No abstract

Galectin-1 (Gal-1), a member of the beta-galactoside-binding animal lectin family, has a wide range of biological activities, which makes it an attractive target for medical applications. Unlike other galectins, Gal-1 is susceptible to oxidation at cysteine residues, which is troublesome for in vitro/vivo studies. To overcome this problem, we prepared a cysteine-less mutant of Gal-1 (CSGal-1) by substituting all cysteine residues with serine residues. In the case of wild-type Gal-1, the formation of covalent dimers/oligomers was evident after 10 days of storage in the absence of a reducing agent with a concomitant decrease in hemagglutination activity, while CSGal-1 did not form multimers and retained full hemagglutination activity after 400 days of storage. Frontal affinity chromatography showed that the sugar-binding specificity and affinity of Gal-1 for model glycans were barely affected by the mutagenesis. Gal-1 is known to induce cell signaling leading to an increase in the intracytoplasmic calcium concentration and to cell death. CSGal-1 is also capable of inducing calcium flux and growth inhibition in Jurkat cells, which are comparable to or more potent than those induced by Gal-1. The X-ray structure of the CSGal-1/lactose complex has been determined. The structure of CSGal-1 is almost identical to that of wild-type human Gal-1, showing that the amino acid substitutions do not affect the overall structure or carbohydrate-binding site structure of the protein. These results indicate that CSGal-1 can serve as a stable substitute for Gal-1.

No abstract

Thermoactinomyces vulgaris R‐47 α‐amylase 1 (TVAI) has unique hydrolyzing activities for pullulan with sequence repeats of α‐(1,4), α‐(1,4), and α‐(1,6) glycosidic linkages, as well as for starch. TVAI mainly hydrolyzes α‐(1,4) glycosidic linkages to produce a panose, but it also hydrolyzes α‐(1,6) glycosidic linkages with a lesser efficiency. X‐ray structures of three complexes comprising an inactive mutant TVAI (D356N or D356N/E396Q) and a pullulan model oligosaccharide (P2; [Glc‐α‐(1,6)‐Glc‐α‐(1,4)‐Glc‐α‐(1,4)]2 or P5; [Glc‐α‐(1,6)‐Glc‐α‐(1,4)‐Glc‐α‐(1,4)]5) were determined. The complex D356N/P2 is a mimic of the enzyme/product complex in the main catalytic reaction of TVAI, and a structural comparison with Aspergillus oryzaeα‐amylase showed that the (–) subsites of TVAI are responsible for recognizing both starch and pullulan. D356N/E396Q/P2 and D356N/E396Q/P5 provided models of the enzyme/substrate complex recognizing the α‐(1,6) glycosidic linkage at the hydrolyzing site. They showed that only subsites −1 and −2 at the nonreducing end of TVAI are effective in the hydrolysis of α‐(1,6) glycosidic linkages, leading to weak interactions between substrates and the enzyme. Domain N of TVAI is a starch‐binding domain acting as an anchor in the catalytic reaction of the enzyme. In this study, additional substrates were also found to bind to domain N, suggesting that domain N also functions as a pullulan‐binding domain.

Background: Helper T (Th) cells are deeply involved in the pathophysiology of bronchial asthma, such as eosinophilic inflammation, bronchial hyperresponsiveness (BHR), airflow limitation and remodeling. It is still unclear whether Th cells contribute to BHR independently of eosinophilic inflammation. The double GATA (dblGATA) site is a high-affinity GATA-binding site in the GATA-1 promoter. dblGATA site-deficient (ΔdblGATA) mice lack eosinophils. Method: Ovalbumin (OVA)-reactive Th clones were transferred into ΔdblGATA and wild-type (WT) mice of BALB/c background. The number of eosinophils in the bronchoalveolar lavage fluid (BALF) and bronchial responsiveness to methacholine were examined after OVA challenge. Results: The number of BALF eosinophils was significantly increased in WT mice, but not detectable in ΔdblGATA mice. BHR was also induced in WT mice, but significantly attenuated in ΔdblGATA mice. Conclusion: Eosinophils are involved in T-cell-mediated BHR.