Characterization of the recognition of blood group B trisaccharide derivatives by the lectin from Marasmius oreades using frontal affinity chromatography-mass spectrometry

A novel lectin from the mushroom Marasmius oreades (MOA) has been shown to bind to human blood group B oligosaccharides [1]. In the present work we examine the binding of a series of analogues of the blood group B-trisaccharide, αGal(1-3)[αFuc(1-2)]βGal-OR (1, R = (CH 2 ) 8 COOMe). MOA was biotinylated and immobilized on a micro column (9.8 μL) for evaluation by Frontal Affinity Chromatography-Mass Spectrometry (FAC-MS) [2]. The trisaccharide 1 was found to be the epitope needed for maximum recognition ( K d = 3.6 μM). A series of synthetic deoxygenated and O-methylated analogues of the B-trisaccharide (R = OMe) were then screened against the lectin, and the key structural elements for binding were determined. OH-4 of the β-Gal residue and OH-2 of the α-Gal residue were found to be critical for recognition. The FAC-MS technique also proved powerful in evaluating mixtures of compounds. Since the solution NMR structure and crystal structure of the B-trisaccharide are known [3], we propose the specific surface of the trisaccharide that is recognized by the lectin. Published in 2003.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45744/1/10719_2004_Article_5125615.pd

Dynein links engulfment and execution of apoptosis via CED-4/Apaf1 in C. <i>elegans</i>

Abstract Apoptosis ensures removal of damaged cells and helps shape organs during development by removing excessive cells. To prevent the intracellular content of the apoptotic cells causing damage to surrounding cells, apoptotic cells are quickly cleared by engulfment. Tight regulation of apoptosis and engulfment is needed to prevent several pathologies such as cancer, neurodegenerative and autoimmune diseases. There is increasing evidence that the engulfment machinery can regulate the execution of apoptosis. However, the underlying molecular mechanisms are poorly understood. We show that dynein mediates cell non-autonomous cross-talk between the engulfment and apoptotic programs in the Caenorhabditis elegans germline. Dynein is an ATP-powered microtubule-based molecular motor, built from several subunits. Dynein has many diverse functions including transport of cargo around the cell. We show that both dynein light chain 1 (DLC-1) and dynein heavy chain 1 (DHC-1) localize to the nuclear membrane inside apoptotic germ cells in C. elegans. Strikingly, lack of either DLC-1 or DHC-1 at the nuclear membrane inhibits physiological apoptosis specifically in mutants defective in engulfment. This suggests that a cell fate determining dialogue takes place between engulfing somatic sheath cells and apoptotic germ cells. The underlying mechanism involves the core apoptotic protein CED-4/Apaf1, as we find that DLC-1 and the engulfment protein CED-6/GULP are required for the localization of CED-4 to the nuclear membrane of germ cells. A better understanding of the communication between the engulfment machinery and the apoptotic program is essential for identifying novel therapeutic targets in diseases caused by inappropriate engulfment or apoptosis

An efficient arabinoxylan-debranching α-L-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site.

An α-L-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; k cat = 178/s, K m = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3-5 (37-80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-L-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Mutants of the predicted general acid (Glu(188)) and base (Asp(28)) catalysts, and the general acid pK a modulator (Asp(136)) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley β-glucan retarded migration of AnAbf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors. Trp(23) and Tyr(44), situated about 30 Å from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus SthAbf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70 % activity towards WAX with K i of WAX substrate inhibition increasing 4-7-folds, but lost 77-96 % activity for the AXOS. The Y44A single mutant had less effect, suggesting Trp(23) is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp(23) and Tyr(44) belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.This work is supported by the Danish Council for Independent Research|Natural Sciences (FNU) [grant number 09-072151], by 1/3 PhD fellowship from the Technical University of Denmark (to CW) and by a Hans Christian Ørsted postdoctoral fellowship from DTU (to DC).This is the author accepted manuscript. The final version is available from Springer via http://dx.doi.org/10.1007/s00253-016-7417-

The α(1,3)fucosyltransferases FucT-IV and FucT-VII Exert Collaborative Control over Selectin-Dependent Leukocyte Recruitment and Lymphocyte Homing

AbstractE-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by α(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV−/−/FucT-VII−/− mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII−/− mice is revoked in FucT-IV−/−/FucT-VII−/− mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing