Molecular Playground/Hemoglobin-Haptoglobin Complex

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One of the CBI Molecules being studied in the University of Massachusetts Amherst
Chemistry-Biology Interface Program
at UMass Amherst and on display at the Molecular Playground.


 Figure 1. Hb-Hp/CD-163 Pathway during Intravascular hemolysis by Ololade Fatunmbi.      Haptoglobin 1-1 (Hp), an abundant glycoprotein in blood binds free hemoglobin (Hb) dimers in one of the strongest non-covalent binding events known in biology. This interaction shields Hb residues that are prone to oxidative modification. Hb-Hp globin complexes bind to the CD163 cell surface receptor on macrophages leading to their internalization and catabolism.
Figure 1. Hb-Hp/CD-163 Pathway during Intravascular hemolysis by Ololade Fatunmbi. Haptoglobin 1-1 (Hp), an abundant glycoprotein in blood binds free hemoglobin (Hb) dimers in one of the strongest non-covalent binding events known in biology. This interaction shields Hb residues that are prone to oxidative modification. Hb-Hp globin complexes bind to the CD163 cell surface receptor on macrophages leading to their internalization and catabolism.

Color Code:
Haptoglobin (Hp)
Magenta
Hemoglobin α-chain Cyan Hemoglobin β-Chain
Lime Green
Hp:(silver) Hb: (blue spheres) HbHp complex: Residues
involved in
& (bold)

Contents

Summary

Hemoglobin (Hb) is arguably one of the most studied proteins of all time. Hb is essential for our lives and nearly all other vertebrates because it transports oxygen from organs to tissues so that we can have energy. However, like most entities in life, too much of something may actually be harmful. In a process called intravascular hemolysis, high concentrations of Hb are released from red blood cells into the extracellular environment, which could cause oxidative damage to our tissues [1]. Haptoglobin (Hp), an acute phase glycoprotein, counteracts the negative physiological consequences of intravascular hemolysis by binding Hb [2-4] in one of strongest non-covalent events known in nature (Kd ~1 × 10–15 mol/L) [5,6]. See Figure 1.

Function

Hb physiologically exists as a (Hbα1β1α2β2) found in red blood cells. During intravascular hemolysis, red blood cells (erythrocytes) rupture. Hb is then released into the extracellular environment, and could be come very toxic to the body by causing severe oxidative tissue damage. Hb subunits dissociate into dimers (Hbα1β1) in the extracellular environment exposing [10]. In addition, heme, the prosthetic group on Hb could react with hydrogen peroxide to generate reactive oxygen species (ROS) through Fenton and Haber-Weiss reactions and the iron present in heme catalyzes these reactions [3]. See Figure 2. Moreover, both the protein and heme regions of free Hb could cost excessive oxidative damage to the body by reacting with small molecules in circulation such as hydrogen peroxide. Extracellular Hb, could also react irreversibly with nitric oxide (NO), a critical regulator of smooth muscle tone and platelet activation [2]. The consumption of NO by Hb leads to limited bioavailability of NO and the production of nitrate and methemoglobin [2].and exposes a recognized by the multifunctional receptor, CD163. The Hb-Hp complex binds CD-163 with high affinity and mediates haptoglobin-hemoglobin endocytosis and degradation [4].
 Figure 2. Reactions of iron and hydrogen peroxide  generate reactive oxygen species generated by [3].  Blue spheres indicate radicals.
Figure 2. Reactions of iron and hydrogen peroxide generate reactive oxygen species generated by [3]. Blue spheres indicate radicals.

Hp is found in nearly all mammals and some vertebrates. In humans, there are two allelic forms, Hp1 and Hp2, which manifest three phenotypes [2]. Hp1 is responsible for the Hp1-1 phenotype. Partial intragenic duplication in Hp1 gives rise to Hp2 allele, which is responsible for two phenotypes, Hp2-1 and Hp2-2 [1,2]. Both in vitro and in vivo studies have established that subjects with the Hp1-1 phenotype are more likely to resist cellular oxidative stress than those with the Hp2-2 phenotype, with Hp2-1 being intermediate [7].

