S. Drouillard sophie.drouillard
Centre de recherche sur Macromolecules Vegetales (CERMAV-CNRS), BP 53, F-38041 Grenoble Cedex 9, France
The α-Gal epitope is a carbohydrate structure, Galα-3Galβ-4GlcNAc-R, expressed on glycoconjuguates in many mammals, but not in humans. Species that do not express this epitope have present in their serum large amounts of natural anti-Gal antibodies, which contribute to organ hyperacute rejection during xenotransplantation. We first describe the efficient conversion of lactose into isoglobotriaose (Galα-3Galβ-4Glc) using high cell density cultures of a genetically engineered Escherichia coli strain expressing the bovine gene for α-1,3-galactosyltransferase. Attempts to produce the Galili pentasaccharide (Galα-3Galβ-4GlcNAcβ-3Galβ-4Glc) by additionally expressing the Neisseria meningitis lgtA gene for β-1,3-N-acetylglucosaminyltransferase and the Helicobacter pylori gene for β-1,4-galactosyltransferase were unsuccessful and led to the formation of a series of long chain oligosaccharides formed by the repeated addition of the trisaccharide motif [Galβ-4GlcNAcβ-3Galα-3] onto a lacto-N-neotetraose primer. The replacement of LgtA by a more specific β-1,3-N-acetylglucosaminyltransferase from H. pylori, which was unable to glycosylate α-galactosides, prevented the formation of these unwanted compounds and allowed the successful formation of the Galili pentasaccharide and longer α-Gal epitopes.
α-1,3-galactosyltransferase (recombinant α-3GalT), lgtA, β-1,4-galactosyltransferase (recombinant β-4GalT)
29178, 29490, 29491, 29492, 29493, 29494, 29495, 29496, 29497, 29499, 29500, 29501, 29502, 29503, 29504, 29505, 29506, 29507, 29508, 29509, 29510, 29511, 29512, 29513, 29514
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6
4,3,4,3,3,4,3,3,4 bDGalp 104.08 ? ? ? ? 62.24
4,3,4,3,3,4,3,3,2 Ac 176.18 23.44
4,3,4,3,3,4,3,3 bDGlcpN 103.95 56.50 ? 79.36 ? 61.13
4,3,4,3,3,4,3 aDGalp 96.89 ? 80.30 70.18 71.63 62.02
4,3,4,3,3,4 bDGalp 103.86 ? 78.41 66.09 ? 62.24
4,3,4,3,3,2 Ac 176.18 23.44
4,3,4,3,3 bDGlcpN 103.95 56.50 ? 79.36 ? 61.13
4,3,4,3 aDGalp 96.89 ? 80.30 70.18 71.63 62.02
4,3,4 bDGalp 103.86 ? 78.41 66.09 ? 62.24
4,3,2 Ac 176.18 23.41
4,3 bDGlcpN 104.03 56.42 ? 79.46 ? 61.13
4 bDGalp 104.08 ? 83.28 69.59 ? 62.24
bDGlcp 96.98 75.01 ? 79.46 ? 61.27
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6
4,3,4,3,3,4,3,3,4 bDGalp 4.51 3.58 ? ? ? ?
4,3,4,3,3,4,3,3,2 Ac - 2.06
4,3,4,3,3,4,3,3 bDGlcpN 4.76 3.84 ? ? ? ?
4,3,4,3,3,4,3 aDGalp 5.14 3.94 4.06 4.27 4.25 ?
4,3,4,3,3,4 bDGalp 4.58 3.69 3.82 4.22 ? ?
4,3,4,3,3,2 Ac - 2.06
4,3,4,3,3 bDGlcpN 4.76 3.84 ? ? ? ?
4,3,4,3 aDGalp 5.14 3.94 4.06 4.27 4.25 ?
4,3,4 bDGalp 4.58 3.69 3.82 4.22 ? ?
4,3,2 Ac - 2.06
4,3 bDGlcpN 4.73 3.84 ? 3.79 ? ?
4 bDGalp 4.47 3.62 3.76 4.18 ? ?
bDGlcp 4.69 3.31 ? 3.69 ? ?
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6
4,3,4,3,3,4,3,3,4 bDGalp 104.08/4.51 ?/3.58 ?/? ?/? ?/? 62.24/?
4,3,4,3,3,4,3,3,2 Ac 23.44/2.06
4,3,4,3,3,4,3,3 bDGlcpN 103.95/4.76 56.50/3.84 ?/? 79.36/? ?/? 61.13/?
4,3,4,3,3,4,3 aDGalp 96.89/5.14 ?/3.94 80.30/4.06 70.18/4.27 71.63/4.25 62.02/?
4,3,4,3,3,4 bDGalp 103.86/4.58 ?/3.69 78.41/3.82 66.09/4.22 ?/? 62.24/?
4,3,4,3,3,2 Ac 23.44/2.06
4,3,4,3,3 bDGlcpN 103.95/4.76 56.50/3.84 ?/? 79.36/? ?/? 61.13/?
4,3,4,3 aDGalp 96.89/5.14 ?/3.94 80.30/4.06 70.18/4.27 71.63/4.25 62.02/?
4,3,4 bDGalp 103.86/4.58 ?/3.69 78.41/3.82 66.09/4.22 ?/? 62.24/?
4,3,2 Ac 23.41/2.06
4,3 bDGlcpN 104.03/4.73 56.42/3.84 ?/? 79.46/3.79 ?/? 61.13/?
4 bDGalp 104.08/4.47 ?/3.62 83.28/3.76 69.59/4.18 ?/? 62.24/?
bDGlcp 96.98/4.69 75.01/3.31 ?/? 79.46/3.69 ?/? 61.27/?
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