Snmp-1, a Novel Membrane Protein of Olfactory Neurons of the Silk Moth Antheraea polyphemus with Homology to the CD36 Family of Membrane Proteins
1997; Elsevier BV; Volume: 272; Issue: 23 Linguagem: Inglês
10.1074/jbc.272.23.14792
ISSN1083-351X
AutoresMatthew E. Rogers, Ming Sun, Michael R. Lerner, Richard G. Vogt,
Tópico(s)Insect Pheromone Research and Control
ResumoWhile olfactory neurons of silk moths are well known for their exquisite sensitivity to sex pheromone odorants, molecular mechanisms underlying this sensitivity are poorly understood. In searching for proteins that might support olfactory mechanisms, we characterized the protein profile of olfactory neuron receptor membranes of the wild silk moth Antheraea polyphemus. We have purified and cloned a prominent 67-kDa protein which we have named Snmp-1 (sensory neuron membrane protein-1). Northern blot analysis suggests that Snmp-1 is uniquely expressed in antennal tissue; in situ hybridization and immunocytochemical analyses show that Snmp-1 is expressed in olfactory neurons and that the protein is localized to the cilia, dendrites, and somata but not the axons. Snmp-1 mRNA expression increases significantly 1–2 days before the end of adult development, coincident with the functional maturation of the olfactory system. Sequence analysis suggests Snmp-1 is homologous with the CD36 protein family, a phylogenetically diverse family of receptor-like membrane proteins. CD36 family proteins are characterized as having two transmembrane domains and interacting with proteinaceous ligands; Snmp-1 is the first member of this family identified in nervous tissue. These findings argue that Snmp-1 has an important role in olfaction; possible roles of Snmp-1 in odorant detection are discussed. While olfactory neurons of silk moths are well known for their exquisite sensitivity to sex pheromone odorants, molecular mechanisms underlying this sensitivity are poorly understood. In searching for proteins that might support olfactory mechanisms, we characterized the protein profile of olfactory neuron receptor membranes of the wild silk moth Antheraea polyphemus. We have purified and cloned a prominent 67-kDa protein which we have named Snmp-1 (sensory neuron membrane protein-1). Northern blot analysis suggests that Snmp-1 is uniquely expressed in antennal tissue; in situ hybridization and immunocytochemical analyses show that Snmp-1 is expressed in olfactory neurons and that the protein is localized to the cilia, dendrites, and somata but not the axons. Snmp-1 mRNA expression increases significantly 1–2 days before the end of adult development, coincident with the functional maturation of the olfactory system. Sequence analysis suggests Snmp-1 is homologous with the CD36 protein family, a phylogenetically diverse family of receptor-like membrane proteins. CD36 family proteins are characterized as having two transmembrane domains and interacting with proteinaceous ligands; Snmp-1 is the first member of this family identified in nervous tissue. These findings argue that Snmp-1 has an important role in olfaction; possible roles of Snmp-1 in odorant detection are discussed. The antennae of silk moths are well known for their exquisite sensitivity to pheremonal odorants (1Boeckh J. Kaissling K.E. Schneider D. Cold Spring Harbor Symp. Quant. Biol. 1965; 30: 263-280Google Scholar, 2Schneider D. Science. 1969; 163: 1031-1037Google Scholar, 3Kaissling K.E. Annu. Rev. Neurosci. 1986; 9: 121-145Google Scholar). Early reports demonstrated that the males of the wild silk moth Samia cynthia could locate a sex pheromone source over 2 miles away within several hours of their release (4Rau P. Rau N.L. Trans. Acad. Sci. St. Louis. 