Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells

The analysis of many natural glycoproteins and their recombinant counterpar ts from mammalian hosts has revealed that the basic oligosaccharide structu res and the site occupancy of glycosylated polypeptides are primarily dicta ted by the protein conformation. The equipment of many frequently used host cells (e.g. BHK-21 and CHO-cells ) with glycosyltransferases, nucleotide-sugar synthases and transporters ap pears to be sufficient to guarantee complex-type glycosylation of recombina nt proteins with a high degree of terminal alpha 2-3 sialylation even under high expression conditions. Some human tissue-specific terminal carbohydra te motifs are not synthesized by these cells since they lack the proper sug ar-transferring enzymes (e.g. alpha 1-3/4 fucosyltransferases, alpha 2-6 si alyltransferases). Glycosylation engineering of these hosts by stable trans fection with genes encoding terminal human glycosyltransferases allows to o btain products with tailored (human tissue-specific) glycosylation in high yields. Using site-directed mutagenesis, unglycosylated polypeptides can be success fully converted in N- and/or O-glycoproteins by transferring glycosylation domains (consisting of 7-17 amino acids) from donor glycoproteins to differ ent loop regions of acceptor proteins. The genetic engineering of glycoproteins and of host cell lines are conside red to provide a versatile tool to obtain therapeutic glyco-products with n ovel/improved in-vivo properties, e.g. by introduction of specific tissue-t argeting signals by a rational design of terminal glycosylation motifs.