Human Apolipoprotein E Resequencing by Proteomic Analysis and Its Application to Serotyping

Background

Apolipoprotein E (ApoE) typing is considered important because of the association between ApoE and Alzheimer’s disease and familial dyslipidemia and is currently performed by genetic testing (APOE genotyping). ApoE levels in plasma and serum are clinically determined by immunoassay.

Methods

Combining an ApoE immunoassay reagent with proteomic analysis using an Orbitrap mass spectrometer, we attempted to resequence ApoE from trace amounts of serum for typing (serotyping). Most (24 of 33) ApoE mutant proteins registered to date with Online Mendelian Inheritance in Man, such as ApoE2 and ApoE4, involve lysine and arginine mutations. Digestion of mutant ApoE with trypsin will thus result in fragments that differ substantially from wild-type ApoE3 in terms of mass, making serotyping ideally suited to mass spectrometry analysis.

Results

The mean coverage of the amino acid sequence of full-length ApoE was 91.6% in the protein resequence. Residues 112 and 158 (which are mutated in ApoE2 and ApoE4) were covered in all samples, and the protein sequences were used for serotyping. Serotypes including all heterozygous combinations (ApoE2/E3, E2/E4, E3/E4) corresponded exactly to the APOE genotyping results in each of the subjects.

Conclusion

Our novel ApoE serotyping method with protein resequencing requires no synthesis of stable isotope-labeled peptides or genome analysis. The method can use residual blood from samples collected for routine clinical tests, thus enabling retrospective studies with preserved body fluids. The test could be applied to samples from subjects whose DNA is unavailable. In future studies, we hope to demonstrate the capability of our method to detect rare ApoE mutations.     

Increased Metabolite Levels of Glycolysis and Pentose Phosphate Pathway in Rabbit Atherosclerotic Arteries and Hypoxic Macrophage

Aims

Inflammation and possibly hypoxia largely affect glucose utilization in atherosclerotic arteries, which could alter many metabolic systems. However, metabolic changes in atherosclerotic plaques remain unknown. The present study aims to identify changes in metabolic systems relative to glucose uptake and hypoxia in rabbit atherosclerotic arteries and cultured macrophages.

Methods

Macrophage-rich or smooth muscle cell (SMC)-rich neointima was created by balloon injury in the iliac-femoral arteries of rabbits fed with a 0.5% cholesterol diet or a conventional diet. THP-1 macrophages stimulated with lipopolysaccharides (LPS) and interferon-γ (INFγ) were cultured under normoxic and hypoxic conditions. We evaluated comprehensive arterial and macrophage metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) and pimonidazole, a marker of hypoxia.

Results

The levels of many metabolites increased in the iliac-femoral arteries with macrophage-rich neointima, compared with those that were not injured and those with SMC-rich neointima (glycolysis, 4 of 9; pentose phosphate pathway, 4 of 6; tricarboxylic acid cycle, 4 of 6; nucleotides, 10 of 20). The uptake of ¹⁸F-FDG in arterial walls measured by autoradiography positively correlated with macrophage- and pimonidazole-immunopositive areas (r = 0.76, and r = 0.59 respectively; n = 69 for both; p<0.0001). Pimonidazole immunoreactivity was closely localized with the nuclear translocation of hypoxia inducible factor-1α and hexokinase II expression in macrophage-rich neointima. The levels of glycolytic (8 of 8) and pentose phosphate pathway (4 of 6) metabolites increased in LPS and INFγ stimulated macrophages under hypoxic but not normoxic condition. Plasminogen activator inhibitor-1 protein levels in the supernatant were closely associated with metabolic pathways in the macrophages.

Conclusion

Infiltrative macrophages in atherosclerotic arteries might affect metabolic systems, and hypoxia but not classical activation might augment glycolytic and pentose phosphate pathways in macrophages.     

A role of the sphingosine-1-phosphate (S1P)–S1P receptor 2 pathway in epithelial defense against cancer (EDAC)

At the initial step of carcinogenesis, transformation occurs in single cells within epithelia, where the newly emerging transformed cells are surrounded by normal epithelial cells. A recent study revealed that normal epithelial cells have an ability to sense and actively eliminate the neighboring transformed cells, a process named epithelial defense against cancer (EDAC). However, the molecular mechanism of this tumor-suppressive activity is largely unknown. In this study, we investigated a role for the sphingosine-1-phosphate (S1P)–S1P receptor 2 (S1PR2) pathway in EDAC. First, we show that addition of the S1PR2 inhibitor significantly suppresses apical extrusion of RasV12-transformed cells that are surrounded by normal cells. In addition, knockdown of S1PR2 in normal cells induces the same effect, indicating that S1PR2 in the surrounding normal cells plays a positive role in the apical elimination of the transformed cells. Of importance, not endogenous S1P but exogenous S1P is involved in this process. By using FRET analyses, we demonstrate that S1PR2 mediates Rho activation in normal cells neighboring RasV12-transformed cells, thereby promoting accumulation of filamin, a crucial regulator of EDAC. Collectively these data indicate that S1P is a key extrinsic factor that affects the outcome of cell competition between normal and transformed epithelial cells.     

