Accelerated Enzymatic Galactosylation of N-Acetylglucosaminolipids in Lipid Microdomains

Abstract

A fluoro-tagged N-acetylglucosamine-capped glycolipid that can form lipid microdomains in fluid phospholipid bilayers has been shown to be enzymatically galactosylated by bovine β(1,4)-galactosyltransferase. MALDI MS, HPLC, and LC-MS revealed that the rate of enzymatic transformation was significantly enhanced by lipid clustering; at a 1% mol/mol loading, clustered glycolipids were galactosylated 9-fold faster than glycolipids dispersed across the bilayer surface. The transformation of the GlcNAc "glycocalyx" into a Gal(β1-4)GlcNAc "glycocalyx" relabeled these vesicles, making them susceptible to agglutination by Erythrina cristagalli lectin (ECL). The kinetic parameters for this transformation revealed a lower apparent Km when the substrate lipids were clustered, which is attributed to multivalent binding to an extended substrate cleft around the active site. These observations may have important implications where soluble enzymes act on substrates embedded within cellular lipid rafts.

Unprecedented inhibition of glycosidase-catalyzed substrate hydrolysis by nanodiamond-grafted O-glycosides

“…1 This has demonstrated that the multivalent presentation of a particular glycotope on an appropriate scaffold (i.e. [2][3][4][5][6][7][8][9][10][11][12][13] Indeed, the promise that specic lectin-mediated processes (i.e., pathogen recognition, viral entry, tumor migration and metastasis events, etc.) can affect signicantly its potency and selectivity of binding with a given target protein, relative to that of a monovalent counterpart.…”

“…cellmembrane, dendrimer, polymer, nanoparticle, etc.) [8][9][10][11]13,[15][16][17][43][44][45][46] Glycosidases constitute a large and important family of enzymes that are responsible for assuring the proper biosynthesis and/or biodegradation of glycoproteins, glycolipids and also proteoglycansthe very entities that serve as ligands for complimentary lectins in vivo. [2][3][4][5][6][7][8][9][10][11][12][13] Indeed, the promise that specic lectin-mediated processes (i.e., pathogen recognition, viral entry, tumor migration and metastasis events, etc.)…”

Multivalent Inhibitors for Carbohydrate‐Processing Enzymes: Beyond the “Lock‐and‐Key” Concept

“…Webb and co‐workers have developed models of “lipid rafts”, which are liquid‐ordered microdomains within the phospholipid bilayer, concentrating cell‐adhesion molecules (Figure 1). 4 GlcNAc sugars were covalently attached to perfluoroalkyl–pyrene motifs, which were previously shown to form vesicle aggregates with significant phase separation 5. These ordered fluorolipid domains within the membranes mimicked the naturally occurring “lipid raft” at cell membranes.…”

Application of Biocatalysis to on‐DNA Carbohydrate Library Synthesis

“…[9,10] Given the complexity of carbohydrate structure and the need for highly selective transformations, carbohydrate-basedt arget libraries are an excellent test for the application of biocatalysis to synthesis of DNA-encoded ligand libraries. [15][16][17][18][19] The overall strategy to accessalibrary of DNA-carbohydrate glycoconjugates by using ac ombination of chemical methods and biocatalysis is outlined in Scheme1.T he initial step involvesc hemical ligation of the first carbohydrate building block,f ollowed by biocatalytic transformationt oe ither elongate the glycan structure by glycosylationo rg enerate an aldehyde by oxidation of the primary sugar hydroxyl groupsf or the site-specific modification of the carbohydrate by reductive amination or hydrazone ligation. [12] Given the lack of precedent, we decided to study biocatalysts that had previously been shown to tolerate diverse conjugates and focus on glycosylationr eactions catalyzed by glycosyltransferases [13] and selectivef unctionalization of carbohydrates.…”

“…Moreover, the reductivea mination approach was generally associated with both high conversiona nd purity when using av ast variety of amine derivatives, makingt his reactions uitablef or DEL synthesis. In contrast, high conversion was obtained for secondary amines 48-51 (Table S2, entries [13][14][15][16], particularly with pyrrolidine 48 (95 %, entry 13). [31] Briefly,t he aldehyde in MOPS buffer (300 mm, pH 7.4) was mixed with al arge excesso fa mine and sodium cyanoborohydride (NaBH 3 CN), followed by incubation for 16 h at 37 8C.…”