Genetic gradual reduction of OGT activity unveils the essential role of O-GlcNAc in the mouse embryo
Fig 1
Structure-guided generation of Ogt’s hypomorphic allelic series.
(A) Crystal structure of human OGT isoform 1 (UniProt O15294-1) in complex with UDP-5SGlcNAc (PDB 4GYY). The area in the square is shown as an insert with higher details. Except for two single amino acid substitutions outside the catalytic core, human OGT1 is identical to Mus musculus OGT isoform 1 (UniProt Q8CGY8-1), hence residues’ numbering is the same. The residues mutated in this study are shown in sticks representation in blue and UDP-5SGlcNAc in sticks representation, coloured by heteroatom. T931 and Q849 establish direct interactions with the donor substrate. Y851 interacts with the donor substrate via hydrogen bonds with an intermediate water molecule. H568 is considered to be the catalytic base and in the crystal structure it coordinates different water molecules near the binding site. (B) Scheme of the murine Ogt genomic context with the location of the introduced point mutations. The exons of the longest transcript Ogt-201 are numbered. (C) Catalytic rate measured in vitro for the four studied OGT single amino acid substitutions. Data are from Martinez-Fleites et al. 2008 [46] and for H568A also from Lazarus et al. 2011 [111]. (D) For the four hypomorphic mutants of OGT described in (A-C): number of animals (F0) bearing the correct point mutation after CRISPR-targeted mutagenesis in the zygote (founders); number of mutants (F1) produced by female founders (female germline transmission, i.e. maternal transmission); lethal phenotypes observed from the F1 generation onwards.