.0.5.5 without picture pre-processing. (G,H) The colocalization analysis reveals a partial overlap between GM130 and TGN38 positive particles in WT skeletal muscle fibers, which is reduced in KO samples (n = 3 mice per group, 5 images per animal). The colocalization analysis was performed either with the Coloc2 ImageJ plugin (G) that measures the Manderson overlap coefficient or the ComDet v.0.5.5 ImageJ plugin (H). 2-way-ANOVA with Bonferroni post-hoc analysis. (I,J) Representative images of Purkinje cells from 3-month-old mice stained for GM130 and TGN38 (scale bars: 5 µm) for (I) WT and (J) KO sections. White dashed lines indicate cell borders. White arrows indicate co-localized structures. (K,L) Representative images of (K) WT and (L) GMPPA KO MEFs stained for GM130 and TGN38 (scale bars: 5 µm). White arrows indicate co-localized structures. (M) ImageJ analysis (ComDet plugin) reveals increased numbers of TGN38 positive particles in Purkinje cells of KO mice (n = 4–5 mice per group, 10 images per animal). (N) The colocalization analysis (ComDet plugin) reveals a significantly reduced overlap between the cis-Golgi protein GM130 and the trans-Golgi protein TGN38 in Purkinje cells of KO mice (n = 4–5 mice per group, 10 images per animal, 2-way-ANOVA with Bonferroni post-hoc analysis). (O,P) ImageJ analysis (ComDet plugin) reveals an increased number of TGN38 positive particles (O) as well as a reduced colocalization between GM130 and TGN38 (P) in GMPPA KO MEFs (n = 4 experiments per group, 5 images per genotype per experiment). (Q) Representative electron microscopy images of skeletal muscle cells from 12-month-old WT and GMPPA KO mice (scale bars: 500 nm) showing the Golgi complex. Asterisks indicate abnormal secretory vesicles or Golgi cisterna. (R) Representative electron microscopy images of hippocampal neuronal cells from 12-month-old WT and GMPPA KO mice (scale bars: 500 nm) showing the Golgi complex. Asterisks indicate abnormal secretory vesicles or Golgi cisterna. (S) ImageJ analysis reveals reduced number of Golgi stacks and enlarged secretory vesicles in hippocampal neurons from KO mice (n = 3 mice per group, 4–10 images per animal, two-tailed Student’s t-test). * indicates p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.0001.

Figure 2

Quantitative changes of proteins necessary for ER and Golgi organization and function in GMPPA KO skeletal muscle compared to WT before and upon Con A pulldown. (A) Violin plots for proteins involved in selected biological processes (“Endoplasmic reticulum organization”, “Golgi organization”, “protease activity”, “protein maturation” and “protein processing”, and “vesicle mediated transport”) associated with the ER or the Golgi complex before and upon Con A enrichment (full: whole tissue lysates or Con A: Con A enriched samples). (B) Heatmaps for significantly altered proteins in either whole tissue lysates (full) or Con A enriched samples (Con A) (q-value below 0.05). Proteins significantly up-regulated between WT and GMPPA KO are shown in red and those down-regulated in blue (n = 3 mice per group). Not identified proteins are shown in grey. Green labeling indicates glycoproteins.

Figure 3

Quantitative changes of proteins necessary for ER and Golgi organization and function in GMPPA KO brain compared to WT before and upon Con A pull-down. (A) Violin plots for selected biological processes (“Endoplasmic reticulum organization”, “Golgi organization”, “protease activity”, “protein maturation” and “protein processing”, and “vesicle mediated transport”) associated with the ER or the Golgi complex before and upon Con A enrichment. (B) Heatmaps for significantly altered proteins found in either whole tissue lysates (full) or Con A enriched samples (Con A) (q-value below 0.05). Proteins significantly up-regulated between WT and GMPPA KO are shown in red and those down-regulated in blue (n = 3 mice per group). Not identified proteins are shown in grey. Green labeling indicates glycoproteins.