1929; 26: 80-221Google Scholar). Studies of the silk moth Bombyx morisuggested that a single pheromone molecule was sufficient to activate olfactory neurons in the antenna (5Schneider D. Kasang G. Kaissling K.E. Naturwissenschaften. 1968; 55: 395Google Scholar). In insects, odors are detected by sensilla, small hair-like structures arrayed along the antennae. The sensilla are hollow, fluid-filled cuticular structures that contain the receptor cilia of olfactory neurons. Small holes penetrate through the wall of a sensillum, permitting entry of odor molecules; odorant-binding proteins are then thought to transport the odor molecules through the fluid-filled lumen to receptor proteins in the receptor membranes of the olfactory neurons (3Kaissling K.E. Annu. Rev. Neurosci. 1986; 9: 121-145Google Scholar, 6Vogt R.G. Prestwich G.D. Blomquist G.J. Pheromone Biochemistry. Academic Press, Inc., New York1987: 385-431Google Scholar, 7Vogt R.G. Goldsmith M.R. Wilkins A.S. Molecular Model Systems in the Lepidoptera. Cambridge University Press, New York1995: 341-367Google Scholar, 8Pelosi P. J. Neurobiol. 1996; 30: 3-19Google Scholar, 9Steinbrecht R.A. Chem. Senses. 1996; 21: 719-727Google Scholar). In searching for proteins that might support olfactory mechanisms, we characterized the protein profile of olfactory neuron receptor membranes of the wild silk moth Antheraea polyphemus. The morphology of the A. polyphemus antenna permits the relatively easy isolation of olfactory sensilla in a manner yielding olfactory receptor cilia as the only cellular component (10Vogt R.G. Prestwich G.D. Riddiford L.M. J. Biol. Chem. 1988; 263: 3952-3959Google Scholar). This preparation is free of other parts of the olfactory neurons as well as of nonneuronal cells of the antenna, and it was previously used to identify a pheromone-binding membrane protein using a radiolabeled photoaffinity analog of the A. polyphemus sex pheromone (10Vogt R.G. Prestwich G.D. Riddiford L.M. J. Biol. Chem. 1988; 263: 3952-3959Google Scholar). This protein co-migrated with bovine serum albumin on SDS gels (around 67 kDa) and appeared to be uniquely expressed in antennal tissue and to be a principal component of the olfactory cilia membrane. We have now used this A. polyphemus sensilla preparation to isolate and clone a 67-kDa membrane protein that is a major component of the ciliary receptor membranes of olfactory neurons; we have named this protein Snmp-1, or sensory neuron membrane protein-1. Molecular analyses suggest that Snmp-1 mRNA is uniquely expressed in olfactory neurons and that the protein is localized to the cilia, dendrites, and soma but is absent from the axons. Snmp-1 expression appears to initiate with the developmental appearance of the olfactory neurons, and it increases significantly late in antennal development, coincident with the acquisition of olfactory function. Sequence analysis indicates that Snmp-1 is homologous with the CD36 family of proteins, an as yet small family of receptor-like membrane proteins with identified members in vertebrates, arthropods, and nematodes. The few vertebrate CD36 proteins characterized thus far are thought to have two transmembrane domains and to interact with proteinaceous ligands (11Silverstein R.L. Asch A.S. Nachman R.L. J. Clin. Invest. 1989; 84: 546-552Google Scholar, 12Vega M.A. Segui-Real B. Garcia J.A. Cales C. Rodriguez F. Vanderkerckhove J. Sandoval I.V. J. Biol. Chem. 1991; 266: 16818-16824Google Scholar, 13Leung L.L.K. Li W.-X. McGregor J.L. Albrecht G. Howard R.J. J. Biol. Chem. 1992; 267: 18244-18250Google Scholar, 14Hart K. Wilcox M. J. Mol. Biol. 1993; 234: 249-253Google Scholar, 15Frieda S. Pearce S.F.A. Wu J. Silverstein R.L. J. Biol. Chem. 1995; 270: 2981-2986Google Scholar, 16Hunziker W. Geuze H.J. BioEssays. 