Kouki Kitagawa

Plant defense–related enzymes as latex antigens☆☆☆★★★♢

BACKGROUND Latex allergy is an increasing hazard to people who frequently come into contact with latex products. Of interest concerning this immediate-type allergy is the cross-reactivity to various vegetable foods and pollen. Despite its high prevalence, no adequate explanation has been provided for the cross-reactive antigens. OBJECTIVE We have hypothesized that a series of plant defense-related proteins act as latex allergens, as well as vegetable food allergens. To evaluate this hypothesis, hydrolytic enzymes that are very likely to take on defensive roles in rubber trees were examined for their antigenicity. METHODS By applying chromatographic procedures, defense-related enzymes were separated from nonammoniated latex (NAL). Their antigenicity was examined by immunoblotting and ELISA with sera containing IgE antibodies to crude latex proteins. RESULTS Three kinds of hydrolytic enzymes (basic beta-1,3-glucanases [35, 36.5, and 38 kd], a basic chitinase/lysozyme [29.5 kd], and an acidic esterase [44 kd]) were separated from NAL. They were recognized by IgE antibodies from a significant number of patients allergic to latex. The basic beta-1,3-glucanases and the acidic esterase were also strongly recognized by IgE antibodies from several atopic subjects who were allergic to various vegetable foods rather than latex products. CONCLUSION It was ascertained that the three defense-related enzymes separated from NAL constituted part of the latex antigens. Taking together the well-known serologic or immunologic relationships and amino acid sequence similarities of defense-related proteins coming from phylogenetically distant plant species, we can suspect their universal antigenicity and cross-reactivity.

Artificial collagen gels via self‐assembly of de novo designed peptides

Development of artificial collagens to replace the animal‐derived collagens presents a challenge in the formation of safer and functional biomaterials. We report here the development of collagen‐like gels by means of the self‐assembly of chemically synthesized peptides. The peptides are disulfide‐linked trimers of collagenous Gly‐X‐Y triplet repeats with self‐complementary shapes. Upon cooling the peptide solutions, hydrogels of peptide supramolecules are formed by spontaneous intermolecular triple helix formation. The thermal gel–sol transition appeared to be reversible, and the transition temperatures were found to be tunable by the design of the peptides. Our systems for the formation of artificial collagen‐like gels will offer possibilities for novel types of biomaterials.

Specific recognition of the collagen triple helix by chaperone HSP47: minimal structural requirement and spatial molecular orientation.

The unique folding of procollagens in the endoplasmic reticulum is achieved with the assistance of procollagen-specific molecular chaperones. Heat-shock protein 47 (HSP47) is an endoplasmic reticulum-resident chaperone that plays an essential role in normal procollagen folding, although its molecular function has not yet been clarified. Recent advances in studies on the binding specificity of HSP47 have revealed that Arg residues at Yaa positions in collagenous Gly-Xaa-Yaa repeats are critical for its interactions (Koide, T., Takahara, Y., Asada, S., and Nagata, K. (2002) J. Biol. Chem. 277, 6178-6182; Tasab, M., Jenkinson, L., and Bulleid, N. J. (2002) J. Biol. Chem. 277, 35007-35012). In the present study, we further examined the client recognition mechanism of HSP47 by taking advantage of systems employing engineered collagen model peptides. First, in vitro binding studies using conformationally constrained collagen-like peptides revealed that HSP47 only recognized correctly folded triple helices and that the interaction with the corresponding single-chain polypeptides was negligible. Second, a binding study using heterotrimeric model clients for HSP47 demonstrated a minimal requirement for the number of Arg residues in the triple helix. Finally, a cross-linking study using photoreactive collagenous peptides provided information about the spatial orientation of an HSP47 molecule in the chaperone-collagen complex. The obtained results led to the development of a new model of HSP47-collagen complexes that differs completely from the previously proposed “flying capstan model” (Dafforn, T. R., Della, M., and Miller, A. D. (2001) J. Biol. Chem. 276, 49310-49319).

