Accelerated enzymatic galactosylation of N-acetylglucosaminolipids in lipid microdomains.
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.
Manchester Interdisciplinary Biocentre and the School of Chemistry, University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Gavin T
GT
Noble
Faye L
FL
Craven
Josef
J
Voglmeir
Robert
R
Sardzík
Sabine L
SL
Flitsch
Simon J
SJ
Webb
D000117:Acetylglucosamine / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain / Q000502:physiology*; D002417:Cattle; D004134:Dimyristoylphosphatidylcholine; D006017:Glycolipids / Q000737:chemistry*; D008051:Lipid Bilayers / Q000378:metabolism*; D037121:Plant Lectins / Q000378:metabolism; D013379:Substrate Specificity; D053835:Unilamellar Liposomes / Q000737:chemistry; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase / Q000378:metabolism*
D006017:Glycolipids; D008051:Lipid Bilayers; D037121:Plant Lectins; D053835:Unilamellar Liposomes; C045629:erythrina lectin; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase; D004134:Dimyristoylphosphatidylcholine; D000117:Acetylglucosamine