Variability of haptoglobin beta-chain proteoforms.

Existing knowledge on changes of the haptoglobin (Hp) molecule suggests that it may exist in multiple proteoforms, which obviously exhibit different functions. Using two-dimensional electrophoresis (2DE) in combination with mass spectrometry and immunodetection, we have analyzed blood plasma samples from both healthy donors and patients with primary grade IV glioblastoma (GBM), and obtained a detailed composite 2DE distribution map of ß-chain proteoforms, as well as the full-length form of Hp (zonulin). Although the total level of plasma Hp exceeded normal values in cancer patients (especially patients with GBM), the presence of particuar proteoforms, detected by their position on the 2DE map, was very individual. Variability was found in both zonulin and the Hp ß-chain. The presence of an alkaline form of zonulin in plasma can be considered a conditional, but insufficient, GBM biomarker. In other words, we found that at the level of minor proteoforms of Hp, even in normal conditions, there was a high individual variability. On the one hand, this raises questions about the reasons for such variability, if it is present not only in Hp, but also in other proteins. On the other hand, this may explain the discrepancy between the number of experimentally detected proteoforms and the theoretically possible ones not only in Hp, but also in other proteins.

Development and validation of a model and nomogram for breast cancer diagnosis based on quantitative analysis of serum disease-specific haptoglobin N-glycosylation.

BACKGROUND: A better diagnostic marker is in need to distinguish breast cancer from suspicious breast lesions. The abnormal glycosylation of haptoglobin has been documented to assist cancer diagnosis. This study aims to evaluate disease-specific haptoglobin (DSHp)-ß N-glycosylation as a potential biomarker for breast cancer diagnosis. METHODS: DSHp-ß chains of 497 patients with suspicious breast lesions who underwent breast surgery were separated from serum immunoinflammatory-related protein complexes. DSHp-ß N-glycosylation was quantified by mass spectrometric analysis. After missing data imputation and propensity score matching, patients were randomly assigned to the training set (n = 269) and validation set (n = 113). Logistic regression analysis was employed in model and nomogram construction. The diagnostic performance was analyzed with receiver operating characteristic and calibration curves. RESULTS: 95 N-glycopeptides at glycosylation sites N207/N211, N241, and N184 were identified in 235 patients with benign breast diseases and 262 patients with breast cancer. DSHp-ß N-tetrafucosyl and hexafucosyl were significantly increased in breast cancer compared with benign diseases (p < 0.001 and p = 0.001, respectively). The new diagnostic model and nomogram included GN2F2, G6N3F6, GN2FS at N184, G-N&G2S2, G2&G3NFS, G2N3F, GN3 at N207/N211, CEA, CA153, and could reliably distinguish breast cancer from benign diseases. For the training set, validation set, and training and validation sets, the area under the curves (AUCs) were 0.80 (95% CI: 0.75-0.86, specificity: 87%, sensitivity: 62%), 0.77 (95% CI:0.69-0.86, specificity: 75%, sensitivity: 69%), and 0.80 (95% CI:0.76-0.84, specificity: 77%, sensitivity: 68%), respectively. CEA, CA153, and their combination yielded AUCs of 0.62 (95% CI: 0.56-0.67, specificity: 29%, sensitivity: 90%), 0.65 (95% CI: 0.60-0.71, specificity: 74%, sensitivity: 51%), and 0.67 (95% CI: 0.62-0.73, specificity: 60%, sensitivity: 68%), respectively. CONCLUSIONS: The combination of DSHp-ß N-glycopeptides, CEA, and CA153 might be a better serologic marker to differentiate between breast cancer and benign breast diseases. The dysregulated N-glycosylation of serum DSHp-ß could provide insights into breast tumorigenesis.

In-depth investigation of altered glycosylation in human haptoglobin associated cancer by mass spectrometry.

