A new saponin has been isolated from a tropical plant Chrysantellum procumbens Rich. which is thought to be useful in the therapy of digestive troubles. The structure of this saponin was determined by chemical methods, nuclear magnetic resonance and mass spectrometry. The aglycone moiety is a triterpene, echinocystic acid; D-glucose, D-xylose and L-rhamnose are carbohydrate components. The structure of saponin was established as 3-O-beta-D-glucopyranosyl [L-rhamnopyranosyl-(alpha 1 leads to 3)-D-xylopyranosyl-)beta 1 leads to 4)-L-rhamnopyranosyl-(alpha 1 leads to 2)-D-xylopyranosyl]-(alpha 1 leads to 28)-echinocystyl. The name, chrysantellin A, is proposed for this new saponin.
Collapsin response mediator proteins (CRMPs) are believed to play a crucial role in neuronal differentiation and axonal outgrowth. Among them, CRMP2 mediates axonal guidance by collapsing growth cones during development. This activity is correlated with the reorganization of cytoskeletal proteins. CRMP2 is implicated in the regulation of several intracellular signaling pathways. Two subtypes, A and B, and multiple cytosolic isoforms of CRMP2B with apparent masses between 62 and 66 kDa have previously been reported. Here, we show a new short isoform of 58 kDa, expressed during brain development, derived from C-terminal processing of the CRMP2B subtype. Although full-length CRMP2 is restricted to the cytoplasm, using transfection experiments, we demonstrate that a part of the short isoform is found in the nucleus. Interestingly, at the tissue level, this short CRMP2 is also found in a nuclear fraction of brain extract. By mutational analysis, we demonstrate, for the first time, that nuclear translocation occurs via nuclear localization signal (NLS) within residues Arg471-Lys472 in CRMP2 sequence. The NLS may be unmasked after C-terminal processing; thereby, this motif may be surface-exposed. This short CRMP2 induces neurite outgrowth inhibition in neuroblastoma cells and suppressed axonal growth in cultured cortical neurons, whereas full-length CRMP2 promotes neurite elongation. The NLS-mutated short isoform, restricted to the cytoplasm, abrogates both neurite outgrowth and axon growth inhibition, indicating that short nuclear CRMP2 acts as a dominant signal. Therefore, post-transcriptional processing of CRMP2 together with its nuclear localization may be an important key in the regulation of neurite outgrowth in brain development. Collapsin response mediator proteins (CRMPs) are believed to play a crucial role in neuronal differentiation and axonal outgrowth. Among them, CRMP2 mediates axonal guidance by collapsing growth cones during development. This activity is correlated with the reorganization of cytoskeletal proteins. CRMP2 is implicated in the regulation of several intracellular signaling pathways. Two subtypes, A and B, and multiple cytosolic isoforms of CRMP2B with apparent masses between 62 and 66 kDa have previously been reported. Here, we show a new short isoform of 58 kDa, expressed during brain development, derived from C-terminal processing of the CRMP2B subtype. Although full-length CRMP2 is restricted to the cytoplasm, using transfection experiments, we demonstrate that a part of the short isoform is found in the nucleus. Interestingly, at the tissue level, this short CRMP2 is also found in a nuclear fraction of brain extract. By mutational analysis, we demonstrate, for the first time, that nuclear translocation occurs via nuclear localization signal (NLS) within residues Arg471-Lys472 in CRMP2 sequence. The NLS may be unmasked after C-terminal processing; thereby, this motif may be surface-exposed. This short CRMP2 induces neurite outgrowth inhibition in neuroblastoma cells and suppressed axonal growth in cultured cortical neurons, whereas full-length CRMP2 promotes neurite elongation. The NLS-mutated short isoform, restricted to the cytoplasm, abrogates both neurite outgrowth and axon growth inhibition, indicating that short nuclear CRMP2 acts as a dominant signal. Therefore, post-transcriptional processing of CRMP2 together with its nuclear localization may be an important key in the regulation of neurite outgrowth in brain development. Collapsin response mediator proteins (CRMPs) 3The abbreviations used are: CRMP, collapsin response mediator protein; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; NLS, nuclear localization signal; Px, postnatal day x;Ex, embryonic day x; c-ter, C-terminal region; DAPI, 4′,6-diamidino-2-phenylindole. constitute a family of five cytosolic proteins, abundantly expressed in the developing nervous system but down-regulated in the adult brain. They play important roles in neurite outgrowth and axonal guidance (1Minturn J.E. Fryer H.J. Geschwind D.H. Hockfield S. J. Neurosci. 