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

Abstract

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 a HS mutant mouse lung endothelial cell library by systematic deletion of HS genes expressing in the cell. We applied this library to answer several fundamental questions about HS biology including: 1) determining that strictly defined fine structure of HS, not its overall sulfation degree, is more important for FGF2-FGFR1 signaling; 2) defining the epitope features of commonly used anti-HS phage display antibodies; and 3) delineating the fine inter-regulation networks of HS modification and chain length by HS genes in mammalian cells and at a cell type specific level. Our mutant cell library will enable robust and systematic interrogation of the roles and related structures of HS in a cellular context.

3‐O‐Sulfation of Heparan Sulfate Enhances Tau Interaction and Cellular Uptake

“…The selection of Hs3st1 was based on the expression profiles of HS 3‐ O ‐sulfotransferases in primary mouse cerebral cortex neurons determined by RNA‐seq, with the highest expression level observed for Hs3st1 among all Hs3st s (see Figure S4). The Hs3st1 −/− MLEC line was derived from the WT parent line using CRISPR‐Cas9 gene‐editing and expressed normal levels of NS, 6‐ O ‐S, and 2‐ O ‐S (see Figure S5A), but reduced level of 3‐ O ‐S (confirmed by significantly reduced cell surface binding to antithrombin III requiring a 3‐ O ‐S for binding, see Figure S5B) . Biotinylated tau was generated and incubated with cells, followed by washing and detection of surface‐bound tau with streptavidin‐HRP.…”

Shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides

“…Purification of GAG oligosaccharides from tissues with current chromatography methods is notoriously challenging due to the complex mixtures obtained from such heterogenous sources. However, an emerging source for standards may be cell lines with engineered capacities for producing limited subsets of GAG features 3,48 , which should simplify separation and isolation of homogeneous and heterogenous oligosaccharides. In particular, the GAGOme method employing the robust Chinese hamster ovary (CHO) cell line to engineer and produce specifically restricted repertoires of GAGs with distinct features can help reduce complexities.…”

3‐O‐Sulfation of Heparan Sulfate Enhances Tau Interaction and Cellular Uptake

“…The Hs3st1 À/À MLEC line was derived from the WT parent line using CRISPR-Cas9 gene-editing and expressed normal levels of NS,6 -O-S,a nd 2-O-S (see Figure S5A), but reduced level of 3-O-S (confirmed by significantly reduced cell surface binding to antithrombin III requiring a3 -O-S for binding,s ee Figure S5B). [25] Biotinylated tau was generated and incubated with cells,f ollowed by washing and detection of surface-bound tau with streptavidin-HRP.T au bound strongly to the surface of WT MLECs,while the binding was greatly diminished on the Hs3st1 À/À MLECs surface,showing that 3-O-S strongly enhances HS binding of tau on the cell surface ( Figure 3A). We next incubated both WT and Hs3st1 À/À cells with Alexa488-labeled full-length tau (tau-Alexa) for 12 hours,f ollowed by detection with both flow cytometry ( Figure 3B)a nd confocal imagining (Figure 3C)tofurther investigate the effects of 3-O-S deletion on the cellular uptake of tau.…”