Cellular localization and functions of DGAT1

A protein BLAST search with human DGAT1 (UniProt ID O7590) was performed to find homologous proteins in the Dictyostelium genome. Five homologues were identified, DDB0304727, DDB0238278, DDB0304757, DDB0308488 and DDB0220436 (Fig. 50A and

Fig. 49: No alteration of phagocytosis in DGAT2-KO cultivated in the medium with PA. Two independent DGAT2-KO clones 1-7 and 2-5 were applied in the phagocytosis assay together with AX2 cells. The assay was performed and exhibited as Fig. 24. The result was derived from 3 replicates.

Tab. 10) of which DDB0304727 had the highest score. Except the N-terminal approximate 200 aa which are special to Dictyostelium DGAT1, the remaining sequence (the blue line with gene ID DDB_G0271342, Fig. 50) spans 34% identical and 54% similar sequence regions (Tab. 10) to the human DGAT1 protein. Therefore, this protein was selected and named as DGAT1 to study its functions, focusing on the biosynthesis of TAG.

Fig. 50: Five homologous proteins in Dictyostelium to human DGAT1 and the degree of homology for each protein. These 5 homologues to human DGAT1 were identified by protein BLAST research at Dictybase. The similar regions of each protein to human DGAT1 are shown in blue lines and exact value of identical and similar sequences in the region are shown in Tab. 10. The gene ID of each protein is shown in this figure, and the corresponding Dictybase ID can be also read in Tab. 10.

Tab. 10: Protein sequence comparison of human DGAT1 protein with Dictyostelium homologues Dictybase ID Gene ID Identities aa Positives Score(bits) E-value DDB0304727 DDB_G0271342 141/414 (34%) 617 223/414 (54%) 246 2e-65 DDB0238278 DDB_G0282197 77/237 (32%) 480 123/237 (51%) 120 2e-27 DDB0304757 DDB_G0276523 43/175 (24%) 518 77/175 (44%) 62.8 3e-10 DDB0308488 DDB_G0284259 17/55 (30%) 907 26/55 (47%) 35 0.07 DDB0220436 DDB_G0290621 21/57 (36%) 967 32/57 (56%) 34.7 0.087

The full length genomic dgat1 sequence comprises 2,219 bp with 3 introns, and is located on chromosome 2 of Dictyostelium. The DGAT1 protein is composed of 617 aa, corresponding to a molecular weight of 72 kDa.

2.4.3.3.2 GFP-tagged DGAT1 protein localizes to ER in Dictyostelium cells in the presence and absence of PA

In order to find out the cellular localization of DGAT1, plasmids expressing GFP-DGAT1 and DGAT1-GFP were constructed and transformed into WT Dictyostelium cells. Both cell lines showed a rather weak expression in Western-blot (Fig. 51).

Fig. 51: Western-blot of GFP-tagged DGAT1. The protein samples were extracted from 3x10⁶ GFP-DGAT1 (#751), DGAT1-GFP (#752) and AX2 cell strains. Because of the very low expression, the GFP-tagged DGAT1 cells were grown in G30 selection medium to increase the expression level of fusion protein. The calculated GFP-tagged DGAT1 is 99 kDa and their expressions were detected by anti-GFP antibody. Porin (30 kDa) serves as loading control. Protein sample extracted from AX2 was used as negative control as it lacks the GFP protein.

Molecular weights of protein marker (M) are shown on the left.

Unlike tagged DGAT2 stimulating LD accumulation (Diploma thesis, 2009), GFP-DGAT1 and GFP-DGAT1-GFP stains did not show increased LD formation as compared to WT cells, none or 1-2 LDs per cell (data not shown). The majority of GFP-tagged DGAT2 localized to LD and a small fraction to the ER in normal and PA-supplemented medium, while GFP-tagged DGAT1 localized to the ER exclusively, but did not move to LDs even after stimulation with PA (Fig. 52 A and C). Besides the ER localization, both fusion-proteins also showed some aggregation: some GFP-DGAT1 cells had a strong signal in some parts of nuclear envelope, and/or 1-3 small dots or tiny rings in the cytoplasm (Fig. 52B); most DGAT1-GFP cells showed similar dots or rings in the cytoplasm but lacked nuclear membrane accumulation (Fig. 52A lower panel and C), possibly due to the lower expression levels (see Fig. 51). Since no stainable structure could be observed by LD540 in the normal medium, the small dots or rings might not reflect association with LDs. They also did not stain with LD540 when the cells were induced by PA-treatment (Fig. 52C).

