A unique serpin P1′ glutamate and a conserved β-sheet C arginine are key residues for activity, protease recognition and stability of serpinA12 (vaspin)

Abstract

SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1') of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1' glutamate (Glu(379)) with an arginine residue (Arg(302)) of the β-sheet C. A P1' glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1' residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within β-sheet C impeded the serpin-protease interaction. Arg(302) is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1' Glu(379), which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu(379) and Arg(302) influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin.

Basic Residues of β-Sheet A Contribute to Heparin Binding and Activation of Vaspin (Serpin A12)

“…inhibition by vaspin most likely by bridging both proteins in a ternary complex rather than inducing conformational changes in the inhibitor (15). As also implied by the binding data, neither the 5-mer fondaparinux nor an 8-mer dp8 accelerated complex formation.…”

“…We have shown previously that both vaspin and its target protease KLK7 are heparin binding molecules (15). Microscale thermophoresis revealed high affinity binding of heparin by vaspin (K D ϭ 21 nM), which is comparable with that of plasma AT (K D ϭ 10 nM (18)) and protein Z-dependent protease inhibitor (K D ϭ 25 nM (19)).…”

“…The heparin-induced acceleration of KLK7 inhibition by vaspin is dose-dependent with a bell-shaped dose-response curve (15). This indicates a major contribution of the template effect of longer heparin chains to the acceleration of the vaspin-KLK7 reaction, whereby binding of one heparin chain by both the serpin and the protease induces spatial proximity and an increase in reaction rate.…”

“…Inhibition of KLK7 by vaspin is furthermore accelerated by heparin (15). Yet, the nature of the interaction between glycosaminoglycans and vaspin has not been explored in much detail.…”

“…We have recently reported that the inhibition of KLK7 by vaspin is critically dependent on an arginine residue in proximity to the reactive center loop (RCL), 2 because the native P1Ј glutamate residue is essentially repressing inhibition of KLK7 (15). Inhibition of KLK7 by vaspin is furthermore accelerated by heparin (15).…”