ABSTRACT
ß(1,4)-Galactosyltransferase (ß4Gal-T1) and T. cruzi trans-sialidase (TcTS) have been used in a 'one-pot' cascade to provide vesicles (liposomes) with a trisaccharide coating. These soluble enzymes catalysed the transfer of galactose then sialic acid onto a synthetic N-acetylglucolipid embedded in the bilayers. Clustering of this substrate into microdomains increased the rate of sialylated lipid production, showing that an increase in ß4Gal-T1 activity is carried through the enzymatic cascade. These coatings modulated cell recognition. Hepatocellular carcinoma cells took up vesicles modified by ß4Gal-T1 alone more extensively than sialylated vesicles produced by 'one-pot' sequential enzymatic modification.
Subject(s)
Galactosyltransferases/chemistry , Glycolipids/metabolism , Glycoproteins/chemistry , Liposomes/metabolism , Neuraminidase/chemistry , Trisaccharides/chemical synthesis , Carbohydrate Sequence , Endocytosis/physiology , Glycolipids/chemistry , Glycosylation , Hep G2 Cells , Humans , Liposomes/chemistry , Trisaccharides/chemistryABSTRACT
A synthetic perfluoroalkyl-tagged lactosyl glycolipid has been shown to form lipid microdomains in fluid phospholipid bilayers. When embedded in the membranes of phospholipid vesicles, this glycolipid was trans-sialylated by soluble T. cruzi trans-sialidase (TcTS) to give a perfluoroalkyl-tagged glycolipid that displayed the ganglioside GM3 epitope, with up to 35% trans-sialylation from fetuin after 18 h. Following sialylation, vesicles bearing this Neu5Ac(α2-3)Gal(ß1-4)Glc sequence in their "glycocalyx" were recognised and agglomerated by the lectin M. amurensis leukoagglutinin. Monitoring TcTS-mediated trans-sialylation by HPLC over the first 6 h revealed that enzymatic transformation of bilayer-embedded substrate was much slower than that of a soluble lactosyl substrate. Furthermore, clustering of the lactose-capped glycolipid into "acceptor" microdomains did not increase the rate of sialic acid transfer from fetuin by soluble TcTS, instead producing slight inhibition.
Subject(s)
Glycoproteins/metabolism , Lactose/chemistry , Lipids/chemistry , Membrane Microdomains/chemistry , Neuraminidase/metabolism , Sialic Acids/chemistry , Trypanosoma cruzi/enzymology , Binding Sites , Membrane Microdomains/enzymology , Models, Molecular , Molecular StructureABSTRACT
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 ß(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(ß1-4)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.