Structural Highlights

Haptoglobin
The alone crystal structure of Hp has not yet been elucidated. Hp1-1 is ~90kda and exists as a consisting of the two light chains and two heavy chains. The light chains are linked by the disulfide bond formed between . Altogether there are 4 disulfides bonds on Hp and the bond hold the light and heavy chains of Hp together. There are also found on each monomer of Hp1-1 [8].
The light chain of Hp shares high homology with complement control proteins while the heavy chain is very homologous with serine proteases [1]. Although Hp is not an active protease, the Hb-binding site in Hp is located in the region responsible for substrate specificity in other serine proteases (17). In serine proteases, the typical active-site residues are -histidine-57 and serine-195. In Hp these residues are replaced by lysine and alanine, respectively [9]). However, a substrate specific residue in trypsin does occur in Hp [9].

Hemoglobin-Haptoglobin Complex
What makes the binding between Hp so tight and nearly irreversible? Recently, the crystal structure of porcine Hb-Hp was solved and revealed interactions involved in Hb-Hp interface [1]. Porcine Hp shares 82% homology with human Hp 1-1 [1,10]. The interaction between Hb and Hp is composed of various hydrophobic and and [10].

Disease

Prevention of Renal Damage: Intravascular hemolysis occurs in several diseases including sickle-cell anemia and malaria [1]. Another consequence caused by free hemoglobin is oxidative damage in renal tissues following intravascular hemolysis [7]. Yet when haptoglobin binds to hemoglobin, the complex is too large to pass through the glomeruli of the kidney and will be removed via the reticuloendothelial system [7]. Therefore Hb induced injury to the parenchyma is prevented by haptoglobin [11].

Antibacterial Activity: When hemoglobin becomes non-covalently bound to haptoglobin, Hb and iron are no longer available to Escherichia coli and other bacteria that require iron [7]. Eaton was able to demonstrate that when Hp was given to rats that have been intraperitoneally injected with E. Coli and hemoglobin, Hp was able to prevent fatal effects [12].

Antioxidant Activity: Haptoglobin has a significant role as an antioxidant [13]. Free Hb also increases the peroxidation of purified arachidonic acid and other polyunsaturated fatty acids within neuronal cell membranes (10). Iron released from heme proteins can catalyze oxidative injury to neuronal cell membranes and might have a role in posttraumatic central nervous system (CNS) damage [14]. Haptoglobin, binds to Hb and removes it from the circulation and prevents iron-stimulated formation of oxygen radicals [15].

Relevance

Hp has developed of a lot interest in drug therapy development recently because of its effectiveness in detoxifying free Hb activity when hemolytic related events occur in diseases [16-20] such as sickle cell anemia [16].

Research Interests

Kaltashov Lab: We are a Biological Mass Spectrometry group at the University of Massachusetts, Amherst. Our research is focused on developing mass spectrometry-based strategies to study protein architecture, dynamics, and interactions with small molecules and other biopolymers.

Ololade Fatunmbi' (Graduate Assistant Researcher)
Studies of the Hp mediated Hb clearance pathway suggest that Hp may be used for targeted drug delivery. This use requires a detailed understanding of conformational dynamics and interactions in this protein/receptor system. However, Hb-Hp/CD163 complex crystal structure and conformational dynamics have not yet been determined. I focus on studyingatomic level predictions of Hb-Hp/CD163 protein interactions and conformational dynamics using bioinformatics techniques and native mass spectrometry.

Chibueze Egeruoh (Undergraduate Assistant Researcher)
African trypanosomiasis or sleeping sickness is a parasitic disease of humans and other animals. On the structures of African trypanosomes, there is a coat of surface monolayer of variant surface glycoprotein (VSG) that protects the parasite. Within the VSG coat there are HbHp receptors that have the purposes of binding Hb-Hp acquisition heme through endocytosis so that the parasite would have nutrients such as iron present in Hb. Understanding the interaction of Hb-Hp complexes and trypanosome receptors will improve drug delivery to susceptible trypanosomes parasite. I conduct molecular modeling studies on trypanosome receptors and docking studies on Hb-Hp african trypanosoma receptors in complex with Hb-Hp complexes.

