Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation

Abstract

Asparagine linked glycosylation of proteins is an essential protein modification reaction in most eukaryotic organisms. Metazoan organisms express two oligosaccharyltransferase complexes that are composed of a catalytic subunit (STT3A or STT3B) assembled with a shared set of accessory subunits and one to two complex specific subunits. siRNA mediated knockdowns of STT3A and STT3B in HeLa cells have shown that the two OST complexes have partially non-overlapping roles in N-linked glycosylation. However, incomplete siRNA mediated depletion of STT3A or STT3B reduces the impact of OST complex loss, thereby complicating the interpretation of experimental results. Here, we have used the CRISPR/Cas9 gene editing technology to create viable HEK293 derived cells lines that are deficient for a single catalytic subunit (STT3A or STT3B) or two STT3B-specific accessory subunits (MagT1 and TUSC3). Analysis of protein glycosylation in the STT3A, STT3B and MagT1/TUSC3 null cell lines revealed that these cell lines are superior tools for investigating the in vivo role and substrate preferences of the STT3A and STT3B complexes.

Figure 1. siRNA-mediated depletion of OST subunits is relatively ineffective in HEK293 cells.

HEK293 cells were treated with negative control (NC) siRNA or siRNAs specific for MagT1, TUSC3, STT3A or STT3B for 72 h. (a,b) HEK293 cell extracts were resolved by SDS-PAGE and analyzed by protein immunoblotting using the specified antisera. Sec61α served as gel loading control. Expression values relative to cells treated with the NC siRNA are for the displayed image that is representative of two or more experiments. (a) The asterisk designates a nonspecific product recognized by the anti-STT3A sera. (b) Anti-TUSC3 sera crossreacts with MagT1. (c,d) Cells were transfected with expression vectors for pSAP-DDK-His (c) or SHBG (d) 48 h after siRNA treatment and pulse labeled 24 h later for either 4 min (SHBG) or 10 min (pSAP-DDKHis) and chased for either 10 min (pSAP-DDKHis) or 20 min (SHBG). Proteins were immunoprecipitated with anti-DDK or anti-SHBG sera. EH designates digestion with endoglycosidase H. Glycoforms resolved by PAGE in SDS are labeled to indicate the number of N-linked glycans. Quantified values below gel lanes (c,d) are for the displayed image that is representative of two or more experiments. (a–d) Immunoblots and phosphorimages were cropped to display the region of interest. Full-length immunoblot images and full length gel images are shown in Supplemental Fig. S2.

Figure 2. CRISPR/Cas9 genome editing in HEK293 cells.

(a) A mismatch-specific T7E1 endonuclease assay was used to analyze DNA flanking the CRISPR target sites. Cleavage products are designated by arrowheads. Agarose gel images were cropped to display the region of interest. Full length gel images are shown in Supplemental Fig. S3. (b) Sequence alignment of the WT allele from control HEK293 cells and mutant alleles identified from each cell line by DNA sequencing of genomic PCR products. Nucleotide numbers are expressed relative to the initiation codon. The sequences corresponding to the 20-nt srRNA target are underligned, the 3-nt PAM is highlighted in cyan. The intron sequence adjacent to the target site in STT3A (−/−) clone is in magenta.

Figure 3. Validation of HEK293 derived cell lines.

(a) Total protein extracts from WT or mutant HEK293 cells (50 ug) were resolved by SDS-PAGE and analyzed by protein immunoblotting using the specified sera. Downward pointing arrowheads in the STT3B blot designate non-specific background bands. Anti-TUSC3 sera cross-reacts with MagT1. Expression of the OST subunits and BiP were adjusted relative to the gel loading control (GAPDH), and were normalized to the relevant wild type control to compare the relative expression of OST subunits. (b) Actin, TUSC3 and STT3A mRNA expression in knockout HEK293 cells was examined by reverse transcription PCR. Amplification products were resolved on 1.5% agarose gels. Immunoblot images and agarose gel were cropped to display the region of interest. Full-length immunoblot images for panel (a) and the full length agarose gel image for panel (b) are shown in Supplemental Fig. S4.

Figure 4. Hypoglycosylation of proteins in knockout HEK293 cell lines.

Cell lines were transfected with expression vectors for the following human glycoproteins: (a) SHBG, (b) SHBG derivatives (see diagram) with single glycosylation sites (SHBG N380Q and N396Q), (c) a cathepsin C derivative with a single glycosylation site (CatCΔ234-HA), (d) prosaposin (pSAP-DDKHis), (e) haptoglobin (Hp-DDKHis), (f) hemopexin (Hpx-DDKHis). Endogenous progranulin (pGran) (d) was analyzed using non-transfected cells. The cells were pulse labeled for 10 min and chased for 10 min (pSAP, pGran and CatCΔ234-HA) or pulse labeled for 5 min and chased for 20 min (Hp, SHBG and Hpx). Glycoproteins precipitated with anti-DDK, anti-HA or anti-SHBG were resolved by PAGE in SDS. EH designates treatment with endoglycosidase H. Quantified values below gel lanes (a–f) are for the displayed image, which is representative of two or more experiments. (b) The vertical line indicates the excision of three intervening gel lanes. (d) Resolution of pGran glycoforms is not sufficient for quantification. Nonglycosylated forms of pSAP, Hp, and Hpx that comigrate with the EH-digested form of the substrate in HEK293 cells correspond to a non-translocated precursor and were not used to calculate the average number of glycans. (a–f) Phosphorimages were cropped to display the region of interest. Full-length phosphorimages are shown in Supplemental Fig. S5.

Figure 5. Restoration of N-glycosylation of proteins in knockout cell lines.

(a) Wild type or mutant HEK293 cells were cotransfected with an OST subunit expression vector (empty vector (e.v.), MagT1-V5 His, STT3B-myc DDK, or MagT1-V5 His plus TUSC3-DDKHis) and an expression vector for CatCΔ234-HA. CatCΔ234-HA was immunoprecipitated with anti-HA sera from total cell extracts after a 10 min pulse, 10 min chase labeling period. (b) Wild type and mutant HEK293 cells were transfected with an OST expression vector (empty vector or STT3A-DDKHis). After 48 h of culture the cells were cotransfected with the OST expression plasmid and a pSAP-DDKHis expression vector. Glycosylation of pSAP-DDKHis was evaluated by a 10 min pulse, 10 min chase labeling procedure 24 h after the second transfection. pSAP-DDKHis was immunoprecipitated with anti-DDK sera. Phosphorimages were cropped to display the region of interest. Full-length phosphorimages are shown in in Fig. S6.