The heparin-binding domain of extracellular superoxide dismutase C and formation of variants with reduced heparin affinity.
A fundamental property of the secretory tetrameric extracellular superoxide dismutase (EC-SOD) is its affinity for heparin and analogues, in vivo, mediating attachment to heparan sulfate proteoglycans located on cell surfaces and in the connective tissue matrix. EC-SOD is in vivo heterogeneous with regard to heparin affinity and can be divided into subclasses; A which lacks heparin affinity, B with intermediate affinity, and C with strong heparin affinity. The EC-SOD C subunits contain 222 amino acids and among the last 20 carboxyl-terminal amino acids, 10 are positively charged and six of these are located in a cluster in positions 210-215. To analyze if this local accumulation of basic amino acids is responsible for heparin binding we produced three series of recombinant EC-SOD (rEC-SOD) variants, six containing amino acid exchanges in the carboxyl-terminal end, four with truncations, and two with both truncations and substitutions. Exchange of positively or negatively charged amino acids on the carboxyl-terminal side of the cluster results in only minor modifications in heparin affinity, whereas substitution of three of the amino acids in the cluster abrogates the heparin binding. Insertions of stop codons at different positions resulted in either C or A but not B class EC-SOD. In an attempt to produce EC-SODs with intermediate heparin affinities, plasmids defining C and A class EC-SOD were cotransfected into Chinese hamster ovary cells. In addition to the parental A and C class EC-SOD forms, two variants with intermediate heparin affinities were formed. Coincubation of EC-SOD C and A resulted in the appearance of one heterotetramer with intermediate affinity for heparin. We conclude that the cluster of six basic amino acids forms the essential part of the heparin-binding domain and that the composition of the four subunits in the EC-SOD tetramer determines the affinity for heparin. This domain is different from heparin-binding domains of other proteins, and its localization allows the distribution of EC-SOD in vivo to be regulated by proteolytic processing.
Sandström
J
J
Department of Microbiology, University of Umeå, Sweden.
Carlsson
L
L
Marklund
S L
SL
Edlund
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006224:Cricetinae; D005109:Extracellular Matrix / Q000201:enzymology*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; D006493:Heparin; D013482:Superoxide Dismutase