Protein Conformational Change Delayed by Steric Hindrance from an N-Linked Glycan

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  • René Bager
  • ,
  • Jesper Sanderhoff Johansen, Denmark
  • Jan K Jensen
  • Allan Stensballe, Sektion for Bioteknologi, Denmark
  • Agnieszka Jendroszek, Denmark
  • Linette Buxbom Nielsen, Denmark
  • Hans Peter Sørensen, Denmark
  • Peter A Andreasen, Denmark
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 centre loop (RCL) 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 RCL passes during latency transition. While the latency transition of human PAI-1 is unaffected by deglycosylation, deglycosylated zebrafish PAI-1 goes latent about 50-fold faster than the glycosylated zebrafish PAI-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 zebrafish PAI-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.
Original languageEnglish
JournalJournal of Molecular Biology
Volume425
Issue16
Pages (from-to)2867–2877
Number of pages11
ISSN0022-2836
DOIs
Publication statusPublished - 23 Aug 2013

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