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Haptoglobin (Hpt), primarily synthesized by hepatocytes, can be produced in central nervous system (CNS) under inflammatory conditions. In fact, increased Hpt was found in Cerebrospinal Fluids from patients with neurodegenerative diseases. We previously reported that Hpt binds Apolipoprotein E (ApoE), and influences ApoE key roles in cholesterol homeostasis. ApoE is the major constituent of lipoproteins into CNS, and modulates Amyloid-beta (A-beta) deposition and clearance. A-beta is known to trigger an inflammatory response in the brain, which is involved in the pathogenesis of Alzheimer’s disease. Our aim was to verify whether Hpt affects the ApoE function in the regulation of A-beta accumulation in the brain. We report here that the abilities of Hpt and ApoE to bind A-beta are similar. In addition, our data demonstrate that Hpt enhances the binding of ApoE to A-beta, thus suggesting that the two proteins and A-beta might interact with each other to form a trimeric complex. Further, a SDS-stable complex between the two proteins (ApoE and Hpt) and A-beta is formed following incubation at 37°C, and A-beta aggregation is reduced when it is incubated in presence of Hpt and ApoE. Finally, we found that Hpt can bind astrocytes. In conclusion Hpt, as forming a trimeric complex with ApoE and A-beta, and binding astrocytes, might play a critical role, participating in the binding and clearance of A-beta, thus alleviating its toxicity and promoting its removal from the brain.

INTRODUCTION: ApoA-1, the major apolipoprotein of the high density lipoprotein (HDL), plays a key role in the removal of cholesterol excess from peripheral cells in a recognized anti-atherosclerotic process called “reverse cholesterol transport (RCT)”. Among known ApoA-1 variants two, with one amino acid change (Zavalla, L159P; Zaragoza, L144R), are associated to poor RCT. Both mutations are located within the domain of ApoA1 (Leu141 – Ala164) binding Haptoglobin (Hpt) 1, a protein of the acute phase of inflammation. Hpt binding to ApoA1 was suggested to impair the role of HDL in RCT1. AIMS: Our aims were to study whether peptides containing the mutations of Zavalla (Pep Zav) or Zaragoza (Pep Zara) are able to bind Hpt or compete with native ApoA1, exposed on HDL, for binding Hpt. METHODS: The binding of biotinylated peptides to Hpt and the experiments of competition of acetylated peptides with HDL for Hpt binding were performed by ELISA, as previously published1. RESULTS: Both Pep-Zav and Pep-Zara bound to Hpt with efficiency similar to that of the peptide harbouring the native sequence of ApoA1 (pep 141–164). Nevertheless, when the same peptides were analysed for their ability to influence Hpt binding to HDL, only Pep Zav effectively displaced (50% at 15μM) Hpt from HDL (fig), as was the case for the pep. 141-164. CONCLUSIONS: We suggest that the L>P conversion, in the Zavalla variant, enhances hydrophilicity in ApoA1, improving the binding to Hpt. Therefore, as ApoA-1 binding to Hpt negatively affects RCT, individuals with Zavalla mutation might be exposed to higher risk of cardiovascular disease. 1.Spagnuolo M.S. et al (2005) J. Biol. Chem. 280, 1193-1198

Haptoglobin (Hp) can be purified by affinity chromatography using hemoglobin(Hb)-linked Sepharose. Elution with 8 M urea is generally performed, resulting in Hp heavy contamination by the 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 the Hp binding to ApoAI. The results suggest that changes in the plasma level of ApoAI or Hb affect the Hp role in regulating the cholesterol reverse transport or preventing Hb-dependent oxidative damages.

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) binds the Apolipoprotein A-I (ApoA-I), and impairs its stimulation on lecithin:cholesterol acyl-transferase (LCAT), an enzyme playing a major role in the reverse cholesterol transport (RCT). The Apolipoprotein E (ApoE), like ApoA-I, promotes different steps of RCT, including LCAT stimulation. ApoE contains aminoacid sequences which are homologous to the ApoA-I region bound by Hpt and involved in the interaction with LCAT. Therefore Hpt was expected to bind also ApoE, and inhibit the ApoE stimulatory effect on LCAT. Western Blotting and enzyme-linked immunosorbent assay experiments demonstrated that the Hpt subunit  actually binds ApoE. The Hpt affinity for ApoE was higher than that for ApoA-I. High ratios of Hpt with either apolipoprotein, as those associated with the acute phase of inflammation, inhibited in vitro ApoE in stimulating the cholesterol esterification activity of LCAT. Hpt also impaired human hepatoblastoma-derived cells in uptaking 3H-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 different from RCT.

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 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, α-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 (H2O2 with ferrous ions, spermine–NO, KO2, 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.