1996; 18: 379-389Google Scholar). Snmp-1 is the first member of the CD36 family isolated from neural tissue. A. polyphemus silk moths were obtained as diapausing pupae from D. Bantz of Racine, Wisconsin, or collected in Wisconsin as wild males lured to caged females emitting sex pheromone. Pupae were held in diapause at 4 °C and allowed to develop to adults by incubation at 27 °C. Antennae from wild males were excised upon capture onto dry ice and were stored (−70 °C) for subsequent processing. Olfactory sensilla were collected from 800 A. polyphemus antennae (wild caught males). Antennal branches were isolated and processed in batches (40 antenna equivalents) as described previously (10Vogt R.G. Prestwich G.D. Riddiford L.M. J. Biol. Chem. 1988; 263: 3952-3959Google Scholar). Dried sensillar material was recovered (200 mg), and debris was thoroughly removed under a microscope. Branch material was collected for later mRNA isolation and cDNA library construction. Frozen sensilla were lyophilized, suspended in homogenization buffer (10 ml of TEM-P; 10 mm Tris-HCl, 10 mm EGTA, 1 mm MgCl2, pH 7.0, 0.1 mm phenylmethylsulfonyl fluoride) using a Dounce homogenizer and transferred to a 30-ml Corex centrifuge tube. Sensilla suspension was sonicated (5 min, Beckman microprobe, setting 45, in ice/water) and centrifuged three times (2,500 rpm; Sorvall HB-4 rotor; 10-ml resuspensions in TEM-P). The pooled supernatants were ultracentrifuged (35,000 rpm, Sorvall TH641 swinging bucket rotor, 4 °C, 90 min), the supernatant was collected, and the pellet was resuspended (TEM-P) and ultracentrifuged a second time. The final pellet was suspended in H2O with brief sonication and stored in aliquots (−70 °C). For electrophoresis, samples were lyophilized, dissolved in SDS sample buffer, and denatured as described previously (10Vogt R.G. Prestwich G.D. Riddiford L.M. J. Biol. Chem. 1988; 263: 3952-3959Google Scholar). For direct sequencing, proteins were electrophoretically transferred (Bio-Rad Trans-Blot 170-3910) as described by Vogt et al.(18Vogt R.G. Prestwich G.D. Lerner M.R. J. Neurobiol. 1991; 22: 74-84Google Scholar), either to polyvinylidene difluoride membranes (Immobilon-P, Millipore Corp.; Refs. 19Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Google Scholar and 20LeGendren N. Matsudaira P. BioTechniques. 1988; 6: 154-159Google Scholar) or to glass fiber filters (Whatman GF/C) modified by soaking in trifluoroacetic acid (21Aebersold R.H. Teplow D.B. Hood L.E. Kent S.B.H. J. Biol. Chem. 1986; 261: 4229-4238Google Scholar). For transfer to GF/C filters, SDS was first removed by washing the gel in four changes of 2% Nonidet P-40 in H2O (200 ml each wash); the gel was then rinsed in H2O (three times) and transferred in 1% acetic acid overnight at 80 V and 4 °C. GF/C and polyvinylidene difluoride blots were stained in Coomassie Blue and destained in acetic acid/methanol (18Vogt R.G. Prestwich G.D. Lerner M.R. J. Neurobiol. 1991; 22: 74-84Google Scholar). Stained bands were excised and sequenced using an Applied Biosystems model 470A gas phase microsequencer equipped with a model 120A on-line phenylthiohydantoin detector (by Dr. Kenneth Williams and Kathy Stone, Protein and Nucleic Acid Chemistry Facility, Yale University School of Medicine). Three blots and subsequent sequence analyses were performed; repetitive yields ranged from 91.2 to 91.8%. The 67-kDa protein yielded the N-terminal sequence MLLPKPLKYAAIGGGVFVFGILIGXVIFPV. The lack of ambiguity in the obtained sequences indicated that these sequences derived from the abundant protein shown (Fig. 1 A) rather than from a minor co-migrating protein. polymerase chain reaction polyacrylamide gel electrophoresis phosphate-buffered saline ribonuclease protection assay odorant-binding proteins lysosomal integral membrane protein II epithelial membrane protein scavenger receptor-type BI All RNA isolations utilized the acid guanidinium thiocyanate-phenol-chloroform method (22Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Google Scholar, 23Vogt R.G. Rybczynski R. Lerner M.R. J. Neurosci. 