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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...

No abstract

TT1466 is a hypothetical protein from the extremely thermophilic bacterium Thermus thermophilus HB8 and is highly conserved in bacteria and archaea. The selenomethionyl protein was synthesized by a cell-free system and the crystal structure was determined at 2.0 A by MAD phasing. A native crystal was used for structure refinement to 1.7 A. The structure is highly homologous to that of the CoA-binding domain of the succinyl-CoA synthetase from Escherichia coli, despite the protein having only 14% sequence identity to this domain. An isothermal titration calorimetry experiment was performed to investigate whether TT1466 binds CoA and revealed high-affinity CoA binding of TT1466.