Hp-Hb Complex Research Articles

Assessment of Haptoglobin 2-2 Genotype in Type 2 Diabetes and Cardiovascular Patients in North Indian Population: A Case-control Study

Introduction: Haptoglobin (Hp), an acute phase protein, is a plasma inflammatory marker. It is important in both infectious and non infectious illnesses. When intravascular haemolysis occurs, the dead red blood cells are discharged into the circulation and link to Hp to form the haemoglobin (Hb)-haptoglobin (Hb-Hp) complex that is recognised by the macrophage scavenger receptor CD163. The Hp1 and Hp2 are its two alleles. The Hp2 is formed by a partial duplication of the Hp1 gene. The Hp1 has five exons, but Hp2 has seven exons owing to the duplication of Hp1 third and fourth exon. There are three genotypes of Hp: Hp1-1, Hp2-1, and Hp2-2. The encoded products of genotypes are attached to haemoglobin with varying degrees of affinity: Hp1-1 has the most affinity, Hp2-1 has medium affinity, and Hp2-2 has the lowest affinity. Hp allelic and genotypic variation differs amongst populations and different geographic regions of the world. Aim: To study the haptoglobin 2-2 genotype in type 2 diabetes and cardiovascular patients in north Indian population. Materials and Methods: It was a case-control study and a total of 200 participants were recruited randomly including 50 participants of type 2 Diabetes Mellitus (DM), 50 participants of Cardiovascular Disorders (CVD), 50 participants with Cardiovascular Disorders With type 2 Diabetes Mellitus (CVDWDM) and 50 normal healthy controls from Punjab Institute of Medical Sciences (PIMS) Hospital and clinics and Aashirwad laboratory of Jalandhar, Punjab, India in February 2021 to January 2022. Anthropometric variables were determined by using standard methods and biochemical parameters were measured by commercial available kits. Allele specific Polymerase Chain Reaction (PCR) or Amplification Refractory Mutation System (ARMS) PCR was used to detect Hp genotypes. Data were represented as mean±Standard Deviation and the statistical difference i.e. p<0.05 between all parameters was determined by using the Mann-Whitney U test. Results: Anthropometric parameters i.e. Body Mass Index (BMI) was found to be statistically significant in CVDWDM as compared to CVD, N and T2DM i.e. p=0.023, p=0.01 and p=0.014. Waist Circumference (WC) was found to be statistically significant in CVDWDM as compared to CVD, N and T2DM i.e. p=0.02, p=0.013 and p=0.012 and Hip Circumference (HC) was found to be statistically significant in CVDWDM as compared to CVD, N and T2DM i.e. p=0.02, p=0.017 and p=0.014. However, Apolipoprotein A1 mean value was found to be higher and statistically significant i.e. p=0.03 in Type 2 Diabetes Mellitus (T2DM) as compared to healthy participants. Triglycerides mean value was increased in T2DM as compared to CVD, CVD as compared to N and CVDWDM as compared to healthy controls (p-value=0.012,0.04,0.015, respectively). The findings had shown that a 349 bp band was detected in patients with T2DM, CVD and CVDWDM, implying that the Hp2-2 genotypes were observed but not in healthy controls. Conclusion: The study finds that people with DM, CVD, or CVDWDM all have a 349bp band. It means that the genotypes of the Hp2-2 allele were found in patients of diabetes, CVD and diabetics with CVD but not in the healthy individuals.

Hyperglycemia Induces Inflammatory Response of Human Macrophages to CD163-Mediated Scavenging of Hemoglobin-Haptoglobin Complexes.

Hyperglycemia, a hallmark of diabetes, can induce inflammatory programming of macrophages. The macrophage scavenger receptor CD163 internalizes and degrades hemoglobin-haptoglobin (Hb-Hp) complexes built due to intravascular hemolysis. Clinical studies have demonstrated a correlation between impaired scavenging of Hb-Hp complexes via CD163 and diabetic vascular complications. Our aim was to identify whether hyperglycemia is able to amplify inflammation via Hb-Hp complex interactions with the immune system. M(IFNγ), M(IL-4), and control M0 macrophages were differentiated out of primary human monocytes in normo- (5 mM) and hyperglycemic (25 mM) conditions. CD163 gene expression was decreased 5.53 times in M(IFNγ) with a further decrease of 1.99 times in hyperglycemia. Hyperglycemia suppressed CD163 surface expression in M(IFNγ) (1.43 times). Flow cytometry demonstrated no impairment of Hb-Hp uptake in hyperglycemia. However, hyperglycemia induced an inflammatory response of M(IFNγ) to Hb-Hp1-1 and Hb-Hp2-2 uptake with different dynamics. Hb-Hp1-1 uptake stimulated IL-6 release (3.03 times) after 6 h but suppressed secretion (5.78 times) after 24 h. Contrarily, Hb-Hp2-2 uptake did not affect IL-6 release after 6h but increased secretion after 24 h (3.06 times). Our data show that hyperglycemia induces an inflammatory response of innate immune cells to Hb-Hp1-1 and Hb-Hp2-2 uptake, converting the silent Hb-Hp complex clearance that prevents vascular damage into an inflammatory process, hereby increasing the susceptibility of diabetic patients to vascular complications.

