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 for the first time in a mammalian host cell line. Stable BHK-21 cell lines exhibiting altered terminal sialylation properties were constructed by cotransfection of cells with the plasmids pMT-beta TP or pAB3-1 which contain the cDNAs encoding the human secretory glycoproteins beta-trace protein or antithrombin III and pABSial containing the human Golgi enzyme CMP-NeuAc:Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase (ST6N) gene. The beta-trace protein was purified by immunoaffinity chromatography and N-linked oligosaccharides were subjected to carbohydrate structural analysis. The enzymically liberated oligosaccharides were found to consist of 90% of diantennary chains as is the case for natural beta-trace protein from human cerebrospinal fluid. About 90% of the total oligosaccharides were recovered in the monosialo and disialo fractions 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 1H-NMR analysis as well as calculations of peak areas obtained by HPLC, 60% of the molecules of the disialo fraction were found to contain NeuAc in both 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 shown to be attached preferentially to the Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3) branch of the diantennary structure. Therefore the in vivo specificity of the newly introduced recombinant human ST6N observed in this study supports the previously reported in vitro branch specificity of the bovine colostrum ST6N activity. Furthermore, these studies demonstrate the suitability of genetically engineered mammalian host cell lines with novel glycosylation properties for the production of human-type glycosylated secretory recombinant polypeptides.
Grabenhorst
E
E
Department of Gene Regulation and Differentiation, Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany.
Hoffmann
A
A
Nimtz
M
M
Zettlmeissl
G
G
Conradt
H S
HS
D000818:Animals; D000990:Antithrombin III / Q000096:biosynthesis; D001483:Base Sequence; D001609:Beta-Globulins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D002240:Carbohydrate Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D019746:Intramolecular Oxidoreductases; D054834:Lipocalins; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D001609:Beta-Globulins; D006023:Glycoproteins; D054834:Lipocalins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012794:Sialic Acids; D000990:Antithrombin III; D012799:Sialyltransferases; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase