Modulated glycosylation of proteoglycans during differentiation of human B lymphocytes

AbstractProteoglycans are mediators of cellular adhesion and regulate growth factor activities. Proteoglycans of B lymphocytes undergo structural changes during B cell ontogeny which may correspond to the specific requirements of the respective microenvironment of the maturing cell. We analyzed three human B cell lines representing pre-B cells (Nalm-6), activated B cells (Jok-1) and plasma cells (U266) for their cellular proteoglycans. Gel filtration of the 35S-labeled macromolecules of the three cell lines revealed an increase in size in the order Nalm-6 < Jok-1 < U266. In Jok-1 and U266 cells the major pool of proteoglycans consisted of proteochondroitin sulfates of 50 to 90 kDa. These proteoglycans carried a protein core of approx. 30 kDa to which 1 to 3 glycosaminoglycan chains in the range of 28 to 32 kDa were attached. In Nalm-6 cells only free chondroitin sulfate chains of 23 kDa, but no intact proteoglycans, were detected. Chondroitin sulfate chains were predominantly composed of chondroitin-4-sulfate, those of Nalm-6 and U266 cells additionally contained 10–20% of unsulfated disaccharides. In U266 cells 30% of glycosaminoglycans consisted of heparan sulfate either bound to pure proteoheparan sulfate or to chondroitin sulfate/heparan sulfate hybrid-proteoglycans. Earlier, syndecan-1 was described as a hybrid proteoglycan containing heparan sulfate/chondroitin sulfate chains which is transcribed by murine B cells at early and late maturation stages. In order to see whether syndecan is transcribed by the human B cell lines used here, we measured expression of syndecan mRNA by the reverse transcriptase polymerase chain reaction. Similar to murine lymphocytes, syndecan-specific mRNA was detected in Nalm-6 and U266 cells, equivalent to early and late B cells, but not in lymphoblastoid Jok-1 cells. However, Nalm-6 cells do not produce proteoheparan sulfate. In these cells, syndecan synthesis may be blocked at the translational level. Also, the proteoglycans of U266 are different from syndecan-1 in their composition of glycosaminoglycans and in size of protein cores. Together, these results indicate that the major pool of proteoglycans produced by human B cells consists of proteochondroitin sulfate and additionally in later stages of a smaller proportion of proteoheparan sulfate which is not identical to syndecan-1. During distinct phases of B cell differentiation, modulations in the glycosaminoglycan moiety concerning size and sulfation of glycosaminoglycan chains were also found

Monoclonal antibodies against the human lymphocyte differentiation antigen CD 76 bind to gangliosides

AbstractTwo monoclonal antibodies, HD 66 and CRIS-4, by which the new CD 76 B-cell-associated cluster was defined, bound to several gangliosides (sialic acid containing glycolipids) of different polarity. One of the gangliosides recognized by HD 66 could be identified as NeuAcα2-6Galβl-4GlcNAcβl-3Galβl-4Glc-βl-l'Cer. This antigen was enzymatically synthesized. Sialidase treatment of the ganglioside antigens abolished binding of HD 66 and CRIS-4

Modulation of the CD95-Induced Apoptosis: The Role of CD95 N-Glycosylation

Protein modifications of death receptor pathways play a central role in the regulation of apoptosis. It has been demonstrated that O-glycosylation of TRAIL-receptor (R) is essential for sensitivity and resistance towards TRAIL-mediated apoptosis. In this study we ask whether and how glycosylation of CD95 (Fas/APO-1), another death receptor, influences DISC formation and procaspase-8 activation at the CD95 DISC and thereby the onset of apoptosis. We concentrated on N-glycostructure since O-glycosylation of CD95 was not found. We applied different approaches to analyze the role of CD95 N-glycosylation on the signal transduction: in silico modeling of CD95 DISC, generation of CD95 glycosylation mutants (at N136 and N118), modulation of N-glycosylation by deoxymannojirimycin (DMM) and sialidase from Vibrio cholerae (VCN). We demonstrate that N-deglycosylation of CD95 does not block DISC formation and results only in the reduction of the procaspase-8 activation at the DISC. These findings are important for the better understanding of CD95 apoptosis regulation and reveal differences between apoptotic signaling pathways of the TRAIL and CD95 systems