Quantification of human plasma metalloproteins in multiple sclerosis, ischemic stroke and healthy controls reveals an association of haptoglobin-hemoglobin complexes with age.

Advanced analytical methods play an important role in quantifying serum disease biomarkers. The problem of separating thousands of proteins can be reduced by analyzing for a ‘sub-proteome’, such as the ‘metalloproteome’, defined as all proteins that contain bound metals. We employed size exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic emission spectrometer (ICP-AES) to analyze plasma from multiple sclerosis (MS) participants (n = 21), acute ischemic stroke (AIS) participants (n = 17) and healthy controls (n = 21) for Fe, Cu and Zn-metalloproteins. Using ANOVA analysis to compare the mean peak areas among the groups revealed no statistically significant differences for ceruloplasmin (p = 0.31), α2macroglobulin (p = 0.51) and transferrin (p = 0.31). However, a statistically significant difference was observed for the haptoglobin-hemoglobin (Hp-Hb) complex (p = 0.04), being driven by the difference between the control group and AIS (p = 0.012), but not with the MS group (p = 0.13), based on Dunnes test. A linear regression model for Hp-Hb complex with the groups now adjusted for age found no statistically significant differences between the groups (p = 0.95), but was suggestive for age (p = 0.057). To measure the strength of association between the Hp-Hb complex and age without possible modifications due to disease, we calculated the Spearman rank correlation in the healthy controls. The latter revealed a positive association (r = 0.39, 95% Confidence Interval = (-0.05, 0.83), which suggests that either the removal of Hp-Hb complexes from the blood circulation slows with age or that the release of Hb from red blood cells increases with age. We also observed that the Fe-peak corresponding to the Hp-Hb complex eluted ~100 s later in ~14% of all study samples, which was not correlated with age or disease diagnosis, but is consistent with the presence of the smaller Hp (1–1) isoform in 15% of the population.

Articles of Significant Interest in This Issue.

The HbpA protein in Corynebacterium diphtheriae is crucial for iron acquisition from the hemoglobin-haptoglobin (Hb-Hp) complex. Lyman et al. (e00196-21) show that the C-terminal domain of HbpA is essential for cellular localization, hemin iron acquisition, and optimal hemoprotein binding. The C-terminal domain is also required for the formation of large, secreted complexes of HbpA that are involved in iron uptake from Hb-Hp. HbpA lacks homology to known hemin transport proteins and has exceptional affinity for hemoglobin. The multiple functions associated with this unusual C-terminal domain expand our understanding of the mechanism of iron acquisition by bacterial pathogens.

Sample preparation of blood plasma enables baseline separation of iron metalloproteins by SEC-GFAAS

The analysis of human plasma for biomarkers holds promise to revolutionize disease diagnosis, but is hampered by the inherent complexity of the plasma proteome. One way to overcome this problem is to analyze plasma for a sub-proteome, such as the metalloproteome. Previous studies employing size-exclusion chromatography (SEC) coupled on-line to an inductively coupled plasma-atomic emission spectrometer (ICP-AES) have revealed that plasma contains ~12 copper, iron and zinc metalloproteins. This included the iron metalloproteins transferrin (Tf) and a recently identified haptoglobin-hemoglobin (Hp-Hb) complex, which is formed in plasma when red blood cells rupture. Since this SEC-ICP-AES method required a sample volume of 500 µL to generate diagnostically useful results, we sought to develop an alternative SEC-based hyphenated approach using a smaller SEC column (150 × 5 mm I.D.) and a graphite furnace atomic absorption spectrometer (GFAAS) as the iron-specific detector. A designed interface enabled the integration of the SEC system with the GFAAS. Baseline separation between the Hp-Hb complex and Tf was achieved by developing a sample preparation procedure which involved the chelating agent-based mobilization of Fe from Tf to a small molecular weight Fe complex. Spiking of human plasma (1.0 mL) with red blood cell lysate (1–2 µL) increased only the intensity of the Fe peak corresponding to the Hp-Hb complex, but not that of Tf. Since the developed SEC-GFAAS method requires only 50 µL of plasma for analysis, it can now be employed for the cost-effective quantification of the clinically relevant Hb-Hp complex in human plasma in <50 min.

