Identification of NIPSNAP1 as a nocistatin-interacting protein involving pain transmission

Abstract

4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) is a molecule of physiologically unknown function, although it is predominantly expressed in the brain, spinal cord, liver, and kidney. We identified NIPSNAP1 as a protein that interacts with the neuropeptide nocistatin (NST) from synaptosomal membranes of mouse spinal cord using high-performance affinity latex beads. NST, which is produced from the same precursor protein as an opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ), has opposite effects on pain transmission evoked by N/OFQ. The calculated full-length pre-protein of NIPSNAP1 was 33 kDa, whereas the N-terminal truncated form of NIPSNAP1 (29 kDa) was ubiquitously expressed in the neuronal tissues, especially in synaptic membrane and mitochondria of brain. The 29-kDa NIPSNAP1 was distributed on the cell surface, and NST interacted with the 29-kDa but not the 33-kDa NIPSNAP1. Although intrathecal injection of N/OFQ induced tactile allodynia in both wild-type and NIPSNAP1-deficient mice, the inhibition of N/OFQ-evoked tactile allodynia by NST seen in wild-type mice was completely lacking in the deficient mice. These results suggest that NIPSNAP1 is an interacting molecule of NST and plays a crucial role in pain transmission.

FIGURE 1.

Purification and identification of NST-interacting proteins. A, NST-conjugated SG beads at the indicated concentrations of NST and unconjugated SG beads were incubated overnight at 4 °C with synaptosomal membrane extracts of the spinal cord. B, quantification of protein bands in the right two lanes of A (NST-beads 4.8 μm with or without NST) using ImageJ. C, displacement of NST-conjugated SG beads binding protein bands by 10 μm NST and 10 μm N/OFQ. D, proteins from COS-7 cells transfected with expression vectors for mouse NIPSNAP1 and NIPSNAP2 were purified with NST-conjugated SG beads. The eluates were subjected to SDS-PAGE followed by silver staining. These experiments were performed at least 4 times, and similar results were obtained. Arrowheads in A–D indicate the positions of the interacting proteins. E, amino acid sequences of mouse NIPSNAP1, NIPSNAP2, and NIPSNAP3A. The parts of NIPSNAP1 in red correspond to the peptide fragments obtained by trypsin digestion. The peptide fragments used as immunogens are indicated by the blue underlines. Conserved residues are boxed. A possible transmembrane helix is shadow boxed. F–I, [³H]NST binding to NIPSNAP1. F, the membrane fraction prepared from COS-7 cells expressing mouse NIPSNAP1 was incubated with [³H]NST (8 nm) at 4 and 30 °C. Specific binding was determined as described under “Experimental Procedures.” Data are expressed as the mean ± S.E. (n = 3–6). G, the membrane fractions prepared from COS-7 cells expressing mouse NIPSNAP1 and NIPSNAP2 were incubated with the indicated concentrations of [³H]NST at 30 °C. Data are expressed as the mean ± S.E. (n = 3–9). **, p < 0.01; *, p < 0.05 versus vector-transfected cells (mock). H, [³H]NST (15 nm) binding was determined in the presence of the indicated concentrations of unlabeled NST or N/OFQ (mean ± S.E., n = 3). *, p < 0.05 versus vehicle. I, NIPSNAP1-HA and NOP-HA expressed in COS-7 cells were immunoprecipitated with anti-HA antibody-conjugated agarose and applied to a Whatman GF/C filter. Data are expressed as the mean ± S.E. (n = 3). **, p < 0.01 versus mock.

FIGURE 2.

Characterization of NIPSNAP1 protein. A, schematic diagram depicting the structure and antibodies against NIPSNAP1. B, specificity of NIPSNAP1 antibodies. COS-7 cells expressing NIPSNAP1, NIPSNAP2, or NIPSNAP3A were subjected to immunoblot analysis with anti-NSP1-N, anti-NSP1-I, and anti-NSP1-C antibodies. C, immunoblotting of the membrane fraction of NIPSNAP1-transfected COS-7 cells with anti-NSP1-C antibody. D and E, immunoblotting of the eluates from COS-7 cells transfected with NIPSNAP1 in Fig. 1D (D) and synaptosomal membrane extracts of the spinal cord in Fig. 1A (E) with anti-NSP1-C antibody. These experiments were performed at least 4 times, and similar results were obtained. The open arrowheads indicate the full-length form of NIPSNAP1 (B and C); the closed arrowhead indicate a mature form of NIPSNAP1 (B–E). The asterisk indicate NIPSNAP1 (B). Arrows indicate NIPSNAP1-related proteins (C–E).

