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Beta-amyloid accumulation in brain is a driving force for Alzheimer's disease pathogenesis. Apolipoprotein E (ApoE) represents a critical player in beta-amyloid homeostasis, but its role in disease progression is controversial. We previously reported that the acute-phase protein haptoglobin binds ApoE and impairs its function in cholesterol homeostasis. The major aims of this study were to characterize the binding of haptoglobin to beta-amyloid, and to evaluate whether haptoglobin affects ApoE binding to beta-amyloid. Haptoglobin is here reported to form a complex with beta-amyloid as shown by immunoblotting experiments with purified proteins, or by its immunoprecipitation in brain tissues from patients with Alzheimer's disease. The interaction between ApoE and beta-amyloid was previously shown to be crucial for limiting beta-amyloid neurotoxicity and for promoting its clearance. We demonstrate that haptoglobin, rather than impairing ApoE binding to beta-amyloid, promotes to a different extent the formation of the complex between beta-amyloid and ApoE2 or ApoE3 or ApoE4. Our data suggest that haptoglobin and ApoE functions in brain should be evaluated taking into account their mutual interaction with beta-amyloid. Hence, the risk of developing Alzheimer's disease might not only be linked to the different ApoE isoforms, but also rely on the level of critical ligands, such as haptoglobin.

Improved diagnosis of psoriasis, by new biomarkers, is required for evaluating the progression rate of the disease and the response to treatment. Haptoglobin (Hpt), a glycoprotein secreted by hepatocytes and other types of cells including keratinocytes, was found with glycan changes in psoriasis and other diseases. We previously reported that Hpt isolated from plasma of psoriatic patients is more fucosylated than Hpt of healthy subjects. The aim of this study was to compare the glycosylation pattern of Hpt isolated from skin scales or plasma of patients with psoriasis with that of Hpt from cornified epidermal layer or plasma of healthy subjects. High performance liquid chromatography analysis of the glycans isolated from the protein backbone revealed that glycan patterns from skin and plasma of patients were similar, and mostly displayed quantitative rather than qualitative differences from normal pattern. Biotin-labeled lectins were used to evaluate quantitative differences in the glycoforms of Hpt from plasma and psoriatic skin scales. Hpt from skin and plasma of patients showed more fucosylated and branched glycans than Hpt from plasma of healthy subjects. Tryptic glycopeptides of Hpt were also analyzed by mass spectrometry, and a decreased amount of sialylated glycan chains was found in glycopeptides of skin Hpt, as compared with Hpt from plasma. High levels of glycans with fucosylated and tetra-antennary chains were detected on the peptide NLFLNHSENATAK from Hpt of psoriatic patients. Our data demonstrate that specific changes in glycan structures of Hpt, such as enhanced glycan branching and fucose content, are associated with psoriasis, and that differences between circulating and skin Hpt do exist. A lower extent of glycan fucosylation and branching was found in Hpt from plasma of patients in disease remission. Altered glycoforms might reflect changes of Hpt function in the skin, and could be used as markers of the disease.

Haptoglobin (Hpt) is known to capture circulating free hemoglobin (Hb) and bind apolipoprotein (Apo) A-I or E. Here, we report that Hb can be tightly bound by most of Hpt molecules (TB-Hpt, 80%), whereas loosely bound by a minor part of them (LB-Hpt, 20%). LB-Hpt amount was significantly increased (over 60%) in patients with acute coronary syndrome. LB-Hpt bound ApoA-I and ApoE less efficiently than TB-Hpt (8- and 4-fold less, respectively) and did not affect their activity of stimulating the enzyme lecithin-cholesterol acyltransferase. LB-Hpt and TB-Hpt displayed comparable levels of nitrotyrosine residues, but differences in glycan chains. Changes in LB-Hpt level might be associated with changes in Hpt functions.

Haptoglobin is an acute phase glycoprotein, secreted by hepatocytes and other types of cells including keratinocytes. Haptoglobin has been suggested to impair the immune response, inhibit gelatinases in the extracellular matrix and promote angiogenesis, but its role in psoriasis is obscure to date. Changes in haptoglobin glycan structure were observed in several diseases. The aim of this study was to investigate whether haptoglobin displays glycan variations in psoriasis. We found that the pattern of plasma haptoglobin glycoforms, following two-dimensional electrophoresis, exhibited significant quantitative differences in spot intensities between patients and controls. Quantitative and qualitative differences in glycan mass, between patients and controls, were found by mass spectrometry of glycopeptides from tryptic digests of protein isolated from both patients and controls. The number of distinct fucosylated glycoforms of peptides NLFLNHSENATAK and MVSHHNLTTGATLINEQWLLTTAK was higher in patients than in controls, but no fucosylated glycan was detected on peptide VVLHPNYSQ-VDIGLIK in either case. The number of peptides with distinct triantennary and tetraantennary glycans was higher in patients than in controls. Abundance or structure of specific glycans, which are present in haptoglobin from patients and are different or missing in normal haptoglobin, might be associated with disease activity.

