Soluble leptin receptor represents the main leptin binding activity in human blood.
In human blood leptin circulates both free and bound to high molecular weight proteins. Hypothesising that these proteins may modulate ligand bioavailability and bioactivity of leptin, we investigated their molecular nature. Therefore, leptin binding activity was partially purified from human plasma using a leptin affinity column. Subjecting this preparation to size exclusion chromatography (SEC) we observed a coelution of leptin binding activity with levels of the soluble leptin receptor (sOB-R) determined by a newly developed ligand immunofunctional assay. In Western blot analysis the partially purified leptin binding activity exhibited sOB-R immunoreactivity in two bands of 110 and 140 kD. Following N-deglycosylation these bands were replaced by two bands with the molecular weight of 90 and 60 kD, suggesting two isoforms which are capable of leptin binding, as determined by cross-linking. Furthermore, different ratios of these isoforms were detectable in fractions of the leptin binding activity after separation by SEC. These findings indicate the formation of heterodimers and homodimers complexed with and without leptin. As the two sOB-R bands from Western blot analysis correspond to only two specific bands in cross-linking experiments with 125l-leptin, the role of both isoforms as leptin binding proteins appears to be exclusive. Therefore, our results indicate that sOB-R is the major leptin binding protein in the circulating human blood.
Lammert
A
A
Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics, University of Leipzig, Leipzig, 04103, Germany.
Kiess
W
W
Bottner
A
A
Glasow
A
A
Kratzsch
J
J
D015153:Blotting, Western; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D020738:Leptin / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D012995:Solubility
D002352:Carrier Proteins; C515448:LEPR protein, human; D020738:Leptin; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin