Chemical Cross-linking and Hydrogen-Deuterium Exchange –Tools for Precise Definition of Human Haptoglobin Structure

Z. Kukačka^1,2, P. Man^1,2, P. Novák^1,2 and P. Pompach^1,2

¹Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic

²Department of Biochemistry, Faculty of Sciences, Charles University in Prague, Prague, Czech Republic

Haptoglobin (Hp) is acute phase plasma glycoprotein that binds hemoglobin (Hb) dimer to extraordinarily strong complex. The forming Hb-Hp complex is subjected to CD163-mediated endocytosis by macrophages and also prevents renal filtration of Hb in kidneys. Humans possess three phenotypes of Hp, reffered as Hp(1-1), Hp(2-1), and Hp(2-2). These variants differ in structure and function. The Hp(1-1) phenotype is composed of one α1 and β subunits, the Hp(2‑1) phenotype consists of α1β monomer and α2β monomer and Hp(2-2) phenotype is created by α2 and β subunits. Although molecular model of human haptoglobin based on the X‑ray structure of porcine hemoglobin-haptoglobin complex was described, unfortunately no structure of human haptoglobin has been revealed so far. The only structure was created based on the molecular modeling and CD spectroscopy.

In this study we present a workflow for detail structural characterization of human haptoglobin by using homobifunctional cross-linking reagents (BS2G, BS3, DSG, DSS and ADH) in combination with reverse-phase chromatography coupled to FT-ICR mass spectrometer. The knowledge of distance constrains between modified amino acids allowed us to re-model the structure of human haptoglobin monomer. On the other hand, cross-linkers between α subunits enabled us to reconstitute the structure of haptoglobin multimer. The hydrogen-deuterium exchange approach was used to monitor the haptoglobin surface, which is partially covered by complex glycans. Combination of protein chemistry with analytical tools mentioned above can lead to better design of the structure of human haptoglobin and its multimers in solution.

This work was supported by grants from Grant Agency of the Czech Republic (P207/10/1934), Grant Agency of Charles University (800413), the Ministry of Education, Youth and Sports of the Czech Republic (AMVIS LH13051, CZ.1.07/2.3.00/20.0055 and CZ.1.07/2.3.00/30.0003), and by the Project UNCE204025/2012.