Serum haptoglobin (Hp), a highly sialylated biomolecule with four N-glycosylation sites, is a positive acute-phase response glycoprotein that acts as an immunomodulator. Hp has gained considerable attention due to its potential as a signature molecule that exhibits aberrant glycosylation in inflammatory disorders and malignancies. Its glycosylation can be analyzed qualitatively and quantitatively by various methods using mass spectrometry. In this review, we have provided a brief overview of Hp structure and biological function and described mass spectrometry-based techniques for analyzing glycosylation ranging from macroheterogeneity to microheterogeneity of Hp in diseases and cancer. The sugars on haptoglobin can be a sweet bridge to link the potential of cancer-specific biomarkers to clinically relevant applications.

Haptoglobin-related protein in human plasma correlates to haptoglobin concentrations and phenotypes.

Haptoglobin-related protein (Hpr) is a plasma protein with high sequence similarity to haptoglobin (Hp). Like Hp, Hpr also binds hemoglobin (Hb) with high affinity, but it does not bind to the Hb-Hp receptor CD163 on macrophages. The Hpr concentration is markedly lower than Hp in plasma and its regulation is not understood. In the present study, we have developed non-crossreactive antibodies to Hpr to analyze the Hpr concentration in 112 plasma samples from anonymized individuals and compared it to Hp. The results show that plasma Hpr correlated with Hp concentrations (rho = 0.46, p = .0001). Hpr accounts for on average 0.35% of the Hp/Hpr pool but up to 29% at low Hp levels. Furthermore, the Hpr concentrations were significantly lower in individuals with the Hp2-2 phenotype compared to those with the Hp2-1 or Hp1-1 phenotypes. Experimental binding analysis did not provide evidence that Hpr associates with Hp and in this way is removed via CD163. In conclusion, the Hpr concentration correlates to Hp concentrations and Hp-phenotypes by yet unknown mechanisms independent of CD163-mediated removal of Hb-Hp complexes.

Evidence of haptoglobin in the porcine female genital tract during oestrous cycle and its effect on in vitro embryo production.

Recent evidence supports involvement of the acute phase protein haptoglobin in numerous events during mammalian reproduction. The present study represents an in-depth investigation of haptoglobin expression and secretion in the porcine oviduct and uterus, and assesses its effect on porcine in vitro embryo production. A systematic study was made of sows in different oestrous stages: late follicular, early luteal and late luteal stages. Relative haptoglobin mRNA abundance was quantified by RT-qPCR. In addition, expression of the protein was analysed by immunohistochemistry and the results were complemented by Western-blot and proteomic analyses of the oviductal and uterine fluids. In vitro porcine fertilization and embryo culture were carried out in the presence of haptoglobin. The results indicate that haptoglobin mRNA expression in the porcine oviduct and uterus is most abundant during the late luteal stage of the oestrous cycle. By means of Western blot and proteomic analyses haptoglobin presence was demonstrated in the oviduct epithelium and in the oviductal and uterine fluids in different stages of the oestrous cycle. The addition of haptoglobin during gamete co-incubation had no effect on sperm penetration, monospermy or efficiency rates; however, compared with the control group, blastocyst development was significantly improved when haptoglobin was present (haptoglobin: 64.50% vs. control: 37.83%; p < 0.05). In conclusion, the presence of haptoglobin in the oviduct and uterus of sows at different stages of the oestrous cycle suggests that it plays an important role in the reproduction process. The addition of haptoglobin during in vitro embryo production improved the blastocyst rates.

Haptoglobin: From hemoglobin scavenging to human health.

