Tosifusa Toda

Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer's disease

Citrullinated proteins are the products of a posttranslational process in which arginine residues undergo modification into citrulline residues when catalyzed by peptidylarginine deiminases (PADs) in a calcium ion‐dependent manner. In our previous report, PAD2 expressed mainly in the rat cerebrum became activated early in the neurodegenerative process. To elucidate the involvement of protein citrullination in human neuronal degeneration, we examined whether citrullinated proteins are produced during Alzheimers disease (AD). By Western blot analysis with antimodified citrulline antibody, citrullinated proteins of varied molecular weights were detected in hippocampal tissues from patients with AD but not normal humans. Two of the citrullinated proteins were identified as vimentin and glial fibrillary acidic protein (GFAP) by using two‐dimensional gel electrophoresis and MALDI‐TOF mass spectrometry. Interestingly, PAD2 was detected in hippocampal extracts from AD and normal brains, but the amount of PAD2 in the AD tissue was markedly greater. Histochemical analysis revealed citrullinated proteins throughout the hippocampus, especially in the dentate gyrus and stratum radiatum of CA1 and CA2 areas. However, no citrullinated proteins were detected in the normal hippocampus. PAD2 immunoreactivity was also ubiquitous throughout both the AD and the normal hippocampal areas. PAD2 enrichment coincided well with citrullinated protein positivity. Double immunofluorescence staining revealed that citrullinated protein‐ and PAD2‐positive cells also coincided with GFAP‐positive cells, but not all GFAP‐positive cells were positive for PAD2. As with GFAP, which is an astrocyte‐specific marker protein, PAD2 is distributed mainly in astrocytes. These collective results, the abnormal accumulation of citrullinated proteins and abnormal activation of PAD2 in hippocampi of patients with AD, strongly suggest that PAD has an important role in the onset and progression of AD and that citrullinated proteins may become a useful marker for human neurodegenerative diseases.

Proteome analysis of mouse brain: Two-dimensional electrophoresis profiles of tissue proteins during the course of aging

Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two‐dimensional gel electrophoresis (2‐DE). 2‐DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino‐terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N‐termini and 122 proteins were identified. Twenty‐seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2‐DE map images and the respective spot data files with 15 related references.

Analysis of glycopeptides using lectin affinity chromatography with MALDI-TOF mass spectrometry.

Glycopeptides prepared from 1 nmol of a mixture of glycoproteins, transferrin, and ribonuclease B by lysylendopeptidase digestion were isolated by lectin and cellulose column chromatographies, and then they were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and MALDI-quadrupole ion trap (QIT)-TOF mass spectrometry which enables the performance of MS ( n ) analysis. The lectin affinity preparation of glycopeptides with Sambucus nigra agglutinin and concanavalin A provides the glycan structure outlines for the sialyl linkage and the core structure of N-glycans. Such structural estimation was confirmed by MALDI-TOF MS and MALDI-QIT-TOF MS/MS. Amino acid sequences and location of glycosylation sites were determined by MALDI-QIT-TOF MS/MS/MS. Taken together, the combination of lectin column chromatography, MALDI-TOF MS, and MALDI-QIT-TOF MS ( n ) provides an easy way for the structural estimation of glycans and the rapid analysis of glycoproteomics.

Proteomic approaches to oxidative protein modifications implicated in the mechanism of aging

Accumulation of oxidatively modified proteins is widely observed in aged animal tissues. Protein carbonyls are mostly derived from lysine, arginine, proline and threonine residues under oxidative conditions. Many groups have investigated carbonylated proteins since a convenient immunochemical procedure was established for detecting dinitrophenyl derivatives of carbonyls and applied to proteomic research. An alternative method of tagging with biotin or fluorescent dyes has been also introduced to proteomic analysis of protein carbonyls. Nitrotyrosine was primarily identified as a biomarker of cellular damage and inflammation under nitrosative stress. Nitrated proteins have been subsequently detected in aged animal tissues and Alzheimers disease affected brains by Western blotting, and identified by mass spectrometry. Protein s‐thiolation, a mixed‐derivatization of cysteine (Cys) by conjugation of low‐molecular‐weight thiol compounds, is recognized as protecting functional proteins from more serious damage. A method of biotin labeling has been used in proteomics for tracing protein s‐thiolation. Among all kinds of amino acid residues, methionine (Met) is the most susceptible to reactive oxygen species, and Met oxidation seems to occur in ordinary cellular circumstances because most cells contain Met sulfoxide reductases, which might prevent serious cellular damage. In proteomic analysis, Met sulfoxide‐containing peptides are generally observed as 16‐Da‐high mass peaks in peptide mass fingerprinting. A modified procedure of two‐dimensional gel electrophoresis, in which proteins are kept under non‐oxidative conditions throughout the procedure, is appropriate for the estimation of the Met sulfoxide level of each protein in aged animal tissues and cells to evaluate the pathophysiological significance of Met oxidation in the mechanism of aging. Geriatr Gerontol Int 2010; 10 (Suppl. 1): S25–S31.

Comparison of hippocampal synaptosome proteins in young-adult and aged rats

The hippocampus is important in learning and memory functions but its ability to aid in these functions declines during aging. In this study, we examined hippocampal proteins whose expressions changed in the aging process. A comparison of synaptosome proteins of hippocampus prepared from young-adult (9-week-old) rats with those from aged (30-month-old) rats by two-dimensional fluorescence difference gel electrophoresis revealed 24 spots that were expressed differently among about 1000 spots detected in both young-adult and aged rat samples. Nineteen of these 24 spots were identified by peptide mass fingerprinting. These proteins included chaperone proteins and proteins related to the cytoskeleton, neurotransmission, signal transduction and energy supply. The cytoskeleton-related proteins included actin and T-complex 1, which is thought to play a role in actin folding. Actin was up-regulated but T-complex 1 was down-regulated in aged rat synapses. These results suggest that age-dependent changes of actin filament formation are related to neuronal dysfunction associated with aging.

