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MFG-E8 (milk fat globule-EGF factor 8) is a peripheral membrane glycoprotein, which is expressed abundantly in lactating mammary glands and is secreted in association with fat globules. This protein consists of two-repeated EGF-like domains, a mucin-like domain and two-repeated discoidinlike domains (C-domains), and contains an integrin-binding motif (RGD sequence) in the EGF-like domain. To clarify the role of each domain on the peripheral association with the cell membrane, several domain-deletion mutants of MFG-E8 were expressed in COS-7 cells. The immunofluorescent staining of intracellular and cell-surface proteins and biochemical analyses of cell-surface-biotinylated and secreted proteins demonstrated that both of the two C-domains were required for the membrane association. During the course of these studies for domain functions, MFG-E8, but not C-domain deletion mutants, was shown to be secreted as membrane vesicle complexes. By size-exclusion chromatography and ultracentrifugation analyses, the complexes were characterized to have a high-molecular mass, low density and higher sedimentation velocity and to be detergentsensitive. Not only such a exogenously expressed MFG-E8 but also that endogenously expressed in a mammary epithelial cell line, COMMA-1D, was secreted as the membrane vesicle-like complex. Scanning electron microscopic analyses revealed that MFG-E8 was secreted into the culture medium in association with small membrane vesicles with a size from 100 to 200 nm in diameter. Furthermore, the expression of MFG-E8 increased the number of these membrane vesicle secreted into the culture medium. These results suggest a possible role of MFG-E8 in the membrane vesicle secretion, such as budding or shedding of plasma membrane (microvesicles) and exocytosis of endocytic multivesicular bodies (exosomes).

Familial exudative vitreoretinopathy (FEVR) is a hereditary blinding disorder that features defects in retinal vascular development. The mutations in the genes encoding the Wnt receptor pair, frizzled 4 (FZD4) and low-density-lipoprotein receptor-related protein 5 (LRP5), have been shown to cause FEVR. In this study we screened 56 unrelated patients with FEVR (31 familial and 25 simplex cases) for possible mutations in LRP5 and FZD4. Six novel mutations in either LRP5 or FZD4 were identified in six familial cases. Four novel mutations in LRP5 and one known mutation in FZD4 were detected in three simplex cases, and two of these patients carried compound heterozygous mutations in LRP5. Remarkably, c.1330C>T [p.R444C] in LRP5 was found in the family in which c.1250G>A [p.R417Q] in FZD4 had previously been identified. The phenotype of these patients suggested a synergistic effect of the two mutations in the independent FEVR-causing genes. We also demonstrated that reduced bone density is a common feature in patients with FEVR who harbor LRP5 mutations. The profile of the mutations obtained in the current study further illustrates the complexity of the disease and provides a better understanding of the spectrum, frequencies, and genotype-phenotype correlation.

Degradation of arylglycerol-␤-aryl ether is the most important process in bacterial lignin catabolism. Sphingobium sp. strain SYK-6 degrades guaiacylglycerol-␤-guaiacyl ether (GGE) to ␣-(2-methoxyphenoxy)-␤-hydroxypropiovanillone (MPHPV), and then the ether linkage of MPHPV is cleaved to generate ␣-glutathionyl-␤-hydroxypropiovanillone (GS-HPV) and guaiacol. We have characterized three enantioselective glutathione S-transferase genes, including two genes that are involved in the ether cleavage of two enantiomers of MPHPV and one gene that is involved in the elimination of glutathione from a GS-HPV enantiomer. However, the first step in the degradation of four different GGE stereoisomers has not been characterized. In this study, three alcohol dehydrogenase genes, ligL, ligN, and ligO, which conferred GGE transformation activity in Escherichia coli, were isolated from SYK-6 and characterized, in addition to the previously cloned ligD gene. The levels of amino acid sequence identity of the four GGE dehydrogenases, which belong to the short-chain dehydrogenase/ reductase family, ranged from 32% to 39%. Each gene was expressed in E. coli, and the stereospecificities of the gene products with the four GGE stereoisomers were determined by using chiral high-performance liquid chromatography with recently synthesized authentic enantiopure GGE stereoisomers. LigD and LigO converted (␣R,␤S)-GGE and (␣R,␤R)-GGE into (␤S)-MPHPV and (␤R)-MPHPV, respectively, while LigL and LigN transformed (␣S,␤R)-GGE and (␣S,␤S)-GGE to (␤R)-MPHPV and (␤S)-MPHPV, respectively. Disruption of the genes indicated that ligD is essential for the degradation of (␣R,␤S)-GGE and (␣R,␤R)-GGE and that both ligL and ligN contribute to the degradation of the two other GGE stereoisomers.

Vitreous samples collected in retinopathic surgeries have diverse properties, making proteomics analysis difficult. We report a cluster analysis to evade this difficulty. Vitreous and subretinal fluid samples were collected from 60 patients during surgical operation of non-proliferative diabetic retinopathy, proliferative diabetic retinopathy, proliferative vitreoretinopathy, and rhegmatogenous retinal detachment. For controls, we collected vitreous fluid from patients of idiopathic macular hole, epiretinal, and from a healthy postmortem donor. Proteins from these samples were subjected to quantitative proteomics using two-dimensional gel electrophoresis. We selected 105 proteins robustly expressed among ca. 400 protein spots and subjected them to permutation test. By using permutation test analysis we observed unique variations in the expression of some of these proteins in vitreoretinal diseases when compared to the control and to each other: (i) the levels of inflammation-associated proteins such as alpha1-antitrypsin, apolipoprotein A4, albumin, and transferrin were significantly higher in all four types of vitreoretinal diseases, and (ii) each vitreoretinal disease elevated a unique set of proteins, which can be interpreted based on the pathology of retinopathy. Our protocol will be effective for the study of protein expression in other types of clinical samples of diverse properties.

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This systematic approach facilitated the identification of genes that cause arRP, and the results provide a widened spectrum of the mutation severity associated with a broader range of phenotypic manifestations of arRP.