Figure 4

Loss of GMPPA results in functional alterations of the ER and the Golgi apparatus. (A–C) Furin activity assay for (A) serum (n = 4 mice per group), (B) brain lysates (n = 4 mice per group), and (C) MEF lysates (n = 3 experiments per group, 2-way-ANOVA with Bonferroni post-hoc analysis). (D) Representative images of WT and GMPPA KO MEFs stained for PDI and calnexin (scale bars: 5 µm). (E) Representative images of WT and GMPPA KO MEFs transfected with α-DG-GFP and stained for PDI (scale bars: 8 µm). White arrows indicate colocalized structures. (F,G) ImageJ analysis (ComDet plugin) does not show any differences between genotypes in (F) particle numbers as well as (G) colocalization between PDI and calnexin (n = 4 experiments per group, 5 images per genotype per experiment, 2-way-ANOVA with Bonferroni post-hoc analysis). (H) ImageJ analysis (ComDet plugin) shows increased colocalization between α-DG-GFP and PDI in KO cells (n = 3 experiments per group, 5 images per genotype per experiment, two tailed Student’s t-Test). (I,J) Colocalization analysis with the Coloc2 ImageJ plugin that measures the (I) Pearson’s colocalization coefficient as well as the (J) Manderson overlap coefficient showing increased colocalization between α-DG-GFP and PDI in KO cells (n = 3 experiments per group, 5 images per genotype per experiment, two tailed Student’s t-Test, and 2-way-ANOVA with Bonferroni post-hoc analysis). * indicates p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 5

Effects of mannose supplementation on ER and Golgi morphology. (A) Left panel: relative GDP-mannose levels in arbitrary units (A.U.) measured in WT MEFs either cultured with 4.5 g/L glucose or mannose for different time points. Right panel: relative GDP-mannose levels in arbitrary units (A.U.) normalized to GNP levels (GMP + GDP + GTP) measured in WT MEFs either treated with 4.5 g/L glucose or mannose for different time points. (B) Representative images of WT and GMPPA KO MEFs stained for GM130 and TGN38 upon treatment with 4.5 g/L glucose or mannose (scale bars: 8 µm). White arrows indicate co-localized structures. (C) ImageJ analysis (ComDet plugin) for particle numbers and colocalization (n = 3 experiments per group, 9 images per genotype and treatment per experiment). (D) Representative images of WT and GMPPA KO MEFs stained for CLIMP63 and RTN4 upon treatment with either 4.5 g/L glucose or mannose (scale bars: 8 µm). White arrows indicate colocalized structures. (E) ImageJ analysis (ComDet plugin) for particle numbers and colocalization (n = 3 experiments per group, 2-way-ANOVA with Bonferroni post-hoc analysis). * indicates p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.0001.

Figure 6

Culture of WT cells with mannose supplementation causes similar alterations of the secretory pathway as observed in GMPPA KO cells. (A) Representative images of WT and GMPPA KO MEFs transfected with α-DG-GFP and stained for PDI upon treatment with either 4.5 g/L glucose or mannose (scale bars: 8 µm). White arrows indicate colocalized structures. (B) Representative images of WT and GMPPA KO MEFs transfected with α-DG-GFP and stained for GLG1 cultured with either 4.5 g/L glucose or mannose (scale bars: 8 µm). White arrows indicate colocalized structures. (C) ImageJ analysis shows increased colocalization between α-DG-GFP and PDI in KO cells and upon mannose treatment (n = 3 experiments per group, 9 images per genotype and treatment per experiment). To analyze the colocalization between PDI and αDG, we either used the ComDet v.0.5.5 plugin (left), Pearson’s colocalization coefficient (middle), or the Manderson colocalization coefficient (right). (D) ImageJ analysis (ComDet plugin) shows decreased colocalization between α-DG-GFP and GLG1 upon mannose treatment (n = 3 experiments per group, 9 images per genotype and treatment per experiment). To analyze the colocalization between GLG1 and αDG, we either used the ComDet v.0.5.5 plugin (left), Pearson’s colocalization coefficient (middle), or the Manderson colocalization coefficient (right). 1-way ANOVA with Bonferroni post-hoc analysis. (E) Furin activity is reduced in WT MEFs upon replacement of glucose by mannose (n = 3 experiments per group, 2-way ANOVA with Bonferroni post-hoc analysis). * indicates p < 0.05, ** p < 0.01, and *** p < 0.001.