FIGURE 3.

Cell surface expression of NIPSNAP1. A, total and surface levels of NIPSNAP1 and NOP were examined in NIPSNAP1- and NOP-HA-transfected COS-7 cells by using biotinylation and immunoblot analysis with anti-NSP1-C, anti-HA, and anti-HSP60 antibodies. B, immunofluorescence analysis using anti-NSP1-I, anti-NSP1-C, anti-HA, and anti-β-tubulin antibodies with permeablized and non-permeablized NIPSNAP1- and NOP-HA-transfected COS-7 cells. These experiments were performed at least 3 times, and similar results were obtained. Bar, 10 μm.

FIGURE 4.

Distribution of NIPSNAP1. A, immunoblot analysis of NIPSNAP1. The homogenates of various mouse tissues were subjected to immunoblot analysis with anti-NSP1-I and anti-NSP1-C antibodies. Arrowheads indicate the mature form of NIPSNAP1, and the asterisk, NIPSNAP2. B, ubiquitous distribution of NIPSNAP1 in the central nervous system. The homogenates of various regions of the brain and spinal cord were subjected to immunoblot analysis with anti-NSP1-I, anti-NSP1-C, and anti-β-tubulin. C, subcellular distribution of NIPSNAP1. The homogenate of mouse brain was subjected to subcellular fractionation. Each fraction (12 μg of protein) was analyzed by immunoblotting with anti-NSP1-C, NR2B, synaptophysin, and HSP60 antibodies. These experiments were performed at least 3 times, and similar results were obtained. P1, nucleus and cell debris; S1, crude synaptosomal fraction; P2, crude synaptosomal pellet fraction; S2, cytosolic synaptosomal fraction; PM, plasma membrane fraction; SM, crude synaptic membrane fraction; SV, crude synaptic vesicle fraction.

FIGURE 5.

Generation and characterization of NIPSNAP1^−/− mice. A, construction of the targeting vector. The wild-type allele of the NIPSNAP1 gene, targeting vector, and mutant allele are shown. Exons are represented by boxes. The neomycin resistance gene (Neo) and the gene coding for diphtheria toxin A (DT-A) are indicated. The probe used for Southern blot analysis in B is indicated by a bold bar; the locations of PCR primers used for genotyping in C are indicated by arrows; and NheI-digested fragments detected by the probe by double-headed arrows. B, Southern blot analysis of the mutant mice. NheI-cleaved tail DNA of wild-type (+/+), NIPSNAP1^−/− (−/−), and heterozygous (+/−) littermates were hybridized with the probe indicated in A (10.0 kb for NIPSNAP1^+/+ and 6.6 kb for NIPSNAP1^−/−). C, PCR genotyping of the mutant mice. The wild-type allele band (174 bp) and the mutant allele band (483 bp) were amplified by PCR with the primers indicated in A. D, immunoblot analysis of the mutant mice. The homogenates of the adult tissues were immunoblotted with anti-NSP1-C antibody. The arrowhead indicates NIPSNAP1, and the asterisk, NIPSNAP2. E, [³H]NST binding to the membrane fraction from spinal cord of the mutant mice. The membrane fraction was incubated with [³H]NST (6.4 and 11 nm) at 30 °C for 60 min (mean ± S.E., n = 3). **, p < 0.01, *, p < 0.05 versus wild-type value. F, histological analysis of wild-type and NIPSNAP1^−/− mice. Nissl staining of the spinal cord. Bar, 500 μm. G, fluorescence micrographs of the spinal dorsal horn of the mutant mice stained with anti-NST antibody. These experiments were performed at least 3 times, and similar results were obtained. Bar, 100 μm.

FIGURE 6.

Tactile allodynia induced by N/OFQ and NST in NIPSNAP1^−/− mice. A, N/OFQ (50 pg; ○, ●) or NST (500 pg; □, ■) was injected intrathecally into wild-type (+/+; ○, □) and NIPSNAP1^−/− (−/−; ●, ■) mice. Allodynia was assessed once every 5 min for 50 min, and the values (mean ± S.E., n = 6) are expressed as % of the maximal possible cumulative score over the 50-min experimental period. B, NST (500 pg) and N/OFQ (50 pg) were simultaneously injected intrathecally into wild-type (△, +/+) and NIPSNAP1^−/− (▴, −/−) mice. C, the values shown are expressed as a percent of the maximum possible cumulative score for allodynia over the 50-min observation period at the indicated concentration of NST and N/OFQ (mean ± S.E., n = 5). **, p < 0.01, versus wild-type value; ##, p < 0.01, N/OFQ-injected value in wild-type mice.