Haptoglobin (Hp) belongs to the family of acute-phase plasma proteins and represents the most important plasma detoxifier of hemoglobin (Hb). The basic Hp molecule is a tetrameric protein built by two α/ß dimers. Each Hp α/ß dimer is encoded by a single gene and is synthesized as a single polypeptide. Following post-translational protease-dependent cleavage of the Hp polypeptide, the α and ß chains are linked by disulfide bridge(s) to generate the mature Hp protein. As human Hp gene is characterized by two common Hp1 and Hp2 alleles, three major genotypes can result (i.e., Hp1-1, Hp2-1, and Hp2-2). Hp regulates Hb clearance from circulation by the macrophage-specific receptor CD163, thus preventing Hb-mediated severe consequences for health. Indeed, the antioxidant and Hb binding properties of Hp as well as its ability to stimulate cells of the monocyte/macrophage lineage and to modulate the helper T-cell type 1 and type 2 balance significantly associate with a variety of pathogenic disorders (e.g., infectious diseases, diabetes, cardiovascular diseases, and cancer). Alternative functions of the variants Hp1 and Hp2 have been reported, particularly in the susceptibility and protection against infectious (e.g., pulmonary tuberculosis, HIV, and malaria) and non-infectious (e.g., diabetes, cardiovascular diseases and obesity) diseases. Both high and low levels of Hp are indicative of clinical conditions: Hp plasma levels increase during infections, inflammation, and various malignant diseases, and decrease during malnutrition, hemolysis, hepatic disease, allergic reactions, and seizure disorders. Of note, the Hp:Hb complexes display heme-based reactivity; in fact, they bind several ferrous and ferric ligands, including O2, CO, and NO, and display (pseudo-)enzymatic properties (e.g., NO and peroxynitrite detoxification). Here, genetic, biochemical, biomedical, and biotechnological aspects of Hp are reviewed.

Differential Quantitative Determination of Site-Specific Intact N-Glycopeptides in Serum Haptoglobin between Hepatocellular Carcinoma and Cirrhosis Using LC-EThcD-MS/MS.

Intact N-glycopeptide analysis remains challenging due to the complexity of glycopeptide structures, low abundance of glycopeptides in protein digests, and difficulties in data interpretation/quantitation. Herein, we developed a workflow that involved advanced methodologies, the EThcD-MS/MS fragmentation method and data interpretation software, for differential analysis of the microheterogeneity of site-specific intact N-glycopeptides of serum haptoglobin between early hepatocellular carcinoma (HCC) and liver cirrhosis. Haptoglobin was immunopurified from 20 µL of serum in patients with early HCC, liver cirrhosis, and healthy controls, respectively, followed by trypsin/GluC digestion, glycopeptide enrichment, and LC-EThcD-MS/MS analysis. Identification and differential quantitation of site-specific N-glycopeptides were performed using a combination of Byonic and Byologic software. In total, 93, 87, and 68 site-specific N-glycopeptides were identified in early HCC, liver cirrhosis, and healthy controls, respectively, with high confidence. The increased variety of N-glycopeptides in liver diseases compared to healthy controls was due to increased branching with hyper-fucosylation and sialylation. Differential quantitation analysis showed that 5 site-specific N-glycopeptides on sites N184 and N241 were significantly elevated in early HCC compared to cirrhosis ( p < 0.05) and normal controls ( p ≤ 0.001). The result demonstrates that the workflow provides a strategy for detailed profiles of N-glycopeptides of patient samples as well as for relative quantitation to determine the level changes in site-specific N-glycopeptides between disease states.

The Diversity of Mammalian Hemoproteins and Microbial Heme Scavengers Is Shaped by an Arms Race for Iron Piracy.

Iron is an essential micronutrient for most living species. In mammals, hemoglobin (Hb) stores more than two thirds of the body's iron content. In the bloodstream, haptoglobin (Hp) and hemopexin (Hpx) sequester free Hb or heme. Pathogenic microorganisms usually acquire iron from their hosts and have evolved complex systems of iron piracy to circumvent nutritional immunity. Herein, we performed an evolutionary analysis of genes coding for mammalian heme-binding proteins and heme-scavengers in pathogen species. The underlying hypothesis is that these molecules are engaged in a molecular arms race. We show that positive selection drove the evolution of mammalian Hb and Hpx. Positively selected sites in Hb are located at the interaction surface with Neisseria meningitidis heme scavenger HpuA and with Staphylococcus aureus iron-regulated surface determinant B (IsdB). In turn, positively selected sites in HpuA and IsdB are located in the flexible protein regions that contact Hb. A residue in Hb (S45H) was also selected on the Caprinae branch. This site stabilizes the interaction with Trypanosoma brucei hemoglobin-haptoglobin (HbHp) receptor (TbHpHbR), a molecule that also mediates trypanosome lytic factor (TLF) entry. In TbHpHbR, positive selection drove the evolution of a variant (L210S) which allows evasion from TLF but reduces affinity for HbHp. Finally, selected sites in Hpx are located at the interaction surface with the Haemophilus influenzae hemophore HxuA, which in turn displays fast evolving sites at the Hpx-binding interface. These results shed light into host-pathogens conflicts and establish the importance of nutritional immunity as an evolutionary force.