The human protein haptoglobin inhibits IsdH-mediated heme-sequestering by Staphylococcus aureus

Iron is an essential nutrient for all living organisms. To acquire iron, many pathogens have developed elaborate systems to steal it from their hosts. The iron acquisition system in the opportunistic pathogen Staphylococcus aureus comprises nine proteins, called iron-regulated surface determinants (Isds). The Isd components enable S. aureus to extract heme from hemoglobin (Hb), transport it into the bacterial cytoplasm, and ultimately release iron from the porphyrin ring. IsdB and IsdH act as hemoglobin receptors and are known to actively extract heme from extracellular Hb. To limit microbial pathogenicity during infection, host organisms attempt to restrict the availability of nutrient metals at the host-pathogen interface. The human acute phase protein haptoglobin (Hp) protects the host from oxidative damage by clearing hemoglobin that has leaked from red blood cells and also restricts the availability of extracellular Hb-bound iron to invading pathogens. To investigate whether Hp serves an additional role in nutritional immunity through a direct inhibition of IsdH-mediated iron acquisition, here we measured heme extraction from the Hp-Hb complex by UV-visible spectroscopy and determined the crystal structure of the Hp-Hb-IsdH complex at 2.9 Å resolution. We found that Hp strongly inhibits IsdH-mediated heme extraction and that Hp binding prevents local unfolding of the Hb heme pocket, leaving IsdH unable to wrest the heme from Hb. Furthermore, we noted that the Hp-Hb binding appears to trap IsdH in an initial state before heme transfer. Our findings provide insights into Hp-mediated IsdH inhibition and the dynamics of IsdH-mediated heme extraction.

Tyrosyl radical in haemoglobin and haptoglobin-haemoglobin complex: how does haptoglobin make haemoglobin less toxic?

One of the difficulties in creating a blood substitute on the basis of human haemoglobin (Hb) is the toxic nature of Hb when it is outside the safe environment of the red blood cells. The plasma protein haptoglobin (Hp) takes care of the Hb physiologically leaked into the plasma – it binds Hb and makes it much less toxic while retaining the Hb's high oxygen transporting capacity. We used Electron Paramagnetic Resonance (EPR) spectroscopy to show that the protein bound radical induced by H2O2 in Hb and Hp-Hb complex is formed on the same tyrosine residue(s), but, in the complex, the radical is found in a more hydrophobic environment and decays slower than in unbound Hb, thus mitigating its oxidative capacity. The data obtained in this study might set new directions in engineering blood substitutes for transfusion that would have the oxygen transporting efficiency typical of Hb, but which would be non-toxic.

Anti-Inflammatory Effects of Hydroxyurea in a Murine Model of Chronic Intravascular Hemolysis

