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.
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References
van Denderen BJ, Salvaris E, Romanella M, Aminian M, Katerelos M, Tange MJ, Pearse MJ, d'Apice AJ (1979) Transplantation 64: 882–88.
Osman N, McKenzie IFC, Ostenried K, Ionnou YA, Desnick RJ, Sandrin MS (1997) Proc Natl Acad Sci (USA) 94: 14677–82.
Ohdan H, Yang YG, Sykes M (1999) Transplant Proc 31: 945–46.
Vanhove B, Charreau B, Cassard A, Pourcel C, Soulillou JP (1998) Transplantation 66: 1477–85.
Pearse MJ, Witort E, Mottram P, Han W, Murray-Segal L, Romanella M, Salvaris E, Shinkel TA, Goodman DJ, d'Apice AJ (1998) Transplantation 66: 748–54.
Bracy JL, Sachs DH, Iacomini J (1998) Science 281: 1845–47.
Sandrin MS, Vaughan HA, Dabkowski PL, McKenzie LF (1993) Proc Natl Acad Sci (USA) 90: 11391–95.
Galili U, Shohet SB,Kobrin E, Stults CL, Macher BA(1988) J Biol Chem 263: 17755–62.
Vaughan HA, Loveland BE, Sandrin MS (1994) Transplantation 58: 879–82.
Galili U, Macher BA, Buehler J, Shohet SB (1985) J Exp Med 162: 573–82.
Good AH, Cooper DK, Malcolm AJ, Ippolito RM, Koren E, Neethling FA, Ye Y, Zuhdi N, Lamontagne LR (1992) Transplant Proc 24: 559–62.
Dalmasso AP, Vercellotti GM, Fischel RJ, Bolman RM, Bach FH, Platt JL (1992) Am J Pathol 140: 1157–66.
Ferguson MAJ, Homans SW, Dwek RA, Rademacher TW (1988) Science 239: 753–59.
Pingel S, Duszenko M (1992) Biochem J 283: 479–85.
Pingel S, Field RA, Güther MLS, Duszenko M, Ferguson MAJ (1995) Biochem J 309: 877–82.
Cross, GAM (1996) BioEssays 18: 283–91.
Pingel S, Rheinweiler U, Kolb V, Duszenko M (1998) Biochem J 338: 545–51.
Smith TK, Cottaz S, Brimacombe JS, Ferguson MAJ (1996) J Biol Chem 271: 6476–82.
Ziegler T, Dettmann R, Duszenko M, Kolb V (1996) Carbohydr Res 295: 7–23.
Frische K, Schmidt RR (1994) Liebigs Ann Chem 297–303.
Dettinger H, Kurz G, Lehmann J (1979) Carbohydr Res 74: 301–07.
Meier C, Laux WHG, Bats JW, (1995) Liebigs Ann Chem 1963–1979; and references therein
Dale JA, Mosher HS (1973) J Am Chem Soc 95: 512–19.
Hammerschmidt F, Li YF (1994) Tetrahedron 50: 10253–64; and references therein.
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Kolb, V., Amann, F., Schmidt, R.R. et al. Specific inhibition of an α-galactosyltransferase from Trypanosoma brucei by synthetic substrate analogues. Glycoconj J 16, 537–544 (1999). https://doi.org/10.1023/A:1007026122209
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DOI: https://doi.org/10.1023/A:1007026122209
- 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)