Maria Stefania Spagnuolo

Assignment of the Binding Site for Haptoglobin on Apolipoprotein A-I

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 binds the antiatherogenic protein apolipoprotein E – impairment of apolipoprotein E stimulation of both lecithin:cholesterol acyltransferase activity and cholesterol uptake by hepatocytes

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 β‐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 [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 other than RCT.

Evaluation of oxidative damage in mozzarella cheese produced from bovine or water buffalo milk

Abstract Technological processes are the main sources of protein and lipid oxidation in food. The oxidative status was determined in a soft Italian cheese, namely mozzarella, produced from water buffalo or bovine milk. The amount of protein-bound carbonyls, dityrosine and α-lactalbumin aggregates were measured to evaluate the extent of protein oxidation. The α-tocopherylquinone/α-tocopherol ratio and the trolox-equivalent antioxidant capacity were used as redox markers in the fat fraction. The levels of protein-bound carbonyls and α-lactalbumin aggregates were found significantly higher in bovine mozzarella than in buffalo mozzarella. On the other hand, higher amounts of redox markers were found in buffalo mozzarella. The levels of dityrosine aggregates were similar in the two types of cheese. The data suggest that protein and fat are more protected against oxidative structure alterations in buffalo mozzarella than in bovine mozzarella.

Estradiol esterification in the human preovulatory follicle

In the preovulatory follicle, the LH surge stimulates progesterone production, reduces estradiol synthesis, and scales up the permeability of the blood-follicle barrier. The purpose of this study was to investigate whether the extent of these changes is correlated with the levels of estradiol, estradiol esters, and cholesteryl esters in the follicular fluid. The follicular levels of progesterone, estradiol, estradiol linoleate, cholesterol, and cholesteryl linoleate were measured by HPLC. The estradiol linoleate/estradiol ratio, which reflects the efficiency of in vivo estradiol esterification, and the cholesteryl linoleate/cholesterol ratio were calculated and found negatively correlated. The estradiol level was positively correlated with the cholesteryl linoleate/cholesterol ratio while negatively correlated with the estradiol linoleate/estradiol ratio. The in vitro activity of lecithin-cholesterol acyltransferase, the enzyme esterifying both cholesterol and estradiol, was assayed by incubating the fluid with labeled substrates. This activity was not correlated with either the estradiol linoleate/estradiol or the cholesteryl linoleate/cholesterol ratio. The enzyme K(m) and V(max) values were lower with estradiol than with cholesterol. Higher estradiol linoleate/estradiol ratios and lower cholesteryl linoleate/cholesterol ratios were associated with higher level of Haptoglobin penetration into the follicle. This level, which was determined by ELISA, was found increased with increased progesterone concentration and, therefore, used as a marker of the LH-stimulated permeability of the blood-follicle barrier. Our data suggest that early preovulatory follicles contain more cholesteryl esters and less estradiol esters than follicles closer to ovulation.

The binding of haptoglobin to apolipoprotein AI: influence of hemoglobin and concanavalin A.

Abstract 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.

High Fat Diet and Inflammation – Modulation of Haptoglobin Level in Rat Brain

Obesity and dietary fats are well known risk factors for the pathogenesis of neurodegenerative diseases. The analysis of specific markers, whose brain level can be affected by diet, might contribute to unveil the intersection between inflammation/obesity and neurodegeneration. Haptoglobin (Hpt) is an acute phase protein, which acts as antioxidant by binding free haemoglobin (Hb), thus neutralizing its pro-oxidative action. We previously demonstrated that Hpt plays critical functions in brain, modulating cholesterol trafficking in neuroblastoma cell lines, beta-amyloid (Aβ) uptake by astrocyte, and limiting Aβ toxicity on these cells. A major aim of this study was to evaluate whether a long term (12 or 24 weeks) high-fat diet (HFD) influences Hpt and Hb expression in rat hippocampus. We also assessed the development of obesity-induced inflammation by measuring hippocampal level of TNF-alpha, and the extent of protein oxidation by titrating nitro-tyrosine (N-Tyr). Hpt concentration was lower (p < 0.001) in hippocampus of HFD rats than in control animals, both in the 12 and in the 24 weeks fed groups. HFD was also associated in hippocampus with the increase of Hb level (p < 0.01), inflammation and protein oxidative modification, as evidenced by the increase in the concentration of TNF-alpha and nitro-tyrosine. In fact, TNF-alpha concentration was higher in rats receiving HFD for 12 (p < 0.01) or 24 weeks (p < 0.001) compared to those receiving the control diet. N-Tyr concentration was more elevated in hippocampus of HFD than in control rats in both 12 weeks (p = 0.04) and 24 weeks groups (p = 0.01), and a positive correlation between Hb and N-Tyr concentration was found in each group. Finally, we found that the treatment of the human glioblastoma-astrocytoma cell line U-87 MG with cholesterol and fatty acids, such as palmitic and linoleic acid, significantly impairs (p < 0.001) Hpt secretion in the extracellular compartment. We hypothesize that the HFD-dependent decrease of Hpt in hippocampus, as associated with Hb increase, might enhance the oxidative stress induced by free Hb. Altogether our data, identifying Hpt as a molecule modulated in the brain by dietary fats, may represent one of the first steps in the comprehension of the molecular mechanisms underlying the diet-related effects in the nervous system.

Quantitative variations of the isoforms in haptoglobin 1-2 and 2-2 individual phenotypes

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.

The enzyme lecithin-cholesterol acyltransferase esterifies cerebrosterol and limits the toxic effect of this oxysterol on SH-SY5Y cells

Cholesterol is mostly removed from the CNS by its conversion to cerebrosterol (24(S)‐hydroxycholesterol, 24(S)OH‐C), which is transported to the circulation for bile formation in liver. A neurotoxic role of this oxysterol was previously demonstrated in cell culture. Here, we provide evidence that the enzyme lecithin‐cholesterol acyltransferase, long known to esterify cholesterol, also produces monoesters of 24(S)OH‐C. Proteoliposomes containing apolipoprotein A‐I or apolipoprotein E were used to stimulate the enzyme activity and entrap the formed esters. Proteoliposomes with apolipoprotein A‐I were found to be more active than those with apolipoprotein E in stimulating the production of oxysteryl esters. Cholesterol and 24(S)OH‐C were found to compete for enzyme activity. High levels of haptoglobin, as those circulating during the acute inflammatory phase, inhibited 24(S)OH‐C esterification. When highly neurotoxic 24(S)OH‐C was treated with enzyme and proteoliposomes before incubation with differentiated SH‐SY5Y cells, the neuron survival improved. The esters of 24(S)OH‐C, embedded into proteoliposomes by the enzyme and isolated from unesterified 24(S)OH‐C by gel filtration chromatography, did not enter the neurons in culture. These results suggest that the enzyme, in the presence of the apolipoproteins, converts 24(S)OH‐C into esters restricted to the extracellular environment, thus preventing or limiting oxysterol‐induced neurotoxic injuries to neurons in culture.

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 non-genetic 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 pathophysiology.

Haptoglobin interacts with Apolipoprotein e and beta-amyloid and influences their crosstalk

Beta-amyloid accumulation in brain is a driving force for Alzheimers 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 Alzheimers 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 Alzheimers disease might not only be linked to the different ApoE isoforms, but also rely on the level of critical ligands, such as haptoglobin.