Specific recognition of the collagen triple helix by chaperone HSP47. II. The HSP47-binding structural motif in collagens and related proteins

The endoplasmic reticulum-resident chaperone heat-shock protein 47 (HSP47) plays an essential role in procollagen biosynthesis. The function of HSP47 relies on its specific interaction with correctly folded triple-helical regions comprised of Gly-Xaa-Yaa repeats, and Arg residues at Yaa positions have been shown to be important for this interaction. The amino acid at the Yaa position (Yaa-3) in the N-terminal-adjoining triplet containing the critical Arg (defined as Arg0) was also suggested to be directly recognized by HSP47 (Koide, T., Asada, S., Takahara, Y., Nishikawa, Y., Nagata, K., and Kitagawa, K. (2006) J. Biol. Chem. 281, 3432-3438). Based on this finding, we examined the relationship between the structure of Yaa-3 and HSP47 binding using synthetic collagenous peptides. The results obtained indicated that the structure of Yaa-3 determined the binding affinity for HSP47. Maximal binding was observed when Yaa-3 was Thr. Moreover, the required relative spatial arrangement of these key residues in the triple helix was analyzed by taking advantage of heterotrimeric collagen-model peptides, each of which contains one Thr-3 and one Arg0. The results revealed that HSP47 recognizes the Yaa-3 and Arg0 residues only when they are on the same peptide strand. Taken together, the data obtained led us to define the HSP47-binding structural epitope in the collagen triple helix and also define the HSP47-binding motif in the primary structure. A motif search against human protein database predicted candidate clients for this molecular chaperone. The search result indicated that not all collagen family proteins require the chaperoning by HSP47.

Isolation of a novel analgesic pentapeptide, neo-kyotorphin, from bovine brain

Abstract A novel peptide isolated from the bovine brain was found to contain the Tyr-Arg (kyotorphin) unit at the C-terminal portion. This peptide in the methanol soluble fraction was isolated by gel filtration and cation exchange chromatography. We termed this peptide “neo-kyotorphin” and the amino acid sequence is Thr-Ser-Lys-Tyr-Arg. The synthetically prepared neo-kyotorphin proved to have a dose-dependent analgesic effect in mice.

Cathepsin L Plays a Major Role in Cholecystokinin Production in Mouse Brain Cortex and in Pituitary AtT-20 Cells: Protease Gene Knockout and Inhibitor Studies

Cholecystokinin (CCK) is a peptide neurotransmitter whose production requires proteolytic processing of the proCCK precursor to generate active CCK8 neuropeptide in brain. This study demonstrates the significant role of the cysteine protease cathepsin L for CCK8 production. In cathepsin L knockout (KO) mice, CCK8 levels were substantially reduced in brain cortex by an average of 75%. To evaluate the role of cathepsin L in producing CCK in the regulated secretory pathway of neuroendocrine cells, pituitary AtT-20 cells that stably produce CCK were treated with the specific cathepsin L inhibitor, CLIK-148. CLIK-148 inhibitor treatment resulted in decreased amounts of CCK secreted from the regulated secretory pathway of AtT-20 cells. CLIK-148 also reduced cellular levels of CCK9 (Arg-CCK8), consistent with CCK9 as an intermediate product of cathepsin L, shown by the decreased ratio of CCK9/CCK8. The decreased CCK9/CCK8 ratio also suggests a shift in the production to CCK8 over CCK9 during inhibition of cathepsin L. During reduction of the PC1/3 processing enzyme by siRNA, the ratio of CCK9/CCK8 was increased, suggesting a shift to the cathepsin L pathway for the production of CCK9. The changes in ratios of CCK9 compared to CCK8 are consistent with dual roles of the cathepsin L protease pathway that includes aminopeptidase B to remove NH2-terminal Arg or Lys, and the PC1/3 protease pathway. These results suggest that cathepsin L functions as a major protease responsible for CCK8 production in mouse brain cortex, and participates with PC1/3 for CCK8 production in pituitary cells.

A collagen-mimetic triple helical supramolecule that evokes integrin-dependent cell responses.

