Fig 4.
Mutagenesis identifies one PglA amino acid responsible for diminished galactosyltransferase activity. Expression of different glycans was monitored by immunoblotting with the glycan-specific antibodies npg1, npg2, and npg3. (A) Various amino acid substitutions were introduced into PglAFA1090, and the following glycosylation patterns were examined. Mutating only one amino acid, A350G, was sufficient to obtain the same glycosylation pattern obtained when pglAN400 was introduced into FA1090 (sixth lane). All amino acids mutated are shown for each lane. Strains used were KS300, KS100, KS462, KS475, KS496, KS490, KS465, KS468, KS469, KS467, KS472, KS470, KS466, KS471, KS476, and KS491. (B) An assortment of amino acid mutations was introduced into PglAN400, and the glycosylation patterns that followed were examined. The analogous G350A mutation in PglAN400 changed the glycosylation pattern, which was similar to that of pglAFA1090 expressed in N400 (last lane). All amino acids mutated are shown for each respective lane. Strains used were KS300, KS100, KS463, KS495, KS473, KS474, and KS492. (C) An alignment of PglA protein sequences from FA1090 and N400, where all dissimilar amino acids are shown in bold.