Localization of D-ß-aspartyl residue-containing proteins in various tissues.

Prior to the emergence of life, it is believed that only l-amino acids were selected for formation of protein and that d-amino acids were eliminated on the primitive Earth. Whilst homochirality is essential for life, the occurrence of proteins containing d-beta-aspartyl (Asp) residues in various tissues from elderly subjects has been reported recently. Here, we demonstrate the presence of a d-beta-Asp-containing protein in the cardiac muscle of heart, blood vessels of the lung, chief cells of the stomach, longitudinal and circular muscle of the stomach, small intestine and large intestine. Since the d-beta-Asp residue occurs through a succinimide intermediate, this isomer may potentially be generated in proteins more easily than initially thought. Formation of the d-beta-Asp residue in proteins may be related to stress.

A protein-permeable scaffold of a collagen vitrigel membrane useful for reconstructing crosstalk models between two different cell types.

Soft and turbid collagen gel disks were previously converted into strong and transparent gel membranes utilizing a concept for the vitrification of heat-denatured of proteins. This novel stable and transparent gel has been termed 'vitrigel'. By encompassing the collagen vitrigel membrane in a nylon frame, it can be easily handled with tweezers, and functions as an excellent scaffold for three-dimensional cell culture models, as cells can be cultured on both sides. Here, we investigated the molecular permeability of the collagen vitrigel membrane in a time course-dependent manner using glucose and serum proteins. Glucose penetrated through the collagen vitrigel membrane to the opposite side, and concentrations on each side were found to be equilibrated within 24 h. Serum proteins up to a molecular weight >100 kDa also gradually passed through the collagen vitrigel membrane. In addition, human microvascular endothelial cells (HMVECs) were cultured on one surface of the collagen vitrigel membrane with a nylon frame, and human dermal fibroblasts (HDFs) or HT-29 (a human colon carcinoma cell line) cells were cocultured on the opposite surface. Histomorphological observations revealed the formation of three-dimensional crosstalk models composed of HMVECs and HDFs or HMVECs and HT-29 cells. Resulting data suggest that the protein-permeable scaffold composed of the collagen vitrigel membrane is useful for the reconstruction and/or modeling of 'crosstalk' between two different cells types. Hereafter, such crosstalk models in vitro could be applied to research not only of paracrine factors, but also to epithelial- or endothelial-mesenchymal transitions.

Differential analysis of D-beta-Asp-containing proteins found in normal and infrared irradiated rabbit lens.

Although proteins are generally composed of l-alpha-amino acids, d-beta-aspartic acid (Asp)-containing proteins have been reported in various elderly tissues. Our previous study detected several d-beta-Asp-containing proteins in a rabbit lens derived from epithelial cell line by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for d-beta-Asp-containing proteins. The identity of each spot was subsequently determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Ms-Fit online database searching algorithm. In this study, we discovered novel d-beta-Asp-containing proteins from rabbit lens. The results indicate that beta-crystallin A3, beta-crystallin A4, beta-crystallin B1, beta-crystallin B2, beta-crystallin B3, gamma-crystallin C, gamma-crystallin D, and lambda-crystallin in rabbit lens contain d-beta-Asp residues. Furthermore, the occurrence of d-beta-Asp residues increases with infrared ray (IR) irradiation. Additionally, some d-beta-Asp-containing proteins only appear after IR irradiation. One such protein is the alpha-enolase, which shows homology to tau-crystallin.

Characterization of new D-beta-aspartate-containing proteins in a lens-derived cell line.

Although proteins are generally composed of l-alpha-amino acids, biologically uncommon D-beta-aspartic acid (Asp)-containing proteins have been reported in various tissues from elderly individuals. Our previous study indicated that the N/N1003A cell line, derived from rabbit lens, includes D-beta-Asp-containing proteins of approximately 50 kDa by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for D-beta-Asp-containing proteins. In this study, we identified the D-beta-Asp-containing proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm. The results indicate that one of these 50 kDa proteins is an enolase showing homology with tau-crystallin. Other D-beta-Asp-containing proteins, which we have recently discovered include lamin A/C, cytoplasmic NADP+-dependent isocitrate dehydrogenase, fructose-bisphosphate aldolase A, aldose reductase, L-lactate dehydrogenase A or calponin H2, phosphoglycerate mutase 1, phosphatidylethanolamine-binding protein, alpha-B-crystallin, and peptidyl-prolyl cis-trans isomerase A (PPlase).

