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...
In current plant biotechnology, the introduction of exogenous DNA encoding desired traits is the most common approach used to modify plants. However, general plant transformation methods can cause random integration of exogenous DNA into the plant genome. To avoid these events, alternative methods, such as a direct protein delivery system, are needed to modify the plant. Although there have been reports of the delivery of proteins into cultured plant cells, there are currently no methods for the direct delivery of proteins into intact plants, owing to their hierarchical structures. Here, we demonstrate the efficient fusion-peptide-based delivery of proteins into intact Arabidopsis thaliana. Bovine serum albumin (BSA, 66 kDa) was selected as a model protein to optimize conditions for delivery into the cytosol. The general applicability of our method to large protein cargo was also demonstrated by the delivery of alcohol dehydrogenase (ADH, 150 kDa) into the cytosol. The compatibility of the fusion peptide system with the delivery of proteins to specific cellular organelles was also demonstrated using the fluorescent protein Citrine (27 kDa) conjugated to either a nuclear localization signal (NLS) or a peroxisomal targeting signal (PTS). In conclusion, our designed fusion peptide system can deliver proteins with a wide range of molecular weights (27 to 150 kDa) into the cells of intact A. thaliana without interfering with the organelle-targeting peptide conjugated to the protein. We expect that this efficient protein delivery system will be a powerful tool in plant biotechnology.
A two-step PCR method has been developed for the robust, high-throughput production of linear templates ready for cell-free protein synthesis. The construct made from the cDNA expresses a target protein region with N- and/or C-terminal tags. The procedure consists only of mixing, dilution, and PCR steps, and is free from cloning and purification steps. In the first step of the two-step PCR, a target region within the coding sequence is amplified using two gene-specific forward and reverse primers, which contain the linker sequences and the terminal sequences of the target region. The second PCR concatenates the first PCR product with the N- and C-terminal double-stranded fragments, which contain the linker sequences as well as the sequences for the tag(s) and the initiation and termination, respectively, for T7 transcription and ribosomal translation, and amplifies it with the universal primer. Proteins can be fused with a variety of tags, such as natural poly-histidine, glutathione-S-transferase, maltose-binding protein, and/or streptavidin-binding peptide. The two-step PCR method was successfully applied to 42 human target protein regions with various GC contents (38-77%). The robustness of the two-step PCR method against possible fluctuations of experimental conditions in practical use was explored. The second PCR product was obtained at 60-120 microg/ml, and was used without purification as a template at a concentration of 2-4 microg/ml in an Escherichia coli coupled transcription-translation system. This combination of two-step PCR with cell-free protein synthesis is suitable for the rapid production of proteins in milligram quantities for genome-scale studies.
Polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae FA440 (PhaC(Ac), BAA21815) is one of the most valuable PHA synthase, because of its function to synthesize a practical bioplastic, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)]. However, biochemical activity and active intermediates of PhaC(Ac) have not been clarified until now. In the present study, a gene of PhaC(Ac) was cloned and overexpressed by a cell-free protein expression system. Both the polymerization activity and oligomerization behavior of the purified PhaC(Ac) were characterized in order to clarify the active intermediates of PhaC(Ac) based on the hydrodynamic diameters and specific activities of PhaC(Ac). The influences of a substrate, (R)-3-hydroxybutyryl-CoA (3HB-CoA), on the oligomerization of PhaC(Ac) (7.5 μM) were also investigated, and then the Hill coefficient (n = 2.6 ± 0.4) and the microscopic dissociation constant (K(m) = 77 ± 5 μM) were determined. Based on the results, the active intermediate of PhaC(Ac) was concluded to be the dimeric PhaC(Ac) containing 3HB-CoA as an activator for its dimerization. This information is critical for revealing the relationships between its dimerization and function in PHA synthesis.
Microtubule-associated protein/microtubule affinity-regulating kinases (MARKs)/PAR-1 are common regulators of cell polarity that are conserved from nematode to human. All of these kinases have a highly conserved C-terminal domain, which is termed the kinase-associated domain 1 (KA1), although its function is unknown. In this study, we determined the solution structure of the KA1 domain of mouse MARK3 by NMR spectroscopy. We found that approximately 50 additional residues preceding the previously defined KA1 domain are required for its proper folding. The newly defined KA1 domain adopts a compact alpha+beta structure with a betaalphabetabetabetabetaalpha topology. We also found a characteristic hydrophobic, concave surface surrounded by positively charged residues. This concave surface includes the highly conserved Glu-Leu-Lys-Leu motif at the C terminus, indicating that it is important for the function of the KA1 domain.
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