Intravascular hemolysis is associated with a number of disorders, including sickle cell anemia (SCA). In addition to diminishing red blood cell (RBC) counts, clinical manifestations associated with intravascular hemolysis include leg ulcers, pulmonary hypertension and priapism. Recent studies indicate that intravascular hemolysis propagates the inflammatory processes associated with many hemolytic diseases. This study aimed to establish a murine model of chronic hemolysis (CH) that elevates circulating cell-free heme to concentrations similar to those seen in SCA and observe the effects of CH on inflammatory parameters. We also evaluated the effects of concomitant administration of hydroxyurea (HU), a drug commonly used as therapy for SCA, on hemolytic inflammation. For induction of CH, C57BL/6 mice received twice weekly low concentration doses (10 mg/Kg/day) of phenylhydrazine (PHZ, i.p.) for four weeks (wks). Control mice received saline (CON) and some mice also received HU (50 mg/Kg/day, 5 times/wk for 4 wks, i.p.). Plasma cell-free hemoglobin (Hb) and heme were elevated to 0.4±0.1 g/L and 51.4±8.1 µM, respectively (N=4, P&lt;0.05 compared to CON), after four wks of PHZ; concentrations similar to those observed in humans and mice with SCD (data not shown). Four wks of CH induced significant leukocytosis and reduced RBC counts (WBC; 6.1±0.8, 20.2±3.2 x103/µL: RBC; 6.6±0.4, 4.8±0.3 x106/µL for CON and CH, respect., P&lt;0.01, N=4-5), and also significantly increased spleen weight in mice (0.33±0.01, 1.00±0.09 % of body weight for CON and CH, respect., P&lt;0.0001, N=4-5). CH mice displayed substantial depletion of the Hb- and heme-binding proteins, haptoglobin (HP) and hemopexin (HPX) (HP; 129.9±5.2, 1.01±0.2 µg/ml: HPX; 1.98±0.22, 0.44±0.13 mg/ml for CON and CH, respect., P&lt;0.0001, N=5), and increased plasma hemoxygenase-1 (HO-1) (17.4±1.7, 37.8±3.3 ng/ml, P&lt;0.0001, N=5). With regard to inflammatory markers, four wks of CH significantly increased circulating levels of cytokines IL-1β and IL-10 (IL-1β; 3.77±0.53, 6.06±1.14 pg/mL: IL-10, 6.75±0.28, 8.46±0.87 pg/mL for CON and CH, respect., P&lt;0. 05, N=4-5), but not TNF-α (0.70±0.17, 0.76±0.24 pg/mL for CON and CH, respect., N=4-5). Western blotting demonstrated that CH elevated hepatic protein expressions of CD163 (Hb-HP complex receptor), HO-1 and LRP1 (CD91, Heme-HPX complex membrane receptor), while depleting hepatic HPX (CD163; 0.3±0.02, 1.8±0.4 AU: LRP1; 0.4±.0.1, 1.2±0.4 AU: HO-1; 0.93±0.03, 2.66±1.01 AU: HPX; 0.7±0.2, 0.14±0.02 AU relative to GAPDH, for CON and CH, respect., N=3-8, P&lt;0.05 for CD163, HO-1, HPX). Concomitant treatment of mice with 50 mg/Kg/day HU throughout hemolysis induction (CHHU) did not significantly alter hematological data, spleen weight or plasma markers of hemolysis, compared to CH (WBC, 20.6±1.91 x103/µL: RBC, 4.6±0.1 x106/µL: spleen weight; 1.01±0.06 % body weight: HP; 0.71±0.04 pg/mL: HPX; 0.34±0.64 mg/mL, respect., P&gt;0.05 compared to CH, N=5), although a slight decrease in plasma HO-1 was observed (30.7 ± 2.2 ng/ml for CHHU, P&gt;0.05). Notably, HU therapy significantly decreased plasma levels of cytokines IL-1β and IL-10, in mice undergoing CH (IL-1β; 2.22±0.24 pg/mL: IL-10; 5.81±0.29 pg/mL, N=5, P&lt;0.01 compared to CH) and partially abolished the effects of CH on hepatic protein levels of HO-1 (1.39±0.23 AU, N=3) and HPX (0.4±0.08 AU, N= 7, P&lt;0.05 compared to CH). In summary, using repeated low dose PHZ administrations, we successfully induced moderate and continuous hemolysis in mice, similar to that observed in SCA. This CH was associated with the depletion of hemoglobin and heme scavenger proteins, and with the production of pro- and anti-inflammatory cytokines, in addition to higher expression of scavenger receptors in the livers of these mice. Concomitant therapy of mice with HU, frequently used to induce fetal hemoglobin (HbF) in SCA patients, did not alter hematological data or markers of hemolysis, indicating that this drug does not inhibit hemolysis in our CH model. However, HU had significant anti-inflammatory effects, reducing markers of inflammation and elevating hepatic HPX protein expression. As such, HU has anti-inflammatory effects during long term use that are independent of its HbF-inducing properties, providing further evidence of the mechanism of action HU in SCA and further supporting its use in other hemolytic disorders. Disclosures No relevant conflicts of interest to declare.

Identification of a haptoglobin-hemoglobin complex in human blood plasma

Blood plasma metalloproteins that contain copper (Cu), iron (Fe), zinc (Zn) and/or other metals/metalloids are potential disease biomarkers because the bloodstream is in permanent contact with organs. Their quantification and/or the presence of additional metal-entities or the absence of certain metalloproteins in blood plasma (e.g. in Wilson's disease) may provide insight into the dyshomeostasis of the corresponding metal (s) to gain insight into disease processes. The first step in investigating if the determination of plasma metalloproteins is useful for the diagnosis of diseases is their definitive qualitative identification. To this end, we have added individual highly pure Cu, Fe or Zn-containing metalloproteins to plasma (healthy volunteer) and analyzed this mixture by size-exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic spectrometer (ICP-AES), simultaneously monitoring the emission lines of Cu, Fe and Zn. The results clearly identified ceruloplasmin (Cp), holo-transferrin (hTf), and α2-macroglobulin (α2M), which verifies our previous assignments. Interestingly, another major Fe-peak in plasma was identified as a haptoglobin (Hp)-hemoglobin (Hb) complex. This Hp-Hb complex is formed after Hb, which is released during the hemolysis of erythrocytes, binds to the plasma protein Hp. The Hp-Hb complex formation is known to be one of the strongest interactions in biochemistry (Kd≈1pmol/L) and is critical because it prevents kidney toxicity of free Hb. Hence, the simultaneous determination of Cp, hTf, α2M and the Hp-Hb complex in plasma in <25min has the potential to provide new insight into disease processes associated with the bioinorganic chemistry of Cu, Fe and Zn.

