Soluble macrophage-derived CD163: A homogenous ectodomain protein with a dissociable haptoglobin–hemoglobin binding

BackgroundThe soluble form of the haptoglobin–hemoglobin (Hp–Hb) scavenger receptor (sCD163) is a specific plasma/serum marker for macrophage activity. Here, we have characterized molecular forms in serum and investigated a role of sCD163 as a binder of Hp–Hb complexes.MethodsThe sCD163 species in serum (from 50 healthy subjects and 29 patients) were measured with domain-specific ELISAs, purified from serum (from 6 individuals) by affinity chromatography and identified by western blotting and MALDI-TOF/TOF mass spectrometry. Binding to Hp–Hb complexes was investigated by gel-chromatography, surface plasmon resonance (SPR) analyses, and inhibition of Hp–Hb endocytosis in CD163-transfected Chinese hamster ovary (CHO) cells.ResultsBy using C- and N-terminal-specific ELISAs, no sCD163 concentration differences in plasma were seen, thus indicating a homogenous sCD163 species. Affinity-purified sCD163 from serum migrated as a single band of 130 kDa, and spanned at least 945 amino acids (94%) of the total extra-cellular part of CD163. In solution sCD163 only weakly competed for Hp–Hb uptake in CD163-expressing cells, and Hp–Hb saturation of sCD163 in serum was only seen with large excess of Hp–Hb complexes. However, upon immobilisation, recombinant sCD163 bound Hp–Hb with high affinity. This suggests that Hp–Hb is less dissociable when bound to the membrane form of CD163, presumably because of the di- or multivalent nature of Hp–Hp complexes in terms of CD163 binding.ConclusionsSerum sCD163 is a homogenous protein covering more than 94% of the CD163 ectodomain including the Hp–Hb-binding region. However, CD163 is a poor competitor of Hp–Hb uptake, probably because of its soluble nature, where Hp–Hb cannot take advantage of receptor cross-linkage.