Proteomics

Dataset Information

0

Structure of the human heparan sulfate polymerase complex EXT1-EXT2


ABSTRACT: Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating Nacetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Human EXT1 and EXT2, lacking the N-terminal transmembrane helix, were co-expressed as a secreted complex using human embryonic kidney cells (FreeStyle 293-F). We purified 0.5 mg of the complex from 900 mL suspension culture using two immobilized metal affinity chromatography steps and an interspersed protease treatment to cleave off the alkaline phosphatase fusion protein serving as a secretion signal. Mass spectrometry-based proteomic analysis confirmed the quality of the sample with EXT1 and EXT2 found to be the most abundant proteins, and being present in comparable amounts.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yohann Couté  

LAB HEAD: Yohann Couté

PROVIDER: PXD034871 | Pride | 2022-12-09

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
EXT1-EXT2.mgf Mgf
EXT1-EXT2.mzid.gz Mzid
EXT1-EXT2.raw Raw
checksum.txt Txt
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Publications


Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating N-acetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Here, we present the cryo-electron microscopy structure o  ...[more]

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