Lysine at position 329 within a C-terminal dilysine motif is crucial for the ER localization of human SLC35B4

Abstract

SLC35B4 belongs to the solute carrier 35 (SLC35) family whose best-characterized members display a nucleotide sugar transporting activity. Using an experimental model of HepG2 cells and indirect immunofluorescent staining, we verified that SLC35B4 was localized to the endoplasmic reticulum (ER). We demonstrated that dilysine motif, especially lysine at position 329, is crucial for the ER localization of this protein in human cells and therefore one should use protein C-tagging with caution. To verify the importance of the protein in glycoconjugates synthesis, we generated SLC35B4-deficient HepG2 cell line using CRISPR-Cas9 approach. Our data showed that knock-out of the SLC35B4 gene does not affect major UDP-Xyl- and UDP-GlcNAc-dependent glycosylation pathways.

Fig 1. Subcellular localization of HA- …KDSKKN, human SLC35B4 with HA-epitope at the N-terminus in the wild-type HepG2 cells.

Stably transfected wild-type (SLC35B4-WT) cells, overexpressing SLC35B4 variants, were subjected to indirect immunofluorescent staining with anti-HA (green) and anti-organelle markers (Table 3) (red) antibodies. Cell nuclei were counterstained with DAPI. Scale bar 10 μm.

Fig 2. Subcellular localization of …KDSKKN-HA, human SLC35B4 with HA-epitope at the C-terminus in the wild-type HepG2 cells.

Stably transfected wild-type (SLC35B4-WT) cells, overexpressing SLC35B4 variants, were subjected to indirect immunofluorescent staining with anti-HA (green) and anti-organelle markers (Table 3) (red) antibodies. Cell nuclei were counterstained with DAPI. Scale bar 10 μm.

Fig 3. Subcellular localization of HA- …KDSAAN, human SLC35B4 K329AK330A with HA-epitope at the N-terminus in the wild-type HepG2 cells.

Stably transfected wild-type (SLC35B4-WT) cells, overexpressing SLC35B4 variants, were subjected to indirect immunofluorescent staining with anti-HA (green) and anti-organelle markers (Table 3) (red) antibodies. Cell nuclei were counterstained with DAPI. Scale bar 10 μm.

Fig 4. Subcellular localization of HA- …KDSAKN, human SLC35B4 K329A with HA-epitope at the N-terminus in the wild-type HepG2 cells.

Stably transfected wild-type (SLC35B4-WT) cells, overexpressing SLC35B4 variants, were subjected to indirect immunofluorescent staining with anti-HA (green) and anti-organelle markers (Table 3) (red) antibodies. Cell nuclei were counterstained with DAPI. Scale bar 10 μm.

Fig 5. Subcellular localization of HA-…KDSKAN, human SLC35B4 K330A with HA-epitope at the N-terminus in the wild-type HepG2 cells.

Stably transfected wild-type (SLC35B4-WT) cells, overexpressing SLC35B4 variants, were subjected to indirect immunofluorescent staining with anti-HA (green) and anti-organelle markers (Table 3) (red) antibodies. Cell nuclei were counterstained with DAPI. Scale bar 10 μm.

Fig 6. CRISPR-Cas9 knock-out of the SLC35B4 gene in HepG2 cells.

Genomic DNA (A) and total RNA (B) were isolated from SLC35B4-deficient (B4KO) and wild-type (WT) HepG2 cells and PCR (genomic DNA) or RT-PCR (mRNA) reaction was performed using SLC35B4 gene-specific primers. Products were separated in 2% agarose gel, containing ethidium bromide.

Fig 7. Subcellular localization of HA- …KDSKKN, human SLC35B4 with HA-epitope at the N-terminus in the knock-out HepG2 cells.

Stably transfected cells, lacking functional SLC35B4 gene (SLC35B4-KO), overexpressing SLC35B4 variants, were subjected to indirect immunofluorescent staining with anti-HA (green) and anti-organelle markers (Table 3) (red) antibodies. Cell nuclei were counterstained with DAPI. Scale bar 10 μm.

