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
E. Grabenhorst et al., 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, European journal of biochemistry, 232(3), 1995, pp. 718-725
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.