ISSN:
1573-4986
Keywords:
Trypanosoma brucei
;
α-galactosyltransferases
;
sugar donor analogs
;
competitive inhibitors
;
UDP, uridine-5′-diphosphate
;
UDP-Gal, uridine-5′-diphosphate galactose
;
VSG, variant surface glycoprotein
;
αGalT, α-galactosyltransferase
;
Galα1,3GalT, UDP-Gal:β-d-Gal(1 → 4)-D-GlcNAc-α(1,3)-galactosyltransferase (EC 2.4.1.51)
;
Galα1,3ManT, UDP-Gal:GPI-anchor-α(1,3)-galactosyltransferase
;
Man, D-mannose
;
Gal, D-galactose
;
Man2-S-C8, Manα(1-6)Manα1S-(CH2)7-CH3
;
GlcNAc, D-N-acetylglucosamine
;
LacNAc, D-N-acetyllactosamine
;
Galα1,3Gal, α-d-Gal(1→3)-D-Gal epitope
;
GPI, glycosyl-phosphatidylinositol
;
DTT, dithiothreitol
;
PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid)
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract Since the α-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 α-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 α-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 Galα1→3Gal epitope. In order to study inhibition of galactosylation on the VSG from Trypanosoma brucei, we designed, synthesized and tested substrate analogues of trypanosomal α-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 Manα1→6Manα1S-(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 α-galactosyltransferases prepared from T. brucei membranes and lactose synthetase from bovine milk. While the KM-value for UDP-galactose was determined as 59 µM on bovine lactose synthetase, the KI-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 α-galactosyltransferase from trypanosomes, the KM-value was determined as 20 µM, while the KI-values were 34 µM and 21 µM respectively. Interestingly, other trypanosomal α-galactosyltransferases, which modify the GPI membrane anchor, are 2 orders of magnitude less effected by the inhibitor.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1007026122209
