Generation of lpgat1 mutant zebrafish by CRISPR/Cas9 system. (A) Domain structure and targeting sites for disruption of zebrafish Lpgat1 and schematic diagram of mutant Lpgat1 proteins. The active domain (motif 1) and the three acyl-acceptor binding domains (motif 2–4), which are highly conserved in AGPAT family molecules, are indicated by a pink box and blue boxes, respectively. For efficient disruption of Lpgat1 function, two sites near the functional motif were targeted. Both mutants 1 and 2 are predicted to be truncated proteins. The changed amino acid sequences are indicated by black bars. (B) Sequence alignment of the mutant alleles with predicted amino acids. Mutated nucleotide sequences and the resulted stop codons are in red. Targeting sites are in green. PAM sequences are underlined. Predicted amino acid sequences are in gray. (C) Lysophosphatidylglycerol acyltransferase (LPGAT) activities of wild-type and mutant Lpgat1 proteins. The LPGAT assay was performed using oleoyl lysophosphatidylglycerol (C18:1 LPG), palmitoyl-CoA (C16:0-CoA) and the microsomal fraction from HEK293A cells expressing wild-type or mutant Lpgat1 proteins. Relative LPGAT activities (activity of the microsomal fraction from vector-transfected cells = 1) are shown. The data represents the mean ± S.D. of triplicate measurements. Statistically significant differences are marked with asterisk. **p < 0.01. Two-way ANOVA, Holm’s multiple comparison test was used.
|
