Human CA125, encoded by the MUC16 gene, is an ovarian cancer antigen widely used for a serum assay. Its extracellular region consists of tandem repeats of SEA domains. In this study we determined the three-dimensional structure of the SEA domain from the murine MUC16 homologue using multidimensional NMR spectroscopy. The domain forms a unique ␣/␤ sandwich fold composed of two ␣ helices and four antiparallel ␤ strands and has a characteristic turn named the TY-turn between ␣1 and ␣2. The internal mobility of the main chain is low throughout the domain. The residues that form the hydrophobic core and the TY-turn are fully conserved in all SEA domain sequences, indicating that the fold is common in the family. Interestingly, no other residues are conserved throughout the family. Thus, the sequence alignment of the SEA domain family was refined on the basis of the three-dimensional structure, which allowed us to classify the SEA domains into several subfamilies. The residues on the surface differ between these subfamilies, suggesting that each subfamily has a different function. In the MUC16 SEA domains, the conserved surface residues, Asn-10, Thr-12, Arg-63, Asp-75, Asp-112, Ser-115, and Phe-117, are clustered on the ␤ sheet surface, which may be functionally important. The putative epitope (residues 58 -77) for anti-MUC16 antibodies is located around the ␤2 and ␤3 strands. On the other hand the tissue tumor marker MUC1 has a SEA domain belonging to another subfamily, and its GSVVV motif for proteolytic cleavage is located in the short loop connecting ␤2 and ␤3.CA125 is a serum marker that is widely used to monitor ovarian cancer because it is overexpressed in ovarian cancer cells and secreted into the blood. An elevated serum CA125 level is a useful indicator of ovarian cancer, but it is also observed in a number of benign conditions (1, 2). CA125 is a mucin-type O-linked glycoprotein (3, 4), but other details about its molecular nature remain unclear. Recently two research groups cloned CA125 (5-8), revealing that CA125 is a membrane protein with some splicing variants. The splicing variants have the same intracellular and transmembrane regions. The extracellular domain consists of the SEA 1 domains, which are repeated 7, 12, or 60 times, according to the variant. The gene was named MUC16, after the mucin-like nature of CA125. The elucidation of the amino acid sequence has made it possible to specify the approximate position of the epitope. A previous study showed that the peptide epitope position of CA125 is located between two conserved cysteines in the SEA domain (7).A cDNA of the murine MUC16 homologue, cloned in the RIKEN FANTOM project (9), has a total of 258 amino acids and a transmembrane domain. It is 66% identical to the C terminus of human MUC16 and has only one SEA domain in its extracellular region. However, our investigation of the mouse and human genomic sequences showed that they share the same characteristic repeat structure of MUC16. Thus, the murine MUC16 appears to have splicing variants, as i...

The food-poisoning bacterium Clostridium perfringens produces an enterotoxin (~35 kDa) that specifically targets human claudin-4, among the 26 human claudin proteins, and causes diarrhea by fluid accumulation in the intestinal cavity. The C-terminal domain of the Clostridium perfringens enterotoxin (C-CPE, ~15 kDa) binds tightly to claudin-4, and disrupts the intestinal tight junction barriers. In this study, we determined the 3.5-Å resolution crystal structure of the cell-free synthesized human claudin-4•C-CPE complex, which is significantly different from the structure of the off-target complex of an engineered C-CPE with mouse claudin-19. The claudin-4•C-CPE complex structure demonstrated the mechanism underlying claudin assembly disruption. A comparison of the present C-CPE-bound structure of claudin-4 with the enterotoxin-free claudin-15 structure revealed sophisticated C-CPE-induced conformation changes of the extracellular segments, induced on the foundation of the rigid four-transmembrane-helix bundle structure. These conformation changes provide a mechanistic model for the disruption of the lateral assembly of claudin molecules. Furthermore, the present novel structural mechanism for selecting a specific member of the claudin family can be used as the foundation to develop novel medically important technologies to selectively regulate the tight junctions formed by claudin family members in different organs.