1995; 15: 6757-6766Crossref PubMed Google Scholar, 2Goshima Y. Nakamura F. Strittmatter P. Strittmatter S.M. Nature. 1995; 376: 509-514Crossref PubMed Scopus (641) Google Scholar). Among them, CRMP2 was originally identified as a signaling molecule, required for growth cone collapse of dorsal root ganglion neurons, in response to a repulsive guidance cue, semaphorin-3A (2Goshima Y. Nakamura F. Strittmatter P. Strittmatter S.M. Nature. 1995; 376: 509-514Crossref PubMed Scopus (641) Google Scholar). CRMP2 was also reported to have a positive effect on axonal extension and to play a crucial role in axon-dendrite specification and axon regeneration, since the overexpression of CRMP2 induces the formation of multiple axons, and the expression of the dominant-negative form of CRMP2 or the knockdown of CRMP2 suppresses axon formation (3Inagaki N. Chuhara K. Arimura N. Menager C. Kawano Y. Matsuo N. Nishimura T. Amano M. Kaibuchi K. Nat. Neurosci. 2001; 4: 781-782Crossref PubMed Scopus (466) Google Scholar, 4Fukata Y. Itoh T.J. Kimura T. Ménager C. Nishimura T. Shiromizu T. Watanabe H. Inagaki N. Iwamatsu A. Hotani H. Kaibuchi K. Nat. Cell Biol. 2002; 4: 583-591Crossref PubMed Scopus (637) Google Scholar, 5Suzuki Y. Nakagomi S. Namikawa K. Kiryu-Seo S. Inagaki N. Kaibuchi K. Aizawa H. Kibuchi K. Kiyama H. J. Neurochem. 2003; 86: 1042-1050Crossref PubMed Scopus (73) Google Scholar, 6Nishimura T. Fukata Y. Kato K. Yamaguchi T. Matsuura Y. Kamiguchi H. Kaibuchi K. Nat. Cell Biol. 2003; 5: 819-826Crossref PubMed Scopus (220) Google Scholar, 7Yoshimura T. Kawano Y. Arimura N. Kawabata S. Kibuchi A. Kaibuchi K. Cell. 2005; 120: 137-149Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar). Morphological changes and the motility of neuronal growth cones are closely related to the reorganization of actin, tubulin, and other cytoskeletal proteins (8Buck K.B. Zheng J.Q. J. Neurosci. 2002; 22: 9358-9367Crossref PubMed Google Scholar, 9Zhou F.Q. Cohan C.S. J. Neurobiol. 2004; 58: 84-91Crossref PubMed Scopus (104) Google Scholar). Accordingly, CRMP2 co-localizes with F-actin in the growth cones of different types of neurons (1Minturn J.E. Fryer H.J. Geschwind D.H. Hockfield S. J. Neurosci. 1995; 15: 6757-6766Crossref PubMed Google Scholar, 2Goshima Y. Nakamura F. Strittmatter P. Strittmatter S.M. Nature. 1995; 376: 509-514Crossref PubMed Scopus (641) Google Scholar, 10Yuasa-Kawada J. Suzuki R. Kano F. Ohkawara T. Murata M. Noda M. Eur. J. Neurosci. 2003; 17: 2329-2343Crossref PubMed Scopus (119) Google Scholar). Moreover, CRMP2 is thought to regulate axonal growth through its interaction with tubulin dimers to promote microtubule assembly. The CRMP2-tubulin interaction seems to be mediated by a microtubule-binding domain, spanning amino acids 323-381 within CRMP2 (4Fukata Y. Itoh T.J. Kimura T. Ménager C. Nishimura T. Shiromizu T. Watanabe H. Inagaki N. Iwamatsu A. Hotani H. Kaibuchi K. Nat. Cell Biol. 2002; 4: 583-591Crossref PubMed Scopus (637) Google Scholar). Besides, CRMP2 was shown to bind to the kinesin-1 light chain, thereby regulating the transport of soluble tubulin to the distal parts of growing axons (11Kimura T. Arimura N. Fukata Y. Watanabe H. Iwamatsu A. Kaibuchi K. J. Neurochem. 