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.

Designation of fingerprint glycopeptides for targeted glycoproteomic analysis of serum haptoglobin: insights into gastric cancer biomarker discovery.

Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide, largely because of difficulties in early diagnosis. Despite accumulating evidence indicating that aberrant glycosylation is associated with GC, site-specific localization of the glycosylation to increase specificity and sensitivity for clinical use is still an analytical challenge. Here, we created an analytical platform with a targeted glycoproteomic approach for GC biomarker discovery. Unlike the conventional glycomic approach with untargeted mass spectrometric profiling of released glycan, our platform is characterized by three key features: it is a target-protein-specific, glycosylation-site-specific, and structure-specific platform with a one-shot enzyme reaction. Serum haptoglobin enriched by immunoaffinity chromatography was subjected to multispecific proteolysis to generate site-specific glycopeptides and to investigate the macroheterogeneity and microheterogeneity. Glycopeptides were identified and quantified by nano liquid chromatography-mass spectrometry and nano liquid chromatography-tandem mass spectrometry. Ninety-six glycopeptides, each corresponding to a unique glycan/glycosite pairing, were tracked across all cancer and control samples. Differences in abundance between the two groups were marked by particularly high magnitudes. Three glycopeptides exhibited exceptionally high control-to-cancer fold changes along with receiver operating characteristic curve areas of 1.0, indicating perfect discrimination between the two groups. From the results taken together, our platform, which provides biological information as well as high sensitivity and reproducibility, may be useful for GC biomarker discovery. Graphical abstract ᅟ.

LC-MS/MS isomeric profiling of permethylated N-glycans derived from serum haptoglobin of hepatocellular carcinoma (HCC) and cirrhotic patients.

Early stage detection and cancer treatment demand the identification of reliable biomarkers. Over the past decades, efforts have been devoted to assess the variation of glycosylation level as well as the glycan structures of proteins in blood or serum, associated with the development and/or progression of several cancers, including liver. Herein, an LC-MS/MS-based analysis was conducted to define the glycosylation patterns of haptoglobin glycoprotein derived from sera collected from cirrhotic and hepatocellular carcinoma (HCC) patients. The haptoglobin samples were extracted from serum using an antibody-immobilized column prior to the release of N-glycans. A comparison of non-isomeric and isomeric permethylated glycan forms was achieved using C18 and porous graphitic carbon (PGC) columns, respectively. In the case of C18-LC-MS/MS analysis, 25 glycan structures were identified of which 10 sialylated structures were found to be statistically significant between the two cohorts. Also, 8 out of 34 glycan structures identified by PGC-LC-MS/MS were found to be statistically significant, suggesting that isomeric distributions of a particular glycan composition were different in abundances between the two cohorts. The glycan isoform patterns distinguished early stage HCC from cirrhotic patients. Both retention times and tandem mass spectra were utilized to determine the specific isomeric glycan structures. All of the glycan isomers, which were statistically significant, were either branch fucosylated or composed of α-2,6 linked sialic acid moieties. The result of this study demonstrates the potential importance of isomeric separation for defining disease prompted aberrant glycan changes. The levels of several glycan isoforms effectively distinguished early stage HCC from cirrhosis.

Soluble ectodomain CD163 and extracellular vesicle-associated CD163 are two differently regulated forms of 'soluble CD163' in plasma.