Fig. 52: tagged DGAT1 localizes to the ER. The cell lines used for confocal imaging were from GFP-DGAT1 (#651) clone 1-3 and GFP-DGAT1-GFP (#652) clone 1-4. They were grown in normal medium (A) or in medium containing PA (B and C). After being fixed, the cells were incubated with anti-PDI antibody or LD540 as indicated on the top of images. Scale bar shows the length of 5 µm.

2.4.3.3.3 Generation of DGAT1-KO Dictyostelium cells

A DGAT1-KO was generated by inserting the floxed Bsr cassette into the first exon of DGAT1. Like in the case of GPAT3, the primer upstream to the start codon shows robust amplification together with the Bsr-specific oligonucleotide or using an in-gene primer, across the Bsr cassette (Fig. 53 A and B). The genomic fragment amplified with the Bsr-specific

primer in combination with the DGAT1 upstream primer is 0.9 kb long in the DGAT1-KO and no amplification occurred in AX2 cells (Fig. 53B, left panel). The amplification using primers specific for upstream sequences and in-gene regions achieved a 2.6 kb fragment in DGAT1-KO cells and 1.0 kb in the WT strain (Fig. 53B, right panel).

Fig. 53: Construction and PCR confirmation of the DGAT1-KO. A) Schematic representation of dgat1 inserted with the floxed Bsr cassette (from plasmid #840) in the genome. The Bsr cassette was inserted in the first exon of dgat1 and transcribed in the same direction as the dgat1 gene. The positions of primers used for PCR verification are shown and the arrows indicate their directions to be amplified. Exons are coloured bright blue and floxed Bsr cassette was shown in red. Thin lines indicate genome regions outside the target gene. I1, I2 and I3 indicate 3 introns. B) The PCR verification of DGAT1-KO. Two primer combinations used for verification are indicated at the bottom of each DNA-gel. TRX, the quantity control for template gDNA, was amplified by primers #216 5’-TRX and #217 3’-TRX. A DNA-marker (M) is shown on the left side of each image.

2.4.3.3.4 The TAG and UKL content are strongly decreased in DGAT1-KO cells

Unlike what we had seen in the DGAT2-KO strains, DGAT1-KO cells showed a dramatically reduced cellular TAG content as measured by the enzymatic TAG assay when cells were grown in medium supplemented with PA (Fig. 54A). The decreased TAG content was further verified by TLC lipid separation (Fig. 54B). Furthermore, the unknown lipid, UKL, is lost in DGAT1-KO cells (Fig. 54B). This result indicated that DGAT1 is the major enzyme mediating TAG and UKL synthesis in Dictyostelium cells.

2.4.3.3.5 DGAT1-KO decreases the phagocytosis in the medium supplemented with PA Again, it is important to know if the strongly reduced TAG content in the cells can affect phagocytosis of Dictyostelium cells as previously observed with the GPAT3-KO (Fig. 36).

This could give another hint on the relationship between the cellular TAG content and its connection to phagocytic function.

Fig. 55: Phagocytic activity of DGAT1-KO in the medium with or without PA. The phagocytosis assay was performed and presented as in Fig. 24. A) DGAT1-KO showed no influence on phagocytosis in regular medium.

The mean and SD were calculated from 5 replicates. B) Decreased phagocytic rate in DGAT1-KO incubated in PA-containing medium. This assay was repeated for 4 times.

Fig. 54: DGAT1-KO cells exhibit reduced TAG and UKL content. Two independent DGAT1-KO clones 1-17 and 2-10 were employed for TAG measurement together with AX2 in normal medium (PA-) or medium with PA (PA+). A) DGAT1-KO cells have a decreased TAG content in medium with PA as determined by the enzymatic assay. The experiments were repeated for 3 times and shown as mean ± SD. *** indicates p<0.001 versus AX2. B) Lipid composition of WT and DGAT1-KO strains on a TLC plate. The black arrow indicates TAG while the red arrow shows UKL migrating position on the TLC plate. The solvent system used for the neutral lipid separation and the lipid standard are specified in Fig. 12B.

The two assays are shown correspondingly.

Phagocytosis was measured with 2 independent DGAT1-KO clones in the medium with or without PA. This time, DGAT1-KO cells showed no effect on phagocytosis in normal growth medium (Fig. 55A). Furthermore, DGAT1-KO even decreased the phagocytosis capacity of the cells compared wild type in the medium added by PA significantly by about 27% (Fig.

55B).

2.4.3.4 Investigating the functional relationship between DGAT1 and DGAT2 in double