Abstract
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.
Highlights
From the $Department of Microbiology, University of UmeQ S-90187 Ume& Sweden and the VDepartment of Clinical Chemistry, Umei University Hospital, S-901 85 Umeci, Sweden
In the acids is responsible for heparin binding we produced vasculature, such binding to endothelial cell surfaces is evithree series of recombinant EC-SOD var- denced by a prompt release of EC-SOD C to plasma upon iants, six containing amino acid exchanges in the cairn-travenous injection of heparin [7, 8, 11].The effect is seen boxyl-terminal end, four with truncations, and two only forplasma EC-SOD subtypewshich elute from aheparinwith both truncations and substitutions
A highly positively charged domain is located in the carboxylterminal end of EC-SOD C(13), andit has earlier been suggested that this region could mediate the binding to heparin
Summary
From the $Department of Microbiology, University of UmeQ S-90187 Ume& Sweden and the VDepartment of Clinical Chemistry, Umei University Hospital, S-901 85 Umeci, Sweden. Extracellular superoxide dismutas(Ee C-SOD)is its aff- is a secretory tetrameric copper- and zinc-containing glycoinity for heparin and analogues, in vivo, mediating protein [1,2] It is the major SOD isoenzyme in extracellular attachment to heparan sulfate proteoglycans located fluids such as plasma, lymph, and synovialfluid[3,4], alon cell surfaces and in the connective tissue matrix. The major portion of EC-SOD C charged and six of these are located in a cluster in binds to heparan sulfapteroteoglycan in theglycocalyx of cell positions 210-215 To analyze if this local accumulation of basic amino equilibrium with the extracellular fluid phase [8, 10]. Exchange of Sepharose column at a NaCl concentration above 0.38 M [8]
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