Tamiko Minamisawa

Autonomous Silica Encapsulation and Sustained Release of Anticancer Protein

We present a novel method for preparing a silica carrier for the sustained release of a proteinaceous pharmaceutical. This method makes use of the silicification activity of the protein itself, which autonomously formed a protein-silica composite upon simple incubation with a silica precursor. The composite was dissolved, and the encapsulated protein was released into a culture medium, thereby sustaining the proteins activity for a long period of time.

Motif programming: a microgene-based method for creating synthetic proteins containing multiple functional motifs

The presence of peptide motifs within the proteins provides the synthetic biologist with the opportunity to fabricate novel proteins through the programming of these motifs. Here we describe a method that enables one to combine multiple peptide motifs to generate a combinatorial protein library. With this method, a set of sense and antisense oligonucleotide primers were prepared. These primers were mixed and polymerized, so that the resultant DNA consisted of combinatorial polymers of multiple microgenes created from the stochastic assembly of the sense and antisense primers. With this motif-mixing method, we prepared a protein library from the BH1-4 motifs shared among Bcl-2 family proteins. Among the 41 clones created, 70% of clones had a stable, presumably folded expression product in human cells, which was detectable by immunohistochemistry and western blot. The proteins obtained varied with respect to both the number and the order of the four motifs. The method enables homology-independent polymerization of DNA blocks that coded motif sequences, and the frequency of each motif within a library can be adjusted in a tailor-made manner. This motif programming has a potential for creating a library with a large proportion of folded/functional proteins.

Distinct macroscopic structures developed from solutions of chemical compounds and periodic proteins.

By controlling the growth of inorganic crystals, macro‐biomolecules, including proteins, play pivotal roles in modulating biomineralization. Natural proteins that promote biomineralization are often composed of simple repeats of peptide sequences; however, the relationship between these repetitive structures and their functions remains largely unknown. Here we show that an artificial protein containing a repeated peptide sequence allows NaCl, KCl, CuSO4 and sucrose to form a variety of macroscopic structures, as represented by their dendritic configurations. Mutational analyses revealed that the physicochemical characteristics of the protein, not the peptide sequence per se, were responsible for formation of the dendritic structures. This suggests that proteins that modulate crystal growth may have evolved as repeat‐containing forms at a relatively high rate. These observations could serve as the basis for developing new genetic programming systems for creation of artificial proteins able to modulate crystal growth from inorganic compounds, and may thus provide a new tool for nano‐biotechnology.

Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations.

Diagnostic methods that focus on the extracellular vesicles (EVs) present in saliva have been attracting great attention because of their non-invasiveness. EVs contain biomolecules such as proteins, messenger RNA (mRNA) and microRNA (miRNA), which originate from cells that release EVs, making them an ideal source for liquid biopsy. Although there have been many reports on density-based fractionation of EVs from blood and urine, the number of reports on EVs from saliva has been limited, most probably because of the difficulties in separating EVs from viscous saliva using density gradient centrifugation. This article establishes a protocol for the isolation of EVs from human saliva using density gradient centrifugation. The fractionated salivary EVs were characterized by atomic force microscopy, western blot and reverse transcription polymerase chain reaction. The results indicate that salivary EVs have a smaller diameter (47.8±12.3 nm) and higher density (1.11 g/ml) than EVs isolated from conditioned cell media (74.0±23.5 nm and 1.06 g/ml, respectively). Additionally, to improve the throughput of density-based fractionation of EVs, the original protocol was further modified by using a fixed angle rotor instead of a swinging rotor. It was also confirmed that several miRNAs were expressed strongly in the EV-marker-expressing fractions.

Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment

Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell‐platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation‐inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS‐8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C‐type lectin‐like receptor 2 (CLEC‐2). Here, we generated a novel single‐chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti‐human Aggrus monoclonal antibody MS‐1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus‐induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high‐affinity scFv, K‐11. K‐11 scFv was able to suppress Aggrus‐induced platelet aggregation in vitro. Moreover, K‐11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K‐11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus‐targeted cancer therapies.

Effect of Motif-Programmed Artificial Proteins on the Calcium Uptake in a Synthetic Hydrogel

Motif-programmed artificial proteins with mineralization-related activity were covalently immobilized onto the surface of a hydrogel, poly(2-hydroxyethyl methacrylate) (PHEMA). We investigated the influence of assaying conditions upon the ability of three selected proteins (PS64, PS382 and PS458) to modulate calcification in vitro. A long-term assay measuring the real amount of calcium phosphate phase in the protein-modified PHEMA showed that all proteins enhanced the uptake of calcium by the hydrogel. For PS382 and PS458, this is a behaviour opposite to that displayed when the same proteins were tested in a free state by a rapid solution assay. Such difference may be attributed to a restricted mobility of the proteins due to immobilization.