System A amino acid transporter SNAT2 shows subtype-specific affinity for betaine and hyperosmotic inducibility in placental trophoblasts

Betaine uptake is induced by hypertonic stress in a placental trophoblast cell line, and involvement of amino acid transport system A was proposed. Here, we aimed to identify the subtype(s) of system A that mediates hypertonicity-induced betaine uptake. Measurement of [¹⁴C]betaine uptake by HEK293 cells transiently transfected with human or rat sodium-coupled neutral amino acid transporters (SNATs), SNAT1, SNAT2 and SNAT4 revealed that only human and rat SNAT2 have betaine uptake activity. The Michaelis constants (K_m) of betaine uptake by human and rat SNAT2 were estimated to be 5.3 mM and 4.6 mM, respectively. Betaine exclusively inhibited the uptake activity of SNAT2 among the rat system A subtypes. We found that rat SNAT1, SNAT2 and SNAT4 were expressed at the mRNA level under isotonic conditions, while expression of SNAT2 and SNAT4 was induced by hypertonicity in TR-TBT 18d-1 cells. Western blot analyses revealed that SNAT2 expression on plasma membrane of TR-TBT 18d-1 cells was more potently induced by hypertonicity than that in total cell lysate. Immunocytochemistry confirmed the induction of SNAT2 expression in TR-TBT 18d-1 cells exposed to hypertonic conditions and indicated that SNAT2 was localized on the plasma membrane in these cells. Our results indicate that SNAT2 transports betaine, and that tonicity-sensitive SNAT2 expression may be involved in regulation of betaine concentration in placental trophoblasts.     

Prostaglandin E2 Stimulates the Production of Amyloid-�� Peptides through Internalization of the EP4 Receptor