1991; 11: 2972-2984Google Scholar); tissue was initially ground under liquid nitrogen in a mortar and pestle in the presence of guanidinium thiocynate solution. cDNA template was synthesized using cloned Moloney murine leukemia virus RNase H− reverse transcriptase (Life Technologies, Inc.), following recommended protocols and including 400 units of RNasin (Promega) and 5 μg of total RNA in a 40-μl reaction. For PCR, degenerate primers were designed against four regions of the derived N-terminal amino acid sequence; degeneracy was reduced by making four primers to each region (Table I). PCR (100 μl) usedTaq DNA polymerase (3 units; Promega), supplied buffer containing Triton and 1.5 mm magnesium, 2 mmdNTP, 2 μCi of [32P]dCTP, 50 pmol of each primer, and 1 μl of cDNA from the above reaction. PCR was performed on a Perkin-Elmer thermocycler under oil overlay: 3 min 94 °C followed by 35 cycles at 94 °C (30 s), 52 °C (1 min), and 72 °C (3 min). PCR products were analyzed by 8% PAGE in TBE (24Maniatis, T., Fritsch, E. F., Sambrook, J., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar); products were visualized by autoradiography on x-ray film (Kodak X-Omat).Table IOligonucleotides used for PCRN-terminalMLLPKP1ATGCTNCTNCCNAATCC2ATGCTNTTRCCNAARCC3ATGTTRCTNCCNAARCC4ATGTTRTTRCCNAARCCC-terminalGIAAYK5CCDATRGCRGCRTAYTT6CCDATYGCRGCRTAYTT7CCDATRGCYGCRTAYTT8CCDATYGCYGCRTAYTTC-terminalIGFVFV9ATNCCRAARACRAARAC10ATNCCRAARACRAAYAC11ATNCCRAAYACRAARAC12ATNCCRAAYACRAAYACC-terminalVPFIV13ACRGGRAADATRAC14ACYGGRAADATYAC15ACYGGRAADATRAC16ACRGGRAADATYACOligonucleotides 1–4 are sense; 5–16 are antisense. All oligonucleotide sequences are 5′–3′ under amino acid sequences. Open table in a new tab Oligonucleotides 1–4 are sense; 5–16 are antisense. All oligonucleotide sequences are 5′–3′ under amino acid sequences. A “best” primer pair was determined based on the PCR results and used to amplify a 65-base pair product; primers were constructed withEcoRI sites: TTTTGAATTCATGTTRTTRCCNAARCC (sense) and TTTTGAATTCATNCCRAATACTAAYAC (antisense). The resulting PCR product was digested with EcoRI, gel-purified (8% PAGE), ligated into M13 vector, and sequenced (dideoxynucleotide method; Sequenase kit, U. S. Biochemical Corp.). A random primed adult male antennal cDNA library was constructed in λ Zap II (Stratagene) from mRNA isolated from the antennal branch material (described above) employing the RNase H method. cDNA (300 ng) was synthesized (described above) from 4 μg of mRNA (selected four times on oligo(dT)-cellulose) and 0.8 μg of random hexamer primer. Prior to ligation, cDNA larger than 800 bp was gel-purified and precipitated with excess glycogen to yield 36 ng of cDNA. The resulting library had a titer of 300,000 plaque-forming units. The library (180,000 plaque-forming units) was screened in XL1 Blue bacteria (Stratagene) following recommended protocols. Probe was a 32-base synthetic oligonucleotide (see Fig. 2) end-labeled with32P ATP using a T4 polynucleotide kinase (24Maniatis, T., Fritsch, E. F., Sambrook, J., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar) and used at 5 × 106 cpm/ml. Nylon membrane (ICN) lifts were prehybridized (15 h) and hybridized (24 h) in 5 × SSC (24Maniatis, T., Fritsch, E. F., Sambrook, J., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar), 