Engineering protein O-glycosilation for the production of receptor blockers

• Research unit: EU RFP
• Project number: 95.0206

Key words (English)

Selectins; N-acetylglucosaminyltransferase; fucosyltransferase; sialyl-Lewisx; PSGL-1; multicistronic vectors

Alternative project number (English)

EU project number: BIO4CT950138

Research programs (English)

EU-programme: 4. Frame Research Programme - 4.1 Biotechnology

Short description (English)

See abstract

Partners and International Organizations (English)

Forschungszentrum Jülich GmbH (D), Physiologisches Institut, Univ. Zürich (CH), Copenhagen Univ., Faculty of health sciences (DK), Utrecht Univ., Dept. Bio-organic Chemistry (NL), Institut für Enzymtechnologie der Heinrich-Heine-Univ. Düsseldorf (D), Glybiology Institute, Department of Biochemistry, Univ. of Oxford (GB)

Abstract (English)

Work from many laboratories has shown that the first step of inflammation involves selectin binding by 2,3sialylated and 1,3fucosylated oligosaccharides (e.g. sialyl-Lewisx, SLex) expressed on defined proteins. The goal of our collaborative group was the synthesis in vitro of O-glycosidically linked selectin ligands as well as the expression in CHO cells of P-selectin-binding glycoprotein 1 (PSGL-1), one of the main selectin-binding proteins identified thus far.
The O-linked hexasaccharide involved in P-selectin binding NeuAca2->3Galb1->4[Fuca1->3]GlcNAcb1->6Galb1->3GalNAca1->pNp has been synthesised by stepwise enzymatic reactions using a panel of crude and purified natural or recombinant glycosyltransferases. These latter included core 2 b1,6N-acetylglucosaminyltransferase which was expressed in CHO cells, a2,3sialyltransferase expressed in insect cells and a1,3fucosyltransferase VI expressed in Pichia pastoris. The product was analysed by 1H-NMR in collaboration with the group in Utrecht and shown to be SLex.
Production of recombinant glycosyltransferase in CHO cell factories was optimised by transfecting the cells at confluency in G0/G1 phase instead of subconfluency in S/G2 phase. Cells thus transfected proved to be five times more productive when grown in batch cultures.
To express PSGL-1 in CHO cells with P-selectin binding activity, CHO cells had to stably express core 2 b1,6N-acetylglucosaminyltransferase and a a1,3fucosyltransferase. This was achieved by using bi- and tricistronic constructs with neoR in the third cistron and the resp. enzymes in the first and second cistron. The cells resisting the selection were sorted by FACS using an anti-SLex-mAB. After 4 consecutive rounds of this bulk sorting process a homogeneous cell population exhibiting activity of both glycosyltranferases was obtained which was then transfected with a vector encoding soluble PSGL-1. The emerging cell population was tested for SLex binding, anti PSGL-1 binding and P-selectin binding was obtained in about 80 % of the cells.
In addition, a gene family of b1,3galactosyltransferases (b3GalTs) was discovered. One enzyme homologous to the b3GalT was found to have a different specificity for the donor substrate, i.e. for UDP-GlcNAc while the glycosidic bond formed remained the same. This enzyme represents one of the poly-N-acetyllactosamine forming enzymes and is thus involved in structuring selectin ligands of the polylactosaminyl type.

References in databases (English)

Swiss Database: Euro-DB of the
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Swiss Project-Number: 95.0206