CD163 is the macrophage receptor for uptake of hemoglobin-haptoglobin complexes. The human receptor can be shed from the macrophage surface owing to a cleavage site for the inflammation-inducible TACE/ADAM17 enzyme. Accordingly, plasma 'soluble CD163' (sCD163) has become a biomarker for macrophage activity and inflammation. The present study disclosed that 10% of sCD163 in healthy persons is actually extracellular vesicle (EV)-associated CD163 not being cleaved and shed. Endotoxin injection of human volunteers caused a selective increase in the ectodomain CD163, while septic patients exhibited high levels of both soluble ectodomain CD163 and extracellular vesicle (EV) CD163, the latter representing up 60% of total plasma CD163. A poor prognosis of septic patients measured as the sequential organ failure assessment (SOFA) score correlated with the increase in membrane-associated CD163. Our results show that soluble ectodomain CD163 and EV CD163 in plasma are part of separate macrophage response in the context of systemic inflammation. While that soluble ectodomain CD163 is released during the acute systemic inflammatory response, this is not the case for EV CD163 that instead may be released during a later phase of the inflammatory response. A separate measurement of the two forms of CD163 constituting 'soluble CD163' in plasma may therefore add to the diagnostic and prognostic value.

Rapid diagnosis of hepatocellular carcinoma using a haptoglobin ELISA assay based on AAL-coated magnetic beads.

Fucosylated glycans play a critical role in cell biology processes during the occurrence and development of tumor, such as cell signal transduction, cellular differentiation, ligand-receptor interactions, and metastasis formation. Fucosylated haptoglobin (Hp) was reported to be increased in hepatocellular carcinoma (HCC). In this study, we aimed to develop a convenient and sensitive AAL@Magbeads-based Hp ELISA assay to rapidly detect fucosylated Hp in 270 serum samples from HCC patients and normal controls. Fucosylated Hp exhibited an area under the ROC curve (AUC) of 0.818 with sensitivity of 72.59% and specificity of 79.26% as well as accuracy of 75.93% in distinguishing HCC patients from healthy controls. For 40 AFP- HCC (AFP <=20 ng/mL) cases and 135 normal volunteers, AUC of fucosylated Hp was 0.892 with sensitivity of 80%, specificity of 81.48% and accuracy of 81.14%. The results of present study demonstrated that AAL@Magbeads-based Hp ELISA assay could efficiently determine fucosylated Hp, which may serve as a promising glycobiomarker in the diagnosis of HCC, including AFP- HCC patients.

Site-specific and linkage analyses of fucosylated N-glycans on haptoglobin in sera of patients with various types of cancer: possible implication for the differential diagnosis of cancer.

Fucosylation is an important type of glycosylation involved in cancer, and fucosylated proteins could be employed as cancer biomarkers. Previously, we reported that fucosylated N-glycans on haptoglobin in the sera of patients with pancreatic cancer were increased by lectin-ELISA and mass spectrometry analyses. However, an increase in fucosylated haptoglobin has been reported in various types of cancer. To ascertain if characteristic fucosylation is observed in each cancer type, we undertook site-specific analyses of N-glycans on haptoglobin in the sera of patients with five types of operable gastroenterological cancer (esophageal, gastric, colon, gallbladder, pancreatic), a non-gastroenterological cancer (prostate cancer) and normal controls using ODS column LC-ESI MS. Haptoglobin has four potential glycosylation sites (Asn184, Asn207, Asn211, Asn241). In all cancer samples, monofucosylated N-glycans were significantly increased at all glycosylation sites. Moreover, difucosylated N-glycans were detected at Asn 184, Asn207 and Asn241 only in cancer samples. Remarkable differences in N-glycan structure among cancer types were not observed. We next analyzed N-glycan alditols released from haptoglobin using graphitized carbon column LC-ESI MS to identify the linkage of fucosylation. Lewis-type and core-type fucosylated N-glycans were increased in gastroenterological cancer samples, but only core-type fucosylated N-glycan was relatively increased in prostate cancer samples. In metastatic prostate cancer, Lewis-type fucosylated N-glycan was also increased. These data suggest that the original tissue/cell producing fucosylated haptoglobin is different in each cancer type and linkage of fucosylation might be a clue of primary lesion, thereby enabling a differential diagnosis between gastroenterological cancers and non-gastroenterological cancers.