5 × Denhardt's solution, 0.1% SDS, and 0.2 mg/ml salmon sperm DNA at 50 °C without formamide. Following hybridization, membranes were washed twice in 2 × SSPE (24Maniatis, T., Fritsch, E. F., Sambrook, J., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar), 0.1% SDS at room temperature, once in 2 × SSPE, 0.1% SDS at 50 °C, and once in 0.2 × SSPE, 0.1% SDS at 50 °C, all for 20 min. Membranes were exposed to Kodak X-Omat film. Positive plaques were eluted into 0.5 ml of SM buffer (24Maniatis, T., Fritsch, E. F., Sambrook, J., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar) and rescreened at low density. Final positive clones were subjected to plasmid rescue following recommended protocols (Stratagene) yielding 24 cDNA clones in pBluescript II (SK+). Following PCR analysis using the degenerate primer pairs 4–10 and 4–13 (Table I), a clone designated RP11 (random prime 11) was chosen for further analysis. snmp-1(RP11) was sequenced as double-stranded DNA by the dideoxynucleotide termination method (Sequenase kit, U. S. Biochemical Corp.). The entire clone was sequenced in both sense and antisense directions following exonuclease deletions from both 5′ and 3′ directions (ExoIII/mung bean nuclease deletion system, Stratagene). RNA was in vitro transcribed from the full-length RP11 cDNA clone linearized with KpnI, following protocols described below. Transcribed RNA (2 μg) was in vitro translated to protein using nuclease-treated rabbit reticulocyte lysate (Promega) in the presence of [35S]methionine (ICN Tran35S-label), subjected to SDS-PAGE, and visualized by fluorography (17Vogt R.G. Kohne A.C. Dubnau J.T. Prestwich G.D. J. Neurosci. 1989; 9: 3332-3346Google Scholar). Proteins with sequence homology to the deduced amino acid sequence of snmp-1 were identified by using the NCBI BLAST network server (25Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Google Scholar); additional analysis of BLAST identified homologues employed the FASTA algorithm (26Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Google Scholar, 27Pearson W.R. Methods Enzymol. 1990; 183: 63-98Google Scholar). Snmp-1 and CD36 family member amino acid sequences were aligned with the Clustal W multiple alignment program (28Thompson J.D. Higgins G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Google Scholar) for all group and pairwise comparisons. Snmp-1 RNA probe used in the Northern blot analyses and in situ hybridizations wasin vitro transcribed from the snmp-cr subclone (base pairs 1–1578, Fig. 2). Snmp-cr was prepared by PCR amplification of the coding region using primers specific to the extreme 5′ and 3′ ends; product was gel-purified and ligated into pCR-Script vector (Stratagene). Digoxigenin-incorporated RNA probes (antisense and sense) were synthesized from linearized plasmid using T7 or T3 RNA polymerase (Stratagene) following recommended protocols (Boehringer Mannheim) and in the presence of 40 units of RNasin (Promega). For in situ hybridization studies, RNA was alkaline-degraded to an approximately 160-base length (29Byrd C.A. Jones J.T. Quattro J.M. Rogers M.E. Brunjes P.C. Vogt R.G. J. Neurobiol. 