Comparative study of the sugar chains of factor VIII purified from human plasma and from the culture media of recombinant baby hamster kidney cells

• Published in: The Journal of biological chemistry Vol. 267; no. 12; pp. 8012 - 8020
• Main Authors: HIRONAKA, T, FURUKAWA, K, ESMON, P. C, FOURNEL, M. A, SAWADA, S, KATO, M, MINAGA, T, KOBATA, A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25.04.1992
• Subjects: Animals; Biological and medical sciences; Blood coagulation. Blood cells; Blotting, Western; Carbohydrate Sequence; cells; Cells, Cultured; Chromatography, Gel; coagulation factor VIII; Coagulation factors; comparison; Cricetinae; Culture Media; Electrophoresis, Paper; Factor VIII - isolation & purification; Factor VIII - metabolism; Fundamental and applied biological sciences. Psychology; hamsters; Humans; Kidney - metabolism; man; Methylation; Molecular and cellular biology; Molecular Sequence Data; oligosaccharides; Oligosaccharides - genetics; Oligosaccharides - metabolism; plasma; structure; Human; Blood coagulation; Molecular structure; Oligosaccharide; Factor VIII; Glycoproteins; Glycosylation; Recombinant protein; BHK cell line; Coagulation factor; Comparative study
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)42401-5

The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1---3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1---4(Fuc alpha 1---3)GlcNAc beta 1---4 (Gal beta 1---4GlcNAc beta 1---2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.