Production of microhomogeneous glycopeptide by a mutated NGT according FuncLib with unique sugar as substrate

N-glycosylation is one of the most important post-translational modifications of proteins. Cytoplasmic soluble N-glycosyltransferase (NGT) exists in bacteria, which is able to transfer different monosaccharide from sugar nucleotide to the NXS/T(X not equal Pro) consensus sequence in a polypeptide. At present, the NGT enzymes reported could transfer a variety of different sugars to protein, which will lead to the heterogeneity of the sugar chain and the complexity and instability of the structure and function of glycopeptides. According to the FuncLib algorithm, we obtained mutant ApNGT-P1 from ApNGT (the NGT from Actinobacillus pleuropneumoniae) with increased substrate specificity. Compared with the wild-type ApNGT, mutant ApNGT-P1 could only utilize UDP-Glc as sugar donors. The optimum temperature of ApNGT-P1 was about 40 degrees C and the optimum pH was 7.5-8.0 in PBS buffer. ApNGT-P1 exhibited better tolerance for K+, Mn2+, Ca2+, and Mg2+, but was strongly inhibited by Na+, Cu2+ and Zn2+. The mutant can be applied to the efficient production of glycosylated peptides or proteins with uniform glucose at their glycosylation sites. Besides, this work provided a feasible pathway for further studies on the improving donor substrates selectivity of NGTs.

Automated summary

N-glycosylation is an important post-translational modification of proteins. A cytoplasmic soluble N-glycosyltransferase (NGT) in bacteria can transfer different sugars to proteins, leading to heterogeneity and complexity of glycopeptides. A mutant ApNGT-P1 with increased substrate specificity was obtained, which can be used for efficient production of glycosylated peptides or proteins with uniform glucose at glycosylation sites. This study also provides a feasible pathway for further research on improving donor substrate selectivity of NGTs.