No abstract

It has been assumed that the pi-electrons of aromatic residues in the catalytic sites of triterpene cyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene or oxidosqualene, but no definitive experimental evidence has been given. To validate this cation-pi interaction, natural and unnatural aromatic amino acids were site-specifically incorporated into squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius and the kinetic data of the mutants were compared with that of the wild-type SHC. The catalytic sites of Phe365 and Phe605 were substituted with O-methyltyrosine, tyrosine, and tryptophan, which have higher cation-pi binding energies than phenylalanine. These replacements actually increased the SHC activity at low temperature, but decreased the activity at high temperature, as compared with the wild-type SHC. This decreased activity is due to the disorganization of the protein architecture caused by the introduction of the amino acids more bulky than phenylalanine. Then, mono-, di-, and trifluorophenylalanines were incorporated at positions 365 and 605; these amino acids reduce cation-pi binding energies but have van der Waals radii similar to that of phenylalanine. The activities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the number of the fluorine atoms on the aromatic ring and clearly correlated with the cation-pi binding energies of the ring moiety. No serious structural alteration was observed for these variants even at high temperature. These results unambiguously show that the pi-electron density of residues 365 and 605 has a crucial role for the efficient polycyclization reaction by SHC. This is the first report to demonstrate experimentally the involvement of cation-pi interaction in triterpene biosynthesis.

γ-Secretase is an intramembrane-cleaving protease responsible for the generation of amyloid-β (Aβ) peptides. Recently, a series of compounds called γ-secretase modulators (GSMs) has been shown to decrease the levels of long toxic Aβ species (i.e., Aβ42), with a concomitant elevation of the production of shorter Aβ species. In this study, we show that a phenylimidazole-type GSM allosterically induces conformational changes in the catalytic site of γ-secretase to augment the proteolytic activity. Analyses using the photoaffinity labeling technique and systematic mutational studies revealed that the phenylimidazole-type GSM targets a previously unidentified extracellular binding pocket within the N-terminal fragment of presenilin (PS). Collectively, we provide a model for the mechanism of action of the phenylimidazole-type GSM in which binding at the luminal side of PS induces a conformational change in the catalytic center of γ-secretase to modulate Aβ production.Alzheimer's disease | intramembrane proteolysis | allosteric modulator | chemical biology | amyloid-β protein

The BolA-like proteins are widely conserved from prokaryotes to eukaryotes. The BolA-like proteins seem to be involved in cell proliferation or cell-cycle regulation, but the molecular function is still unknown. Here we determined the structure of a mouse BolA-like protein. The overall topology is alphabetabetaalphaalphabetaalpha, in which beta(1) and beta(2) are antiparallel, and beta(3) is parallel to beta(2). This fold is similar to the class II KH fold, except for the absence of the GXXG loop, which is well conserved in the KH fold. The conserved residues in the BolA-like proteins are assembled on the one side of the protein.

Four hundred and ninety-five Japanese primary-school children aged from 8 (Grade-2) to 12 (Grade-6) were tested for their abilities to read/write in Hiragana, Katakana, and Kanji, for their size of vocabulary and for other cognitive abilities including arithmetic, visuo-spatial and phonological processing. Percentages of the children whose reading/writing scores fell below the −1.5SD cut-off differ according to the scripts-Hiragana: 0.2% for reading and 1.6% for writing, Katakana: 1.4% and 3.8%, and Kanji: 6.9% and 6%, respectively. Further, for the normal children, the older the age, the better they performed on cognitive tasks, while the reading/writing disability (RWD) group (below −1.5SD) showed a weaker relationship between the age and the performance level. It was also revealed that for A. Uno the normal children, the "vocabulary size" was the most potent predictor variable in accounting for Kanji word reading performance for all grades except Grade-6, for whom nonword reading/repetition were also significant predictor variables. In contrast, for Kanji word writing, generally other writing related variables were better predictor variables. The RWD group however showed different patterns of results. Thus the data from the normal and RWD children in the current cohort were discussed in terms of the current theories of reading, and developmental dyslexia.

The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits. These proteins were produced at levels of about 0.1–1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin.