Haptoglobin polymorphisms and iron homeostasis in health and in disease

Abstract

Haptoglobin (Hpt) is a plasma protein with hemoglobin-binding capacity. It is a well-known marker of hemolysis. Hpt is also an acute-phase protein that functions as a bacteriostatic agent, an inhibitor of prostaglandin synthesis and angiogenesis. However, the best-known biological function of Hpt is capture of hemoglobin (Hb).

The identification of functional differences in haptoglobin molecules resulting from relatively common polymorphisms has further elucidated the importance of haptoglobin in iron homeostasis and in disease processes influenced by iron metabolism. In this review the effect of Hpt polymorphism on these different disease entities will be discussed.

Introduction

Haptoglobin (Hpt) is a plasma protein with hemoglobin-binding capacity. It is a well-known marker of hemolysis. Hpt is also an acute-phase protein that functions as a bacteriostatic agent, an inhibitor of prostaglandin synthesis and angiogenesis [1]. However, the best-known biological function of Hpt is capture of hemoglobin (Hb). After destruction of erythrocytes, free Hb in the circulation passes through the glomerular filter and renal damage may occur. The binding of Hpt with Hb prevents both iron loss and kidney damage during intravascular hemolysis [2].

The identification of functional differences in haptoglobin molecules resulting from relatively common polymorphisms has further elucidated the importance of haptoglobin in iron homeostasis and in disease processes influenced by iron metabolism including hemochromatosis—a disease of iron overload—atherosclerosis and cardiovascular disease, and infectious diseases. In this review, the effect of Hpt polymorphism on these different disease entities will be discussed.

Haptoglobin (Hpt) is an α₂-sialoglycoprotein with hemoglobin (Hb)-binding capacity [3], [4] and is characterised by a molecular heterogeneity with three major phenotypes: Hpt 1-1, Hpt 2-1 and Hpt 2-2 [2], [3], [4], [5]. Using starch gel electrophoresis, the three major phenotypes can be identified [6]. These phenotypes are genetically determined by two alleles: Hpt¹ and Hpt² [3], [4]. The homozygote, Hpt¹/Hpt¹ shows a single fast-migrating Hpt 1-1 protein band on starch gel electrophoresis. The homozygote Hpt²/Hpt² shows a series of slower migrating bands. The heterozygote Hpt¹/Hpt² displays another series of slow bands and a weak Hpt 1-1 band. Hpt consists of two different polypeptide chains, the α-chain and the β-chain [3], [4]. The β-chain (40 kDa) is heavier than the α-chain and is identical in all Hpt types. The α-chain shows three major forms: α^1s, α^1f (s=slower, f=faster) and the slow migrating α²-chain. The Hpt 1-1 phenotype has α¹-chains, while α²-chains are present in Hpt from individuals with the Hpt 2-1 or Hpt 2-2 phenotype [3], [4]. The loci involved for the Hpt synthesis are located on chromosome 16q22. The Hpt 1-1 protein is a small molecule (86 kDa) with formula (α¹β)₂. Heterozygote Hpt 2-1, (α¹β)₂+(α²β)_n (n=0, 1, 2,…), is characterised by polymerisation. Hpt 2-2 comprises higher molecular mass forms (>200 kDa) with formula (α²β)_n (n=3, 4, 5,…) [5], [7] (Table 1) (Fig. 1).

The synthesis of Hpt is considerably lower in foetal than in adult liver [3]. The hepatic synthesis of Hpt is induced by cytokines such as interleukin-6, interleukin-1 and tumour necrosis factor [3], [8]. The haptoglobin concentration is Hpt phenotype-dependent. The reference range for haptoglobin concentration is lower in individuals carrying the Hpt 2-2 phenotype than individuals carrying the Hpt 1-1 and Hpt 2-1 phenotype [9].

The haptoglobin phenotype distribution differs according to geographical localisation of the population studied [1]. The haptoglobin allele frequencies show marked geographical differences, with the lowest Hpt¹ allele frequency (0.10) in Southeast Asia and the greatest Hpt¹ frequency (0.80) in indigenous populations of South America [1]. The phenotypic distribution in European populations shows that ∼15% individuals are Hpt 1-1, 50% Hpt 2-1, and 35% Hpt 2-2, corresponding with a Hpt1 allele frequency of ∼0.40 [1], [10] (Table 1).

Haptoglobin polymorphisms, directly or indirectly, influence pathways involved in iron metabolism:

Hpt forms a soluble complex with Hb. The binding of Hpt with Hb is the strongest known noncovalent interaction among the plasma transport proteins, with the complex having a very high affinity and stability [11]. Circulating Hpt is saturated when 500–1500 mg/l free Hb is present. The half-life of Hb–Hpt complexes in plasma is ∼20 min [12]. Hepatocellular uptake of Hpt–Hb complexes reduces the loss of haem iron through the kidney [1]. Unlike hemopexin and transferrin, Hpt is not recycled after endocytosis but the Hb–Hpt complex is instead degraded by lysosomes [13]. Hb binding depends not only on the serum concentration of Hpt but also on the Hpt phenotype [14]. The hemoglobin binding capacity is lowest among Hpt 2-2 subjects [15] due to lower serum concentrations, as discussed above, as well as to a lower ability to bind Hb [11]. After destruction of erythrocytes, free Hb in the circulation passes through the glomerular filter and renal damage may occur. Hpt reduces the loss of Hb and iron, because the Hb–Hp complex is not filtered through the glomeruli but is transported to the liver. The lower binding capacity of Hb in individuals with the Hpt 2-2 phenotype results in more renal damage and higher serum iron levels [1].

Free Hb promotes the accumulation of hydroxyl radicals [16] and harmful reactive oxygen species (free radicals), because iron (Fe²⁺) can generate extremely reactive hydroxyl radicals in the presence of H₂O₂ (Fenton reaction). Haem iron catalyses the oxidation of low-density lipoproteins, which can damage vascular endothelial cells [17]. In addition, the breakdown of erythrocytes in the interstitial fluid results in Hb-mediated hydroxyl radical formation. Plasma Hpt can be regarded as a major antioxidant protecting against Hb-driven lipid peroxidation [18], [19]. The ability of haptoglobin to reduce hemoglobin-induced free radical damage is phenotype-dependent [14], [18]. The distribution of highly polymeric Hpt 2-2 proteins in extravascular fluids is restricted by their molecular mass [5]. Consequently, the antioxidative capacity of body fluids is less effective in Hpt 2-2 individuals [5].

The physiological importance of Hpt–Hb complex formation has become evident by the increased susceptibility to Hb-driven oxidative tissue damage demonstrated in conditions of hypo- or anhaptoglobinemia (Hpt0 phenotype) or in haptoglobin-deficient mice [20], [21], [22]. In Hpt knockout (−/−) mice, induction of severe hemolysis by phenylhydrazine caused extensive hemoglobin precipitation in the renal tubular cells of both −/− and +/+ mice, with death occurring in 55% of −/− mice and in 18% of +/+ mice. In general, phenylhydrazine-treated −/− mice suffered greater tissue damage, as evidenced by the induction of hepatic acute phase response resulting in increased plasma alpha 1-acid glycoprotein (AGP) levels. Among −/− and +/+ mice that survived, −/− mice tend to suffer greater oxidative damage and failed to repair or regenerate damaged renal tissues, as indicated by their higher plasma malonaldehyde (MDA) and 4-hydroxy-2(E)-nonenal (HNE) levels and lower mitotic indices in their kidneys, respectively [20]. Hpt 2-2 concentrations in cerebrospinal fluid are very low because diffusion into the interstitial compartment is restricted by the high molecular mass of the polymers. An increased frequency of Hpt 2-2 and Hpt0 was observed in familial and posttraumatic epilepsy and was explained by a less efficient inhibition of Hb-driven brain-lipid peroxidation after hemorrhage within the central nervous system [22].

