Haptoglobin Modulates Beta-Amyloid Uptake by U-87 MG Astrocyte Cell Line

Accumulation of beta-amyloid (Aβ) in the extracellular space, which is one of the hallmarks of Alzheimer’s disease (AD), depends on the balance between its synthesis and clearance. The physiological role of extracellular chaperones, capable of affecting early events in the amyloid cascade, is increasingly being investigated by many research groups. Among these proteins, we focused on haptoglobin, which we recently found to form a complex with beta-amyloid in brain tissues or cerebrospinal fluids from patients with AD.We also previously reported that haptoglobin increases with age in rat hippocampus. Major aimof this study was to evaluate whether haptoglobin influences Aβ interaction with astrocytes and its internalization into these cells. Haptoglobin effect on Aβ- induced cell death was also explored. We report here that haptoglobin impairs Aβ uptake by human glioblastoma–astrocytoma cell line U-87 MG and limits the toxicity of this peptide on these cells. Of note, our data also show that Aβ can stimulate haptoglobin release by astrocyte cell lines. The study of the risk of developing AD should be focused not only on the analysis ofAβ but also on the level of critical ligands, such as haptoglobin, able to influence peptide aggregation or clearance

Differences between the glycosylation patterns of haptoglobin isolated from skin scales and plasma of psoriatic patients.

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 increases with age in rat hippocampus and modulates Apolipoprotein E mediated cholesterol trafficking in neuroblastoma cell lines

Alteration in cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative disorders. Apolipoprotein E (ApoE) is the major component of brain lipoproteins supporting cholesterol transport. We previously reported that the acute-phase protein Haptoglobin (Hpt) binds ApoE, and influences its function in blood cholesterol homeostasis. Major aim of this study was to investigate whether Hpt influences the mechanisms by which cholesterol is shuttled from astrocytes to neurons. In detail it was studied Hpt effect on ApoE-dependent cholesterol efflux from astrocytes and ApoE-mediated cholesterol incorporation in neurons. We report here that Hpt impairs ApoE-mediated cholesterol uptake in human neuroblastoma cell line SH-SY5Y, and limits the toxicity of a massive concentration of cholesterol for these cells, while it does not affect cholesterol efflux from the human glioblastoma-astrocytoma cell line U-87 MG. As aging is the most important nongenetic risk factor for various neurodegenerative disorders, and our results suggest that Hpt modulates ApoE functions, we evaluated the Hpt and ApoE expression profiles in cerebral cortex and hippocampus of adolescent (2 months), adult (5 and 8 months), and middle-aged (16 months) rats. Hpt mRNA level was higher in hippocampus of 8 and 16 month-old than in 2-month old rats (p<0.05), and Hpt concentration increased with the age from adolescence to middle-age (p<0.001). ApoE concentration, in hippocampus, was higher (p<0.001) in 5 month-old rats compared to 2 month but did not further change with aging. No age-related changes of Hpt (protein and mRNA) were found in the cortex. Our results suggest that aging is associated with changes, particularly in the hippocampus, in the Hpt/ApoE ratio. Age-related changes in the concentration of Hpt were also found in human cerebrospinal fluids.The age-related changes might affect neuronal function and survival in brain, and have important implications in brain pathophy

Quantitative determination of haptoglobin glycoform variantsin psoriasis

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 VVLHPNYSQVDIGLIK 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

Binding of lectins to Hpt.

<p>The wells of a microtiter plate were coated with different amounts of pHpt-N (white bars), pHpt-P (grey bars), or sHpt-P (black bars). Solutions containing 1 µM biotinylated LTA (panel A), MAA (panel B), ConA (panel C), or SNA (panel D) were separately incubated into the wells. Avidin-HRP and hydrogen peroxide were used to develop color from OPD. Color intensity was determined by measuring the absorbance at 492 nm (A₄₉₂). Five equal aliqouts of each sample were processed, and means ± SEM are shown. Asterisk: significant difference between the linked bars (P<0.0001). Triangle: not significant difference (P>0.05). Data from one experiment are shown. Inter-assay CV for each sample, from three separate experiments, was less than 5%.</p

Relative amounts of the glycan peaks eluted by HPLC of pHpt-N, pHpt-P and sHpt-P.

1<p>GU value represents the peak elution as compared to that of 2-aminobenzamide-labelled oligomers of glucose. A mixture of standards (1 to 20 units of glucose) was used for the chromatography calibration.</p>2<p>The HPLC pattern of Hpt glycans, shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052040#pone-0052040-g001" target="_blank">Figure 1</a>, was analyzed. The relative area of each peak is expressed as ratio with peak <b><i>c</i></b> area. Data from one experiment are shown. Inter-assay CV of each peak, from three separate experiments, was less than 5%.</p

Glycopeptides in the P2 repertoire of Hpt tryptic digest from plasma of patients in disease remission.

a<p>The asparagine residue harbouring the glycan is underlined.</p>b<p>Quantitative analysis for relative abundance of species was obtained taking into account all the observed multicharged ions. The values are expressed as means (from three technical replicates) ± standard deviations. Glycan species are indicated with the same nomenclature previously used for pHpt-P and pHpt-N <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052040#pone.0052040-Maresca1" target="_blank">[16]</a>.</p

Mass spectrum of the P2 glycopeptide repertoire from skin of patients.

<p>Purified sHpt-P was digested by trypsin, and the resulting fragments were fractionated by UPLC and analyzed by ESI-MS. Positive ions of P2 (NLFL<u>N</u> HSE<u>N</u>ATAK) glycopeptides from Hpt of skin of patients is shown. Glycopeptide peaks are indicated with their observed mass value (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052040#pone-0052040-t003" target="_blank">Table 3</a>). The glycoforms found only in sHpt-P and not in pHpt-N and in pHpt-P are indicated. Peaks with mass attributable to non-glycosylated species were ignored.</p