Identification and characterization of emGalaseE, a β-1,4 galactosidase from Elizabethkingia meningoseptica, and its application on living cell surface

β-Galactosidase (EC 3.2.1.23) is an important biocatalyst with both transglycosylation and hydrolysis activities[1]. The first galactosidase was reported in 1889[2], and the first sequence and crystal structure of galactosidase was published in 1983 and 1994 respectively[3, 4]. Galactosidase is widely presented in bacteria, yeast, fungi, plant and mammals[5]. Based on the similarity of protein sequences, β -galactosidases can be classified into 4 glycoside hydrolase (GH) families, including GH -1, GH -2, GH - 35, and GH -42[6]. The substrates for galactosidase is the non -reducing β - D -galactose on oligosaccharides and glycans, such as lactose, galacto -oligosaccharides (GOS), free glycans and glycans on glycoproteins[7]. Galactosidase has been widely used in the food industry, especially for the dairy products. Galactosidase -treated milk is a solution for lactose intolerant people[8 -11]. The galactosidase -generated GOS is an important prebiotic ingredient in dairy products[12 -15] . Although N -glycosylation is a common post -translational modification (PTM) observed in all eukaryotic cells[16], report on galactosidase directly on glycoproteins is rare. Butler et al. firstly applied bovine testes β -galactosidase in detailed structure analysis for free N -glycans in 2003[17]. Subsequently the method was used for diagnosis of congenital disorders of glycosylation (CDG) and identification of antibody glycoforms[18, 19]. A limitation of the reported method is that N -glycans must be released by PNGaseF from denatured proteins to be a substrate for galactosidase. In 2018, Butler et al. reported that a galactosidase from Bacteroides fragilis could remove terminal galactose directly from naïve antibody thus could be applied for high yields of single glycoform antibodies[20, 21]. However, all the previous studies on galactosidase activity were conducted in a cell -free system. It is noteworthy that a significant proportion of cell surface proteins are glycoproteins[22, 23]. Some N -glycans on cell surface glycoproteins contain terminal galactose[24]. Studies on galectins have illustrated that galactose on cell surface may play a critical biological function[25, 26]. Galectins could bind to the galactose molecules present on cell surface to regulate cell viability, function, proliferation, and differentiation[27 - 34]. Nevertheless, research on the function of cell surface galactose is limited partially because of the lack of tool to cleave galactose on living cells. In this investigation, we isolated a galactosidase from Elizabethkingia meningoseptica, demonstrated its activity directly on terminal galactose on living cell surface, conducted preliminary study on its regulatory action on targeted cells, and named it emGalaseE or glycan-editing galactosidase I (csgeGalaseI).