Milk haptoglobin detection based on enhanced chemiluminescence of gold nanoparticles

The suggested research specifically addresses the major source of economic loss of the dairy industry, the bovine mastitis (BM), an inflammatory disease of mammary gland caused by bacterial intramammary infection. During udder inflammation, the concentrations of acute phase proteins (APP) in both plasma and milk are escalated, which can be distinctively utilized as predicting diagnostic biomarkers of cattle's BM clinical status. Herein, we demonstrate a liquid-phase luminol chemiluminescence (CL) system for sensitive detection of haptoglobin (Hp), a predictive APP of BM, by utilizing the binding capacity of hemoglobin (Hb). The CL intensity is linearly proportional to Hb-Hp complex formation, resulting in peroxidase-like activity inhibition of luminol-H2O2-Hb CL system. Enhanced CL, at least 10-fold effect within real samples, is attained by the addition of catalytically active cross-linked gold nanoparticles (GNPs) onto the luminol-H2O2 solution. Moreover, the influence of different somatic cell counts (representing subclinical and clinical BM status) and pathogen types (i.e., CNS and Streptococcus dysgalactiae) on the secreted milk Hp levels obtained from Holstein cows are established. The analyzed Hp concentrations are in agreement with a commercial enzyme-linked immunosorbent assay kit. The proposed CL sensing concept offers cost-effective, simple, label-free and reliable systematic analysis of Hp biomarker for BM, potentially initiating a positive effect on animals’ health and overall economy of the dairy farms.

Generation of Pigs Resistant to Highly Pathogenic-Porcine Reproductive and Respiratory Syndrome Virus through Gene Editing of CD163.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease and the most economically important disease of the swine industry worldwide. Highly pathogenic-PRRS virus (HP-PRRSV) is a variant of PRRSV, which caused high morbidity and mortality. Scavenger receptor CD163, which contains nine scavenger receptor cysteine-rich (SRCR) domains, is a key entry mediator for PRRSV. A previous study demonstrated that SRCR domain 5 (SRCR5), encoded by exon 7, was essential for PRRSV infection in vitro. Here, we substituted exon 7 of porcine CD163 with the corresponding exon of human CD163-like 1 (hCD163L1) using a CRISPR/Cas9 system combined with a donor vector. In CD163Mut/Mut pigs, modifying CD163 gene had no adverse effects on hemoglobin-haptoglobin (Hb-Hp) complex clearance or erythroblast growth. In vitro infection experiments showed that the CD163 mutant strongly inhibited HP-PRRSV replication by inhibiting virus uncoating and genome release. Compared to wild-type (WT) pigs in vivo, HP-PRRSV-infected CD163Mut/Mut pigs showed a substantially decreased viral load in blood and relief from PRRSV-induced fever. While all WT pigs were dead, there of four CD163Mut/Mut pigs survived and recovered at the termination of the experiment. Our data demonstrated that modifying CD163 remarkably inhibited PRRSV replication and protected pigs from HP-PRRSV infection, thus establishing a good foundation for breeding PRRSV-resistant pigs via gene editing technology.

Is Magnetic Resonance Imaging Detection of Kidney Iron Deposition Increased in Haptoglobin 2-2 Genotype Carriers with Type 1 Diabetes?

Haptoglobin's (Hp) main role is to bind free hemoglobin (Hb), reducing its oxidative potential. The Hp-Hb complex formed is cleared from the circulation by macrophage receptor CD163. In diabetes, impaired Hp 2-2-Hb CD163 clearance and abnormal glomerular permeability allow the large Hp 2-2-Hb complex to cross the barrier, where its redox active iron leads to cellular toxicity. Although Hp 2-2 predicts renal function decline, whether renal iron deposition differs by Hp is unknown. We used renal quantitative T2* magnetic resonance imaging to estimate iron level in the cortex and medullar of type 1 diabetes (T1D) adults [15 Hp 1-1 and 15 Hp 2-2 carriers of similar age (53 years), duration (45 years), and gender]. Total kidney iron level was estimated as the sum of the cortex and medullar iron. Albuminuria was defined as urinary albumin to creatinine ratio >30 mg/g in two of three samples. Total kidney iron did not differ by gender or Hp but was higher in those with albuminuria (p = 0.05), an association confined to Hp 2-2 carriers (p = 0.04 vs. p = 0.51 in Hp 1-1). These data lead to the hypothesis that kidney iron deposition is increased among Hp 2-2 carriers with albuminuria in T1D. Antioxid. Redox Signal. 29, 735-741.