Haptoglobin (Hpt) binds apolipoprotein A-I (ApoA-I), and impairs its stimulation of lecithin:cholesterol acyltransferase (LCAT). LCAT plays a major role in reverse cholesterol transport (RCT). Apolipoprotein E (ApoE), like ApoA-I, promotes different steps of RCT, including LCAT stimulation. ApoE contains amino acid sequences that are homologous with the ApoA-I region bound by Hpt and are involved in the interaction with LCAT. Therefore, Hpt was expected to also bind ApoE, and inhibit the ApoE stimulatory effect on LCAT. Western blotting and ELISA experiments demonstrated that the Hpt beta-subunit binds ApoE. The affinity of Hpt for ApoE was higher than that for ApoA-I. High ratios of Hpt with either apolipoprotein, such as those associated with the acute phase of inflammation, inhibited, in vitro, the stimulatory effect of ApoE on the cholesterol esterification activity of LCAT. Hpt also impaired human hepatoblastoma-derived cell uptake of [(3)H]cholesterol from proteoliposomes containing ApoE or ApoA-I. We suggest that the interaction between Hpt and ApoE represents a mechanism by which inflammation affects atherosclerosis progression. Hpt might influence ApoE function in processes other than RCT.

Haptoglobin (Hpt) binds the apolipoprotein (Apo) A-I domain, which is involved in stimulating the enzyme lecithin-cholesterol acyltransferase (LCAT) for cholesterol esterification. This binding was shown to protect ApoA-I against hydroxyl radicals, thus preventing loss of ApoA-I function in enzyme stimulation. In this study, we report that Hpt is also able to bind ApoE. The Hpt binding site on the ApoE structure was mapped by using synthetic peptides, and found homologous to the Hpt binding site of ApoA-I. Hydroxyl radicals promoted in vitro the formation of ApoE-containing adducts which were detected by immunoblotting. Hpt impaired this oxidative modification whereas albumin did not. CSF from patients with multiple sclerosis or subjects without neurodegeneration contains oxidized forms of ApoE and ApoA-I similar to those observed in vitro. CSF was analyzed for its level of ApoA-I, ApoE, Hpt, cholesteryl esters, and unesterified cholesterol. The ratio of esterified with unesterified cholesterol, assumed to reflect the LCAT activity ex vivo, did not correlate with either analyzed protein, but conversely correlated with the ratio [Hpt]/([ApoE]+[ApoA-I]). The results suggest that Hpt might save the function of ApoA-I and ApoE for cholesterol esterification, a process contributing to cholesterol elimination from the brain.

The high-density lipoprotein apolipoprotein A-I (ApoA-I) stimulates the enzyme lecithin-cholesterol acyltransferase (LCAT) in the reverse cholesterol transport pathway. Two ApoA-I variants, Zaragoza (L144R) and Zavalla (L159P), are associated with low levels of HDL-cholesterol but normal LCAT activity. Haptoglobin interacts with ApoA-I, impairing LCAT stimulation. Synthetic peptides matching the haptoglobin-binding site of native or variant ApoA-I (native, P2a; variants, Zav-pep and Zar-pep) bound haptoglobin with different activity: Zar-pep>P2a>Zav-pep. They also differently rescued LCAT in vitro activity in the presence of haptoglobin (P2a=Zar-pep>Zav-pep). Therefore, both amino acid conversions affect haptoglobin binding and LCAT regulation. We highlight the role of haptoglobin in LCAT regulation in subjects with ApoA-I variants.

