Multidimensional, multinuclear nuclear magnetic resonance spectroscopy combined with dynamical simulated annealing has been used to determine the structure of a 110 amino acid cellulose-binding domain (CBD) from Cex, a beta-1,4-glycanase from the bacterium Cellulomonas fimi (CBDcex). An experimental data set comprising 1795 interproton NOE-derived restraints, 50 phi, 34 chi 1, and 106 hydrogen bond restraints was used to calculate 20 final structures. The calculated structures have an average root-mean-square (rms) deviation about the mean structure of 0.41 A for backbone atoms and 0.67 A for all heavy atoms when fitted over the secondary structural elements. Chromatography, ultracentrifugation, and 15N NMR relaxation experiments demonstrate that CBDcex is a dimer in solution. While attempts to measure NOEs across the dimer interface were unsuccessful, a computational strategy was employed to generate dimer structures consistent with the derived data set. The results from the dimer calculations indicate that, while the monomer topologies produced in the context of the dimer can be variable, the relative positioning of secondary structural elements and side chains present in the monomer are restored upon dimer formation. CBDcex forms an extensive beta-sheet structure with a beta-barrel fold. Titration with cellohexaose, [beta-D-glucopyranosyl-(1,4)]5-D-glucose, establishes that Trp 54 and 72 participate in cellulose binding. Analysis of the structure shows that these residues are adjacent in space and exposed to solvent. Together with other proximate hydrophilic residues, these residues form a carbohydrate-binding cleft, which appears to be a feature common to all CBDs of the same family.

Mucin-type O-glycans are classified according to their core structures. Among them, cores 2 and 4 are important for having N-acetyllactosamine side chains, which can be further modified to express various functional oligosaccharides. Previously, we discovered by cloning cDNAs that the core 2 branching enzyme, termed core 2 ␤-1,6-N-acetylglucosaminyltransferase-leukocyte type (C2GnT-L), is highly homologous to the I branching ␤-1,6-N-acetylglucosaminyltransferase (IGnT) (Bierhuizen, M. F. A., Mattei, M.-G., and Fukuda, M. (1993) Genes Dev. 7, 468 -478). Using these homologous sequences as probes, we identified an expressed sequence tag in dbEST, which has significant homology to C2GnT-L and IGnT. This approach, together with 5and 3 rapid amplification of cDNA ends, yielded a human cDNA that encompasses a whole coding region of an enzyme, termed C2GnT-mucin type (C2GnT-M). C2GnT-M has 48.2 and 33.8% identity with C2GnT-L and IGnT at the amino acid levels. The expression of C2GnT-M cDNA directed the expression of core 2 branched oligosaccharides and I antigen on the cell surface. Moreover, a soluble chimeric C2GnT-M had core 4 branching activity in addition to core 2 and I branching activities. A soluble chimeric C2GnT-L, in contrast, almost exclusively contains core 2 branching activity. Northern blot analysis demonstrated that the C2GnT-M transcripts are heavily expressed in colon, small intestine, trachea, and stomach, where mucin is produced. In contrast, the transcripts of C2GnT-L were more widely detected, including the lymph node and bone marrow. These results indicate that the newly cloned C2GnT-M plays a critical role in O-glycan synthesis in mucins and might have distinctly different roles in oligosaccharide ligand formation compared with C2GnT-L.Mucin-type glycoproteins are unique in having clusters of large numbers of O-glycans. These O-glycans contain N-acetylgalactosamine residues at reducing ends, which are linked to serine or threonine in a polypeptide (1). These attached O-glycans can be classified into several different groups according to the core structures (2). In many cells, core 1, Gal␤133GalNAc, is the major constituent of O-glycans. Core 1 oligosaccharides are converted to core 2 oligosaccharides Gal␤133(GlcNAc␤136)GalNAc when core 2 ␤-1,6-N-acetylglucosaminyltransferase (C2GnT) 1 is present (3, 4). Various ligand carbohydrates can be formed in core 2 branched oligosaccharides. For example, sialyl Le x in mucin-type glycoproteins of blood cells can be formed only in core 2 branched oligosaccharides such as NeuNAc␣233Gal␤134(Fuc␣133) GlcNAc␤136(NeuNAc␣233Gal␤133)GalNAc␣3serine/ threonine (3, 5-7).Those sialyl Le x and sulfated sialyl Le x in O-glycans have been shown to be preferential ligands for P-and L-selectin (6 -10). It has been also shown that poly-N-acetyllactosamines can be extended from core 2 branches (3,5,7,11,12). Poly-Nacetyllactosamines provide the backbone for additional modifications such as sialyl Le x . Moreover, a linear poly-N-acetyllactosamine (Gal␤134GlcNAc␤133) n can ...

Isothermal titration microcalorimetry is combined with solution-depletion isotherm data to analyze the

A delicate balance of signals regulates cell survival. One set of these signals is derived from integrin-mediated cell adhesion to the extracellular matrix (ECM). Loss of cell attachment to the ECM causes apoptosis, a process known as anoikis. In searching for proteins involved in cell adhesion-dependent regulation of anoikis, we identified Bit1, a mitochondrial protein that is released into the cytoplasm during apoptosis. Cytoplasmic Bit1 forms a complex with AES, a small Groucho/transducin-like enhancer of split (TLE) protein, and induces cell death with characteristics of caspase-independent apoptosis. Cell attachment to fibronectin counteracts the apoptotic effect of Bit1 and AES. Increasing Bit1 expression enhances anoikis, while suppressing the expression reduces it. Thus, we have elucidated an integrin-controlled pathway that is, at least in part, responsible for the cell survival effects of cell-ECM interactions.