Preparative in vitro generation of lacto-series type 1 chain glycolipids catalyzed by beta 1----3-galactosyltransferase from human colonic adenocarcinoma Colo 205 cells.
Lacto-series glycolipids, comprising two isomeric types distinguished as type 1 or 2 based upon the linkage of the terminal galactose of the chains, form the basis for a diversity of cell surface antigens expressed on cells. Experimentally, type 2 chain precursors are generally more abundant in tissues for extractive purposes to yield rather large quantities of material compared to the type 1 chain structures. Conditions have been defined for in vitro conversion of terminal Gal beta 1----4GlcNAc linkages of type 2 chain precursors to yield type 1 lacto-series chain based terminal Gal beta 1----3GlcNAc structures in 5- to 10-mg amounts or higher. The terminal galactose of underivatized type 2 chain structures is removed by hydrolysis with jack bean beta-galactosidase followed by transfer of galactose in beta 1----3 linkage catalyzed by a beta 1----3-galactosyltransferase from human colonic adenocarcinoma Colo 205 cells which was first depleted of beta 1----4-galactosyltransferase by chromatography on alpha-lactalbumin-Sepharose. Scaled-up reaction mixtures provided a final yield of product after isolation of about 90% from the immediate Lc3Cer precursor in the 5-mg product range. The biosynthetic product was subjected to extensive chemical analysis by 1H NMR and mass spectrometric methods. These results indicated the presence of a high purity terminal Gal beta 1----3-linked product. The amount of material was sufficient for nondestructive characterization by 2-D NMR, with subsequent confirmation of structure by +FAB-MS and methylation analysis by GC-MS. The results indicate an effective means to rapidly generate lacto-series type 1 precursors in vitro as a superior alternative to direct tissue extractive procedures.
Holmes
E H
EH
Pacific Northwest Research Foundation, Seattle, Washington 98122.
Levery
S B
SB
D000230:Adenocarcinoma / Q000201:enzymology*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D003110:Colonic Neoplasms / Q000201:enzymology*; D005700:Galactosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006017:Glycolipids / Q000138:chemical synthesis*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D014407:Tumor Cells, Cultured / Q000201:enzymology
D006017:Glycolipids; D005700:Galactosyltransferases; C053372:glucosaminylgalactosylglucosylceramide beta-galactosyltransferase