CD163 has been identified as the monocyte–macrophage receptor that binds the Hb–Hpt complex. Hpt and Hb do not bind to the CD163 receptor separately. CD163 binds only hemoglobin and haptoglobin in complex, which indicates the exposure of a receptor-binding neoepitope. In its function as a Hb scavenger, the CD163 receptor accounts for a substantial transfer of iron into the macrophages. Complexes of hemoglobin and multimeric Hpt 2-2 haptoglobin exhibit higher functional affinity for CD163 than do complexes of hemoglobin and dimeric Hpt 1-1 haptoglobin [23]. In vitro experiments with radiolabeled (¹²⁵I) Hb showed that human peripheral blood monocytes take up Hb–Hpt 2-2 complexes whereas, even in zymosan-activated monocytes, free Hb and Hb bound to Hpt 1-1 or 2-1 are not internalised [24]. When intracellular concentrations of haem increase with the endocytosis of Hb–Hpt 2-2 complexes, there is a rapid induction of ferritin synthesis. In mammals, the cytosolic ferritins are ubiquitous and made of two subunit types, the H- and L-chains, with about 50% sequence identity and very similar three-dimensional (3D) structures. H-chains have ferroxidase activity, which accelerates Fe(II) oxidation, the rate-limiting step of ferritin iron incorporation, in a reaction that consumes one dioxygen molecule per two Fe(II) ions with the production of hydrogen peroxide. The L-subunit has no catalytic activity on its own, but it assists the activity of the H-subunits by offering sites for iron nucleation and mineralisation and increasing the turnover at the ferroxidase centers [25]. Higher cytosolic L-ferritin levels are present in peripheral blood monocytes from Hpt 2-2 subjects (687±152 μg/g protein) compared to Hpt 1-1 and 2-1 subjects (326±83 and 366±109 μg/g protein, respectively) [24]. This effect is presumably due to iron released from haem affecting the iron regulatory protein (IRP) which regulates ferritin mRNA translation by binding to the iron responsive element (IRE) [26], [27]. Cytosolic H-ferritin content in monocytes is not different between Hp phenotypes, similar to what is observed in iron-loaded livers where only the L-ferritin form is upregulated [28]. In males, but not in females, the Hpt 2-2 phenotype is associated with higher serum iron, transferrin saturation and ferritin concentrations than in the Hpt 1-1 and Hpt 2-1 phenotype, whereas soluble transferrin concentrations are lower. Serum ferritin correlated with monocyte L-ferritin content which is also highest in the male Hpt 2-2 subgroup [24]. A positive correlation was observed between serum and monocyte ferritin levels suggesting that increased L-ferritin synthesis in monocyte–macrophages results in higher ferritin levels in the circulation [24].

Haptoglobin has been proposed to be involved in a highly interactive ensemble of lymphocytes, neutrophils, and monocytes participating in inflammatory processes [29], [30], [31], [32], [33], [34]. Kristiansen et al. [23] speculated that the Hpt–Hb complex, like antibodies, may cross-link several CD163 molecules on the surface of macrophages, triggering an internal signalling cascade that results in increased secretion of anti-inflammatory cytokines. Human serum from Hpt 2-2 and Hpt 2-1 individuals agglutinates the bacterial cells of the Streptococcus pyogenes group A strain, carrying the T4 antigen. The Hpt 2-2 serum has higher agglutination titres than the Hpt 2-1 serum. In contrast, serum from individuals with the Hpt 1-1 phenotype has no agglutination effect. Hpt is not a true antibody because it does not possess the highly variable antigen-binding sites characteristic of the Fab moiety of immunoglobulins. The agglutination is probably mediated via binding with lectin-like structures [35], [36].

Comparison of reference values for lymphocyte subsets in the peripheral blood and bone marrow show significant differences between haptoglobin phenotypes. Individuals with the Hpt 2-2 phenotype have higher peripheral B-lymphocyte counts and CD4+ T lymphocytes counts than individuals with the Hpt 1-1 phenotype. In contrast, in the bone marrow, CD4+ T cell percentages are high but B cell percentages are low in individuals with the Hpt 2-2 phenotype. Flow cytometric analysis demonstrates that Hpt binds to the CD22 receptor on human B lymphocytes. Although the affinity of the binding is the same for the three phenotypes, the number of free CD22 binding sites in the circulation is estimated to be higher in Hpt 2-2 individuals. No significant Hp binding has been detected for T cells and NK cells [33].