Understanding of the Contribution of Fetuin O-glycans for the Release of New Bioactive Compounds by a Novel Endo-?-N-acetylglucosaminidase

Bovine fetuin is a model protein to study the activity of variousglycosidases since it contains both N- and O- glycans attached to thepolypeptide chain. We recently showed a novel glycosidase, endo-?-Nacetylglucosaminidaseisolated from an infant gut microbe,Bifidobacterium infantis. This enzyme is capable of cleaving the N-N’-diacetyl chitobiose moiety found in the N-glycan core of a wide varietyof proteins. It is considered a promising approach to release N-glycansfrom complex substrates such as whey proteins due to its high activityand wide substrate specificity. Moreover, it also maintains its activityat high temperatures enabling the use of this enzyme in thermal dairyprocesses such as during the pasteurization. Bovine whey is apotential source of glycans providing million tons of glycoproteinsannually. Application of EndoBI-1 on bovine whey is challenging dueto the complexity of the whey proteins and their O-glycosylationpattern. O-glycans are considered to be a protective agent for Ndeglycosylationthat hinders the isolation of these recently found novelcompounds. In this study, O-glycans were removed from fetuin (bothO- and N- glycosylated model glycoprotein) and the contribution of Oglycansto the accessibility of EndoBI-1 to bovine fetuin N-glycanswere tested. Released glycans were characterized by advanced massspectrometry and 22 different N-glycans (including isomers) weremonitored. According to the results, it was shown that removing Oglycansfrom Fetuin increases the Kcat/Km value 0.52 to 1.54 ml/mgx min-1 and the affinity of EndoBI-1 (Km value from 0.32 to 0.22mg/ml) to target N-glycans enabling more feasible application of thisenzyme in dairy streams.