Carbohydrate structures of soluble human L-selectin recombinantly expressed in baby-hamster kidney cells.
A soluble form of L-selectin was recombinantly produced, which might be an effective therapeutic agent in inflammatory disorders, acting as an inhibitor for leucocyte endothelium adhesion. In the present study the oligosaccharide structures of soluble human L-selectin, recombinantly expressed in baby-hamster kidney cells, were determined. The N-linked glycans were enzymically released and fluorescently labelled with 2-aminobenzamide. Sialylation of the N-glycans was analysed by anion-exchange chromatography followed by rechromatography of the resulting fractions on amino-phase HPLC after release of the sialic acid residues. Desialylated oligosaccharides were separated using two-dimensional HPLC and characterized by digestion with exoglycosidases and MS. More than 30 oligosaccharide structures representing at least 95% of the overall glycosylation of this protein were determined. The results revealed that recombinant soluble human L-selectin carries bi-, tri- and tetra-antennary sugar chains, which are fucosylated on the innermost residue of N-acetylglucosamine. The number of sialic acid residues linked to these glycans ranges from 0 (neutral glycans) to 4 (tetrasialylated oligosaccharides). The sialic acid is found exclusively in the alpha 2-3 linkage to galactose. In addition to the main glycans, different minor structures containing terminal N-acetylgalactosamine, or the H (O) blood-group determinant were also identified. O-Glycosylation of mucin-type sugar chains was not detected in recombinant soluble human L-selectin.
Institut für Diagnostikforschung GmbH an der Freien Universität Berlin, Germany.
M
M
Gohlke
U
U
Mach
R
R
Nuck
M
M
Zimmermann-Kordmann
D
D
Grunow
C
C
Fieger
B
B
Volz
R
R
Tauber
T
T
Petri
N
N
Debus
W
W
Reutter
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000235:genetics*; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D007668:Kidney / Q000166:cytology*; D019041:L-Selectin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D012995:Solubility
D002241:Carbohydrates; D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; D019041:L-Selectin