Functional Roles of N-Linked Glycosylation of Human Matrix Metalloproteinase 9

Abstract

Matrix metalloproteinase-9 (MMP-9) is a secreted endoproteinase with two N-glycosylation sites at residues N38 and N120. Using bimolecular fluorescence complementation, co-immunoprecipitation, fluorescence microscopy and extensive site-directed mutagenesis, we identified the indispensible roles of both N-glycosylation sites for the secretion of MMP-9. The N38-glycosylation-deficient MMP-9 revealed a novel polypeptide-binding domain that interacted with calreticulin (CALR) dependent on the molecular volume of the exposed amino acid. The N120-glycosylation-deficient MMP-9 resulted in reduced secretion stemming from a strong interaction with CALR. 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.