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.

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.