Collagen is an abundantly distributed extracellular matrix protein in mammalian bodies that maintains structural integrity of the organs and tissues. Besides its function as a structural protein, collagen has various biological functions which regulate cell adhesion, migration and differentiation. In order to develop totally synthetic collagen-surrogates, we recently reported a basic concept for preparing collagen-like triple helical supramolecules based on the self-assembly of staggered trimeric peptides with self-complementary shapes. In this paper, we add one of the specific cellular functions of the native collagen to the collagen-mimetic supramolecule. We synthesized a self-assembling peptide unit containing the integrin-binding sequence Gly-Phe-Hyp-Gly-Glu-Arg. The supramolecule carrying the sequence exhibited significant binding activity to human dermal fibroblasts. The supramolecular structure was found to be essential for function in in vitro cell culture. Cell adhesion was shown to be comparable to that of native collagen, and was further demonstrated to be mediated solely by integrin alpha 2 beta 1. Well-grown focal contacts and stress fibers were observed in cells spread on the supramolecular collagen-mimetic. The results demonstrate the potential of peptide-based artificial collagen as a biomaterial for regulating specific cellular function and fate.

Stoichiometry of subunit e in rat liver mitochondrial H+-ATP synthase and membrane topology of its putative Ca2+-dependent regulatory region

Previous studies have revealed that residues 34-65 of subunit e of mitochondrial H(+)-ATP synthase are homologous with the Ca(2+)-dependent tropomysin-binding region for troponin T and have suggested that subunit e could be involved in the Ca(2+)-dependent regulation of H(+)-ATP synthase activity. In this study, we determined the content of subunit e in H(+)-ATP synthase purified from rat liver mitochondria, and we also investigated the membrane topology of a putative Ca(2+)-dependent regulatory region of subunit e using an antibody against peptide corresponding to residues 34-65 of subunit e. Quantitative immunoblot analysis of subunit e in the purified H(+)-ATP synthase revealed that 1 mol of H(+)-ATP synthase contained 2 mol of subunit e. The ATPase activity of mitoplasts, in which the C-side of F(0) is present on the outer surface of the inner membrane, was significantly stimulated by the addition of the antibody, while the ATPase activity of submitochondrial particles and purified H(+)-ATP synthase was not stimulated. The antibody bound to mitoplasts but not to submitochondrial particles. These results suggest that the putative Ca(2+)-dependent regulatory region of subunit e is exposed on the surface of the C-side of F(0) and that subunit e is involved in the regulation of mitochondrial H(+)-ATP synthase activity probably via its putative Ca(2+)-dependent regulatory region.

Syntheses of two tyrosine-sulphate containing peptides. Leucosulfakinin (LSK)-II and cholecystokinin (CCK)-12, using the 0-p-(methylsulphinyl)benzyl serine for the selective sulphation of tyrosine

Abstract A novel approach for the synthesis of tyrosine sulphate [Tyr(SO 3 H)]-containing peptides was developed. In this approach, trifluoroacetic acid stable O-p-(methylsulphinyl)-benzyl group was used as a key protecting group for serine to achieve the selective sulphation of tyrosine.

Immunohistochemical presence of 5 α-reductase rat type I -containing cells in the rat brain

Abstract We showed immunohistochemically the localization of 5 α-reductase-containing cells in the rat brain, using a rabbit antibody generated against 5α-reductase rat type 1. The antibody was produced by injecting the synthetic peptide corresponding to the amino acids 38–53 of 5α-reductase rat type 1, conjugated to keyhole limpet hemocyanin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Western blot analysis revealed that this antiserum recognized the protein with a molecular weight of 29 000 Da. The inununoreactive cells were distributed throughout the brain and they were preferentially located in the white matter rather than in the grey matter. These cells were mostly small and round and had a few fine processes. The immunoreaction was confined to the cytoplasm and processes. These findings indicate that 5α-reductase rat type 1-containing cells are widely distributed in the rat brain and are located preferentially in the white matter rather than in the grey matter.