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ER is the primary site for the maturation and folding of soluble and transmembrane secretory proteins. The calnexin cycle is a specific chaperone system that folds and assesses the confirmation of N-glycosylated proteins before they can exit the ER lumen. The primary players of this quality check pipeline are the lectins, ER-resident chaperones, and a glucosyl transferase enzyme. In case the calnexin system in the lumen fails to salvage a misfolded protein, it is transported to the cytoplasm...
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N-glycosylation regulates ADAM8 processing and activation.

Srimathi Srinivasan1, Mathilde Romagnoli1, Andrew Bohm1

  • 1From the Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.

The Journal of Biological Chemistry
|October 23, 2014
PubMed
Summary

N-glycosylation is crucial for the activation and cell surface presence of A Disintegrin And Metalloproteinase 8 (ADAM8) in aggressive breast cancers. This sugar modification impacts ADAM8 processing, localization, and stability, particularly in ERα-negative tumors.

Keywords:
ADAMBreast CancerN-linked GlycosylationPost-translational Modification (PTM)Protein Turnover

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cancer Research

Background:

  • A Disintegrin And Metalloproteinase 8 (ADAM8) is a transmembrane protein highly expressed in aggressive breast tumors and metastases.
  • ADAM8 plays critical roles in Triple-Negative breast cancers (TNBCs), a subtype lacking estrogen receptor alpha (ERα).
  • While ADAM8 is a glycoprotein, the specific role of its N-glycosylation in its function and processing remained largely uncharacterized.

Purpose of the Study:

  • To investigate the role of N-glycosylation in the processing, activation, localization, and stability of ADAM8.
  • To determine if N-glycosylation patterns differ between ERα-negative and ERα-positive breast cancer cells.
  • To identify specific N-glycosylation sites critical for ADAM8 function.

Main Methods:

  • Site-directed mutagenesis was employed to alter identified N-glycosylation sites on human ADAM8.
  • Analysis of ADAM8 processing, cell surface localization, and stability was performed using various biochemical and cell biology techniques.
  • N-glycosylation types (high mannose vs. complex) were characterized at specific sites.

Main Results:

  • N-glycosylation is essential for the correct processing and activation of ADAM8 in ERα-negative breast cancer cells.
  • ADAM8 dimers were detected on the surface of ERα-negative cells but not ERα-positive cells, correlating with N-glycosylation.
  • Mutations at Asn-91 and Asn-612 impaired processing and cell surface exit, while mutation at Asn-436 reduced stability via lysosomal degradation. Asn-67 had minimal impact.

Conclusions:

  • N-glycosylation is a critical post-translational modification regulating ADAM8 processing, cell surface localization, stability, and ultimately, its activity.
  • Specific N-glycosylation sites dictate distinct aspects of ADAM8 maturation and function.
  • Targeting ADAM8 N-glycosylation may offer a therapeutic strategy for ERα-negative breast cancers.