Protease domain glycans affect oligomerization, disulfide bond formation, and stability of the meprin A metalloprotease homo-oligomer.
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.
Ishmael
Susan S
SS
Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Ishmael
Faoud T
FT
Jones
A Daniel
AD
Bond
Judith S
JS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011134:Polysaccharides / Q000737:chemistry; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D004220:Disulfides; D011134:Polysaccharides; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C053833:meprin A