title: Functional Roles of N-Linked Glycosylation of Human Matrix Metalloproteinase 9.
abstract: Matrix metalloproteinase-9 (MMP-9) is a secreted endoproteinase with a critical role in the regulation of the extracellular matrix and proteolytic activation of signaling molecules. Human (h)MMP-9 has two well-defined N-glycosylation sites at residues N38 and N120; however, their role has remained mostly unexplored partly because expression of the N-glycosylation-deficient N38S has been difficult due to a recently discovered single nucleotide polymorphism-dependent miRNA-mediated inhibitory mechanism. hMMP-9 cDNA encoding amino acid substitutions at residues 38 (modified-S38, mS38) or 120 (N120S) were created in the background of a miRNA-binding site disrupted template and expressed by transient transfection. hMMP-9 harboring a single mS38 replacement secreted well, whereas N120S, or a double mS38/N120S hMMP-9 demonstrated much reduced secretion. Imaging indicated endoplasmic reticulum (ER) retention of the non-secreted variants and co-immunoprecipitation confirmed an enhanced strong interaction between the non-secreted hMMP-9 and the ER-resident protein calreticulin (CALR). Removal of N-glycosylation at residue 38 revealed an amino acid-dependent strong interaction with CALR likely preventing unloading of the misfolded protein from the ER chaperone down the normal secretory pathway. As with other glycoproteins, N-glycosylation strongly regulates hMMP-9 secretion. This is mediated, however, through a novel mechanism of cloaking an N-glycosylation-independent strong interaction with the ER-resident CALR.
authors.affiliation: Molecular and Cellular Pharmacology Graduate Program, University of Wisconsin, School of Medicine and Public Health, Madison, WI, 53705, USA.|Department of Anesthesiology, University of Wisconsin-Madison, Madison, WI, 53705, USA.
authors.forename: Tyler
authors.initials: T
authors.lastname: Duellman
authors.affiliation: Department of Anesthesiology, University of Wisconsin-Madison, Madison, WI, 53705, USA.
authors.forename: John
authors.initials: J
authors.lastname: Burnett
authors.affiliation: Molecular and Cellular Pharmacology Graduate Program, University of Wisconsin, School of Medicine and Public Health, Madison, WI, 53705, USA.|Department of Anesthesiology, University of Wisconsin-Madison, Madison, WI, 53705, USA.
authors.forename: Jay
authors.initials: J
authors.lastname: Yang
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D037282:Calreticulin / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D005109:Extracellular Matrix / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D014162:Transfection / Q000379:methods
chemical_list: D037282:Calreticulin; D018076:DNA, Complementary; D006023:Glycoproteins; D035683:MicroRNAs; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9
keywords: ER retention; N-glycosylation; calreticulin; co-IP assay; complementation assay; matrix metalloproteinase-9; molecular volume; mutagenesis; secretion