Limits...
Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor.

Lyukmanova EN, Shulepko MA, Kudryavtsev D, Bychkov ML, Kulbatskii DS, Kasheverov IE, Astapova MV, Feofanov AV, Thomsen MS, Mikkelsen JD, Shenkarev ZO, Tsetlin VI, Dolgikh DA, Kirpichnikov MP - PLoS ONE (2016)

Bottom Line: It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor.Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes.Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter.

View Article: PubMed Central - PubMed

Affiliation: Biological Department, Lomonosov Moscow State University, Moscow, Russian Federation.

ABSTRACT
SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,--non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

Show MeSH

Related in: MedlinePlus

Effect of rSLURP-1 on the growth of Het-1A cells.(A). Amino acid sequence alignment of human SLURP-1, SLURP-2, ws-Lynx1, and non-conventional toxin WTX from N. kaouthia. Cysteine residues are labeled in gray, and the disulfide linkages are shown; additional N-terminal Met residues are underlined. (B). Influence of rSLURP-1 (diamonds) and ws-Lynx1 (squares) on cell growth. Each point is mean ± S.E. of six independent experiments. The Hill equation (Eq 1) was fitted to rSLURP-1 data (% of control) for each of the six experiments independently. After averaging the following values for EC50, nH and A1 were obtained 4.3 ± 0.6 nM, 1.4 ± 0.2 and 59.5 ± 1.3% (mean ± S.E., n = 6). (C). Effects of rSLURP-1 (1 μM), atropine (1 μM), Mec (10 μM), α-Bgtx (1 μM), polyclonal antibodies against α7 (IgG-α7, 1 μg per 50 μl), and their co-application. Each bar is mean ± S.E. of 4–6 independent experiments. ** and *** indicate significant (p<0.01 and p<0.001, t-test) differences.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4764493&req=5

pone.0149733.g001: Effect of rSLURP-1 on the growth of Het-1A cells.(A). Amino acid sequence alignment of human SLURP-1, SLURP-2, ws-Lynx1, and non-conventional toxin WTX from N. kaouthia. Cysteine residues are labeled in gray, and the disulfide linkages are shown; additional N-terminal Met residues are underlined. (B). Influence of rSLURP-1 (diamonds) and ws-Lynx1 (squares) on cell growth. Each point is mean ± S.E. of six independent experiments. The Hill equation (Eq 1) was fitted to rSLURP-1 data (% of control) for each of the six experiments independently. After averaging the following values for EC50, nH and A1 were obtained 4.3 ± 0.6 nM, 1.4 ± 0.2 and 59.5 ± 1.3% (mean ± S.E., n = 6). (C). Effects of rSLURP-1 (1 μM), atropine (1 μM), Mec (10 μM), α-Bgtx (1 μM), polyclonal antibodies against α7 (IgG-α7, 1 μg per 50 μl), and their co-application. Each bar is mean ± S.E. of 4–6 independent experiments. ** and *** indicate significant (p<0.01 and p<0.001, t-test) differences.

Mentions: In spite of the growing evidences supporting a modulatory action of SLURP-1 on nAChR function, the current knowledge about the mechanism of SLURP-1/nAChR interactions is very limited. The progress in this field is hampered by the inability to extract sufficient amounts of SLURP-1 from natural sources and difficulties in the production of the recombinant protein with native sequence and structure. The majority of previous works on SLURP-1 were done using fusion constructs containing, in addition to SLURP-1, some polypeptide fragments, e.g. N-terminal SUMO protein, MPB protein, GST protein or C-terminal Myc-tag [10–13,16,20–22]. It was shown previously that the native SLURP-1 isolated from blood (MW 8,842 Da, 81 a.a.) and a recombinant SLURP-1 analogue produced in HEK-293 cells (yield ~ 0.1 mg/l of cell culture) were not glycosylated [7,10]. This fact points to the possibility to use bacterial expression systems for recombinant production of SLURP-1. Previously we developed the high-efficient E. coli expression system for protein analogue with the near-native structure (rSLURP-1, MW 8,974 Da, 82 a.a.) [23]. The only difference of rSLURP-1 from the native protein is the additional N-terminal Met residue appearing due the cloning of rslurp-1 gene into the expression vector, Fig 1A. The relatively high yield of the recombinant production (~ 5 mg of the refolded protein from 1 l of cell culture) allowed us to carry out NMR structural study of rSLURP-1, which ultimately confirmed its structural homology with the ‘three-finger’ snake neurotoxins and Lynx1, another ‘three-finger’ human neuromodulator acting on nAChRs (Fig 1A) [23].


Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor.

Lyukmanova EN, Shulepko MA, Kudryavtsev D, Bychkov ML, Kulbatskii DS, Kasheverov IE, Astapova MV, Feofanov AV, Thomsen MS, Mikkelsen JD, Shenkarev ZO, Tsetlin VI, Dolgikh DA, Kirpichnikov MP - PLoS ONE (2016)

Effect of rSLURP-1 on the growth of Het-1A cells.(A). Amino acid sequence alignment of human SLURP-1, SLURP-2, ws-Lynx1, and non-conventional toxin WTX from N. kaouthia. Cysteine residues are labeled in gray, and the disulfide linkages are shown; additional N-terminal Met residues are underlined. (B). Influence of rSLURP-1 (diamonds) and ws-Lynx1 (squares) on cell growth. Each point is mean ± S.E. of six independent experiments. The Hill equation (Eq 1) was fitted to rSLURP-1 data (% of control) for each of the six experiments independently. After averaging the following values for EC50, nH and A1 were obtained 4.3 ± 0.6 nM, 1.4 ± 0.2 and 59.5 ± 1.3% (mean ± S.E., n = 6). (C). Effects of rSLURP-1 (1 μM), atropine (1 μM), Mec (10 μM), α-Bgtx (1 μM), polyclonal antibodies against α7 (IgG-α7, 1 μg per 50 μl), and their co-application. Each bar is mean ± S.E. of 4–6 independent experiments. ** and *** indicate significant (p<0.01 and p<0.001, t-test) differences.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764493&req=5

pone.0149733.g001: Effect of rSLURP-1 on the growth of Het-1A cells.(A). Amino acid sequence alignment of human SLURP-1, SLURP-2, ws-Lynx1, and non-conventional toxin WTX from N. kaouthia. Cysteine residues are labeled in gray, and the disulfide linkages are shown; additional N-terminal Met residues are underlined. (B). Influence of rSLURP-1 (diamonds) and ws-Lynx1 (squares) on cell growth. Each point is mean ± S.E. of six independent experiments. The Hill equation (Eq 1) was fitted to rSLURP-1 data (% of control) for each of the six experiments independently. After averaging the following values for EC50, nH and A1 were obtained 4.3 ± 0.6 nM, 1.4 ± 0.2 and 59.5 ± 1.3% (mean ± S.E., n = 6). (C). Effects of rSLURP-1 (1 μM), atropine (1 μM), Mec (10 μM), α-Bgtx (1 μM), polyclonal antibodies against α7 (IgG-α7, 1 μg per 50 μl), and their co-application. Each bar is mean ± S.E. of 4–6 independent experiments. ** and *** indicate significant (p<0.01 and p<0.001, t-test) differences.
Mentions: In spite of the growing evidences supporting a modulatory action of SLURP-1 on nAChR function, the current knowledge about the mechanism of SLURP-1/nAChR interactions is very limited. The progress in this field is hampered by the inability to extract sufficient amounts of SLURP-1 from natural sources and difficulties in the production of the recombinant protein with native sequence and structure. The majority of previous works on SLURP-1 were done using fusion constructs containing, in addition to SLURP-1, some polypeptide fragments, e.g. N-terminal SUMO protein, MPB protein, GST protein or C-terminal Myc-tag [10–13,16,20–22]. It was shown previously that the native SLURP-1 isolated from blood (MW 8,842 Da, 81 a.a.) and a recombinant SLURP-1 analogue produced in HEK-293 cells (yield ~ 0.1 mg/l of cell culture) were not glycosylated [7,10]. This fact points to the possibility to use bacterial expression systems for recombinant production of SLURP-1. Previously we developed the high-efficient E. coli expression system for protein analogue with the near-native structure (rSLURP-1, MW 8,974 Da, 82 a.a.) [23]. The only difference of rSLURP-1 from the native protein is the additional N-terminal Met residue appearing due the cloning of rslurp-1 gene into the expression vector, Fig 1A. The relatively high yield of the recombinant production (~ 5 mg of the refolded protein from 1 l of cell culture) allowed us to carry out NMR structural study of rSLURP-1, which ultimately confirmed its structural homology with the ‘three-finger’ snake neurotoxins and Lynx1, another ‘three-finger’ human neuromodulator acting on nAChRs (Fig 1A) [23].

Bottom Line: It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor.Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes.Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter.

View Article: PubMed Central - PubMed

Affiliation: Biological Department, Lomonosov Moscow State University, Moscow, Russian Federation.

ABSTRACT
SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,--non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

Show MeSH
Related in: MedlinePlus