Apolipoprotein A-I (ApoA-I), a major component of HDL, binds haptoglobin, a plasma protein transporting to liver or macrophages free Hb for preventing hydroxyl radical production. This work aimed to assess whether haptoglobin protects ApoA-I against this radical. Human ApoA-I structure, as analyzed by electrophoresis and MS, was found severely altered by hydroxyl radicals in vitro. Lower alteration of ApoA-I was found when HDL was oxidized in the presence of haptoglobin. ApoA-I oxidation was limited also when the complex of haptoglobin with both high-density lipoprotein and Hb, immobilized on resin beads, was exposed to hydroxyl radicals. ApoA-I function to stimulate cholesterol esterification was assayed in vitro by using ApoA-I-containing liposomes. Decreased stimulation was observed when liposomes oxidized without haptoglobin were used. Conversely, after oxidative stress in the presence of haptoglobin (0.5 microM monomer), the liposome activity did not change. Plasma of carrageenan-treated mice was analyzed by ELISA for the levels of haptoglobin and ApoA-I, and used to isolate HDL for MS analysis. Hydroxyproline-containing fragments of ApoA-I were found associated with low levels of haptoglobin (18 microM monomer), whereas they were not detected when the haptoglobin level increased (34-70 microM monomer). Therefore haptoglobin, when circulating at enhanced levels with free Hb during the acute phase of inflammation, might protect ApoA-I structure and function against hydroxyl radicals.

Growing evidence suggests that atherogenesis is associated with inflammation or defective removal of cholesterol excess from peripheral cells. Apolipoprotein A-I [ApoA-I] activates the enzyme Lecithin-Cholesterol Acyl-Transferase to esterify cell cholesterol for transport to liver. Haptoglobin [Hpt] was previously found able to bind ApoA-I, and suggested to reduce the enzyme activation. The aim of this study was to demonstrate that enhanced levels of Hpt, as present during inflammation, are associated with low enzyme activity and increased thickness of the arterial wall. Enzyme activity and Hpt concentration were analysed in patients with rheumatoid arthritis having the same plasma levels of antioxidants (ascorbate, urate, alpha-tocopherol, retinol) or oxidation markers (nitrotyrosine, lipoperoxide) of healthy subjects. Cholesterol esterification, determined as ratio of cholesteryl esters with cholesterol in high-density lipoproteins, was lower in patients than in controls, and negatively correlated with the intima-media wall thickness of the common carotid. The ratio of Hpt with ApoA-I was negatively correlated with the enzyme activity, while positively correlated with intima-media wall thickness. The results suggest that high Hpt levels might severely impair the enzyme activity, thus contributing to cholesterol accumulation in vascular cells, and lesion formation in the endothelium.

Haptoglobin (Hpt) was previously found to bind the high density lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed to detect the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine; the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or enzyme-linked immunosorbent assay for their ability to bind Hpt. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was determined by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, containing the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, respectively. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with hemoglobin for Hpt binding in enzyme-linked immunosorbent assay experiments. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding hemoglobin, might be used for treatment of diseases associated with defective LCAT function.

Haptoglobin (Hp) can be purified by affinity chromatography using hemoglobin (Hb)-linked Sepharose. Elution with 8 M urea is generally performed, resulting in heavy contamination of the Hp preparation by apolipoprotein AI (ApoAI), and partial loss of Hb binding activity. Hp, separated from ApoAI, was recovered by elution with glycine-HCl at pH 3. Complexes of the isolated protein with Hb or ApoAI were detected by enzyme-linked immunosorbent assay (ELISA). Competition between the two ligands in their interaction with Hp was observed. Concanavalin A (ConA), which binds the Hp carbohydrate chains, did not influence Hp binding to ApoAI. These results suggest that changes in the plasma levels of ApoAI or Hb affect the Hp role in regulating the reverse transport of cholesterol or preventing Hb-dependent oxidative damage.

Haptoglobin is a hemoglobin-binding protein presenting in humans three distinct phenotypes (Hpt 1-1, Hpt 1-2, or Hpt 2-2). The Hpt 1-2 and Hpt 2-2 phenotypes are in turn represented by populations of isoforms. The relative amounts of the major isoforms of Hpt 1-2 and Hpt 2-2 were found to differ not only in different individuals, but also in the same individual before and after a physical effort. Exercise-dependent changes in the plasma concentrations of ascorbate, urate, alpha-tocopherol, retinol, and glutathione were also observed, but correlations between such changes and those of the amount for any isoform were not found. Samples of Hpt 1-2 or Hpt 2-2 were challenged with oxidants (H(2)O(2) with ferrous ions, spermine-NO, KO(2), and 3-morpholinosydnonimine), but the isoform levels were not altered. Hpt 2-2 isoforms were present in Hpt 1-2, as minor species. Furthermore, different isoforms exhibited different hemoglobin binding abilities. Thus, these parameters should also be taken into consideration in studies correlating Hpt phenotypes prevalence with pathologies or functional differences.