Abstract
Hypoxia is a critical microenvironmental factor that drives cancer progression through angiogenesis and metastasis. Glycoproteins, especially those on the plasma membrane, orchestrate this process; however, questions remain regarding hypoxia-perturbed protein glycosylation in cancer cells. We focused on the effects of hypoxia on the integrin family of glycoproteins, which are central to the cellular processes of attachment and migration and have been linked with cancer in humans. We employed electrostatic repulsion hydrophilic interaction chromatography coupled with iTRAQ labeling and LC-MS/MS to identify and quantify glycoproteins expressed in A431. The results revealed that independent of the protein-level change, N-glycosylation modifications of integrin α 3 (ITGA3) were inhibited by hypoxia, unlike in other integrin subunits. A combination of Western blot, flow cytometry, and cell staining assays showed that hypoxia-induced alterations to the glycosylation of ITGA3 prevented its efficient translocation to the plasma membrane. Mutagenesis studies demonstrated that simultaneous mutation of glycosites 6 and 7 of ITGA3 prevented its accumulation at the K562 cell surface, which blocked integrin α 3 and β 1 heterodimer formation and thus abolished ITGA3's interaction with extracellular ligands. By generating A431 cells stably expressing ITGA3 mutated at glycosites 6 and 7, we showed that lower levels of ITGA3 on the cell surface, as induced by hypoxia, conferred an increased invasive ability to cancer cells in vitro under hypoxic conditions. Taken together, these results revealed that ITGA3 translocation to the plasma membrane suppressed by hypoxia through inhibition of glycosylation facilitated cell invasion in A431.
Highlights
As solid tumors grow, those areas distant from the existing blood vessels can become chronically or intermittently deprived of sufficient oxygen
The Functional Annotation Clustering tool from DAVID Bioinformatics Resources 6.7 was used to group the 146 proteins [58]. It indicated that 101 of the 146 proteins we identified with Nglycosites were known N-linked glycoproteins, with 84 being membrane-associated and 70 of those belonging to the plasma membrane
Within the integrin category we identified seven integrin subunits, and we quantified the glycopeptides of these subunits that were modulated by hypoxia and reoxygenation
Summary
Chemicals and Reagents—Reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified. The biotinylated cells were exposed to lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, CompleteTM EDTA-free protease inhibitor mixture) before incubation with avidin at room temperature for 1 h. For flow cytometry, collected cells were washed three times with ice-cold PBS and incubated with 5% human serum in PBS for 30 min on ice for blocking. Immunoprecipitation and Western Blotting—Cells were washed three times with ice-cold PBS before the addition of lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, CompleteTM EDTA-free protease inhibitor mixture). Transfected cells were selected by culture in stand-
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