Amyloid-β (Aβ) peptides, generated by the proteolysis of β-amyloid precursor protein by β- and γ-secretases, play an important role in the pathogenesis of Alzheimer disease. Inflammation is also important. We recently reported that prostaglandin E₂ (PGE₂), a strong inducer of inflammation, stimulates the production of Aβ through EP₂ and EP₄ receptors, and here we have examined the molecular mechanism. Activation of EP₂ and EP₄ receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP₂, but not EP₄, receptor-mediated stimulation of the Aβ production. In contrast, inhibitors of endocytosis suppressed EP₄, but not EP₂, receptor-mediated stimulation. Activation of γ-secretase was observed with the activation of EP₄ receptors but not EP₂ receptors. PGE₂-dependent internalization of the EP₄ receptor was observed, and cells expressing a mutant EP₄ receptor lacking the internalization activity did not exhibit PGE₂-stimulated production of Aβ. A physical interaction between the EP₄ receptor and PS-1, a catalytic subunit of γ-secretases, was revealed by immunoprecipitation assays. PGE₂-induced internalization of PS-1 and co-localization of EP₄, PS-1, and Rab7 (a marker of late endosomes and lysosomes) was observed. Co-localization of PS-1 and Rab7 was also observed in the brain of wild-type mice but not of EP₄ receptor null mice. These results suggest that PGE₂-stimulated production of Aβ involves EP₄ receptor-mediated endocytosis of PS-1 followed by activation of the γ-secretase, as well as EP₂ receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of Alzheimer disease.Alzheimer disease (AD)² is the most common neurodegenerative disorder of the central nervous system and the leading cause of adult onset dementia. AD is characterized pathologically by the accumulation of tangles and senile plaques. Senile plaques are composed of the amyloid-β (Aβ) peptides Aβ40 and Aβ42 (1, 2). To generate Aβ, β-amyloid precursor protein (APP) is first cleaved by β-secretase and then by γ-secretase. Cleavage of APP by α-secretase produces non-amyloidogenic peptides (3, 4). The γ-secretase is an aspartyl protease complex composed of four core components, including presenilin (PS) 1 and PS2 (5). Early onset familial AD is linked to three genes, APP, PS1, and PS2 (5, 6), strongly suggesting that γ-secretase-dependent production of Aβ is a key factor in the pathogenesis of AD. Therefore, cellular factors that affect the γ-secretase-dependent production of Aβ may be good targets for the development of drugs to prevent and treat AD.Both APP and PS-1 are transmembrane proteins, and their intracellular localization is controlled by secretory and endocytic pathways. These proteins are modified in the endoplasmic reticulum and trafficked to the cell surface through the trans-Golgi network (TGN). Then, they are internalized again and trafficked to early endosomes. Next, they are trafficked to late endosomes and lysosomes (LEL), which are recycling endosomes that are targeted to the cell surface or the TGN (7–11). The production of Aβ seems to occur in a broad range of cellular compartments including the cell surface, TGN, and endosomes (12). Abnormalities of secretory and endocytic pathways have been observed in the brains of AD patients (9, 13). Importantly, factors that control these vesicle transport systems affect the production of Aβ. For example, overproduction of Rab5, a factor essential for traffic of vesicles to early endosomes, has been shown to stimulate the production of Aβ (14), and SorL1 has been shown to reduce the production of Aβ by stimulating the traffic of APP in early endosomes to the TGN (15, 16).It has been suggested that inflammation is important in the pathogenesis of AD; chronic inflammation has been observed in the brains of AD patients, and trauma to the brain and ischemia, both of which can activate inflammation, are major risk factors for AD (17–19). Cyclooxygenase (COX) is essential for the synthesis of prostaglandin E₂ (PGE₂), a potent inducer of inflammation and has two subtypes, COX-1 and COX-2. COX-1 is expressed constitutively, whereas expression of COX-2 is induced under inflammatory conditions and is responsible for the progression of inflammation (20–22). The following evidences of the involvement of PGE₂ (and COX-2) in the progression of AD suggest that they are good targets for the development of AD drugs: (i) Elevated levels of PGE₂ and overexpression of COX-2 have been observed in the brains of AD patients (23–25); (ii) the extent of COX-2 expression correlates with the amount of Aβ and the degree of progression of AD pathogenesis (26); (iii) transgenic mice constitutively overexpressing COX-2 show aging-dependent neural apoptosis and memory dysfunction (27); (iv) prolonged use of nonsteroidal anti-inflammatory drugs, inhibitors of COX, delays the onset and reduces the risk of AD (28); (v) PGE₂ stimulates the production of reactive oxygen species in microglia cells, resulting in activation of β-secretase (29).We recently reported that PGE₂ stimulates the production of Aβ in human embryonic kidney (HEK) 293 and human neuroblastoma (SH-SY5Y) cells that stably express a form of APP with two mutations (K651N/M652L) (APPsw) that elevate cellular and secreted levels of Aβ (30). Similar results were reported by another group (31). Using agonists and antagonists specific for each of the four PGE₂ receptors (EP₁, EP₂, EP₃, and EP₄), we found that EP₄ receptors alone and also both EP₂ and EP₄ receptors are involved in PGE₂-stimulated production of Aβ in HEK293 or SH-SY5Y cells, respectively (30). Furthermore, experiments with transgenic mice suggest that EP₂ and EP₄ receptors are involved in the production of Aβ in vivo (30). Based on these results, we propose that antagonists of the EP₂ and/or EP₄ receptors may be therapeutically beneficial for the treatment of AD. Understanding the mechanism governing EP₂ and EP₄ receptor-mediated stimulation of production of Aβ by PGE₂ will be important for such drug development.Activation of EP₂ and EP₄ receptors causes activation of adenylate cyclase and an increase in the cellular level of cAMP (32). We have shown that an EP₄ receptor agonist or both EP₂ and EP₄ receptor agonists increase the cellular level of cAMP in HEK293 or SH-SY5Y cells, respectively, and that a cAMP analogue, 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP), increases the level of Aβ in HEK293 cells (30). These findings suggest that the cellular level of cAMP is important for PGE₂-stimulated production of Aβ. An increase in the cellular level of cAMP is known to activate protein kinase A (PKA), which is important for cAMP-regulated intracellular signal transduction (33). However, an inhibitor of PKA, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89), does not block PGE₂-stimulated production of Aβ in HEK293 cells (30). Other cAMP-regulated signal transduction factors, such as phosphatidylinositol 3-kinase and Epac (exchange protein directly activated by cAMP), were also shown not to be involved in PGE₂-stimulated production of Aβ in HEK293 cells (30). Thus, the mechanism whereby the activation of EP₂ and EP₄ receptors stimulates the production of Aβ has remained unknown. In this study, by using inhibitors of adenylate cyclase and PKA, we found that activation of the EP₂ receptor stimulates production of Aβ through activation of adenylate cyclase and PKA. We also propose that activation of the EP₄ receptor causes its co-internalization with PS-1 (γ-secretase) into endosomes and that this co-internalization is important for EP₄ receptor-mediated stimulation of Aβ production by PGE₂ through the activation of γ-secretase.     

