Highly sensitive quantification of bacterial chondroitin in a culture based on ELISA techniques.

Some bacteria produce non-sulfated chondroitin (CH). Accurate, rapid, and high throughput methods to quantify CH in fermented cultures helps to improve microbial breeding and fermentation conditions efficiently. In this study, highly sensitive methods to quantify bacterial CH were developed based on ELISA techniques. An assay using an anti-K4 antiserum successfully determined the concentration of fructosylated CH in the range from 9 to 800 ng/mL. The method also enabled the determination of CH concentration exceeding 9 µg/mL. To improve the assay sensitivity for CH, hyaluronan (HA) binding protein (HABP) was applied instead of a capture antibody. HABP was bound to CH, but not to chemically desulfated chondroitin sulfate or fructosylated CH. The quantification limit of CH was 18 µg/mL in the HA assay using HABP. Replacing the HA-coated microplate with a CH-coated microplate increased the sensitivity >1000 times (assay range = 14 to 1000 ng/mL). Pretreatment with hyaluronidase enabled us to accurately quantify CH in samples mixed with HA.

NMR studies on the interaction of sugars with the C-terminal domain of an R-type lectin from the earthworm Lumbricus terrestris.

The R-type lectin EW29, isolated from the earthworm Lumbricus terrestris, consists of two homologous domains (14,500 Da) showing 27% identity with each other. The C-terminal domain (Ch; C-half) of EW29 (EW29Ch) has two sugar-binding sites in subdomains alpha and gamma, and the protein uses these sugar-binding sites for its function as a single-domain-type hemagglutinin. In order to determine the sugar-binding ability and specificity for each of the two sugar-binding sites in EW29Ch, ligand-induced chemical-shift changes in EW29Ch were monitored using (1)H-(15)N HSQC spectra as a function of increasing concentrations of lactose, melibiose, D-galactose, methyl alpha-D-galactopyranoside and methyl beta-D-galactopyranoside. Shift perturbation patterns for well-resolved resonances confirmed that all of these sugars associated independently with the two sugar-binding sites of EW29Ch. NMR titration experiments showed that the sugar-binding site in subdomain alpha had a slow or intermediate exchange regime on the chemical-shift timescale (K(d) = 10(-2) to 10(-1) mM), whereas that in subdomain gamma had a fast exchange regime for these sugars (K(d) = 2-6 mM). Thus, our results suggest that the two sugar-binding sites of EW29Ch in the same molecule retain its hemagglutinating activity, but this activity is 10-fold lower than that of the whole protein because EW29Ch has two sugar-binding sites in the same molecule, one of which has a weak binding mode.

Enrichment strategies for glycopeptides.

In order to understand glycoprotein functionality, information on the structure of both the core proteins and the glycan moieties is necessary. From a practical viewpoint, glycopeptides rather than whole glycoproteins are the general targets for structural analysis, which is primarily carried out by employing mass spectrometry (MS). Using the "glycoproteomics" concept, several techniques have recently been developed to allow the preparation of a series of reference glycopeptides. In this chapter, we describe two selective capturing methods for glycopeptides, i.e., lectin-affinity chromatography and polysaccharide hydrophilic affinity physicochemical chromatography. The combined use of these methods effectively removes non-glycosylated peptides, the inclusion of which substantially interferes with glycopeptide ionization in MS analysis.

Crystallographic snapshots of an entire reaction cycle for a retaining xylanase from Streptomyces olivaceoviridis E-86.

Retaining glycosyl hydrolases, which catalyse both glycosylation and deglycosylation in a concerted manner, are the most abundant hydrolases. To date, their visualization has tended to be focused on glycosylation because glycosylation reactions can be visualized by inactivating deglycosylation step and/or using substrate analogues to isolate covalent intermediates. Furthermore, during structural analyses of glycosyl hydrolases with hydrolytic reaction products by the conventional soaking method, mutarotation of an anomeric carbon in the reaction products promptly and certainly occurs. This undesirable structural alteration hinders visualization of the second step in the reaction. Here, we investigated X-ray crystallographic visualization as a possible method for visualizing the conformational itinerary of a retaining xylanase from Streptomyces olivaceoviridis E-86. To clearly define the stereochemistry at the anomeric carbon during the deglycosylation step, extraneous nucleophiles, such as azide, were adopted to substitute for the missing base catalyst in an appropriate mutant. The X-ray crystallographic visualization provided snapshots of the components of the entire reaction, including the E*S complex, the covalent intermediate, breakdown of the intermediate and the enzyme-product (E*P)complex.

Rational affinity purification of native Streptomyces family 10 xylanase.

Xylanase SoXyn10A from Streptomyces olivaceoviridis E-86 comprises a family 10 catalytic module linked to a family 13 carbohydrate-binding module (SoCBM13). The SoCBM13 has a beta-trefoil structure, with binding sites in each subdomain (alpha, beta and gamma). Subdomain alpha, but not subdomains beta and gamma, binds tightly to lactose. It was, therefore, thought that immobilized lactose could be used for the affinity purification of SoXyn10A. Lactosyl-Sepharose was prepared and tested as an affinity matrix. SoXyn10A produced from the cloned xyn10A gene by Escherichia coli, and native SoXyn10A in culture supernatants from S. olivaceoviridis, were purified to homogeneity in a single step by affinity chromatography using this matrix. This simple purification of SoXyn10A makes the enzyme an attractive candidate for applications requiring xylanase. The CBM also has the potential for use as an affinity tag for the purification of other proteins.