A mutant-cell library for systematic analysis of heparan sulfate structure-function relationships.

Heparan sulfate (HS) is a complex linear polysaccharide that modulates a wide range of biological functions. Elucidating the structure-function relationship of HS has been challenging. Here we report the generation of an HS-mutant mouse lung endothelial cell library by systematic deletion of HS genes expressed in the cell.

Expression system for structural and functional studies of human glycosylation enzymes.

Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes.

Helicobacter pylori chronic infection and mucosal inflammation switches the human gastric glycosylation pathways.

Helicobacter pylori exploits host glycoconjugates to colonize the gastric niche. Infection can persist for decades promoting chronic inflammation, and in a subset of individuals lesions can silently progress to cancer. This study shows that H.

Changes in glycosaminoglycan structure on differentiation of human embryonic stem cells towards mesoderm and endoderm lineages.

Background: Proteoglycans are found on the cell surface and in the extracellular matrix, and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate, and therefore represent an excellent tool to manipulate these pathways. Despite their importance, there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation.

Regulation of glycan structures in murine embryonic stem cells: combined transcript profiling of glycan-related genes and glycan structural analysis.

The abundance and structural diversity of glycans on glycoproteins and glycolipids are highly regulated and play important roles during vertebrate development. Because of the challenges associated with studying glycan regulation in vertebrate embryos, we have chosen to study mouse embryonic stem (ES) cells as they differentiate into embryoid bodies (EBs) or into extraembryonic endodermal (ExE) cells as a model for cellular differentiation. We profiled N- and O-glycan structures isolated from these cell populations and examined transcripts encoding the corresponding enzymatic machinery for glycan biosynthesis in an effort to probe the mechanisms that drive the regulation of glycan diversity.

Transcriptional regulation of the protocadherin β cluster during Her-2 protein-induced mammary tumorigenesis results from altered N-glycan branching.

Changes in the levels of N-acetylglucosaminyltransferase V (GnT-V) can alter the function of several types of cell surface receptors and adhesion molecules by causing altered N-linked glycan branching. Using a her-2 mammary tumor mouse model, her-2 receptor signaling was down-regulated by GnT-V knock-out, resulting in a significant delay in the onset of her-2-induced mammary tumors. To identify the genes that contributed to this GnT-V regulation of early events in tumorigenesis, microarray analysis was performed using her-2 induced mammary tumors from wild-type and GnT-V-null mice.

Transcript analysis of stem cells.

Quantitative real-time polymerase chain reaction (qRT-PCR) is a flexible and scalable method for analyzing transcript abundance that can be used at a single gene or high-throughput (>100 genes) level. Information obtained from this technique can be used as an indicator of potential regulation of glycosylation at the transcript level when combined with glycan structural or protein abundance data. This chapter describes detailed methods to design and perform qRT-PCR analyses and provides examples of information that can be obtained from the technique.