Title :
Protease domain glycans affect oligomerization, disulfide bond formation, and stability of the
meprin A metalloprotease homo-oligomer
Abstract :
- The meprin A homo-oligomer is a highly glycosylated, secreted zinc metalloprotease of the astacin family and metzincin superfamily
- This isoform of meprin is composed of disulfide-bonded dimers of alpha subunits that further associate to form large, secreted megadalton complexes of 10 or more subunits
- The aim of this study was to determine the sites of glycan attachment and to assess their ability to affect the formation and stability of the homo-oligomer
- Nine of the ten potential N-linked glycosylation sites ( Asn-41, Asn-152, Asn-234, Asn-270, Asn-330, Asn-426, Asn-452, Asn-546, and Asn-553 ) were found to be glycosylated in recombinant mouse meprin A using chemical and enzymatic deglycosylation methods and electrospray ionization mass spectrometry
- Chemical cross-linking demonstrated that carbohydrates are at or near the noncovalent subunit interface
- The removal of two glycans in the protease domain at Asn-234 and Asn-270 , as well as one in the tumor necrosis factor receptor-associated factor domain at Asn-452 , by a deglycosidase under nondenaturing conditions decreased the chemical and thermal stability of the homo-oligomer without affecting quaternary structure
- Site-directed mutagenesis demonstrated that no single glycan was essential for oligomer formation; however, the combined absence of the glycans at Asn-152 and Asn-270 in the protease domain hindered intersubunit disulfide bond formation, prevented noncovalent associations, and abolished enzymatic activity
- These studies provide insights into the role of glycans in the biosynthesis, activity, and stability of this extracellular protease