Soluble macrophage-derived CD163: a homogenous ectodomain protein with a dissociable haptoglobin-hemoglobin binding.
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.
Møller
Holger Jon
HJ
Department of Clinical Biochemistry, Aarhus University Hospital, Nørrebrogade 44, 8000 Arhus Sygehus, Denmark. holgmoel@rm.dk
Nielsen
Marianne Jensby
MJ
Maniecki
Maciej Bogdan
MB
Madsen
Mette
M
Moestrup
Søren Kragh
SK
D000818:Animals; D015703:Antigens, CD / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D015214:Antigens, Differentiation, Myelomonocytic / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006242:Haptoglobins / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008264:Macrophages / Q000276:immunology*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary / Q000502:physiology; D011956:Receptors, Cell Surface / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012995:Solubility; D000098902:CD163 Antigen
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000098902:CD163 Antigen; D006242:Haptoglobins; D006454:Hemoglobins; D008024:Ligands; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins