Identification of a GH110 Subfamily of α1,3-Galactosidases

Abstract

In search of ␣-galactosidases with improved kinetic properties for removal of the immunodominant ␣1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of ␣-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454 -464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Gal␣1-3(Fuc␣1-2)Gal, whereas linear oligosaccharides terminated by ␣1,3-linked galactose such as the immunodominant xenotransplantation epitope Gal␣1-3Gal␤1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific ␣1,3-galactosidases that act equally well on both branched blood group B and linear ␣1,3Gal structures. We determined by one-dimensional 1 H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known ␣-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant ␣3Gal xenotransplantation epitope.

Thiogalactopyranosides are Resistant to Hydrolysis by α‐Galactosidases

“…To date the FragB gene family has only been found in prokaryotes and no homologues or similar inverting enzymes have yet been found in eukaryotes. [21] As expected, α-1,3-galactosidase did not act on compounds lacking a α-1,3 galactoside linkage ( 1 , 2a , 5 , 6 , 9 and 10 ) (Table 1, right). However, all iGb3 derivatives ( 3 , 4 , 7 and 8 ) including thio-galactoside compounds were hydrolyzed by the enzyme.…”

“…[21] This enzyme catalyzes hydrolysis with inversion of configuration. To date the FragB gene family has only been found in prokaryotes and no homologues or similar inverting enzymes have yet been found in eukaryotes.…”

“…Bacteroides fragilis α-1,3-galactosidase, BfGal110B (FragB) which hydrolyzes blood group B and linear α1,3-Gal structures was expressed and purified as previously reported. [21] The production and purification of recombinant bovine α-1,3-galactosyltransferase [27] and α-1,4-galactosyltransferase from Neisseria meningitidis [28] have been described earlier. Sep-Pak C-18 cartridges were obtained from Waters.…”

“…These were prepared using α(1→3)- and α(1→4)-galactosyltransferases, UDP-galactose (UDP-Gal) or UDP-5′-thio-galactose as the donors, and fluorescently tagged Lac-OR ( 2a and 2b, Figure 2) as acceptors. These were tested to see whether they are resistant to hydrolysis by two different types of galactosidase enzymes, human α-galactosidase A (CAZy GH27, a retaining glycosidase) [20] and Bacteroides fragilis α-1,3-galactosidase (FragB, CAZy GH110, an inverting glycosidase) [21] and enzymes present in mammalian cell extracts which are all retaining α-galactosidases. In addition to the in vitro assays, hydrolytic resistance of 5-thio-galactoside was explored in vivo .…”

Ansätze zur Produktion von universellem Blut durch strukturgerichtete Evolution von Glykosidhydrolasen

“…Andere Versuche wurden seither unternommen, z. B. die Verwendung von bakteriellen Glykosidasen,3c das Problem der geringen Aktivität erwies sich jedoch als hartnäckig 3a,b…”