Enzymatic synthesis of natural and 13C enriched linear poly-N-acetyllactosamines as ligands for galectin-1

Abstract

As part of a study of protein-carbohydrate interactions, linear N-acetyl-polyllactosamines [Galbeta1,4GlcNAcbeta1,3]nwere synthesized at the 10-100 micromol scale using enzymatic methods. The methods described also provided specifically [1-13C]-galactose-labeled tetra- and hexasaccharides ([1-13C]-Galbeta1,4GlcNAcbeta1,3Galbeta1,4Glc and Galbeta1, 4GlcNAcbeta1,3[1-13C]Galbeta1,4GlcNAcbeta1,3Galbeta 1,4Glc) suitable for NMR studies. Two series of oligosaccharides were produced, with either glucose or N-acetlyglucosamine at the reducing end. In both cases, large amounts of starting primer were available from human milk oligosaccharides (trisaccharide primer GlcNAcbeta1,3Galbeta1, 4Glc) or via transglycosylation from N-acetyllactosamine. Partially purified and immobilized glycosyltransferases, such as bovine milk beta1,4 galactosyltransferase and human serum beta1,3 N- acetylglucosaminyltransferase, were used for the synthesis. All the oligo-saccharide products were characterized by1H and13C NMR spectroscopy and MALDI-TOF mass spectrometry. The target molecules were then used to study their interactions with recombinant galectin-1, and initial1H NMR spectroscopic results are presented to illustrate this approach. These results indicate that, for oligomers containing up to eight sugars, the principal interaction of the binding site of galectin-1 is with the terminal N-acetyllactosamine residues.

Galectin-1–Mediated Apoptosis in Mycosis Fungoides: The Roles of CD7 and Cell Surface Glycosylation

“…It is noteworthy that the presence of terminal sialic acid on core 1 O-glycans, added by the ST3Gal I sialyltransferase, inhibits the action of the core 2 GnT enzyme (49). Additional glycosyltransferases, such as the GnT V or the ST6Gal I, also regulate the expression or accessibility of saccharide ligands recognized by galectin-1 (42,43,45,46,51). Thus, in addition to CD7 expression, regulated expression of appropriate glycosyltransferases can also influence susceptibility to galectin-1.…”

“…The presence of a terminal ␣-2,6-linked sialic acid can mask the LacNAc sequences that are preferred galectin-1 ligands (37,51), and SNA reactivity of medullary thymocytes and T-cell lines correlates with loss of susceptibility to galectin-1 (43,45). In all samples, no SNA reactivity was detected on infiltrating Sezary cells or on small round lymphocytes present in the dermis and epidermis (Fig.…”

Dimeric Galectin-1 Binds with High Affinity to α2,3-Sialylated and Non-sialylated Terminal N-Acetyllactosamine Units on Surface-bound Extended Glycans

“…In contrast to these studies, recent data have suggested that dGal-1 may not recognize PL structures with higher affinity compared with its recognition of short non-extended structures (44,45). Interestingly, dGal-1 has been suggested to bind primarily to nonreducing terminal LN residues rather than to mid-chain LN residues within a PL chain (42,43,46).…”

“…dGal-1 has also been suggested to bind primarily to nonreducing terminal LN units rather than to mid-chain LN units within a PL chain (32,46). Other results also suggest that dGal-1 is able to bind to extended glycans containing a terminal LN unit attached to a non-PL backbone (42,43).…”

Expression of a Specific Glycosyltransferase Enzyme Regulates T Cell Death Mediated by Galectin-1

“…Previous studies have shown that lactosamine (Gal␤1-4GlcNAc) is the preferred minimal saccharide ligand bound by galectin-1. Galectin-1 has a relatively low affinity for single N-acetyllactosamine sequences; however, galectin-1 binds with high avidity to glycans containing multiple N-acetyllactosamine units and preferentially binds glycoproteins containing linear polylactosamine sequences (7)(8)(9)(10)(11). These linear polylactosamine sequences can be found on O-linked and N-linked glycans (Scheme 1).…”