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Two forms of erythropoietin, EPO-bi and EPO-tetra, with different biological activities were isolated from the culture medium of a recombinant Chinese hamster ovary cell line, B8-300, into which the human erythropoietin gene had been introduced. EPO-bi, an unusual form, showed only one-seventh the in vivo activity and 3 times higher in vitro activity of the previously described recombinant human EPO (standard EPO). In contrast, EPO-tetra showed both in vivo and in vitro activities comparable to those of the standard EPO. EPO-bi, EPO-tetra, and the standard EPO had the same amino acid composition and immunoreactivity. However, structural analyses of their N-linked sugar chains revealed that EPO-bi contains the biantennary complex type as the major sugar chain, while EPO-tetra and the standard EPO contain the tetraantennary complex type as the major sugar chain. From examination of various preparations of recombinant human EPO, we found a positive correlation between the in vivo activity of EPO and the ratio of tetraantennary to biantennary oligosaccharides. These results suggest that higher branching of the N-linked sugar chains is essential for effective expression of in vivo biological activity of EPO.Human erythropoietin (EPO) is a glycoprotein hormone that plays a major role in regulating the level of circulating erythrocytes (1) by stimulating the maturation of late erythroid progenitor cells into proerythroblasts (2). In the normal human adult, EPO is produced in the kidney (3). EPO was first purified in a small amount from urine of aplastic anemia patients (4). Several recombinant human EPOs produced in mammalian (5-9) and nonmammalian cells (10) have recently become available, but their biological activities differ from cell to cell. Since the structures of their polypeptide moieties are the same, such variation in activity was suspected as being due to the differences in their carbohydrate moieties. The sugar chain structures of urinary human EPO and recombinant human EPO produced in Chinese hamster ovary (CHO) cells were determined independently by us (11) and by Sasaki et al. (12). Both urinary and recombinant human EPOs contain -40% carbohydrate in the form of three N-linked and one O-linked oligosaccharide chain. Important roles of the carbohydrate moiety in the solubility (13, 14), biosynthesis (14), and biological activity of EPO (9, 15) have been reported. Desialylation of EPO caused complete loss of its hormonal activity in vivo (15, 16) as the asialo-EPO was trapped in the liver (17) by the hepatic asialoglycoprotein binding protein (18) and was rapidly cleared from the circulation. Galactose oxidase treatment of asialo-EPO restored part of the biological activity (16). These results suggested that EPO possesses full biological activity only when it is sufficiently sialylated to avoid clearance by the hepatic asialoglycoprotein binding protein. During the course of study of the productivity of EPO in several recombinant CHO cell lines, we found a unique cell line, B8-300, which produced ...

Selectins recognize ligands containing carbohydrate chains such as sialyl Lewis x (sLe x ) that are mainly presented at the terminus of N-acetyl lactosamine repeats on core 2 O-glycans. Several glycosyltransferases act successively to extend the N-acetyl lactosamine repeats and to synthesize sLe x , and ␤-1,4-galactosyltransferase (␤4GalT) plays a key role in these processes. Recently isolated 6 ␤4GalT genes are candidates, but their individual roles, including those in selectin-ligand biosynthesis, remain to be elucidated. More than 80% of the core 2 O-glycans on the leukocyte membrane glycoproteins of ␤4GalT-I-deficient mice lacked galactose residues in ␤-1,4 linkage, and soluble P-selectin binding to neutrophils and monocytes of these mice was significantly reduced, indicating an impairment of selectin-ligand biosynthesis. ␤4GalT-I-deficient mice exhibited blood leukocytosis but normal lymphocyte homing to peripheral lymph nodes. Acute and chronic inflammatory responses, including the contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH) responses, were suppressed, and neutrophil infiltration into inflammatory sites was largely reduced in these mice. Our results demonstrate that ␤4GalT-I is a major galactosyltransferase responsible for selectin-ligand biosynthesis and that inflammatory responses of ␤4GalT-I-deficient mice are impaired because of the defect in selectin-ligand biosynthesis. (Blood. 2003;102: 1678-1685)