Proteome analysis of Epstein-Barr virus-transformed B-lymphoblasts and the proteome database.

The proteome is the entire protein complement of the genome expressed in a particular cell, tissue, or organism at a given time under a specific set of environmental conditions. Proteomics is a combinatorial methodology to comprehensively analyze the proteome. The general protocol of the expression proteomics consists of advanced methods of high-resolution protein separation, high-quality image analysis and high-throughput protein identification. Although Epstein-Barr virus-transformed B-lymphoblastoid cell lines (LCLs) have long been believed to be immortalized, recent studies have provided ample evidence that a large proportion of LCLs have limited life spans due to shortening of telomeres, and that part of them are truly immortalized by developing strong telomerase activity to maintain telomeres. Differential proteome analysis of pre- and post-immortal LCLs would provide a powerful tool to analyze proteins participating in the process of immortalization. We focus in this review on cumulative data of proteomic information on pre- and post-immortal LCLs.

Identification and characterization of an increased glycoprotein in aging: age-associated translocation of cathepsin D.

We found that 14 N-glycosylated proteins were accumulated in the rat cerebral cortex cytosolic fraction in the aging process by a comparative study with two-dimensional gel electrophoresis and concanavalin A staining. All proteins had high mannose and/or hybrid-type N-glycans, as indicated by the fact that they were sensitive to endoglycosidase H digestion. Three of these cytosolic glycoproteins were identified as cathepsin D, a lysosomal protease, by tryptic digestion and nano liquid chromatography electrospray ionization quadrupole time of flight mass spectrometry. The increase of cytosolic cathepsin D during aging was not due to lysosomal membrane disruption, as shown by the fact that the activities of beta-hexosaminidase and beta-glucuronidase, other lysosomal enzymes, did not increase in the cytosolic fraction. Although the total amount of cathepsin D increased during aging, the amount of cathepsin D in the microsomal fraction did not change, indicating a selective increase of cytosolic cathepsin D. This phenomenon was also observed in the hippocampus, cerebellum, kidney, liver, and spleen. Based on these results, we propose that cytosolic cathepsin D is a new biomarker of aging.

Proteomic signatures and aberrations of mouse embryonic stem cells containing a single human chromosome 21 in neuronal differentiation: An in vitro model of down syndrome

Neurodegeneration in fetal development of Down syndrome (DS) patients is proposed to result in apparent neuropathological abnormalities and to contribute to the phenotypic characteristics of mental retardation and premature development of Alzheimer disease. In order to identify the aberrant and specific genes involved in the early differentiation of DS neurons, we have utilized an in vitro neuronal differentiation system of mouse ES cells containing a single human chromosome 21 (TT2F/hChr21) with TT2F parental ES cells as a control. The paired protein extracts from TT2F and TT2F/hChr21 cells at several stages of neuronal differentiation were subjected to two-dimensional polyacrylamide gel electrophoresis protein separation followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to identify the proteins differentially expressed between TT2F and TT2F/hChr21 cells. We provide here a novel set of specific gene products altered in early differentiating DS neuronal cells, which differs from that identified in adult or fetal brain with DS. The aberrant protein expression in early differentiating neurons, due to the hChr21 gene dosage effects or chromosomal imbalance, may affect neuronal outgrowth, proliferation and differentiation, producing developmental abnormalities in neural patterning, which eventually leads to formation of a suboptimal functioning neuronal network in DS.

A comparative analysis of the proteins between the fibroblasts from Werner's syndrome patients and age-matched normal individuals using two-dimensional gel electrophoresis

Werners syndrome (WS) is an autosomal recessive disorder causing symptoms of premature aging. The fibroblasts of WS patients have a shorter life-span than normal fibroblasts. We analyzed the fibroblast proteins from three WS patients and from three age-matched normal individuals using two-dimensional gel electrophoresis and image processing. The expressions of 12 proteins were shown to be augmented or suppressed in WS fibroblasts compared with normal fibroblasts: 11 of 12 spots on the electrophoresis gel of WS fibroblasts were denser than the corresponding spots of normal individual fibroblasts, while the remaining one spot was fainter in WS fibroblasts than in normal fibroblasts. The abundance of these proteins were compared to those of the corresponding proteins from normal fibroblasts at various cell passages in vitro reported in the TMIG-2DPAGE database. The result shows that the change in the protein patterns in in vitro aging did not necessarily correspond to the change in WS fibroblasts, except for three proteins abundant in WS fibroblasts, which increased their abundance during in vitro aging. These results suggest that the premature aging process of WS fibroblasts shares only part of the in vitro aging process of normal fibroblasts.

Two-dimensional electrophoresis on the layers of cellulose acetate membrane☆

Abstract A new type of two-dimensional electrophoresis for analysis of protein using cellulose acetate membrane has been developed. Prior to the separation, proteins in a sample are concentrated to a narrow zone on a strip of cellulose acetate according to “steady-state stacking” of isotachophoresis. Electroendosmotic counterflow on cellulose acetate membranes is advantageous for the isotachophoretic concentration of large sample volumes. The concentrated protein zone is then subjected to electrophoretic separation on the same strip. This first-dimensional separation including the concentrating process is named “concentrating electrophoresis.” Iso-electric focusing on several layers of cellulose acetate membrane is performed in the second-dimensional step. Many kinds of detection methods can be applied to the layers among which proteins are distributed. The novel two-dimensional electrophoresis takes only 5 h to perform.