Unknown,Transcriptomics,Genomics,Proteomics

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To understand how glycosylation on human B cells is regulated during differentiation and activation

ABSTRACT: Our goal is to understand how glycosylation on human B cells is regulated during the differentiation and activation. Accumulating evidences have indicated that functions of immune cells are regulated in a glycan-dependent manner. One of well-known examples is B cell regulation by CD22 through its association with sialylated glycans. While recent studies uncovered that B cell activation leads to loss of high affinity glycan ligands for human CD22 (Neu5Ac-a2,6-Gal-B1,4-GlcNAc(6-sulfo)), the gene expression profile of human B cells before and after activation is unknown. Therefore we would like to perform gene expression analysis of human B cells before and after activation. We already have published the glycan profiling and gene expression of mouse B cells before and after activation done in collaboration with the CFG. This study revealed programmed changes in glycosylation relevant to regulation of B cell signaling by CD22. Since the ligands of human CD22 differ in several respects from that of mouse CD22, these experiments are relevant to the evolution of siglec ligands and their conserved functions in B cell biology. In brief, the experimental protocol is as follows: human peripheral B cells are isolated from human peripheral blood of healthy donors and stimulated for 0, 24, 48 and 72 hrs. Human B cells were isolated from human peripheral blood of healthy donors by the negative selection kit (Miltenyi). For the resting B cell samples, isolated B cells were immediately shock-frozen in the liquid N2 and kept at -80˚C until the RNA extraction. For the activated B cell samples, the cells were activated in the RPMI medium (Invitrogen) supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100 U/mL penicillin, 100 g/mL streptomycin, 1 mM non-essential amino acid, 1 mM sodium pyruvate, and 50 M 2-melcaptoethanol, 10 g/mL anti-human IgA, G, M (Jackson Immunoresearch), and 50 nM CpG ODN 2006 (Invivogen) for 3 days. Total RNA was isolated using the TRIzol reagent (Invitrogen).

ORGANISM(S): Homo sapiens

SUBMITTER: Steven Head

PROVIDER: E-GEOD-31932 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:This data set was downloaded from MetaboLights (http://www.ebi.ac.uk/metabolights/) accession number MTBLS227 Abstract:"Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between a2,3- and a2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation."

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To understand how glycosylation on human B cells is regulated during differentiation and activation

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