Enzymatic Synthesis of N-Acetyllactosamine (LacNAc) Type 1 Oligomers and Characterization as Multivalent Galectin Ligands

Abstract

Repeats of the disaccharide unit N-acetyllactosamine (LacNAc) occur as type 1 (Galβ1, 3GlcNAc) and type 2 (Galβ1, 4GlcNAc) glycosylation motifs on glycoproteins and glycolipids. The LacNAc motif acts as binding ligand for lectins and is involved in many biological recognition events. To the best of our knowledge, we present, for the first time, the synthesis of LacNAc type 1 oligomers using recombinant β1,3-galactosyltransferase from Escherichia coli and β1,3-N-acetylglucosaminyltranferase from Helicobacter pylori. Tetrasaccharide glycans presenting LacNAc type 1 repeats or LacNAc type 1 at the reducing or non-reducing end, respectively, were conjugated to bovine serum albumin as a protein scaffold by squarate linker chemistry. The resulting multivalent LacNAc type 1 presenting neo-glycoproteins were further studied for specific binding of the tumor-associated human galectin 3 (Gal-3) and its truncated counterpart Gal-3∆ in an enzyme-linked lectin assay (ELLA). We observed a significantly increased affinity of Gal-3∆ towards the multivalent neo-glycoprotein presenting LacNAc type 1 repeating units. This is the first evidence for differences in glycan selectivity of Gal-3∆ and Gal-3 and may be further utilized for tracing Gal-3∆ during tumor progression and therapy.

Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System

“…The enzyme production was done as described in our previous studies by Engels et al [15] for UDP-glucose-dehydrogenase (UGDH, EC 1.1.1.22), NADH oxidase (NOX, EC 1.6.99.-) and β1,3glucuronyltransferase (β3GlcAT, EC 2.4.1.17, and by Wahl et al [13] for galactokinase (GalK, EC 2.7.1.6) and for UDP-sugar pyrophosphorylase (USP, EC 2.7.7.64), [12] and by Fischöder et al [14] for β1,4galactosyltransferase (β4GalT, EC 2.4.1.38). The enzyme production was done as described in our previous studies by Engels et al [15] for UDP-glucose-dehydrogenase (UGDH, EC 1.1.1.22), NADH oxidase (NOX, EC 1.6.99.-) and β1,3glucuronyltransferase (β3GlcAT, EC 2.4.1.17, and by Wahl et al [13] for galactokinase (GalK, EC 2.7.1.6) and for UDP-sugar pyrophosphorylase (USP, EC 2.7.7.64), [12] and by Fischöder et al [14] for β1,4galactosyltransferase (β4GalT, EC 2.4.1.38).…”

“…The enzymes studied in this work are fusion constructs with an N-terminal polyhistidine (His 6 )-tag. The enzyme production was done as described in our previous studies by Engels et al [15] for UDP-glucose-dehydrogenase (UGDH, EC 1.1.1.22), NADH oxidase (NOX, EC 1.6.99.-) and β1,3glucuronyltransferase (β3GlcAT, EC 2.4.1.17, and by Wahl et al [13] for galactokinase (GalK, EC 2.7.1.6) and for UDP-sugar pyrophosphorylase (USP, EC 2.7.7.64), [12] and by Fischöder et al [14] for β1,4galactosyltransferase (β4GalT, EC 2.4.1.38). The linker-modified substrate N-acetylglucosamine with a tert-butyloxycarbonyl protected amino group (GlcNAc-linker-tBoc) was kindly provided by Prof. Vladimír Křen (Institute of Microbiology, Czech Academy of Sciences).…”

“…Details of the IR-unit for preheating and the TCM are discussed by Heinzler et al, [11] describing the reactor system. To test different temperatures, the activity of 15 mg/mL MEB was investigated at the respective optimal pH value of the free enzymes according to literature (UGDH/NOX/ GalK, [13] USP, [12] GalT, [14] GlcAT [15] ). For each assay, 15 μg of MEB with a loading of 15 mg immobilized enzyme per mL settled beads were used in a 100 μL compartment.…”

“…Galactokinase (GalK) was combined with UDP-sugar pyrophosphorylase (USP) in an enzyme cascade reaction for the synthesis of UDP-galactose (UDP-Gal, 3) from d-(+)-galactose (Gal,1) via α-d-galactose-1-phosphate (Gal-1-P, 2) in the EM-UDP-Gal. [14] The UDP-GlcA, produced in the EM-UDP-GlcA, was used for glucuronosyltransferase (GlcAT) to transfer the GlcA onto LacNAclinker-tBoc in the EM-GlcAT, resulting in the product non-sulfated human natural killer cell-1 (HNK-1) epitope (8). [13] In the EM-GalT, β1,4-galactosyltransferase (GalT) transfers galactose from UDP-Gal onto a tert-butyloxycarbonyl protected N-acetylglucosamine (GlcNAclinker-tBoc, 6) to synthesize N-Acetyl-d-lactosamine (LacNAc-linker-tBoc, 7).…”

“…[13] In the EM-GalT, β1,4-galactosyltransferase (GalT) transfers galactose from UDP-Gal onto a tert-butyloxycarbonyl protected N-acetylglucosamine (GlcNAclinker-tBoc, 6) to synthesize N-Acetyl-d-lactosamine (LacNAc-linker-tBoc, 7). [14] The UDP-GlcA, produced in the EM-UDP-GlcA, was used for glucuronosyltransferase (GlcAT) to transfer the GlcA onto LacNAclinker-tBoc in the EM-GlcAT, resulting in the product non-sulfated human natural killer cell-1 (HNK-1) epitope (8). [15] In this work, we demonstrate automated enzymatic glycan synthesis by enzyme cascade reactions, conducted in the CFMS, using six different enzymes immobilized on magnetic particles (Scheme 1A).…”