title: 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)α-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 ERα-negative breast cancer cells but not on ERα-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.
authors.affiliation: From the Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.
authors.forename: Srimathi
authors.initials: S
authors.lastname: Srinivasan
authors.affiliation: From the Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.
authors.forename: Mathilde
authors.initials: M
authors.lastname: Romagnoli
authors.affiliation: From the Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.
authors.forename: Andrew
authors.initials: A
authors.lastname: Bohm
authors.affiliation: From the Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts 02111 Gail.Sonenshein@tufts.edu.
authors.forename: Gail E
authors.initials: GE
authors.lastname: Sonenshein
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001943:Breast Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D004795:Enzyme Stability / Q000235:genetics; D047628:Estrogen Receptor alpha; D005260:Female; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics; D059748:Proteolysis
chemical_list: C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D051722:ADAM Proteins; C068490:ADAM8 protein, human
keywords: ADAM; Breast Cancer; N-linked Glycosylation; Post-translational Modification (PTM); Protein Turnover