Reduction of allergenic proteins by the effect of the ripening inhibitor (rin) mutant gene in an F1 hybrid of the rin mutant tomato

The ripening inhibitor (rin) mutant tomato yields non-ripening fruit, and the rin hybrid fruit (RIN/rin) shows an intermediate phenotype between the wild and mutant fruit, that is, red-ripe and extended shelf life. We found by a microarray analysis that the genes encoding possible allergenic proteins were expressed at a significantly lower level in the rin hybrid fruit than in the wild-type fruit. These allergenic proteins, which were beta-fructofuranosidase and polygalacturonase 2A (PG-2A), were confirmed to accumulate at a lower level in the rin hybrid fruit than in the wild-type fruit. The immunoglobulin E (IgE) in serum from a tomato-allergic patient showed lower reactivity to the extract of the rin hybrid fruit than to that of the wild fruit. These results suggest that the rin gene has the potential to regulate allergen accumulation in tomato fruit.     

Substitution of the N-glycan function in glycosyltransferases by specific amino acids: ST3Gal-V as a model enzyme

The sialyltranferase ST3Gal-V transfers a sialic acid to lactosylceramide. We investigated the role of each of the N-glycans modifying mouse ST3Gal-V (mST3Gal-V) by measuring the in vitro enzyme activity of Chinese hamster ovary (CHO) cells transfected with ST3Gal-V cDNA or its mutants. By examining mutants of mST3Gal-V, in which each asparagine was replaced with glutamine (N180Q, N224Q, N334Q), we determined that all three sites are N-glycosylated and that each N-glycan is required for enzyme activity. Despite their importance, N-glycosylation sites in ST3Gal-V are not conserved among species. Therefore, we considered whether the function in the activity that is performed in mST3Gal-V by the N-glycan could be substituted for by specific amino acid residues selected from the ST3Gal-V of other species or from related sialyltransferases (ST3Gal-I, -II, -III, and -IV), placed at or near the glycosylation sites. To this end, we constructed a series of interspecies mutants for mST3Gal-V, specifically, mST3Gal-V-H177D-N180S (medaka or tetraodon type), mST3Gal-V-N224K (human type), and mST3Gal-V-T336Q (zebrafish type). The ST3Gal-V activity of these mutants was quite similar to that of the wild-type enzyme. Thus, we have demonstrated here that the N-glycans on mST3Gal-V are required for activity but can be substituted for specific amino acid residues placed at or near the glycosylation sites. We named this method SUNGA (substitution of N-glycan functions in glycosyltransferases by specific amino acids). Furthermore, we verified that the ST3Gal-V mutant created using the SUNGA method maintains its high activity when expressed in Escherichia coli thereby establishing the usefulness of the SUNGA method in exploring the function of N-glycans in vivo.     

Harmful or Not: Trichostatin A treatment of embryos generated by ICSI or ROSI

Trichostatin A (TSA), a histone deacetylase inhibitor, is a known teratogen causing malformations such as vertebral fusions when applied during the postimplantation period; TSA also causes developmental arrest when applied during the preimplantation period. Regardless of these hindrances, we have succeeded in the establishment of an efficient somatic cloning method for the mouse where reconstructed embryos are treated with TSA. To elucidate this apparent discrepancy, we treated fertilized mouse embryos generated either by intracytoplasmic sperm injection (ICSI) or round spermatid injection (ROSI) with 50 nM TSA for 20 h after fertilization as well as parthenogenetic embryos and found that TSA treatment inhibited the preimplantation development of ICSI embryos but not ROSI or parthenogenetic embryos. And, although we often observed hypomorphism following TSA treatment in embryos grown to full term produced by both ICSI (av. of body weight: 1.7 g vs. 1.5 g) and ROSI (1.6 g vs. 1.2 g), TSA treatment reduced the offspring production rate for ICSI from 57% to 34% but not for ROSI from 30% to 36%. Thus, these data indicate that the effects, harmful or not, of TSA treatment on embryonic development depend on their nuclear derivations. Also, the resulting hypomorphism after TSA treatment is a caveat for this procedure in current Assisted Reproductive Technologies.     

Efficient DNA Extraction from Nail Clippings Using the Protease Solution from <Emphasis Type="Italic">Cucumis melo</Emphasis>

Owing to the increasing importance of genomic information, obtaining genomic DNA easily from biological specimens has become more and more important. This article proposes an efficient method for obtaining genomic DNA from nail clippings. Nail clippings can be easily obtained, are thermostable and easy to transport, and have low infectivity. The drawback of their use, however, has been the difficulty of extracting genomic material from them. We have overcome this obstacle using the protease solution obtained from Cucumis melo. The keratinolytic activity of the protease solution was 1.78-fold higher than that of proteinase K, which is commonly used to degrade keratin. With the protease solution, three times more DNA was extracted than when proteinase K was used. In order to verify the integrity of the extracted DNA, genotype analysis on 170 subjects was performed by both PCR–RFLP and Real Time PCR. The results of the genotyping showed that the extracted DNA was suitable for genotyping analysis. In conclusion, we have developed an efficient extraction method for using nail clippings as a genome source and a research tool in molecular epidemiology, medical diagnostics, and forensic science.