1996; 29: 445-458Google Scholar). RNA probe for the ribonuclease protection assays (RPAs) was transcribed from the snmp-p subclone (base pairs 563–890, Fig. 2). snmp-p was prepared by PstI removal of a 1,850-base pair fragment from the snmp-1(RP11) clone.Snmp-p was linearized with NcoI and transcribed to produce a 408-base (327 bases of snmp-p + 81 bases of vector) 32P-labeled antisense RNA probe using the Maxiscript RNA transcription kit (Ambion) following recommended protocols (specific activity ≥ 3 × 108cpm/μg). Full-length transcript was purified by PAGE (5% acrylamide, 8 m urea) and autoradiography. The control probe used in the Northern blot analysis derived from aManduca sexta 18 S rRNA clone (GenBankTMaccession number U88190) in pBluescript (Stratagene). DNA was amplified by PCR using M13 forward and reverse primers in the presence of 2 mm dATP, dGTP, and dCTP, 0.6 mm dTTP, and 0.3 mm digoxigenin-dUTP (Boehringer Mannheim). Amplification was performed as follows using an Idaho Technology thermocycler: 2 min of denaturation at 94 °C; 35 cycles at 94 °C (15 s), 50 °C (30 s), and 74 °C (25 s). For each sample, 5 μg of poly(A)-enriched mRNA (Fig. 5 A) or 10 μg of total RNA (Fig. 5 C) was electrophoresed on a 1% agarose gel containing formaldehyde (24Maniatis, T., Fritsch, E. F., Sambrook, J., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar) and electrophoretically transferred (Trans-Blot Cell; Bio-Rad) in 1 × TAE (24Maniatis, T., Fritsch, E. F., Sambrook, J., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar) onto nylon membrane (HyBond-N, Amersham Corp.). Membranes were prehybridized for 2.5 h at 68 °C (5 × SSC, 0.1% N-lauroylsarcosine, 2 × Denhardt's solution, 0.02% SDS, 100 μg/ml herring sperm DNA) and hybridized with 25 ng/ml digoxigenin-labeled snmp-cr probe under the same conditions in a solution containing 50% formamide. To show that equivalent amounts of RNA were present in all lanes (Fig.5 C), M. sexta rRNA DNA probe was hybridized to the portion of the blot containing target rRNA and processed separately; hybridization was at 50 °C but at otherwise identical conditions to the snmp-cr blot. Both blots were washed at hybridization temperature in 0.1 × SSC, 0.1% SDS; probes were visualized by luminous detection (Lumiphos 530; Boehringer Mannheim) on x-ray film (Kodak, X-Omat). For each sample, 10 μg of total RNA was hybridized with 32P-labeledsnmp-p(NcoI) probe (8.7 × 104cpm; 14–16 h; 42 °C; 20 μl of hybridization solution (80% formamide, 100 mm Na+-citrate (pH 6.4), 300 mm Na+-acetate, 1 mm EDTA)) following recommended protocols (RPA II kit, Ambion). Unhybridized RNA was degraded by incubation in 200 μl of supplied RNase digestion mixture (containing RNase A and T1) for 30 min at 37 °C. The resulting protected RNA fragments were precipitated and visualized following PAGE (5% acrylamide, 8 m urea) on x-ray film exposed 6–12 h at −70 °C. All histology was done on newly emerged adult tissue; hybridization protocols were modified from Byrdet al. (29Byrd C.A. Jones J.T. Quattro J.M. Rogers M.E. Brunjes P.C. Vogt R.G. J. Neurobiol. 1996; 29: 445-458Google Scholar). Antennae were partially dissected and fixed by perfusion and subsequent incubation in 2% paraformaldehyde in phosphate-buffered saline (PBS), overnight on ice, dehydrated to 70% methanol, and stored at −20 °C. For sectioning, tissue was transferred to 70% ethanol, dehydrated though a graded series of ethanol and toluene, and incubated in melted paraffin (Periplast+) for 2–4 h before being embedded in plastic molds. Longitudinal sections and cross-sections (10 μm) were transferred to water drops on slides coated with albumin (Albumin Fixative, EM Diagnostic Systems). Slides were dewaxed in xylene, and sections were treated with Proteinase K (5 μg/ml in PBST (PBS, 0.1% Tween 20) for 15 min at room temperature), followed by acetic anhydride (0.25% in 100 mmtriethanolamine for 5 min at room temperature); two washes with glycine/PBS (2 mg/ml glycine, 5 min/wash) were applied between treatments. Sections were prehybridized overnight at 42 °C (0.6m NaCl; 10 mm Tris, pH 7.5; 2 mmEDTA; 1 × Denhardt's solution; 50 μg/ml herring sperm DNA; and 50 μg/ml tRNA) and hybridized with 100 ng/ml digoxigenin-labeledsnmp-cr probe or zebrafish odor receptor probe 1A (negative control, Ref. 29Byrd C.A. Jones J.T. Quattro J.M. Rogers M.E. Brunjes P.C. Vogt R.G. J. Neurobiol. 