Functions and biosynthesis of O-acetylated sialic acids

Sialic acids have a pivotal functional impact in many biological interactions such as virus attachment, cellular adhesion, regulation of proliferation, and apoptosis. A common modification of sialic acids is O-acetylation. O-Acetylated sialic acids occur in bacteria and parasites and are also receptor determinants for a number of viruses. Moreover, they have important functions in embryogenesis, development, and immunological processes. O-Acetylated sialic acids represent cancer markers, as shown for acute lymphoblastic leukemia, and they are known to play significant roles in the regulation of ganglioside-mediated apoptosis. Expression of O-acetylated sialoglycans is regulated by sialic acid-specific O-acetyltransferases and O-acetylesterases. Recent developments in the identification of the enigmatic sialic acid-specific O-acetyltransferase are discussed

Understanding the Specificity of Human Galectin-8C Domain Interactions with Its Glycan Ligands Based on Molecular Dynamics Simulations

<div><p>Human Galectin-8 (Gal-8) is a member of the galectin family which shares an affinity for β-galactosides. The tandem-repeat Gal-8 consists of a N- and a C-terminal carbohydrate recognition domain (N- and C-CRD) joined by a linker peptide of various length. Despite their structural similarity both CRDs recognize different oligosaccharides. While the molecular requirements of the N-CRD for high binding affinity to sulfated and sialylated glycans have recently been elucidated by crystallographic studies of complexes with several oligosaccharides, the binding specificities of the C-CRD for a different set of oligosaccharides, as derived from experimental data, has only been explained in terms of the three-dimensional structure for the complex C-CRD with lactose. In this study we performed molecular dynamics (MD) simulations using the recently released crystal structure of the Gal-8C-CRD to analyse the three-dimensional conditions for its specific binding to a variety of oligosaccharides as previously defined by glycan-microarray analysis. The terminal β-galactose of disaccharides (LacNAc, lacto-N-biose and lactose) and the internal β-galactose moiety of blood group antigens A and B (BGA, BGB) as well as of longer linear oligosaccharide chains (di-LacNAc and lacto-N-neotetraose) are interacting favorably with conserved amino acids (H53, R57, N66, W73, E76). Lacto-N-neotetraose and di-LacNAc as well as BGA and BGB are well accommodated. BGA and BGB showed higher affinity than LacNAc and lactose due to generally stronger hydrogen bond interactions and water mediated hydrogen bonds with α1-2 fucose respectively. Our results derived from molecular dynamics simulations are able to explain the glycan binding specificities of the Gal-8C-CRD in comparison to those of the Gal-8N -CRD.</p> </div

Structure superimposition and degree of sequence identity.

<p>Three-dimensional structural alignments and sequence identity of members of the galectin family based on RMSD calculated by using the PDBeFold webserver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059761#pone.0059761-Krissinel1" target="_blank">[49]</a>.</p

Torsional analysis of bound ligands.

<p>Average glycosidic torsion angles for bound ligands in the Gal-8C domain (standard deviation). φ and ψ values for glycosidic linkages using the NMR definition as H1-C1-O1-C_x and C1-O1-C_x-H_x respectively.</p

Multiple sequence alignments of the human galectin members.

<p>Conserved amino acids are shown in bold, amino acids which play important roles in interactions apart from conserved residues in Gal-8C are shown in red and in blue for Gal-8N. This multiple sequence alignment was carried out by MAFFT web server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059761#pone.0059761-Katoh1" target="_blank">[48]</a>.</p

Ligand binding of the galectin-8C domain.

<p>The Gal-8C binding site with (<b>A</b>) LacNAc, II, (<b>B</b>) di-LacNAc, (<b>C</b>) Lactose, (<b>D</b>) Lacto-N-neotetrose, (<b>E</b>) BGA, and (<b>F</b>) BGB. Ligands are shown as stick models and the surface of the protein-binding site in violet color. The ligands are color-coded (β-galactose: red; N-acetyl-glucosamine: green; glucose: blue; fucose: cyan; α-galactose and α- N-acetyl-galactosamine: yellow; downstream hydroxy group: white. Hydrogen bonds are shown as yellow dotted line. A snapshot which contains a maximum number of intermolecular hydrogen bonds is displayed. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059761#pone.0059761.s007" target="_blank"><b>File S1</b></a> for details of hydrogen bond interactions of each complex. The figure was designed using PyMOL Molecular Graphics System (DeLano Scientific, Palo Alto, CA).</p

Oligosaccharides ranked by calculated binding energy towards the Gal-8C domain.

<p>The values are derived from MMGBSA energies and entropy values calculated using NMode.</p