Dataset Information

N-glycosylation regulates ADAM8 processing and activation.

ABSTRACT: The transmembrane ADAM8 (A Disintegrin And Metalloproteinase 8) protein is abundantly expressed in human breast tumors and derived metastases compared with normal breast tissue, and plays critical roles in aggressive Triple-Negative breast cancers (TNBCs). During ADAM8 maturation, the inactive proform dimerizes or multimerizes and autocatalytically removes the prodomain leading to the formation of the active, processed form. ADAM8 is a glycoprotein; however, little was known about the structure or functional role of these sugar moieties. Here, we report that in estrogen receptor (ER)?-negative, but not -positive, breast cancer cells ADAM8 contains N-glycosylation, which is required for its correct processing and activation. Consistently ADAM8 dimers were detected on the surface of ER?-negative breast cancer cells but not on ER?-positive ones. Site-directed mutagenesis confirmed four N-glycosylazhytion sites (Asn-67, Asn-91, Asn-436, and Asn-612) in human ADAM8. The Asn-67 and Asn-91 prodomain sites contained high mannose, whereas complex type N-glycosylation was observed on Asn-436 and Asn-612 in the active and remnant forms. The Asn-91 and Asn-612 sites were essential for its correct processing and cell surface localization, in particular its exit from the Golgi and endoplasmic reticulum, respectively. The N436Q mutation led to decreased ADAM8 stability due to enhanced lysosomal degradation. In contrast, mutation of the Asn-67 site had only modest effects on enzyme stability and processing. Thus, N-glycosylation is essential for processing, localization, stability, and activity of ADAM8.

SUBMITTER: Srinivasan S

PROVIDER: S-EPMC4246118 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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N-glycosylation regulates ADAM8 processing and activation.

Srinivasan Srimathi S Romagnoli Mathilde M Bohm Andrew A Sonenshein Gail E GE

The Journal of biological chemistry 20141021 48

The transmembrane ADAM8 (A Disintegrin And Metalloproteinase 8) protein is abundantly expressed in human breast tumors and derived metastases compared with normal breast tissue, and plays critical roles in aggressive Triple-Negative breast cancers (TNBCs). During ADAM8 maturation, the inactive proform dimerizes or multimerizes and autocatalytically removes the prodomain leading to the formation of the active, processed form. ADAM8 is a glycoprotein; however, little was known about the structure ...[more]

PMID: 25336660

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Project description:Recent advances in targeted treatment for cholangiocarcinoma have focused on fibroblast growth factor (FGF) signaling. There are four receptor tyrosine kinases that respond to FGFs, and posttranslational processing has been demonstrated for each FGF receptor. Here, we investigated the role of N-linked glycosylation on the processing and function of FGFR4. We altered glycosylation through enzymatic deglycosylation, small molecule inhibition of glycosyltransferases, or through site-directed mutagenesis of selected asparagine residues in FGFR4. Signaling was tested through caspase activation, migration, and subcellular localization of FGFR4. Our data demonstrate that FGFR4 has multiple glycoforms, with predominant bands relating to the full-length receptor that has a high mannose- or hybrid-type form and a complex-type glycan form. We further identified a set of faster migrating FGFR4 bands that correspond to the intracellular kinase domain, termed FGFR4 intracellular domain (R4-ICD). These glycoforms and R4-ICD were detected in human cholangiocarcinoma tumor samples, where R4-ICD was predominant. Removal of glycans in intact cells by enzymatic deglycosylation resulted in increased processing to R4-ICD. Inhibition of glycosylation using NGI-1, an oligosaccharyltransferase inhibitor, reduced both high mannose- or hybrid- and complex-type glycan forms of FGFR4, increased processing and sensitized to apoptosis. Mutation of Asn-112, Asn-258, Asn-290, or Asn-311 to glutamine modestly reduced apoptosis resistance, while mutation of Asn-322 or simultaneous mutation of the other four asparagine residues caused a loss of cytoprotection by FGFR4. None of the glycomutants altered the migration of cancer cells. Finally, mutation of Asn-112 caused a partial localization of FGFR4 to the Golgi. Overall, preventing glycosylation at individual residues reduced the cell survival function of FGFR4 and receptor glycosylation may regulate access to an extracellular protease or proteolytic susceptibility of FGFR4.

Project description:Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin(NN163/166AA) is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin(NN163/166AA) is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin(NN163/166AA) exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin(NN163/166AA) participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin(NN163/166AA) shows a significant cholesterol-dependent decrease in cell-surface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin(NN163/166AA). These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by clathrin- and caveolin-dependent mechanisms.

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N-glycosylation regulates ADAM8 processing and activation.

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N-glycosylation regulates ADAM8 processing and activation.

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