title: Autocatalytic cleavage of human gamma-glutamyl transpeptidase is highly dependent on N-glycosylation at asparagine 95.
abstract: γ-Glutamyl transpeptidase (GGT) is a heterodimeric membrane enzyme that catalyzes the cleavage of extracellular glutathione and other γ-glutamyl-containing compounds. GGT is synthesized as a single polypeptide (propeptide) that undergoes autocatalytic cleavage, which results in the formation of the large and small subunits that compose the mature enzyme. GGT is extensively N-glycosylated, yet the functional consequences of this modification are unclear. We investigated the effect of N-glycosylation on the kinetic behavior, stability, and functional maturation of GGT. Using site-directed mutagenesis, we confirmed that all seven N-glycosylation sites on human GGT are modified by N-glycans. Comparative enzyme kinetic analyses revealed that single substitutions are functionally tolerated, although the N95Q mutation resulted in a marked decrease in the cleavage efficiency of the propeptide. However, each of the single site mutants exhibited decreased thermal stability relative to wild-type GGT. Combined mutagenesis of all N-glycosylation sites resulted in the accumulation of the inactive propeptide form of the enzyme. Use of N-glycosylation inhibitors demonstrated that binding of the core N-glycans, not their subsequent processing, is the critical glycosylation event governing the autocleavage of GGT. Although N-glycosylation is necessary for maturation of the propeptide, enzymatic deglycosylation of the mature wild-type GGT does not substantially impact either the kinetic behavior or thermal stability of the fully processed human enzyme. These findings are the first to establish that co-translational N-glycosylation of human GGT is required for the proper folding and subsequent cleavage of the nascent propeptide, although retention of these N-glycans is not necessary for maintaining either the function or structural stability of the mature enzyme.
authors.affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: West
authors.affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Wickham
authors.affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and.
authors.forename: Leslie M
authors.initials: LM
authors.lastname: Quinalty
authors.affiliation: Biophysics Program, College of Pharmacy, Ohio State University, Columbus, Ohio 43210.
authors.forename: Ryan E
authors.initials: RE
authors.lastname: Pavlovicz
authors.affiliation: Biophysics Program, College of Pharmacy, Ohio State University, Columbus, Ohio 43210; Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Ohio State University, Columbus, Ohio 43210.
authors.forename: Chenglong
authors.initials: C
authors.lastname: Li
authors.affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and. Electronic address: marie-hanigan@ouhsc.edu.
authors.forename: Marie H
authors.initials: MH
authors.lastname: Hanigan
mesh_terms: D019943:Amino Acid Substitution; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004795:Enzyme Stability / Q000502:physiology; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017510:Protein Folding; D046188:Protein Modification, Translational / Q000502:physiology*; D013329:Structure-Activity Relationship; D005723:gamma-Glutamyltransferase / Q000235:genetics / Q000378:metabolism*
chemical_list: D001216:Asparagine; D005723:gamma-Glutamyltransferase; C554782:gamma-glutamyltransferase, human