1996; 29: 445-458Google Scholar) under the same conditions but in the presence of 50% formamide. Sections were washed in hybridization solution (minus Denhardt's solution, DNA, tRNA, and probe) progressively diluted with PBST. Hybridized probe was enzymatically detected by phosphatase reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium following recommended protocols (Boehringer Mannheim, Ref.29Byrd C.A. Jones J.T. Quattro J.M. Rogers M.E. Brunjes P.C. Vogt R.G. J. Neurobiol. 1996; 29: 445-458Google Scholar). Slides were coverslipped with Aquamount mounting medium (Lerner Laboratories) and photographed with differential interference contrast optics. Polyclonal antiserum (rabbit) was prepared against recombinant Snmp-1. The snmp-1 coding region was cloned into the expression vector pET 15b (Novagen) and transfected into DE3 (pLysS) cells. Cells were grown to a 0.6 optical density (600 nm) at 37 °C, induced with isopropyl-1-thio-β-d-galactopyranoside (1.0 mm final concentration), and cultured at 27 °C for 4 h. Centrifuged bacterial pellet from a 50-ml culture yielded approximately 1 mg of recombinant Snmp-1 protein. Induced 59.9-kDa protein was isolated from the insoluble fraction of bacterial lysates by a His tag affinity column (Novagen) followed by SDS-PAGE. Gel slices were homogenized through syringes with 10 mm Tris (pH 7.0) and an equal volume of Freund's complete adjuvant and were used to immunize a rabbit by subcutaneous injection (University of South Carolina Institute for Biological Research and Technology Antibody Facility). Preabsorbed antiserum was prepared by incubation with DE3 bacterial lysate. Bacteria were lysed in 50 mm Tris, pH 7.9; 2 mm EDTA; 0.1% Triton X-100; 250 mmNaCl, 0.1 mm phenylmethylsulfonyl fluoride; 100 ng/ml lysozyme; 30 °C, 30 min. The lysate was sheared by passage through a syringe (23 g), incubated with total antiserum (1:5000 serum dilution; 4 °C, overnight), and centrifuged (16,000 × g, 4 °C, 20 min). The resulting supernatant was used as antiserum for Western blot analysis. For Western blot analysis, bacterial lysate (20 μl) containing expressed Snmp-1 and A. polyphemus antennal branch homogenates (2 antennal equivalents) were subjected to SDS-PAGE (10%), transferred to nitrocellulose membrane (Trans-Blot Cell, Bio-Rad; BA-S NC, Schleicher & Schuell), and incubated with Snmp-1 preabsorbed antiserum (36 h, 4 °C). The membrane was then incubated with goat IgG horseradish peroxidase conjugate (ICN; 1:5000, 2 h, room temperature) and stained (VIP substrate; Vector). Preimmune serum was used under otherwise identical conditions as a negative control. All antibody treatments included 3% nonfat dry milk in PBST as a blocking agent, and all washes were in PBST. For immunocytochemistry, tissue sections were prepared as described above for in situ analysis. Sections were dewaxed with toluene and incubated with total Snmp-1 antiserum (1:1000, 15 h, 4 °C) followed by goat IgG horseradish peroxidase conjugate (ICN; 1:100, 2 h, room temperature) and stained with VIP substrate (Vector) following recommended protocols. For a negative control, sections were incubated with preimmune serum under identical conditions. All washes and antibody treatments included 3% nonfat dry milk in PBS as a blocking agent. Slides were coverslipped with Permount (Fisher) and photographed using bright field or differential interference contrast optics. Antisera were immunohistochemically active at dilutions to 1:10,000. All x-ray film and photographic images were digitized and processed using Adobe Photoshop and printed using