Abstract
The human Golgi enzyme CMP-NeuAc:Gal(β1–4)GlcNAc-R α2,6-sialyltransferase (ST6N) was stably coexpressed with human erythropoietin (EPO) from a BHK-21A cell line. The cell line was characterized with respect to the expression and in vitro activity of the ST6N and the endogenous α2,3-sialyltransferase. Detailed structural analysis of the N-linked carbohydrates of the rhuEPO expressed from the new cell line was performed by HPAE-PAD-mapping, MALDI/TOF-MS and methylation analysis after purification of the recombinant protein by immunoaffinity chromatography. This is the first report describing that the human α2,6-sialyltransferase is capable of sialylating, apart from Gal(β1–4)GlcNAc-R, also GalNAc(β1–4)GlcNAc-R motifs in vivo, which is not the case for the endogenous BHK-cell α2,3-sialyltransferase.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bergwerff AA, Van Oostrum J, Kamerling JP and Vliegenthart JFG (1995) The mayor N-linked carbohydrate chains from human urokinase. The occurrence of 4-O-sulfated, (α2–6)-sialylated or(α1–3)-fucosylated N-acetylgalactosamineβ1–4)-N-acetylglucosamine elements. Eur. J. Biochem. 228: 113-126.
Chomczynski P and Sacchi N (1987) Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159.
Conradt HS, Egge J, Peter-Katalinic J, Reiser W, Siklosi T and Schaper K (1987) Structure of the carbohydrate moiety of human Interferon-β secreted by a recombinant Chinese ovary cell line. J. Biol.. Chem. 262: 14600-14605.
Costa J, Grabenhorst E, Nimtz M and Conradt HS (1997) Stable expression of the Golgi form and secretory variants of human fucosyltransferase III from BHK-21 cells. J. Biol. Chem. 272: 11613-11621.
Fibi MR, Hermentin P, Pauly JU, Lauffer L and Zettlmeissl G (1995) N-and O-glycosylation muteins of recombinant human erythropoietin secreted from BHK-21 cells. Blood 85: 1229-1236.
Goldwasser E, Kung CKH and Eliason J (1974) On the mechanism of erythropoietin-induced differentiation, J. Biol. 249: 4202-4206.
Goto M, Akai K, Murakami A, Hashimoto C, Tsuda E, Ueda M, Kawanishi, Takahashi N, Ishimoto A, Chiba H and Sasaki R (1988) Production of recombinant human erythropoietin in mammalian cells: Host-cell dependency of the biological activity of the cloned glycoprotein. Bio/Technology 6: 67-71.
Grabenhorst E, Hoffmann A, Nimtz M, Zettlmeissl G and Conradt HS (1995) Construction of stable BHK-21 cells coexpressing human secretory glycoproteins and human Gal(β1–4)GlcNAc-R α2,6-sialyltransferase. α2,6-linked NeuAc is preferentially attached to the Gal(β1–4)GlcNAc(β1–2)Man(α1–3)-branch of diantennary oligosaccharides from secreted recombinant β-trace protein. Eur. J. Biochem. 232: 718-725.
Graham FL and Van der Eb AJ (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA, Virology 52: 456-467.
Grundmann U, Nerlich C, Rein T and Zettlmeissl G (1990) Complete cDNA sequence encoding human β-galactoside α-2,6-sialyltransferase. Nucleic Acids Res. 18: 667.
Hakomori S (1964) A rapid permerthylation of glycolipid, and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxid. J. Biochem. (Tokyo) 55: 205-207.
Hokke CH, Van der JGM, Kamerling JP and Vliegenthart JFG (1993) Action of rat liver Galβ1-4GlcNAcα(2–6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe, GalNAc β1-4GlcNAcβ-OMe, Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates. Glycoconjugate J. 10: 82-90.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
Lee EU, Roth J and Paulson JV (1989) Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression of β-galactoside α2,6-sialyltransferase. J. Biol. Chem. 264: 13848-13855.
Nemansky M, Schiphorst WECM and Van der Eijnden DH (1995) Branching and elongation with lactosaminoglycan chains of N-linked oligosaccharides result in a shift toward termination with 4α2,3-linked rather than with α2,6-linked sialic acid residues. FEBS Lett. 363: 280-284
Nemansky M and Van den Eijnden DH (1992 Bovine colostrum CMP-NeuAc:Galβ(1→4)GlcNAc-R α(2–6)-sialyltransferase is involved in the synthesis of the terminal NeuAcα(2–6)GalNAc(β1–4)GlcNAc sequence occurring on N-linked glycans of bovine milk glycoproteins. Biochem. J. 287: 311-316.
Nimtz M, Noll G, Pâques E-P and Conradt HS (1990) Carbohydrate structures of a human tissue plasminogen activator variant expressed in recombinant Chinese hamster ovary cells. FEBS Lett. 271: 14-18.
Nimtz M, Martin W, Wray V, Klöppel KD, Augustin J and Conradt HS (1993) Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells. Eur. J. Biochem. 213: 39-56.
Sambrook J, Fritsch EF and Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn, Cold Spring Harbor Laboratory, Cold Spring Harbor NY.
Spivak JL and Hogans BB (1989) In in vivo metabolism of recombinant human erythropoietin. Blood 73: 90-99.
Vara JA, Portela A, Ortin J and Jimeńez A (1986) Expression in mammalian cells of a gene from Streptomyces alboniger conferring puromycon resistance. Nucl. Acids Res. 14: 4617-4624.
Weisshaar G, Hiyama J, Renwick AGC and Nimtz M (1991) NMR investigation of the N-linked oligosacharides at individual glycosylation sites of human lutropin. Eur. J. Biochem 95: 257-268.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schlenke, P., Grabenhorst, E., Nimtz, M. et al. Construction and characterization of stably transfected BHK-21 cells with human-type sialylation characteristic. Cytotechnology 30, 17–25 (1999). https://doi.org/10.1023/A:1008049603947
Issue Date:
DOI: https://doi.org/10.1023/A:1008049603947