Identification of haptoglobin peptide as a novel serum biomarker for lung squamous cell carcinoma by serum proteome and peptidome profiling.

To date, a number of potential biomarkers for lung squamous cell cancer (SCC) have been identified; however, sensitive biomarkers are currently lacking to detect early stage SCC due to low sensitivity and specificity. In the present study, we compared the 7 serum proteomic profiles of 11 SCC patients, 7 chronic obstructive pulmonary disease (COPD) patients and 7 healthy smokers as controls to identify potential serum biomarkers associated with SCC and COPD. Two-dimensional difference gel electrophoresis (2D-DIGE) and mass-spectrometric analysis (MS) using an affinity column revealed two candidate proteins, haptoglobin (HP) and apolipoprotein 4, as biomarkers of SCC, and α-1-antichymotrypsin as a marker of COPD. The iTRAQ technique was also used to identify SCC-specific peptides. HP protein expression was significantly higher in SCC patients than in COPD patients. Furthermore, two HP protein peptides showed significantly higher serum levels in SCC patients than in COPD patients. We established novel polyclonal antibodies for the two HP peptides and subsequently a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of these specific peptides in patient and control sera. The sensitivity of detection by ELISA of one HP peptide (HP216) was 70% of SCC patients, 40% of COPDs patients and 13% of healthy controls. We also measured CYFRA, a cytokeratin fragment clinically used as an SCC tumor marker, in all the 28 cases and found CYFRA was detected in only seven SCC cases. However, when the measurement of HP216 was combined with that of CYFRA, 100% (10 of 10 patients) of SCC cases were detected. Our proteomic profiling demonstrates that the SCC-specific HP peptide HP216 may potentially be used as a diagnostic biomarker for SCC.

Glycosylation-Based Serum Biomarkers for Cancer Diagnostics and Prognostics.

Cancer is the second most common cause of death in developed countries with approximately 14 million newly diagnosed individuals and over 6 million cancer-related deaths in 2012. Many cancers are discovered at a more advanced stage but better survival rates are correlated with earlier detection. Current clinically approved cancer biomarkers are most effective when applied to patients with widespread cancer. Single biomarkers with satisfactory sensitivity and specificity have not been identified for the most common cancers and some biomarkers are ineffective for the detection of early stage cancers. Thus, novel biomarkers with better diagnostic and prognostic performance are required. Aberrant protein glycosylation is well known hallmark of cancer and represents a promising source of potential biomarkers. Glycoproteins enter circulation from tissues or blood cells through active secretion or leakage and patient serum is an attractive option as a source for biomarkers from a clinical and diagnostic perspective. A plethora of technical approaches have been developed to address the challenges of glycosylation structure detection and determination. This review summarises currently utilised glycoprotein biomarkers and novel glycosylation-based biomarkers from the serum glycoproteome under investigation as cancer diagnostics and for monitoring and prognostics and includes details of recent high throughput and other emerging glycoanalytical techniques.

ESI-LC-MS Method for Haptoglobin Fucosylation Analysis in Hepatocellular Carcinoma and Liver Cirrhosis.

A method for the detection of fucosylated glycans from haptoglobin in patient serum has been developed that provides enhanced sensitivity. The workflow involves isolation of the haptoglobin using an HPLC-based affinity column followed by glycan removal, extraction, and desialylation. The fucosylated glycans are then derivatized by Meladrazine, which significantly enhances the detection of the glycans in electrospray ionization. The separation of the derivatized glycans in a HILIC column shows that eight glycans from haptoglobin can be detected using less than 1 µL of a serum sample, with excellent reproducibility and quantitation, where without derivatization the glycans could not be detected. The ratio of the fucosylated peaks to their corresponding nonfucosylated forms shows that the fucosylated glycans are upregulated in the case of hepatocellular carcinoma (HCC) samples versus cirrhosis samples, where the relatively low abundance bifucosylated tetra-antennary form can be detected and may be a particularly good marker for HCC.