N-glycan microheterogeneity regulates interactions of plasma proteins

Altered glycosylation patterns of plasma proteins are associated with autoimmune disorders and pathogenesis of various cancers. Elucidating glycoprotein microheterogeneity and relating subtle changes in the glycan structural repertoire to changes in protein-protein, or protein-small molecule interactions, remains a significant challenge in glycobiology. Here, we apply mass spectrometry-based approaches to elucidate the global and site-specific microheterogeneity of two plasma proteins: α1-acid glycoprotein (AGP) and haptoglobin (Hp). We then determine the dissociation constants of the anticoagulant warfarin to different AGP glycoforms and reveal how subtle N-glycan differences, namely, increased antennae branching and terminal fucosylation, reduce drug-binding affinity. Conversely, similar analysis of the haptoglobin-hemoglobin (Hp-Hb) complex reveals the contrary effects of fucosylation and N-glycan branching on Hp-Hb interactions. Taken together, our results not only elucidate how glycoprotein microheterogeneity regulates protein-drug/protein interactions but also inform the pharmacokinetics of plasma proteins, many of which are drug targets, and whose glycosylation status changes in various disease states.

Haptoglobin Genotype Is a Determinant of Hemoglobin Adducts and Vitamin E Content in HDL.

Haptoglobin (Hp) is an abundant hemoglobin- (Hb-) binding serum protein and a constituent of the HDL proteome. In man, there exists a common polymorphism at the Hp locus with two common alleles defined by the presence (Hp 2 allele) or absence (Hp 1 allele) of a 1.7 kb in-frame partial duplication of exons 3 and 4 of the Hp gene. Numerous studies have demonstrated that the Hp 2-2 genotype is associated with a 3–5-fold increase in vascular disease among individuals with diabetes mellitus (DM). Increased Hp-Hb complex has been shown to be associated with the HDL of Hp 2-2 DM individuals. Hb-associated HDL has been proposed to result in the oxidation of HDL and the consumption of antioxidants in HDL, such as vitamin E, rendering the HDL further susceptible to oxidation. In this study, we set out to identify proteins which become cross-linked to Hb in HDL and to measure vitamin E in HDL as a function of the Hp genotype. We report on the identification of a novel 72 kd Hb reactive species which is cross-linked to HDL and demonstrate that vitamin E in HDL is decreased in Hp 2-2 DM individuals.

Haptoglobin.

Haptoglobin (Hp) is an abundant human plasma protein that tightly captures hemoglobin (Hb) during hemolysis. The Hb-Hp complex formation reduces the oxidative properties of heme/Hb and promotes recognition by the macrophage scavenger receptor CD163. This leads to Hb-Hp breakdown and heme catabolism by heme oxygenase and biliverdin reductase. Gene duplications of a part of or the entire Hp gene in the primate evolution have led to variant Hp gene products that collectively may be designated "the haptoglobins (Hps)" as they all bind Hb. These variant products include the human-specific multimeric Hp phenotypes in individuals, which are hetero- or homozygous for an Hp2 gene allele. The Hp-related protein (Hpr) is another Hp duplication product in humans and other primates. Alternative functions of the variant Hps are indicated by numerous reports on association between Hp phenotypes and disease as well as the elucidation of a specific role of Hpr in the innate immune defense. Recent Advances: Recent functional and structural information on Hp and receptor systems for Hb removal now provides insight on how Hp carries out essential functions such as the Hb detoxification/removal, and how Hpr, by acting as an Hp-lookalike, can sneak a lethal toxin into trypanosome parasites that cause mammalian sleeping sickness. Critical Issues and Future Directions: The new structural insight may facilitate ongoing attempts of developing Hp derivatives for prevention of Hb toxicity in hemolytic diseases such as sickle cell disease and other hemoglobinopathies. Furthermore, the new structural knowledge may help identifying yet unknown functions based on other disease-relevant biological interactions involving Hps. Antioxid. Redox Signal. 26, 814-831.

Plasma levels of Apolipoprotein A1 and Lecithin:Cholesterol Acyltransferase in type 2 diabetes mellitus: Correlations with haptoglobin phenotypes

BackgroundPrevious studies have demonstrated that hemoglobin-haptoglobin (Hb-Hp) complex plays a role in developing vascular complications in type 2 diabetes mellitus (T2DM). The complexes bind with Apolipoprotein A1 (ApoA1) of high-density lipoprotein (HDL), affecting the function of Lecithin:Cholesterol Acyltransferase (LCAT), and impairing the reverse cholesterol transport mechanism (RCT). This study investigated the influence of Hp phenotypes on serum levels of ApoA1 and LCAT in patients with T2DM. MethodsThe study comprised 131 T2DM patients and 111 matching healthy controls. Fasting blood glucose, HbA1C, and lipid profile were determined by chemistry autoanalyzer, LCAT and ApoA1 by ELISA, and Hp phenotypes by gel electrophoresis. ResultsIrrespective of Hp phenotypes, fasting blood glucose, HbA1C, and lipid profile were significantly higher in patients than in controls, while HDL-cholesterol, ApoA1, and LCAT were lower. ApoA1 correlated positively with LCAT (r=0.223, p=0.024) and HDL-cholesterol (r=0.255, 0.003) in patients only.When Hp polymorphism was taken into account, the levels of LCAT and ApoA1 were significantly lower in patients with Hp2-2 than that in patients of Hp1-1 and/or Hp2-1. Correlations between ApoA1 and each of HDL-cholesterol and LCAT (r=0.239, p=0.046, and r=0.252, p=0.040, respectively) were also observed, but only in patients with Hp2-2 phenotype. ConclusionsThe reduced levels of LCAT and ApoA1 observed in this study support the suggestion that T2DM patients with Hp2-2 phenotype could have altered RCT mechanism and increased risk of developing cardiovascular disease.

