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Published on: July 5, 2018
Soluble macrophage-derived CD163: a homogenous ectodomain protein with a dissociable haptoglobin-hemoglobin binding
Holger Jon Møller1, Marianne Jensby Nielsen, Maciej Bogdan Maniecki
1Department of Clinical Biochemistry, Aarhus University Hospital, Nørrebrogade 44, 8000 Arhus Sygehus, Denmark. holgmoel@rm.dk
Background:
The 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.
Methods:
The 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.
Results:
By 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 130kDa, 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.
Conclusions:
Serum 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.