a Kodak ColorEase dye sublimation printer. Membranes of olfactory cilia were isolated from 800 antenna of wild caught A. polyphemus males collected in Wisconsin. SDS-PAGE analysis indicated an approximately 67-kDa protein as a major component of this membrane preparation (Fig. 1 A), here named Snmp-1. N-terminal amino acid analysis yielded a 30-amino acid sequence. PCR primers based on this sequence were used to amplify cDNA derived from leg and antennal mRNA; an antenna-specific product of the expected size was cloned and sequenced and then verified to encode the N-terminal amino acid sequence. A 32-base oligonucleotide was synthesized and used to probe a cDNA library. A random primed cDNA library was constructed from antennal mRNA and screened with the N-terminal 32-base oligonucleotide probe; 24 clones were obtained. Two clones were chosen and sequenced in their entirety. Both clones yielded matching sequences; each contained the N-terminal amino acid sequence obtained from the 67-kDa protein and identical in-frame stop codons following base position 1575. The cDNA sequence of one of these Snmp-1 clones identified as RP11 (random prime clone 11) is presented along with its translated amino acid sequence (Fig.2). The snmp-1(RP11) cDNA consists of 2,726 nucleotides and contains 1,575 nucleotides encoding a protein of 525 amino acids with a derived molecular mass of 59,917 Da. In vitro translation of RNA transcribed from full-length snmp-1(RP11) clone revealed a protein of about 59 kDa by SDS-PAGE (Fig. 1 B). Antiserum generated against 59.9-kDa bacterial expressed Snmp-1 protein recognized a 67-kDa protein isolated from antennal extracts; preimmune serum showed no detectable immunoreactivity against antennal proteins (Fig. 1 C). The approximately 7-kDa difference between cloned and native proteins may be due to post-translational glycosylation. Sequence analysis indicates four possible N-linked glycosylation sites within a generally hydrophilic region of the protein (Fig. 2). Identification of the translational start site is based on the start methionine in the N-terminal amino acid sequence obtained from the purified 67-kDa protein. No additional ATG was observed 5′ to this site in the cDNA sequence, although only 27 5′ nucleotides were present in the analyzed snmp-1(RP11) clone. Support for the stop codon position and size of open reading frame comes from the observed molecular weight of in vitrotranslated and bacterial expressed protein (Fig. 1, B andC) as well as the presence of 23 additional in-frame stop codons downstream from the predicted termination site. A data base homology search using the NCBI BLAST network server identified five candidate SNMP-1 homologous proteins; probability values range from 10−27 to 10−43, where a value ≤0.05 is considered statistically significant (30Karlin S. Altschul S.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2264-2268Google Scholar). FASTA PRDF statistical comparison of Snmp-1 with these five proteins generated a similarly significant value of 10−27 to 10−30 (26Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Google Scholar, 27Pearson W.R. Methods Enzymol. 1990; 183: 63-98Google Scholar). These presumptive homologues compose the CD36 receptor family: human CD36 (31Oquendo P. Hundt E. Lawler J. Seed B. Cell. 1989; 58: 95-101Google Scholar); human CLA I (32Calvo D. Vega M.A. J. Biol. Chem. 1993; 268: 18929-18935Google Scholar); rat LIMP II (12Vega M.A. Segui-Real B. Garcia J.A. Cales C. Rodriguez F. Vanderkerckhove J. Sandoval I.V. J. Biol. Chem. 1991; 266: 16818-16824Google Scholar); Drosophila emp (14Hart K. Wilcox M. J. Mol. Biol. 1993; 234: 249-253G
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