The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen

Hemolysis is a complication in septic infections with Staphylococcus aureus, which utilizes the released Hb as an iron source. S. aureus can acquire heme in vitro from hemoglobin (Hb) by a heme-sequestering mechanism that involves proteins from the S. aureus iron-regulated surface determinant (Isd) system. However, the host has its own mechanism to recapture the free Hb via haptoglobin (Hp) binding and uptake of Hb-Hp by the CD163 receptor in macrophages. It has so far remained unclear how the Isd system competes with this host iron recycling system in situ to obtain the important nutrient. By binding and uptake studies, we now show that the IsdH protein, which serves as an Hb receptor in the Isd system, directly interferes with the CD163-mediated clearance by binding the Hb-Hp complex and inhibiting CD163 recognition. Analysis of truncated IsdH variants including one or more of three near iron transporter domains, IsdHN1, IsdHN2, and IsdHN3, revealed that Hb binding of IsdHN1 and IsdHN2 accounted for the high affinity for Hb-Hp complexes. The third near iron transporter domain, IsdHN3, exhibited redox-dependent heme extraction, when Hb in the Hb-Hp complex was in the oxidized met form but not in the reduced oxy form. IsdB, the other S. aureus Hb receptor, failed to extract heme from Hb-Hp, and it was a poor competitor for Hb-Hp binding to CD163. This indicates that Hb recognition by IsdH, but not by IsdB, sterically inhibits the receptor recognition of Hb-Hp. This function of IsdH may have an overall stimulatory effect on S. aureus heme acquisition and growth.

Haptoglobin increases the vulnerability of CD163-expressing neurons to hemoglobin.

Haptoglobin (Hp) binds hemoglobin (Hb) with high affinity and provides the primary defense against its toxicity after intravascular hemolysis. Neurons are exposed to extracellular Hb after CNS hemorrhage, and a therapeutic effect of Hp via Hb sequestration has been hypothesized. In this study, we tested the hypothesis that Hp protects neurons from Hb in primary mixed cortical cell cultures. Treatment with low micromolar concentrations of human Hb for 24h resulted in loss of 10-20% of neurons without injuring glia. Concomitant treatment with Hp surprisingly increased neuronal loss five-sevenfold, with similar results produced by Hp 1-1 and 2-2 phenotypes. Consistent with a recent invivo observation, neurons expressed the CD163 receptor for Hb and the Hb-Hp complex in these cultures. Hp reduced overall Hb uptake, directed it away from the astrocyte-rich CD163-negative glial monolayer, and decreased induction of the iron-binding protein ferritin. Hb-Hp complex neuronal toxicity, like that of Hb per se, was iron-dependent and reduced by deferoxamine and 2,2' bipyridyl. These results suggest that Hp increases the vulnerability of CD163+ neurons to Hb by permitting Hb uptake while attenuating the protective response of ferritin induction by glial cells. Cover Image for this issue: doi: 10.1111/jnc.13342.

Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense.

BackgroundSince Trypanosoma spp. lack a complete heme synthesis pathway, the parasites are totally dependent on their host for heme throughout all of the stages of their life-cycle. We herein report the identification and characterization of a T. congolense epimastigote form (EMF)-specific hemoglobin (Hb) receptor. The gene was initially reported to encode a T. congolense haptoglobin (Hp)-Hb complex receptor (TcHpHbR) based on its similarity to a gene encoding a T. brucei Hp-Hb complex receptor (TbHpHbR).MethodsTrypanosoma congolense IL3000 was used in this study. A TcHpHbR gene was PCR amplified from the parasite genome. The recombinant protein was used as an immunogen to raise antibodies for immunofluorescence assay and immunoblotting. Hemoglobin uptake by the parasite was examined by using Alexa 488 labelled Hb and visualized by confocal laser scanning microscopy. The qualitative and quantitative interaction between TcHpHbR and its ligand were measured using a surface plasmon resonance assay.ResultsWe found that, unlike TbHpHbR, TcHpHbR was exclusively expressed in the EMF stage at RNA and protein levels. The recombinant TcHpHbR (rTcHpHbR) was co-precipitated with free-Hb in a GST-pull down assay. Surface plasmon resonance revealed that rTcHpHbR binds free-Hb with high affinity (dissociation constant (Kd) = 2.1×10-8 M) but free-Hp with low affinity (Kd = 2.2×10-7 M). Furthermore, Alexa 488-labelled-Hb was only taken up by the EMF and co-localized with tomato lectin, which is a marker of endocytic compartments (flagellar pocket and lysosome).ConclusionWe conclude that the T. congolense EMF takes up free-Hb via TcHpHbR, a receptor which is specific to this developmental stage. We therefore propose renaming TcHpHbR as T. congolense EMF-specific Hb receptor (TcEpHbR).Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1563-9) contains supplementary material, which is available to authorized users.

