Effect of nanocoating with rhamnogalacturonan‐I on surface properties and osteoblasts response.
Gurzawska, K., Svava, R., Syberg, S., Yihua, Y., Haugshøj, K. B., Damager, I., Ulvskov, P., Christensen, L. H., Gotfredsen, K. & Jørgensen, N. R. (2012). Journal of Biomedical Materials Research Part A, 100(3), 654-664.
Long-term stability of titanium implants are dependent on a variety of factors. Nanocoating with organic molecules is one of the methods used to improve osseointegration. Therefore, the aim of this study is to evaluate the in vitro effect of nanocoating with pectic rhamnogalacturonan-I (RG-I) on surface properties and osteoblasts response. Three different RG-Is from apple and lupin pectins were modified and coated on amino-functionalized tissue culture polystyrene plates (aminated TCPS). Surface properties were evaluated by scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy. The effects of nanocoating on proliferation, matrix formation and mineralization, and expression of genes (real-time PCR) related to osteoblast differentiation and activity were tested using human osteoblast-like SaOS-2 cells. It was shown that RG-I coatings affected the surface properties. All three RG-I induced bone matrix formation and mineralization, which was also supported by the finding that gene expression levels of alkaline phosphatase, osteocalcin, and collagen type-1 were increased in cells cultured on the RG-I coated surface, indicating a more differentiated osteoblastic phenotype. This makes RG-I coating a promising and novel candidate for nanocoatings of implants.
Characterization of an exo-β-1,3-galactanase from Clostridium thermocellum.
Ichinose, H., Kuno, A., Kotake, T., Yoshida, M., Sakka, K., Hirabayashi, J., Tsumuraya, Y. & Kaneko, S. (2006). Applied and Environmental Microbiology, 72(5), 3515-3523.
A gene encoding an exo-β-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-β-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50°C and catalyzes hydrolysis only of β-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from β-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a β-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-β-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including β-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a β-1,3-galactobiose, β-1,4-galactosyl glucose, or β-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward β-1,3-galactan and α-L-arabinofuranosidase-treated AGP.
An exo-β-1,3-galactanase having a novel β-1,3-galactan-binding module from Phanerochaete chrysosporium.
Ichinose, H., Yoshida, M., Kotake, T., Kuno, A., Igarashi, K., Tsumuraya, Y., Samejima, M., Hirabayashi, J., Kobayashi, H. & Kaneko, S. (2005). Journal of Biological Chemistry, 280(27), 25820-25829.
An exo-β-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-β-1,3-galactanase indicated that the enzyme consists of an N-terminal catalytic module with similarity to glycoside hydrolase family 43 and an additional unknown functional domain similar to carbohydrate-binding module family 6 (CBM6) in the C-terminal region. The molecular mass of the recombinant enzyme was estimated as 55 kDa based on SDS-PAGE. The enzyme showed reactivity only toward β-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze β-1,4-linked galacto-oligosaccharides, β-1,6-linked galacto-oligosaccharides, pectic galactan, larch arabinogalactan, arabinan, gum arabic, debranched arabinan, laminarin, soluble birchwood xylan, or soluble oat spelled xylan. The enzyme also did not hydrolyze β-1,3-galactosyl galactosaminide, β-1,3-galactosyl glucosaminide, or β-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal β-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from β-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl β-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass β-1,6-linked galactosyl side chains. The C-terminal CBM6 did not show any affinity for known substrates of CBM6 such as xylan, cellulose, and β-1,3-glucan, although it bound β-1,3-galactan when analyzed by affinity electrophoresis. Frontal affinity chromatography for the CBM6 moiety using several kinds of terminal galactose-containing oligosaccharides as the analytes clearly indicated that the CBM6 specifically interacted with oligosaccharides containing a β-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.
Real-time imaging of cellulose reorientation during cell wall expansion in Arabidopsis roots.
Anderson, C. T., Carroll, A., Akhmetova, L. & Somerville, C. (2010). Plant Physiology, 152(2), 787-796.
Cellulose forms the major load-bearing network of the plant cell wall, which simultaneously protects the cell and directs its growth. Although the process of cellulose synthesis has been observed, little is known about the behavior of cellulose in the wall after synthesis. Using Pontamine Fast Scarlet 4B, a dye that fluoresces preferentially in the presence of cellulose and has excitation and emission wavelengths suitable for confocal microscopy, we imaged the architecture and dynamics of cellulose in the cell walls of expanding root cells. We found that cellulose exists in Arabidopsis (Arabidopsis thaliana) cell walls in large fibrillar bundles that vary in orientation. During anisotropic wall expansion in wild-type plants, we observed that these cellulose bundles rotate in a transverse to longitudinal direction. We also found that cellulose organization is significantly altered in mutants lacking either a cellulose synthase subunit or two xyloglucan xylosyltransferase isoforms. Our results support a model in which cellulose is deposited transversely to accommodate longitudinal cell expansion and reoriented during expansion to generate a cell wall that is fortified against strain from any direction.
Family 6 carbohydrate‐binding modules display multiple β1,3‐linked glucan‐specific binding interfaces.
Correia, M. A. S., Pires, V. M. R., Gilbert, H. J., Bolam, D. N., Fernandes, V. O., Alves, V. D., Prates, J. A. M., Ferreira, L. M. A. & Fontes, C. M. G. (2009). FEMS Microbiology Letters, 300(1), 48-57.
Noncatalytic carbohydrate-binding modules (CBMs), which are found in a variety of carbohydrate-degrading enzymes, have been grouped into sequence-based families. CBMs, by recruiting their appended enzymes onto the surface of the target substrate, potentiate catalysis particularly against insoluble substrates. Family 6 CBMs (CBM6s) display unusual properties in that they present two potential ligand-binding sites termed clefts A and B, respectively. Cleft B is located on the concave surface of the β-sandwich fold while cleft A, the more common binding site, is formed by the loops that connect the inner and the outer β-sheets. Here, we report the biochemical properties of CBM6-1 from Cellvibrio mixtus CmCel5A. The data reveal that CBM6-1 specifically recognizes β1,3-glucans through residues located both in cleft A and in cleft B. In contrast, a previous report showed that a CBM6 derived from a Bacillus halodurans laminarinase binds to β1,3-glucans only in cleft A. These studies reveal a different mechanism by which a highly conserved protein platform can recognize β1,3-glucans.