Fig 1.
Leloir pathway of galactose metabolism.
Illustrated are the 2 different pathways in which galactose and GalNAc can be salvaged or synthesized for use in glycosylation. Galactose and GalNAc can be taken up and converted to UDP-galactose and UDP-GalNAc, respectively, via the salvage pathway. UDP-galactose and UDP-GalNAc can also be interconverted from UDP-glucose and UDP-GlcNAc respectively by the enzyme UDP-galactose-4-epimerase (GALE). UDP-galactose and UDP-GalNAc can then be used for glycosylation.
Fig 2.
Generation of knockout cell lines using CRISPR/Cas9.
Three gRNAs per target gene were cloned into an expression vector expressing both Cas9 as well as a GFP tag. A) Following transient transfection of HEK293T cells, GFP expression was analyzed by fluorescent microscopy at 24 hours post-transfection using a Zeiss Axio Observer A1 Microscope (20x) to determine the quality of the transfection. B) Gating strategy for single cell GFP sorts. The 10% highest GFP-expressing cells were sorted into 96-well plates, where one cell was sorted per well. FSC-W by FSC-A and SSC-W by SSC-A were used to reduce the rate of duplets.
Fig 3.
Validation of GALE knockout cell lines.
Following single cell sort, potential GALE KO clones were allowed to grow until they reached confluence in 6 well plates. A) Cell lysate was harvested by incubating cells in RIPA buffer. Clarified lysate was loaded onto a 4–12% SDS-PAGE gel and probed with an anti-GALE antibody. Wild-type HEK293T cell lysate was loaded in the first lane as a control. B) GALE enzymatic activity was analyzed on the protein level. GALE KO cells (clone 4) were transfected with expression vectors for SIVmac239 gp120 made as a truncated secreted product with a C-terminal polyhistidine tag. Secreted gp120 was purified from supernatant 48 hours post transfection using nickel-NTA columns. 3μg of purified gp120 was run on three 4–12% SDS-PAGE gels in triplicate. The first was analyzed by Coomassie Blue staining. The second gel was probed for gp120 using the rhesus anti-gp120 monoclonal antibody 3.11H. The third gel was probed for O-glycosylation using an HRP-labeled Jacalin lectin. For protein production, cells were grown in 10% FBS, 3% LDFBS, or 3% LDFBS with galactose and GalNAc (+sugars) until 12 hours post-transfection, at which point cells were washed and changed to serum free media. For further detail on cell culture conditions, refer to the materials and methods. C) Western blots were repeated as in B, with one exception. Galactose and GalNAc were added to the cell culture media separately to identify whether galactose or GalNAc was the limiting precursor for O-glycosylation in our cell culture conditions.
Fig 4.
Validation of GALK1 and GALE+GALK1 deficient knockout cell lines.
Following single cell sort, potential knockout clones were allowed to grow until they reached confluence in 6 well plates. GALK1 enzymatic activity was analyzed on the protein level. HEK293T, GALE KO, GALK1 KO cells (clone 10), and GALE+GALK1 double KO cells (clone 12) were transfected with expression vectors for SIVmac239 gp120 made as a truncated secreted product with a C-terminal polyhistidine tag. Secreted gp120 was purified from supernatant 48 hours post transfection using nickel-NTA columns. 3μg of purified gp120 was run on two 4–12% SDS-PAGE gels in duplicate. The first gel was probed for gp120 using the rhesus anti-gp120 monoclonal antibody 3.11H. The second gel was probed for O-glycosylation using an HRP labeled Jacalin lectin. For protein production, cells were grown in serum free media, serum free media +galactose, or serum free media +galactose +GalNAc as indicated. For further detail on cell culture conditions, refer to the materials and methods.
Fig 5.
Validation of GALK2 and GALE+GALK2 deficient knockout cell lines.
Following single cell sort, potential knockout clones were allowed to grow until they reached confluence in 6 well plates. A) Cell lysate was harvested by incubating cells in RIPA buffer. Clarified lysate was loaded onto a 4–12% SDS-PAGE gel and probed with an anti-GALK2 antibody. Parental GALE KO cell lysate was loaded in the first lane as a control. B) GALK2 enzymatic activity was analyzed at the protein level. GALK2 KO cells (clone 7) and GALE+GALK2 double KO cells (clone 7) were transfected with expression vectors for SIVmac239 gp120 made as a truncated secreted product with a C-terminal polyhistidine tag. Secreted gp120 was purified from supernatant 48 hours post transfection using nickel-NTA columns. 3μg of purified gp120 was run on three 4–12% SDS-PAGE gels in triplicate. The first was analyzed by Coomassie Blue staining. The second gel was probed for gp120 using the rhesus anti-gp120 monoclonal antibody 3.11H. The third gel was probed for O-glycosylation using an HRP labeled Jacalin lectin. For protein production, cells were grown in serum free media, serum free media +galactose +GalNAc (+Sugars), or serum free media +UDP-galactose +UDP-GalNAc (+UDP) as indicated. For further detail on cell culture conditions, refer to the materials and methods.
Fig 6.
HEK293T cells, GALE KO (clone 4), GALK1 KO (clone 10), GALE+GALK1 KO (clone 12), GALK2 KO (clone 7), and GALE+GALK2 KO (clone 7) were analyzed to determine if disrupting key enzymes of the Leloir pathway impacted cell growth. 4x10^4 HEK293T cells were plated in triplicate in 6-well plates starting on day 0. Cells were harvested and counted using a Beckman Coulter Z1 Coulter Particle Counter every 24 hours.
Fig 7.
Mass spectrometry analysis of total cell lysate.
HEK293T cells, GALE KO (clone 4), GALK1 KO (clone 10), GALE+GALK1 KO (clone 12), GALE+GALK2 KO (clone 7), and GALE+GALK1 KO cells +galactose were grown in standard DMEM-10% FBS media. Cells were washed 3 times in cold PBS before analysis. A) MALDI-TOF mass spectra of permethylated O-glycans isolated from total cell lysates. All molecular ions are [M+Na]+. The sugar symbols are those as described in [28]. Structural assignments are based on monosaccharide composition (obtained by MALDI-TOF MS), fragmentation analyses (MALDI-TOF/TOF MS/MS), and knowledge of glycan biosynthetic pathways. All peakes not labeled with am/z value are not glycans and are either matrix or general chemical background peaks. B) MALDI-TOF MS profiles of the permethylated N-linked glycans from total cell lysate of HEK293T cells, GALE KO, GALK1 KO, GALE+GALK1 KO, GALE+GALK2 KO, and GALE+GALK1 KO cells +galactose. All molecular ions are present in sodiated form [M +Na]+.