Motif-programmed artificial protein induces apoptosis in several cancer cells by disrupting mitochondria

By combinatorially assembling two natural motifs, respectively, associated with protein transduction (PTD) and induction of apoptosis (BH3), we previously synthesized an artificial protein (#284) that is taken up into cells, where it induces apoptosis. Here we used cluster analysis of GI50 (average concentration required for 50% growth inhibition), as well as immunohistochemical and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling analyses to further characterize the capacity of #284 to induce apoptosis in a panel of 39 cancer cell lines. Our results showed that #284 preferentially inhibited the growth of several cancer cells with a GI50 of approximately 5 µM, which is in the range of conventional anticancer drugs such as cisplatin and etoposide. In breast cancer HBC‐4 cells, #284 caused mitochondrial aggregation and induced apoptosis in a BH3 motif‐dependent manner. Moreover, transfection of the artificial gene that encodes #284 led to effective expression of the artificial protein within cells, which in turn caused apoptosis at a level similar to that seen in naturally occurring apoptosis inducers, Noxa/Bax transfectants. These findings suggest that synthetic proteins created by reprogramming peptide motifs have the potential to serve as novel agents useful in the treatment of cancer. (Cancer Sci 2008; 99: 398–406)

Combinatorial Contextualization of Peptidic Epitopes for Enhanced Cellular Immunity

Invocation of cellular immunity by epitopic peptides remains largely dependent on empirically developed protocols, such as interfusion of aluminum salts or emulsification using terpenoids and surfactants. To explore novel vaccine formulation, epitopic peptide motifs were co-programmed with structural motifs to produce artificial antigens using our “motif-programming” approach. As a proof of concept, we used an ovalbumin (OVA) system and prepared an artificial protein library by combinatorially polymerizing MHC class I and II sequences from OVA along with a sequence that tends to form secondary structures. The purified endotoxin-free proteins were then examined for their ability to activate OVA-specific T-cell hybridoma cells after being processed within dendritic cells. One clone, F37A (containing three MHC I and two MHC II OVA epitopes), possessed a greater ability to evoke cellular immunity than the native OVA or the other artificial antigens. The sensitivity profiles of drugs that interfered with the F37A uptake differed from those of the other artificial proteins and OVA, suggesting that alteration of the cross-presentation pathway is responsible for the enhanced immunogenicity. Moreover, F37A, but not an epitopic peptide, invoked cellular immunity when injected together with monophosphoryl lipid A (MPL), and retarded tumor growth in mice. Thus, an artificially synthesized protein antigen induced cellular immunity in vivo in the absence of incomplete Freunds adjuvant or aluminum salts. The method described here could be potentially used for developing vaccines for such intractable ailments as AIDS, malaria and cancer, ailments in which cellular immunity likely play a crucial role in prevention and treatment.

Host Cell Prediction of Exosomes Using Morphological Features on Solid Surfaces Analyzed by Machine Learning

Exosomes are extracellular nanovesicles released from any cells and found in any body fluid. Because exosomes exhibit information of their host cells (secreting cells), their analysis is expected to be a powerful tool for early diagnosis of cancers. To predict the host cells, we extracted multidimensional feature data about size, shape, and deformation of exosomes immobilized on solid surfaces by atomic force microscopy (AFM). The key idea is combination of support vector machine (SVM) learning for individual exosome particles and their interpretation by principal component analysis (PCA). We observed exosomes derived from three different cancer cells on SiO2/Si, 3-aminopropyltriethoxysilane-modified-SiO2/Si, and TiO2 substrates by AFM. Then, 14-dimensional feature vectors were extracted from AFM particle data, and classifiers were trained in 14-dimensional space. The prediction accuracy for host cells of test AFM particles was examined by the cross-validation test. As a result, we obtained prediction of exosome host cells with the best accuracy of 85.2% for two-class SVM learning and 82.6% for three-class one. By PCA of the particle classifiers, we concluded that the main factors for prediction accuracy and its strong dependence on substrates are incremental decrease in the PCA-defined aspect ratio of the particles with their volume.

Encryption of agonistic motifs for TLR4 into artificial antigens augmented the maturation of antigen-presenting cells

Adjuvants are indispensable for achieving a sufficient immune response from vaccinations. From a functional viewpoint, adjuvants are classified into two categories: “physical adjuvants” increase the efficacy of antigen presentation by antigen-presenting cells (APC) and “signal adjuvants” induce the maturation of APC. Our previous study has demonstrated that a physical adjuvant can be encrypted into proteinous antigens by creating artificial proteins from combinatorial assemblages of epitope peptides and those peptide sequences having propensities to form certain protein structures (motif programming). However, the artificial antigens still require a signal adjuvant to maturate the APC; for example, co-administration of the Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) was required to induce an in vivo immunoreaction. In this study, we further modified the previous artificial antigens by appending the peptide motifs, which have been reported to have agonistic activity for TLR4, to create “adjuvant-free” antigens. The created antigens with triple TLR4 agonistic motifs in their C-terminus have activated NF-κB signaling pathways through TLR4. These proteins also induced the production of the inflammatory cytokine TNF-α, and the expression of the co-stimulatory molecule CD40 in APC, supporting the maturation of APC in vitro. Unexpectedly, these signal adjuvant-encrypted proteins have lost their ability to be physical adjuvants because they did not induce cytotoxic T lymphocytes (CTL) in vivo, while the parental proteins induced CTL. These results confirmed that the manifestation of a motif’s function is context-dependent and simple addition does not always work for motif-programing. Further optimization of the molecular context of the TLR4 agonistic motifs in antigens should be required to create adjuvant-free antigens.