2005; 93: 1371-1382Crossref PubMed Scopus (180) Google Scholar). By interacting with the Sra-1 (Rac1-associated protein 1)-Wave1 (WASP family verpolin-homologous protein 1) complex, CRMP2 operates by transporting it to the growth cone of axons via kinesin-1 light chain (12Kawano Y. Yoshimura T. Tsuboi D. Kawabata S. Kaneto-Kawano T. Shirataki H. Takenawa T. Kaibuchi K. Mol. Cell. Biol. 2005; 25: 9920-9935Crossref PubMed Scopus (206) Google Scholar). CRMP2 also participates in the polarized Numb-mediated endocytosis of the neuronal adhesion molecule L1 at the growth cones (6Nishimura T. Fukata Y. Kato K. Yamaguchi T. Matsuura Y. Kamiguchi H. Kaibuchi K. Nat. Cell Biol. 2003; 5: 819-826Crossref PubMed Scopus (220) Google Scholar). Besides the above mentioned proteins, other CRMP2-interacting molecules, such as chimaerin and phospholipase D, have also been identified (13Lee S. Kim J.H. Lee C.S. Kim J.H. Kim Y. Heo K. Ihara Y. Goshima Y. Suh P.G. Ryu S.H. J. Biol. Chem. 2002; 277: 6542-6549Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Brown M. Jacobs T. Eickholt B. Ferrari G. Teo M. Monfries C. Qi R.Z. Leung T. Lim L. Hall C. J. Neurosci. 2004; 24: 8994-9004Crossref PubMed Scopus (182) Google Scholar). In parallel, our group has shown that CRMP2 may act as a regulator of other functions, such as migration and proliferation, since CRMP2 is highly expressed in the developing cerebellum, either in the proliferative external granular layer or in growing fibers in the molecular layer (15Ricard D. Rogemont V. Charrier E. Aguera M. Bagnard D. Belin M.F. Thomasset N. Honnorat J. J. Neurosci. 2001; 15: 7203-7214Crossref Google Scholar). Collectively, these data indicate that by interacting with different molecular partners, CRMP2 may play a pivotal role in regulating several signaling pathways leading to nervous system development. A number of studies have raised the possibility that post-translational modifications of CRMP2 modulate its activity in axonal growth or growth cone dynamics, by preventing its association with other molecules. There is growing evidence that CRMP2-tubulin interaction is regulated by CRMP2 phosphorylation, which inhibits the ability of CRMP2 to promote microtubule assembly and induces growth cone collapse, suggesting that the phosphorylation states of the CRMP2 modulate its activity (7Yoshimura T. Kawano Y. Arimura N. Kawabata S. Kibuchi A. Kaibuchi K. Cell. 2005; 120: 137-149Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar, 16Cole A.R. Knebel A. Morrice N.A. Robertson L.A. Irving A.J. Connolly C.N. Shuterland C. J. Biol. Chem. 2004; 279: 50176-50180Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 17Arimura N. Inagaki N. Chihara K. Ménager C. Nakamura N. Amano M. Iwamatsu A. Goshima Y. Kaibuchi K. J. Biol. Chem. 2000; 275: 23973-23980Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 18Arimura N. Ménager C. Kawano Y. Yoshimura T. Kawabata S. Hattori A. Fukata Y. Amano M. Goshima Y. Inagaki M. Morone N. Usukura J. Kaibuchi K. Mol. Cell. Biol. 2005; 25: 9973-9984Crossref PubMed Scopus (222) Google Scholar). On the other hand, hyperphosphorylation of CRMP2 is implicated in some pathology, such as Alzheimer disease (16Cole A.R. Knebel A. Morrice N.A. Robertson L.A. Irving A.J. Connolly C.N. Shuterland C. J. Biol. Chem. 2004; 279: 50176-50180Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 19Gu Y. Hamajima N. Ihara Y. Biochemistry. 2000; 39: 4267-4275Crossref PubMed Scopus (158) Google Scholar, 20Uchida Y. Ohshima T. Sasaki Y. Suzuki H. Yanai S. Yamashita N. Nakamura F. Takei K. Ihara Y. Mikoshiba K. Kolattukudy P. Honnorat J. Goshima Y. Genes Cell. 2005; 10: 165-179Crossref PubMed Scopus (365) Google Scholar). However, besides phosphorylation, other post-translational modifications may exist, which regulate the function of CRMP2. CRMP2 has the characteristic of being present under different isoforms (21Bretin S. Reibel S. Charrier E. Maus-Moati M. Auvergnon N. Thevenoux A. Glowinski J. Rogemond V. Premont J. Honnorat J. Gauchy C. J. Comp. Neurol. 2005; 486: 1-17Crossref PubMed Scopus (95) Google Scholar); however, little is known about the function of each isoform. Two subtypes (A and B) have been reported for different CRMPs (1Minturn J.E. Fryer H.J. Geschwind D.H. Hockfield S. J. Neurosci. 1995; 15: 6757-6766Crossref PubMed Google Scholar, 2Goshima Y. Nakamura F. Strittmatter P. Strittmatter S.M. Nature. 1995; 376: 509-514Crossref PubMed Scopus (641) Google Scholar, 3Inagaki N. Chuhara K. Arimura N. Menager C. Kawano Y. Matsuo N. Nishimura T. Amano M. Kaibuchi K. Nat. Neurosci. 2001; 4: 781-782Crossref PubMed Scopus (466) Google Scholar, 4Fukata Y. Itoh T.J. Kimura T. Ménager C. Nishimura T. Shiromizu T. Watanabe H. Inagaki N. Iwamatsu A. Hotani H. Kaibuchi K. Nat. Cell Biol. 2002; 4: 583-591Crossref PubMed Scopus (637) Google Scholar), derived from alternative splicing of their N-terminal region (10Yuasa-Kawada J. Suzuki R. Kano F. Ohkawara T. Murata M. Noda M. Eur. J. Neurosci. 