Collagen vitrigel: a novel scaffold that can facilitate a three-dimensional culture for reconstructing organoids.

Three-dimensional reconstructed organoids in vitro are valuable for not only regenerative medicine but also drug development. However, the manipulation of conventional three-dimensional cultures is not simple. We describe a nylon membrane ring-embedded or a pressed silk sheet-embedded scaffold made of collagen "vitrigel" that can facilitate three-dimensional cultures for reconstructing an epithelial-mesenchymal model or a hard connective tissue model, respectively. Here we define vitrigel as a gel in a stable state produced by rehydration after the vitrification of a traditional hydrogel. The collagen vitrigel was successfully prepared in three steps involving a gelation process in which a cold and clear neutral salt solution containing type I collagen formed an opaque and soft gel by incubation at 37 degrees C, a vitrification process in which the gel becomes a rigid material like glass after sufficient drying out, and finally a rehydration process to convert the vitrified material into a thin and transparent gel membrane with enhanced gel strength. The framework-embedded collagen vitrigel scaffold that can be easily reversed by forceps was prepared by inserting a nylon ring or a silk sheet in the collagen solution prior to the gelation. The scaffold enabled culturing anchorage-dependent cells on both surfaces of the collagen vitrigel by the manipulation of two-dimensional cultures and consequently resulted in reconstructing a three-dimensional organoid. An intestinal epithelial-mesenchymal model was reconstructed by coculturing fibroblasts on the opposite side of monolayered Caco-2 cells on the nylon ring-embedded collagen vitrigel. Also, fibroblasts seeded on both surfaces of the silk sheet-embedded collagen vitrigel proliferated well and formed multilayers and some cells invaded into the vitrigel framed by the network of numerous strong silk filaments, suggesting a reconstruction of a hard connective tissue model. These data demonstrate that the collagen vitrigel is a valuable scaffold for tissue engineering.

Immunological detection of D-beta-aspartate-containing protein in lens-derived cell lines.

PURPOSE: Although the presence of biologically uncommon D-beta-aspartate (D-beta-Asp) in lens protein is thought to be related to aging, we recently found this isomer in lens alphaA-crystallin from human newborns. The objective of this study was to examine whether D-beta-Asp occurs in protein from lens-derived cell lines. METHODS: We examined the expression of D-beta-Asp-containing protein in the lens-derived cell lines alphaTN4-1 and N/N1003A, by western blot and immunoprecipitation analysis using a polyclonal antibody against Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr (peptide 3R), which corresponds to three repeats of positions 149-153 in human alphaA-crystallin. The anti-peptide 3R antibody, prepared in a previous study, is a useful tool for investigating D-beta-Asp-containing peptides. RESULTS: Western immunoblot and immunoprecipitation analysis showed that a 50 kDa protein in N/N1003A cells was strongly immunoreactive with the anti-peptide 3R antibody. Antibodies against alphaA- and alphaB-crystallin also stained this protein. On the other hand, the alphaTN4-1 cell line only expressed proteins of about 20 kDa, which also reacted to antibodies against alphaA-crystallin and alphaB-crystallin. CONCLUSIONS: The results indicate that the N/N1003A cell line expressed a 50 kDa D-beta-Asp-containing protein, which may share a common amino acid sequence with alphaA- and alphaB-crystallin.

The presence of D-beta-aspartic acid-containing peptides in elastic fibers of sun-damaged skin: a potent marker for ultraviolet-induced skin aging.

Biologically uncommon d-aspartyl residues have been reported in proteins of various elderly tissues. We prepared a polyclonal antibody against d-beta-Asp-containing peptide and examined its immunoreactivity in the skin. The antibody recognized integrated or disintegrated elastic fibers in the sun-exposed skin but not in the sun-protected skin of the elderly donors. Western blot analysis of the proteins isolated from sun-damaged skin demonstrated that the 50 kDa protein was immunoreactive with both antibodies for d-beta-Asp-containing peptide and elastin. Ultraviolet (UV) irradiation on normal skin caused the appearance of d-beta-Asp-containing peptide-immunoreactive fibers in the dermis. These results suggest that UV irradiation is closely related to the formation of d-beta-Asp in the elastic fibers of skin. We propose that the antibody could be a useful indicator for sun damage of the skin.