ABSTRACT
Most clinically approved biomarkers of cancer are glycoproteins, and those residing on the cell surface are of particular interest in biotherapeutics. We report a method for selective labeling, affinity enrichment, and identification of cell-surface glycoproteins. PC-3 cells and primary human prostate cancer tissue were treated with peracetylated N-azidoacetylgalactosamine, resulting in metabolic labeling of cell surface glycans with the azidosugar. We used mass spectrometry to identify over 70 cell surface glycoproteins and biochemically validated CD146 and integrin beta-4, both of which are known to promote metastatic behavior. These results establish cell-surface glycoproteomics as an effective technique for discovery of cancer biomarkers.
Subject(s)
Carbohydrates/analysis , Prostatic Neoplasms/metabolism , Proteome , Cell Line, Tumor , Electrophoresis, Polyacrylamide Gel , Humans , Male , Mass Spectrometry , Prostatic Neoplasms/chemistry , Prostatic Neoplasms/pathologyABSTRACT
Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked ß-N-acetylglucosamine (O-GlcNAc) to regulate their function, localization, and stability. Despite its broad functional significance, the dynamic and posttranslational nature of O-GlcNAc signaling makes it challenging to study using traditional molecular and cell biological techniques alone. Here, we report that metabolic cross-talk between the N-acetylgalactosamine salvage and O-GlcNAcylation pathways can be exploited for the tagging and identification of O-GlcNAcylated proteins. We found that N-azidoacetylgalactosamine (GalNAz) is converted by endogenous mammalian biosynthetic enzymes to UDP-GalNAz and then epimerized to UDP-N-azidoacetylglucosamine (GlcNAz). O-GlcNAc transferase accepts UDP-GlcNAz as a nucleotide-sugar donor, appending an azidosugar onto its native substrates, which can then be detected by covalent labeling using azide-reactive chemical probes. In a proof-of-principle proteomics experiment, we used metabolic GalNAz labeling of human cells and a bioorthogonal chemical probe to affinity-purify and identify numerous O-GlcNAcylated proteins. Our work provides a blueprint for a wide variety of future chemical approaches to identify, visualize, and characterize dynamic O-GlcNAc signaling.
Subject(s)
Acetylgalactosamine/metabolism , Acetylglucosamine/metabolism , Affinity Labels , Metabolic Networks and Pathways , Receptor Cross-Talk , Cell Line , Chromatography, Affinity , Glycosylation , Humans , Methods , Protein Processing, Post-TranslationalABSTRACT
Fucosylated glycoproteins are involved in many cell-cell recognition events and are markers of embryonic and malignant tissue. Here we report a method for rapid profiling of fucosylated glycoproteins from human cells using 6-azido fucose as a metabolic label.
Subject(s)
Fucose/analogs & derivatives , Fucose/metabolism , Glycoproteins/chemistry , Glycoproteins/metabolism , Azides/chemistry , Azides/metabolism , Flow Cytometry , Fucose/chemistry , Humans , Jurkat Cells , Models, Molecular , Polysaccharides/chemistry , Polysaccharides/metabolismABSTRACT
Sulfate assimilation is a critical component of both primary and secondary metabolism. An essential step in this pathway is the activation of sulfate through adenylation by the enzyme ATP sulfurylase (ATPS), forming adenosine 5'-phosphosulfate (APS). Proteobacterial ATPS overcomes this energetically unfavorable reaction by associating with a regulatory G protein, coupling the energy of GTP hydrolysis to APS formation. To discover the molecular basis of this unusual role for a G protein, we biochemically characterized and solved the X-ray crystal structure of a complex between Pseudomonas syringae ATPS (CysD) and its associated regulatory G protein (CysN). The structure of CysN*D shows the two proteins in tight association; however, the nucleotides bound to each subunit are spatially segregated. We provide evidence that conserved switch motifs in the G domain of CysN allosterically mediate interactions between the nucleotide binding sites. This structure suggests a molecular mechanism by which conserved G domain architecture is used to energetically link GTP turnover to the production of an essential metabolite.