Synthesis of Fucosyl- N -Acetylglucosamine Disaccharides by Transfucosylation Using α-l-Fucosidases from Lactobacillus casei

Abstract

bAlfB and AlfC ␣-L-fucosidases from Lactobacillus casei were used in transglycosylation reactions, and they showed high efficiency in synthesizing fucosyldisaccharides. AlfB and AlfC activities exclusively produced fucosyl-␣-1,3-N-acetylglucosamine and fucosyl-␣-1,6-N-acetylglucosamine, respectively. The reaction kinetics showed that AlfB can convert 23% p-nitrophenyl-␣-L-fucopyranoside into fucosyl-␣-1,3-N-acetylglucosamine and AlfC at up to 56% into fucosyl-␣-1,6-N-acetylglucosamine. Fucosyl-oligosaccharides (FUS) are coming to be of great interest due to their presence in human milk and their antiadhesive activity against pathogens. This last property is characterized by blocking the attachment of intestinal pathogens to host cells due to FUS structural similarity with cell-surface glycoconjugate receptors (1-4). In addition, genes encoding ␣-L-fucosidases (EC 3.2.1.51) have recently been found in genomes of bifidobacteria (5, 6) and lactobacilli (7), which raises issues regarding the role of these enzymes in the metabolism of fucose-containing structures and opens up the possibility of using FUS as novel prebiotics (8).To fully establish the biological function of FUS as antiadhesins and prebiotics, extensive studies are required, for which synthesis of sufficient amounts would be necessary. Chemical synthesis of FUS such as ABH and Lewis blood group antigens has long been performed, but it is a tedious and expensive process since it requires multiple protection and deprotection steps to achieve the desired selectivity (9, 10). The enzymatic synthesis of FUS is an alternative to chemical methods, and it offers the advantage of forming specific glycosidic linkages in the presence of other reactive functional groups. There are two types of enzymes that can carry out fucosylation: fucosyltransferases and fucosidases. Fucosyltransferases present high specificity toward the acceptor and do not hydrolyze the product (11, 12). However, those enzymes are generally difficult to express and they require an expensive sugar nucleotide or a complex multienzymatic system for the regeneration (11, 13). In contrast, oligosaccharides have been largely produced using the transglycosylation activity of glycosidases and engineered glycosynthases (14,15). Only a few works describe the synthesis of FUS by transglycosylation using ␣-L-fucosidases; however, they cover a wide range of sources such as mammalian tissues (16), fungi (17), and bacteria (18,19). The transglycosylation activity of ␣-L-fucosidases is generally moderate compared with the hydrolysis activity, although it is variable and depends on the origin of the enzymes (20).We have previously isolated three ␣-L-fucosidases (AlfA, AlfB, and AlfC) from Lactobacillus casei that cleaved ␣-linked L-fucose from natural FUS (7). AlfB and AlfC showed high specific activity on fucosyl-␣-1,3-N-acetylglucosamine (Fuc-␣-1,3-GlcNAc) and fucosyl-␣-1,6-N-acetylglucosamine (Fuc-␣-1,6-GlcNAc), respectively. These substrate specificities led us in the present work to explore the likely us...

Employment of fucosidases for the synthesis of fucosylated oligosaccharides with biological potential

“…). Fucosylated oligosaccharides are involved in numerous and crucial physiological processes such as the immune response, signal transduction, early embryogenesis and development, growth regulation, apoptosis, adhesion of pathogens, extravasation of leukocytes, maturation and interaction of gametes, and pathological processes, including arteriosclerosis and cancer .…”

“…These fucosidases were named AlfA, AlfB, and AlfC. The last two were evaluated for transfucosylation using pNP‐Fuc as the donor and GlcNAc as the acceptor . It was found that AlfB transfers fucose with the formation of α‐1,3 bonds, synthesizing α‐ l ‐Fuc‐(1–3)‐β‐ d ‐GlcNAc with a yield of 23%.…”

“…The synthesized oligosaccharides are important, since α‐ l ‐Fuc‐(1–3)‐β‐ d ‐GlcNAc can be found in human milk oligosaccharides such as LNFP‐III, the importance of which was discussed in Section 5.2. The disaccharide α‐ l ‐Fuc‐(1–6)‐β‐ d ‐GlcNAc can be found in glycans that are present in N‐glycosylated proteins, for example, in glycan A1F that is present in porcine thyroglobulin .…”

Unique Microbial Catabolic Pathway for the Human Core N -Glycan Constituent Fucosyl-α-1,6- N -Acetylglucosamine-Asparagine

“…Two putative rho-independent terminators were identified downstream of alfC (ΔG, Ϫ13.5 kcal/mol) and sugK (ΔG, Ϫ17.0 kcal/mol). The high specificity of the ␣-L-fucosidase AlfC from L. casei BL23 for ␣1,6 linkages such as those present at fucosyl-oligosaccharides (45,47), together with the sequence analysis of the alf-2 cluster, particularly the presence of a gene coding for a hypothetical N(4)-(␤-N-acetylglucosaminyl)-L-asparaginase, suggested that the alf-2 operon could be involved in the metabolism of the core 6=FN-Asn ( Fig. 1).…”

“…To test this hypothesis, this glycoamino acid was synthesized by transfucosylation with the ␣-L-fucosidase AlfC. We had previously utilized AlfC in transglycosylation reactions with p-nitrophenyl-␣-Lfucopyranoside (pNP-fuc) as the donor and GlcNAc as the acceptor to produce 6=FN (47). Here, the ability of AlfC to use GlcNAc-Asn as the acceptor was tested, and 6=FN-Asn was synthesized and purified.…”