Masayo Ogiso

Enzymatic degradation behavior of porous silk fibroin sheets

We investigated the degradation behavior of porous silk fibroin sheets by in vitro enzymatic experiments with alpha-chymotrypsin, collagenase IA, and protease XIV. With 1.0 U/ml protease XIV, 70% of a silk fibroin sheet was degraded within 15 days at 37 degrees C. When the fibroin sheet was exposed to collagenase IA, the amount of Silk II crystalline structure in the sheets decreased slightly, and a small amount of Silk I crystalline structure was formed. When protease XIV was used, almost all Silk II disappeared, but the crystallinity increased overall because the amount of Silk I increased. During digestion with protease XIV, the pore size of the fibroin sheets increased with increasing degradation time, until the sheets finally collapsed and became totally shapeless. The average molecular weight of the products after degradation with the three enzymes followed the order protease XIV < collagenase IA < alpha-chymotrypsin. More than 50% of the products resulting from degradation with protease XIV were free amino acids.

Carbohydrate immobilized on a dendrimer-coated colloidal gold surface for fabrication of a lectin-sensing device based on localized surface plasmon resonance spectroscopy.

Carbohydrate-mediated functions in biological systems have generated considerable interest in recent years. We have developed a device bearing immobilized carbohydrates on a colloidal gold surface and applied this device to the detection of carbohydrate-binding molecules by using localized surface plasmon resonance (LSPR) spectroscopy. The sensing device was constructed by using cyanuric chloride as an amine-linker between an amino residue of a polyamidoamine (PAMAM) dendrimer-coated colloidal gold surface and the amino residue of a 12-aminododecyl glycoside. After optimizing the construction of the device, we characterized its LSPR-based sensing capability. Binding specificity with lectins and linear range responses were obtained with the device. Our LSPR-based sensing device thus provides a label-free, low-cost detection method for use as a laboratory research tool or in medical glycan arrays.

Immobilization of carbohydrate clusters on a quartz crystal microbalance sensor surface

The immobilization of carbohydrates on gold surfaces is a prerequisite technology for carbohydrate-related studies, including those of carbohydrate-biomolecule interactions. Glycolipid domains in cell membranes, such as lipid rafts, are thought to play an important role in cell biology through their carbohydrate portions. To understand the recognition of glycolipid domains such as receptors for bacterial toxins and viruses, we immobilized clusters of carbohydrates on a gold surface by using polyamidoamine (PAMAM) dendrimers as a scaffold. The PAMAM dendrimers were adsorbed on the gold-coated surface of a quartz crystal microbalance (QCM) sensor and were observed by means of QCM with dissipation (QCM-D). After adsorption of the PAMAM dendrimers, lysoganglioside-GM(1) and 12-aminododecyl-N-acetylglucosaminide (GlcNAc-C12-NH(2)) were immobilized on the amino groups of PAMAM dendrimers by means of an NH(2) cross-linker. Immobilization of the carbohydrates was confirmed by observation of their specific interaction with anti-ganglioside GM(1) antibody or wheat germ agglutinin (WGA). Surfaces with different GlcNAc-C12-NH(2) cluster sizes and densities were prepared by varying the size of the PAMAM dendrimers or the concentration of GlcNAc-C12-NH(2) immobilized on the dendrimers, respectively. Analysis of the binding between the GlcNAc-C12-NH(2)-immobilized surface and WGA revealed that the size of the PAMAM dendrimers influenced the GlcNAc-C12-NH(2)-WGA interaction, with larger dendrimers resulting in higher WGA binding constants.

Intricate Recognition of Glycolipid-Like Compounds by HIV-1 Envelope Proteins Evaluated with Surface Plasmon Resonance Imaging

Fusion of human immunodeficiency virus (HIV) to the cell membrane occurs by the specific binding of an envelope protein of HIV-1 (gp120 and gp160) and a glycosphingolipid of the cell membrane. In this study, quantitative and array-based affinity evaluation of gp120 and gp160 was performed by surface plasmon resonance (SPR) and the SPR imaging technique using a substrate immobilized with glycolipid-like compounds (Gb3, GM3, and Lac). Quantitative affinity evaluation showed that gp160 specifically bound to Gb3 and Lac compared with GM3, whereas gp120 showed lower binding affinity and specificity. Array-based evaluation showed that gp160 binds to Gb3 more favorably than Lac and GM3.