Hepcidin-ferroportin axis controls toll-like receptor 4 dependent macrophage inflammatory responses in human atherosclerotic plaques.

OBJECTIVES: Toll-like Receptor 4 (TLR4) is implicated in modulating inflammatory cytokines though its role in atherosclerosis remains uncertain. We have recently described a non-foam cell macrophage phenotype driven by ingestion of hemoglobin:haptoglobin complexes (HH), via the scavenger receptor CD163, characterized by reduced inflammatory cytokine production. In this study, we examined the role of iron metabolism in modulating TLR4 signaling in these cells. METHODS AND RESULTS: Areas in human atherosclerotic plaque with non-foam cell, CD163 positive macrophages demonstrated reduced expression of tumor necrosis factor alpha (TNF-α) and interferon-beta (INF-ß) compared to foam cells. Human macrophages differentiated in hemoglobin:haptoglobin (HH) complexes expressed the CD163 positive non-foam cell phenotype and demonstrated significantly less TNF-α and INF-ß compared to control macrophages when exposed to oxidized LDL (oxLDL) or lipopolysaccharide (LPS). LPS stimulated expression of TNF-α and INF-ß could be restored in HH macrophages by pretreatment with hepcidin, an endogenous suppressor of ferroportin1 (FPN), or by genetic suppression of FPN in macrophages derived from myeloid specific FPN knockout mice. LPS stimulated control macrophages demonstrated increase in TLR4 trafficking to lipid rafts; this response was suppressed in HH macrophages but was restored upon pretreatment with hepcidin. Using a pharmacologic hepcidin suppressor, we observed a decrease in cytokine expression and TLR4-lipid raft trafficking in LPS-stimulated in a murine macrophage model. CONCLUSION: TLR4 dependent macrophage signaling is controlled via hepcidin-ferroportin1 axis by influencing TLR4-lipid raft interactions. Pharmacologic manipulation of iron metabolism may represent a promising approach to limiting TLR4-mediated inflammatory responses.

Spin trapping combined with quantitative mass spectrometry defines free radical redistribution within the oxidized hemoglobin:haptoglobin complex.

Extracellular or free hemoglobin (Hb) accumulates during hemolysis, tissue damage, and inflammation. Heme-triggered oxidative reactions can lead to diverse structural modifications of lipids and proteins, which contribute to the propagation of tissue damage. One important target of Hb׳s peroxidase reactivity is its own globin structure. Amino acid oxidation and crosslinking events destabilize the protein and ultimately cause accumulation of proinflammatory and cytotoxic Hb degradation products. The Hb scavenger haptoglobin (Hp) attenuates oxidation-induced Hb degradation. In this study we show that in the presence of hydrogen peroxide (H2O2), Hb and the Hb:Hp complex share comparable peroxidative reactivity and free radical generation. While oxidation of both free Hb and Hb:Hp complex generates a common tyrosine-based free radical, the spin-trapping reaction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) yields dissimilar paramagnetic products in Hb and Hb:Hp, suggesting that radicals are differently redistributed within the complex before reacting with the spin trap. With LC-MS(2) mass spectrometry we assigned multiple known and novel DMPO adduct sites. Quantification of these adducts suggested that the Hb:Hp complex formation causes extensive delocalization of accessible free radicals with drastic reduction of the major tryptophan and cysteine modifications in the ß-globin chain of the Hb:Hp complex, including decreased ßCys93 DMPO adduction. In contrast, the quantitative changes in DMPO adduct formation on Hb:Hp complex formation were less pronounced in the Hb α-globin chain. In contrast to earlier speculations, we found no evidence that free Hb radicals are delocalized to the Hp chain of the complex. The observation that Hb:Hp complex formation alters free radical distribution in Hb may help to better understand the structural basis for Hp as an antioxidant protein.