Abstract
Human SLURP-1 is a secreted protein of the Ly6/uPAR/three-finger neurotoxin family that co-localizes with nicotinic acetylcholine receptors (nAChRs) and modulates their functions. Conflicting biological activities of SLURP-1 at various nAChR subtypes have been based on heterologously produced SLURP-1 containing N- and/or C-terminal extensions. Here, we report the chemical synthesis of the 81 amino acid residue human SLURP-1 protein, characterization of its 3D structure by NMR, and its biological activity at nAChR subtypes. Radioligand assays indicated that synthetic SLURP-1 did not compete with [125I]-α-bungarotoxin (α-Bgt) binding to human neuronal α7 and Torpedo californica muscle-type nAChRs, nor to mollusk acetylcholine binding proteins (AChBP). Inhibition of human α7-mediated currents only occurred in the presence of the allosteric modulator PNU120596. In contrast, we observed robust SLURP-1 mediated inhibition of human α3β4, α4β4, α3β2 nAChRs, as well as human and rat α9α10 nAChRs. SLURP-1 inhibition of α9α10 nAChRs was accentuated at higher ACh concentrations, indicating an allosteric binding mechanism. Our results are discussed in the context of recent studies on heterologously produced SLURP-1 and indicate that N-terminal extensions of SLURP-1 may affect its activity and selectivity on its targets. In this respect, synthetic SLURP-1 appears to be a better probe for structure-function studies.
Highlights
The three-finger fold is a protein domain structure comprising a disulfide-stabilized core from which three elongated loops protrude (Fig. 1)
Co-localization studies and functional in vitro activity data have demonstrated that certain Ly6 proteins (Lynx[1] and SLURP-1 and -2) interact with nicotinic acetylcholine receptors (nAChR), suggesting that they might function as endogenous modulators of nAChR signaling in vivo[10,11]
Given the size of the target SLURP-1 molecule, we resorted to a peptide segment ligation approach to overcome the size limitation of traditional stepwise solid phase peptide synthesis (SPPS) (Figs 1 and 2)[15]
Summary
The three-finger fold is a protein domain structure comprising a disulfide-stabilized core from which three elongated loops (fingers) protrude (Fig. 1) It features prominently in two large protein families: snake venom neurotoxins and the Ly6 proteins, the latter first discovered in the mammalian immune system[1,2,3,4]. Recombinant SLURP-1 expressed in E. coli, containing an additional methionine residue at the N-terminus (hereafter referred to as rSLURP-1), exhibited inhibitory activity at α7 nAChR (at micromolar concentrations)[13] To resolve these conflicting data on SLURP-1 activities, we report here the chemical synthesis and biological activity of the 81 amino acid human SLURP-1 identical in amino acid sequence to the human serum-derived protein. Our pharmacological data revealed for the first time the interaction of synthetic SLURP-1 (sSLURP-1) with several neuronal nAChR subtypes
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