CONSTRUCTION OF STABLE BHK-21-CELLS COEXPRESSING HUMAN SECRETORY GLYCOPROTEINS AND HUMAN GAL(BETA-1-4)GLCNAC-R ALPHA-2,6-SIALYLTRANSFERASE ALPHA-2,6-LINKED NEUAC IS PREFERENTIALLY ATTACHED TO THE GAL(BETA-1-4)GLCNAC(BETA-1-2)MAN(ALPHA-1-3)-BRANCH OF DIANTENNARY OLIGOSACCHARIDES FROM SECRETED RECOMBINANT BETA-TRACE PROTEIN

The human beta-trace protein has been cloned and has been expressed fo r the first time in a mammalian host cell Line. Stable BHK-21 cell Lin es exhibiting altered terminal sialylation properties were constructed by cotransfection of cells with the plasmids pMT-beta TP or pAB3-1 wh ich contain the cDNAs encoding the human secretory glycoproteins beta- trace protein or antithrombin III and pABSial containing the human Gol gi enzyme CMP-NeuAc:Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase (ST6N) gene. The beta-trace protein was purified by immunoaffinity chr omatography and N-linked oligosaccharides were subjected to carbohydra te structural analysis. The enzymically liberated oligosaccharides wer e found to consist of 90% of diantennary chains as is the case for nat ural p-trace protein from human cerebrospinal fluid. About 90% of the total oligosaccharides were recovered in the monosialo and disialo fra ctions in a ratio of 1:5. The monosialylated oligosaccharides of beta- trace protein coexpressed with human ST6N were found to contain NeuAc in alpha 2,6- or alpha 2,3-linkage in the same ratio. From H-1-NMR ana lysis as well as calculations of peak areas obtained by HPLC, 60% of t he molecules of the disialo fraction were found to contain NeuAc in bo th alpha 2,3- and alpha 2,6-linkage to Gal beta(1-4)GlcNAc-R, whereas 40% of the molecules of this fraction contained NeuAc in only alpha 2, 3-linkage to Gal(beta 1-4)GlcNAc-R. The alpha 2,6-linked NeuAc was sho wn to be attached preferentially to the Gal(beta 1-4)GlcNAc(beta 1-2)M an(alpha 1-3) branch of the diantennary structure. Therefore the in vi vo specificity of the newly introduced recombinant human ST6N observed in this study supports the previously reported in vitro branch specif icity of the bovine colostrum ST6N activity. Furthermore, these studie s demonstrate the suitability of genetically engineered mammalian host cell lines with novel glycosylation properties for the production of human-type glycosylated secretory recombinant polypeptides.