title: Protein conformational change delayed by steric hindrance from an N-linked glycan.
abstract: Very few studies have attributed a direct, active, functional role to N-linked glycans. We describe here an N-linked glycan with a unique role for maintaining the active conformation of a protein of the serpin family. The distinguishing feature of serpins is the "stressed-to-relaxed" transition, in which the reactive center loop inserts as a β-strand into the central β-sheet A. This transition forms the basis for the conversion of serpins to the inactive latent state. We demonstrate that plasminogen activator inhibitor-1 (PAI-1) from zebrafish converts to the latent state about 5-fold slower than human PAI-1. In contrast to human PAI-1, fish PAI-1 carries a single N-linked glycan at Asn185 in the gate region through which the reactive center loop passes during latency transition. While the latency transition of human PAI-1 is unaffected by deglycosylation, deglycosylated zebrafish PAI-1 (zfPAI-1) goes latent about 50-fold faster than the glycosylated zfPAI-1 and about 25-fold faster than non-glycosylated human PAI-1. X-ray crystal structure analysis of glycosylated fish PAI-1 confirmed the presence of an N-linked glycan in the gate region and a lack of glycan-induced structural changes. Thus, latency transition of zfPAI-1 is delayed by steric hindrance from the glycan in the gate region. Our findings reveal a previously unknown mechanism for inhibition of protein conformational changes by steric hindrance from N-linked glycans.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: René
authors.initials: R
authors.lastname: Bager
authors.forename: Jesper S
authors.initials: JS
authors.lastname: Johansen
authors.forename: Jan K
authors.initials: JK
authors.lastname: Jensen
authors.forename: Allan
authors.initials: A
authors.lastname: Stensballe
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Jendroszek
authors.forename: Linette
authors.initials: L
authors.lastname: Buxbom
authors.forename: Hans Peter
authors.initials: HP
authors.lastname: Sørensen
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D017422:Sequence Analysis, DNA; D015027:Zebrafish
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D011134:Polysaccharides
keywords: LC-MS/MS; MALDI; MS; PAI-1; PEG; PNGase F; RCL; TOF; glycan; liquid chromatography–tandem mass spectrometry; mass spectrometry; matrix-assisted laser desorption/ionization; peptide-N(4) (N-acetyl-β-d-glycosaminyl) asparagine amidase; plasminogen activator inhibitor-1; polyethylene glycol; rainbow trout PAI-1; reactive center loop; rtPAI-1; serpin; time of flight; uPA; urokinase-type plasminogen activator; wild type; wt; x-ray crystallography; zebrafish; zebrafish PAI-1; zebrafish urokinase-type plasminogen activator; zfPAI-1; zfuPA