References

1. Kristiansen M, Graversen JH, Jacobsen C, et al. Identification of the haemoglobin scavenger re- ceptor. Nature. 2001;409:198-201.
2. Marianne Jensby Nielsen and Søren Kragh Moestrup. Receptor targeting of hemoglobin mediated by the haptoglobins: roles beyond heme scavenging. Blood. 2009. 114:764-771
3. Isaac K. Quaye. Haptoglobin, inflammation and disease Trans R Soc Trop Med Hyg. 2008; 102, 735—742.
4. Hanne Van Gorp, et al. Scavenger receptor CD-163, a Jack-of-all-trades and potential target for cell-directed therapy. Molecular Immunology. 2010; 47: 1650–1660.
5. Bowman, BH, Kurosky, A: Haptoglobin: The evolutionary product of duplication, unequal crossing over, and point mutation. Adv Hum Genet 1982 12:189–261
6. McCormick, DJ, Atassi, MZ: Hemoglobin binding with haptoglobin: Delineation of the haptoglobin binding site on the alpha-chain of human hemoglobin. J Protein Chem 1990 9:735–742
7. S.M. Hossein Sadrzadeh, PhD, and Jafar Bozorgmehr, MD. Haptoglobin Phenotypes in Health and Disorders. Am J Clin Pathol. 2004; 121: 97-104
8. Black JA, Dixon GH. Amino-acid sequence of alpha chains of human haptoglobins. Nature. 1968;218:738-741.
9. Alexander Kurosky et al. Covalent structure of human haptoglobin: A serine protease homolog. Biochemistry. 1980; 77: 3388-3392.
10. Christian Brix Folsted Andersen. Structure of the haptoglobin–haemoglobin complex. Nature. 2012; 489: 456-459
11. Javid J. Human haptoglobins. Curr Top Hematol. 1978;1:151- 192.
12. Eaton, et al. Haptoglobin: A natural bacteriostat. Science. 1982. 215: 691–693
13. Lange V. Haptoglobin polymorphism: not only a genetic marker. Anthropol Anz. 1992;50:281-302.
14. Sadrzadeh SMH, Graf E, Panter SS, et al. Hemoglobin: a biologic Fenton reagent. J Biol Chem. 1984;259:14354-14356
15. Vercellotti GM, Balla G, Balla J, et al. Heme and the vasculature: an oxidative hazard that induces antioxidant defenses in the endothelium. Artif Cells Blood Substit Immobil Biotechnol. 1994;22:207-213.
16. A. I. Alayash, C. B. Andersen, S. K. Moestrup, and L. Bülow. (2013) Haptoglobin: the hemoglobin detoxifier in plasma. Trends Biotechno,31. 2–3
17. Gando S, Tedo I. (1994) The effects of massive transfusion and haptoglobin therapy on hemolysis in trauma patients. Surg Today. 9. 785-790.
18. Joseph Bertolini, Neil Goss, John Curling (2012) Production of Plasma Proteins for Therapeutic Use. Biochemistry; Edition 1: 332.
19. Imaizumi H, Tsunoda K, Ichimiya N, Okamoto T, Namiki A. Repeated large-dose haptoglobin therapy in an extensively burned patient: case report. J Emerg Med 1994;12(1):33-37.
20. D. J. Schaer, P. W. Buehler, A. I. Alayash, et,. al Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins. Blood. 121.8:1276–1284, 2013

Acknowledgements

Ololade Fatunmbi and Chibueze Egeruoh (Kaltashov Lab)
PDB ID:4F4O from Anderson, CB et. al (2012. Nature)

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Ololade Fatunmbi

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