Proteoglycan synthesis in conserved oligomeric Golgi subunit deficient HEK293T cells is affected differently, depending on the lacking subunit

Abstract

The Conserved Oligomeric Golgi (COG) complex is an eight subunit protein complex associated with Golgi membranes. Genetic defects affecting individual COG subunits cause congenital disorders of glycosylation (CDGs), due to mislocalization of Golgi proteins involved in glycosylation mechanisms. While the resulting defects in N-and O-glycosylation have been extensively studied, no corresponding study of proteoglycan (PG) synthesis has been undertaken. We here show that glycosaminoglycan (GAG) modification of PGs is significantly reduced, regardless which COG subunit that is missing in HEK293T cells. Least reduction was observed for cells lacking COG1 and COG8 subunits, that bridge the A and B lobes of the complex. Lack of these subunits did not reduce GAG chain lengths of secreted PGs, which was reduced in cells lacking any other subunit (COG2-7). COG3 knock out (KO) cells had particularly reduced ability to polymerize GAG chains. For cell-associated GAGs, the mutant cell lines, except COG4 and COG7 KO, displayed longer GAG chains than wild-type cells, indicating that COG subunits play a role in cellular turnover of PGs. In light of the important roles PGs play in animal development, the effects KO of individual COG subunits have on GAG synthesis could explain the variable severity of COG associated CDGs.

Keywords: congenital disorders of glycosylation; glycosaminoglycan; glycosylation; proteoglycans; the Golgi apparatus; the conserved oligomeric complex.

Figure 1. Synthesis and secretion of sulfate labelled macromolecules in wild type and COG subunit KO HEK293T cells.

HEK293T wild type and cell lines depleted of individual COG subunits were incubated for 24 h in the presence of 0.2 mCi/ml ³⁵S-sulfate in sulfate-depleted medium. The media and cell fractions were processed as described in materials and methods, and the content of sulfated macromolecules was quantified by scintillation counting and is expressed as % of that of wild type HEK293T cells. The figure is based on four experiments with three parallels in each.

Figure 2. Secretion of metabolically labelled proteins from wild type and COG subunit KO HEK293T cells.

HEK293T wild type and cell lines depleted of individual COG subunits were incubated for 20 h in the presence of 0.2 mCi/ml ³⁵S-cys/met in cys/met-depleted medium. The medium fractions were processed as described in Materials and methods, and the content of ³⁵S-labelled macromolecules was quantified by scintillation counting and is expressed as % of that of wild type HEK293T cells. The figure shows one typical experiment of three with three parallels for each cell line.

Figure 3. SDS-PAGE analysis of synthesis and secretion of sulfated macromolecules in wild type and COG subunit KO HEK293T cells.

The eluates from Sephadex G-50 fine gel filtration chromatography columns were concentrated by freeze drying to contain approximately 15-20,000 cpm each in SDS-PAGE sample buffer. Parallel aliquots of each sample were subjected to no treatment (Cnt) or to treatment with cABC to degrade CS/DS (C) or hep to degrade HS and heparin (H) prior to SDS-PAGE (4-12 % gradient gels). Medium samples in panels A and B and cell lysate samples in panels C and D. Samples from COG8 deficient cells (both medium and cell lysate) in panel E.

Figure 4. Gel filtration analysis of GAG chain length of secreted PGs.

Medium samples eluted from Sephadex G50-fine columns were subjected to alkaline treatment (see Materials and methods) to detach GAG chains from their protein cores. The samples were added the V_o and V_t markers Blue Dextran and K₂CrO₄, respectively, for V_e and K_av calculations, and then applied to Sepharose CL-6B columns to compare the chain lengths of GAGs synthesized by HEK293T cells and COG subunit deficient cell lines. Panel A shows two examples (HEK293T control and COG5) runs. The panels show the size distribution of GAG chains in at least three samples for each cell line. The fractionation range of Sepharose CL-6B being 10,000 to 1,000,000 (for dextrans). 0-0.2 are the longer chains, 0.2-0.8 are intermediate chains, while 0.8-1.0 are shorter chains. In the Vt, are very short chains with full access to all of the column material, of less than (approximately) 20 disaccharides.

Figure 5. Gel filtration analysis of GAG chain length of cell-assciated PGs.

Cell lysate samples eluted from Sephadex G50-fine columns were subjected to alkaline treatment (see Materials and methods) to detach GAG chains from their protein cores. The samples were added the Vo and Vt markers Blue Dextran and K₂CrO₄, respectively, for V_e and K_av calculations, and then applied to Sepharose CL-6B columns to compare the chain lengths of GAGs synthesized by HEK293T cells and COG subunit deficient cell lines. Panel A shows two examples (HEK293T control and COG5) runs. The panels show the size distribution of GAG chains in at least three samples for each cell line. The fractionation range of Sepharose CL-6B being 10,000 to 1,000,000 (for dextrans). 0-0.2 are the longer chains, 0.2-0.8 are intermediate chains. In the V_t, are very short chains with full access to all of the column material, of less than (approximately) 20 disaccharides.

Figure 6. Flow cytometry with cell surface bound anti HS-antibody.

HEK293T cells and COG subunit deficient cell lines were suspended in PBS and incubated with the 10E4 anti-HS antibody for 30 min at 4°C before staining with goat anti-mouse IgM FITC. Controls are incubated with goat anti-mouse FITC, only. Cells were analyzed with a FACSort flow cytometer. Flow data collection was carried out with the program CellQuest 3.3 and the data generated using Kaluza Flow Cytometry analysis v 1.2. A total of 20,000 or more cells gated as viable were analyzed per sample. The figure shows data for wild type HEK293T cells (control) and the cell lines deficient in individual COG subunits 1-8.