Therapeutic Targeting of CD163 on Hematopoietic Progenitor Cells By Novel Monoclonal Antibody TBI 304H Demonstrates an Increase in Erythropoiesis in Humanized Mice

▪Hemoglobin (Hb) is known to stimulate erythropoiesis, a process that may be mediated by CD163. CD163 is a receptor for the hemoglobin-haptoglobin (Hb-Hp) complex expressed on monocyte/macrophages as well as a subpopulation of human CD34+ hematopoietic progenitor cells (HPCs). We have demonstrated that administration of ligands to the CD163 receptor can measurably stimulate erythropoiesis in human CD34+ cell-engrafted severe-combined immunodeficiency (SCID) mice. To better elucidate the role of CD163 in hematopoiesis, we investigated the effects of the natural ligand to CD163 (Hb-Hp) as well as a stimulatory antibody, TBI 304H, on HPCs in vivo. SCID mice engrafted with human CD34+ cells were used as a model system were used to investigate the effect of Hb and anti-CD163 monoclonal antibodies (TBI 304 and TBI 304H) on human hematopoiesis in vivo. In an initial study, NOD-SCID IL2R gammanull (NSG) mice were engrafted with human CD34+ cells and animals with < 30% human CD45+ cells in the peripheral blood were administered 2 mg Hb/mouse, or 100 or 500 µg/mouse TBI 304 every 4 days for a total of four doses. At study termination on day 14, bone marrow cells (BMCs) were examined by flow cytometry and CD34+ cells were recovered from the BMCs for enumeration in colony-forming assays. Hemoglobin administration resulted in an increase of human CD34+ cells ranging from 4% to 7% of BMCs and a corresponding 57% increase in colony-forming cells (CFCs) over control animals. In contrast, the monoclonal antibody (mAb), TBI 304, produced a dose-dependent decrease in CD34+ and colonies, possibly reflecting a depletion of CD34+/CD163+ cells as a result of overstimulation due to the much longer circulating half-life of the mAb compared to Hb.. To confirm this hypothesis, human CD34+ cell engrafted animals were given only a single dose of 10 or 100 µg/mouse of TBI 304 and BMCs were examined earlier on day 7. TBI 304 provided a 3.5-fold increase in human CD34+ cells as well as a 1.8 to 6.7-fold increase in bone marrow erythroid lineage engraftment (huGlyA+, huCD36+ and huCD71+) and a 2-fold increase in colony-forming cells. The ability of TBI 304 to stimulate erythropoiesis in preclinical models led to the creation of an anti-CD163 mAb suitable for human clinical use. TBI 304H was generated by grafting the complementarity-determining regions derived from TBI 304 onto a humanized IgG4 framework without altering antigen specificity. An IgG4 framework, as an antibody without Fc effector function, was deemed the most suitable for an agonistic mAb. In the single dose, 7 day Hu-SCID model human CD34+ cells were mobilized from the mouse bone marrow by TBI 304H, as reflected by dose dependent decreases in huCD34+, huCD71+, and huGlyA+ cells in the mouse marrow. At the highest dose tested (500 µg/mouse) the decrease in human HPCs was similar to that found in animals administered Hb (2 mg/mouse). In this model, human hematopoiesis derived from the engrafted human CD34+ cells is not sustained and these date may reflect a mobilization of human HPCs through stimulation by an anti-CD163 antibody. Therapure has received U.S. FDA approval to conduct a Phase I trial of the novel therapeutic antibody TBI 304H. The Phase I clinical trial is a single-center, open-label, intra-subject escalating dose study, which will evaluate the safety, tolerability and pharmacokinetics of TBI 304H following administration to subjects experiencing chemotherapy-induced anemia. DisclosuresMatthews:Therapure Biopharma: Employment. Eberle-Ayres:Therapure Biopharma: Employment. Lu:Therapure Biopharma: Employment. Singh:Therapure Biopharma: Employment. Cutler:Therapure Biopharma: Employment. Bell:Therapure Biopharma: Employment.