Fig 8. Subcellular localization of …KDSKKN-HA, human SLC35B4 with HA-epitope at the C-terminus in the knock-out HepG2 cells.

Stably transfected cells, lacking functional SLC35B4 gene (SLC35B4-KO), overexpressing SLC35B4 variants, were subjected to indirect immunofluorescent staining with anti-HA (green) and anti-organelle markers (Table 3) (red) antibodies. Cell nuclei were counterstained with DAPI. Scale bar 10 μm.

Fig 9. Subcellular localization of HA- …KDSAAN, human SLC35B4 K329AK330A with HA-epitope at the N-terminus in the knock-out HepG2 cells.

Stably transfected cells, lacking functional SLC35B4 gene (SLC35B4-KO), overexpressing SLC35B4 variants, were subjected to indirect immunofluorescent staining with anti-HA (green) and anti-organelle markers (Table 3) (red) antibodies. Cell nuclei were counterstained with DAPI. Scale bar 10 μm.

Fig 10. Subcellular localization of HA- …KDSAKN, human SLC35B4 K329A with HA-epitope at the N-terminus in the knock-out HepG2 cells.

Stably transfected cells, lacking functional SLC35B4 gene (SLC35B4-KO), overexpressing SLC35B4 variants, were subjected to indirect immunofluorescent staining with anti-HA (green) and anti-organelle markers (Table 3) (red) antibodies. Cell nuclei were counterstained with DAPI. Scale bar 10 μm.

Fig 11. Subcellular localization of HA-…KDSKAN, human SLC35B4 K330A with HA-epitope at the N-terminus in the knock-out HepG2 cells.

Stably transfected cells, lacking functional SLC35B4 gene (SLC35B4-KO), overexpressing SLC35B4 variants, were subjected to indirect immunofluorescent staining with anti-HA (green) and anti-organelle markers (Table 3) (red) antibodies. Cell nuclei were counterstained with DAPI. Scale bar 10 μm.

Fig 12. Analysis of N-glycans structures in the wild-type and SLC35B4 knock-out HepG2 cells.

MALDI-TOF-MS characterization of permethylated 2-AB-N-glycans purified from wild-type (WT) (A) and SLC35B4-deficient (B4KO) (B) HepG2 cells. Spectra were offset and scaled. For clarity of presentation mass region was restricted to 2500–4100 Da to contain complex-type N-glycans only. Identified peaks were labeled with mass information and cartoon representations of putative N-glycan chemical structures (based on biosynthetic knowledge). Identification of phosphorylated or sulfated glycans was not attempted. All variations of differently branched and sialylated complex-type N-glycans detected in the wild-type cells were also detected in SLC35B4 knock-out clones showing that N-glycosylation profile was not affected. Representative data from two independent experiments are shown.

Fig 13. Analysis of O-glycans structures in the wild-type and SLC35B4 knock-out HepG2 cells.

MALDI-TOF-MS characterization of permethylated Bn-O-glycans secreted to culture medium by wild-type (WT) (A) and SLC35B4-deficient (B4KO) (B,C) HepG2 cells. Spectra were scaled for maximum value. Identified peaks were labeled with mass information and cartoon representations of putative O-glycan chemical structures (based on biosynthetic knowledge). Identification of phosphorylated or sulfated glycans was not attempted. All variations of core 1- and core 2-type O-glycans detected in the wild-type cells were also detected in SLC35B4 knock-out clones showing that O-glycosylation profile was not affected. Representative data from two independent experiments are shown.

Fig 14. Analysis of proteoglycans.

Proteoglycans present in cell lysates of wild-type (WT) and SLC35B4-deficient (B4KO) cells were subjected to SDS-PAGE, transferred onto nitrocellulose membrane and probed with anti-chondroitin-4-sulfate (C4S) (A) or keratan sulfate (KS) (B) antibodies. Protein loading was assessed using Coomassie Brilliant Blue G-250 (CBB) staining (C).