A Unique Loop Extension in the Serine Protease Domain of Haptoglobin Is Essential for CD163 Recognition of the Haptoglobin-Hemoglobin Complex*
- Marianne Jensby Nielsen ‡ ,
- Steen Vang Petersen § ,
- Christian Jacobsen ‡ ,
- Søren Thirup § ,
- Jan Johannes Enghild § ,
- Jonas Heilskov Graversen ‡ and
- Søren Kragh Moestrup ‡ 1
- Departments of ‡Medical Biochemistry and §Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark
- 1 To whom correspondence should be addressed. Tel.: 45-89422882; Fax: 45-86131160; E-mail: skm{at}biokemi.au.dk.
Abstract
Haptoglobin and haptoglobin-related protein are homologous hemoglobin-binding proteins consisting of a complement control repeat (α-chain) and a serine protease domain (β-chain). Haptoglobin-hemoglobin complex formation promotes high affinity binding of hemoglobin to the macrophage scavenger receptor CD163 leading to endocytosis and degradation of the haptoglobin-hemoglobin complex. In contrast, complex formation between haptoglobin-related protein and hemoglobin does not promote high affinity interaction with CD163. To define structural components of haptoglobin important for CD163 recognition, we exploited this functional difference to design and analyze recombinant haptoglobin/haptoglobin-related protein chimeras complexed to hemoglobin. These data revealed that only the β-chain of haptoglobin is involved in receptor recognition. Substitution of 4 closely spaced amino acid residues of the haptoglobin β-chain (valine 259, glutamate 261, lysine 262, and threonine 264) abrogated the high affinity receptor binding. The 4 residues are encompassed by a part of the primary structure not present in other serine protease domain proteins. Structural modeling based on the well characterized serine protease domain fold suggests that this sequence represents a loop extension unique for haptoglobin and haptoglobin-related protein. A synthetic peptide representing the haptoglobin loop sequence exhibited a pronounced inhibitory effect on receptor binding of haptoglobin-hemoglobin.
Footnotes
-
↵2 The abbreviations used are: Hp, haptoglobin; Hb, hemoglobin; proHp, Hp precursor; Hpr, haptoglobin-related protein; WT, wild type; MS, mass spec-trometry; MALDI-MS, matrix-assisted laser desorption/ionization-MS; SPR, surface plasmon resonance; HPLC, high pressure liquid chromatography.
-
↵3 M. J. Nielsen and S. K. Moestrup, unpublished data.
-
↵* This work was supported by grants from The Lundbeck Foundation, The Danish Medical Research Council, and The Danish Natural Science Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
- Received June 14, 2006.
- Revision received October 25, 2006.
- The American Society for Biochemistry and Molecular Biology, Inc.
