title: Specific inhibition of an alpha-galactosyltransferase from Trypanosoma brucei by synthetic substrate analogues.
abstract: Since the alpha-D-galactose-(1-->3)-D-galactose epitope has been identified to be the major target in the process of hyperacute rejection of xenografts transplanted from nonprimate donors to humans, specific inhibitors of alpha-galactosyltransferases are of broad interest. Using Trypanosoma brucei, a protozoan parasite causing sleeping sickness and Nagana, we have a very useful model system for the investigation of alpha-galactosyltransferase inhibitors, since the variant surface glycoprotein (VSG) accounts for about 10% of the total cell protein an this parasite expresses many different galactosyltransferases including the one catalysing the formation of the Galalpha1-->3Gal epitope. In order to study inhibition of galactosylation on the VSG from Trypanosoma brucei, we designed, synthesized and tested substrate analogues of trypanosomal alpha-galactosyltransferases. Effective inhibitors were a pair of diastereoisomeric UDP-galactose analogs, in which the galactose residue is linked to UDP via a methylene bridge rather than an ester linkage. Hence, galactose cannot be transferred to the respective acceptor substrate VSG or the synthetic acceptor substrate Manalpha1-->6Manalpha1S-(CH2)7-CH3, which was previously proven to replace VSG effectively [Smith et al. (1996) J Biol Chem 271:6476-82]. Inhibitors have been prepared starting from 1-formyl galactal. The final condensation was performed using UMP morpholidate leading to a pair of diastereomeric compounds in 39% or 30% yield, respectively. These compounds were tested using alpha-galactosyltransferases prepared from T. brucei membranes and lactose synthetase from bovine milk. While the K(M)-value for UDP-galactose was determined as 59 microM on bovine lactose synthetase, the K(I)-values for both inhibitors were 0.3 mM and 1.1 mM respectively, showing that these inhibitors are unable to inhibit enzyme activity significantly. However, using the N-glycan specific alpha-galactosyltransferase from trypanosomes, the K(M)-value was determined as 20 microM, while the K(I)-values were 34 microM and 21 microM respectively. Interestingly, other trypanosomal alpha-galactosyltransferases, which modify the GPI membrane anchor, are 2 orders of magnitude less effected by the inhibitor.
authors.affiliation: Physiologisch-chemisches Institut der Universität Tübingen, Germany.
authors.forename: V
authors.initials: V
authors.lastname: Kolb
authors.forename: F
authors.initials: F
authors.lastname: Amann
authors.forename: R R
authors.initials: RR
authors.lastname: Schmidt
authors.forename: M
authors.initials: M
authors.lastname: Duszenko
mesh_terms: D000818:Animals; D002417:Cattle; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry; D005700:Galactosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D007788:Lactose Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry; D051381:Rats; D013237:Stereoisomerism; D014346:Trypanosoma brucei brucei / Q000737:chemistry*; D014530:Uridine Diphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D014531:Uridine Diphosphate Galactose / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; C409213:O(P)-((2,6-anhydro-3-deoxy-talo-hept-2-enitol-1-yl)phosphonyl)uridine-5'-phosphate; D014531:Uridine Diphosphate Galactose; D014530:Uridine Diphosphate; D005700:Galactosyltransferases; D007788:Lactose Synthase