The heparin binding site of human extracellular-superoxide dismutase.
Extracellular-superoxide dismutase (EC-SOD) is a secretory glycoprotein that is major SOD isozyme in extracellular fluids. We revealed the possible structure of the carbohydrate chain of serum EC-SOD with the serial lectin affinity technique. The structure is a biantennary complex type with an internal fucose residue attached to asparagine-linked N-acetyl-D-glucosamine and with terminal sialic acid linked to N-acetyllactosamine. EC-SOD in plasma is heterogeneous with regard to heparin affinity and can be divided into three fractions: A, without affinity; B, with intermediate affinity; and C, with high affinity. It appeared that this heterogeneity is not dependent on the carbohydrate structure upon comparison of EC-SOD A, B, and C. No effect of the glycopeptidase F treatment of EC-SOD C on its heparin affinity supported the results. A previous report showed that both lysine and arginine residues probably at the C-terminal end, contribute to heparin binding. Recombinant EC-SOD C treated with trypsin or endoproteinase Lys C, which lost three lysine residues (Lys-211, Lys-212, and Lys-220) or one lysine residue (Lys-220) at the C-terminal end, had no or weak affinity for the heparin HPLC column, respectively. The proteinase-treated r-EC-SOD C also lost triple arginine residues which are adjacent to double lysine residues. These results suggest that the heparin-binding site may occur on a "cluster" of basic amino acids at the C-terminal end of EC-SOD C. EC-SOD is speculated to be primarily synthesized as type C, and types A and B are probably the result of secondary modifications. It appeared that the proteolytic cleavage of the exteriorized lysine- and arginine-rich C-terminal end in vivo is a more important contributory factor to the formation of EC-SOD B and/or EC-SOD A.
Adachi
T
T
Department of Pharmaceutics, Gifu Pharmaceutical University, Japan.
Kodera
T
T
Ohta
H
H
Hayashi
K
K
Hirano
K
K
D000595:Amino Acid Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D010450:Endopeptidases; D004797:Enzyme-Linked Immunosorbent Assay; D005110:Extracellular Space / Q000201:enzymology*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines; D037102:Lectins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D006898:Hydroxylamines; D037102:Lectins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019811:Hydroxylamine; D006493:Heparin; D013482:Superoxide Dismutase; D010450:Endopeptidases; D002918:Chymotrypsin