2003; 17: 2329-2343Crossref PubMed Scopus (119) Google Scholar). We reported that CRMP2A, present as a 75-kDa protein, was specifically localized in neuronal soma and/or axons but was absent from dendrites, whereas CRMP2B was localized in both axons and dendrites (21Bretin S. Reibel S. Charrier E. Maus-Moati M. Auvergnon N. Thevenoux A. Glowinski J. Rogemond V. Premont J. Honnorat J. Gauchy C. J. Comp. Neurol. 2005; 486: 1-17Crossref PubMed Scopus (95) Google Scholar). The balance between the expressions of the two subtypes is involved in the control of axon branching and elongation. CRMP2B is present in different isoforms with apparent masses varying between 62 and 66 kDa (10Yuasa-Kawada J. Suzuki R. Kano F. Ohkawara T. Murata M. Noda M. Eur. J. Neurosci. 2003; 17: 2329-2343Crossref PubMed Scopus (119) Google Scholar, 19Gu Y. Hamajima N. Ihara Y. Biochemistry. 2000; 39: 4267-4275Crossref PubMed Scopus (158) Google Scholar, 21Bretin S. Reibel S. Charrier E. Maus-Moati M. Auvergnon N. Thevenoux A. Glowinski J. Rogemond V. Premont J. Honnorat J. Gauchy C. J. Comp. Neurol. 2005; 486: 1-17Crossref PubMed Scopus (95) Google Scholar). Despite extensive studies on CRMP2 during the last few years, no attempt has been made to elucidate the spatio-temporal expression and the function of each of these multiple isoforms of CRMP2. Recently, another isoform (58 kDa) resulting from post-translational processing of CRMP2 and of CRMP4 has been reported after brain injury, such as ischemia (21Bretin S. Reibel S. Charrier E. Maus-Moati M. Auvergnon N. Thevenoux A. Glowinski J. Rogemond V. Premont J. Honnorat J. Gauchy C. J. Comp. Neurol. 2005; 486: 1-17Crossref PubMed Scopus (95) Google Scholar, 22Kowara R. Chen Q. Milliken M. Chakravarthy B. J. Neurochem. 2005; 95: 466-474Crossref PubMed Scopus (45) Google Scholar, 23Chung M.A. Lee J.E. Lee J.Y. Ko M.J. Lee S.T. Kim H.J. Neuroreport. 2005; 16: 1647-1653Crossref PubMed Scopus (36) Google Scholar, 24Kobeissy F.H. Otten A.K. Zhang Z. Liu M.C. Denslow N.D. Dave J.R. Tortella C. Hayes R.L. Wang K.K.W. Mol. Cell Proteomics. 2006; 5: 1887-1898Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). We showed an N-methyl-d-aspartate-induced post-translational processing of CRMPs, including CRMP2, in cortical neurons, also leading to the appearance of short isoform (25Bretin S. Rogemond V. Marin P Maus M Torrens Y. Honnorat J. Glowinski J. Prémont J. Gauchy C. J. Neurochem. 2006; 98: 1252-1265Crossref PubMed Scopus (66) Google Scholar). Because CRMP2 exerts various functions, depending on its post-translational modification state, an insight into the spatio-temporal expression and the function of the 58-kDa short isoform offers an important key to elucidating the role of this isoform in neuronal development. In the present study, we show, the developmentally dependent expression of a short isoform (58 kDa) of CRMP2 exclusively present in the embryonic and postnatal stages but absent in the adult brain. We show that this short isoform derives from post-translational C-terminal processing of the CRMP2B subtype, thus unmasking a nuclear localization signal (NLS) within CRMP2. This short isoform is found in the nucleus, at both cell and tissue levels. We define, for the first time, a functional NLS sequence within the CRMP2 isoform. The nuclear short CRMP2 induces neurite outgrowth inhibition, thereby suggesting that CRMP2 plays an important role in the regulation of neurite outgrowth in brain development. Crude Brain Extract—Adult male, postnatal days 1 (P1), 5 (P5), and 15 (P15), rats or mice (OFA; Charles River Laboratories, L'Arbresle, France) were anesthetized with pentobarbital. Embryos at embryonic days 14 (E14), 16 (E16), and 19 (E19) were removed from anesthetized pregnant females. After decapitation, the brains were removed and chilled on ice. All subsequent steps were performed at 4 °C. Cerebral tissues were sonicated in 10 mm Tris-HCl, pH 7.4, 0.02% sodium azide, 1 mm EDTA, 0.2% Triton X-100 supplemented with protease inhibitor mixture (Complete; Roche Applied Science) and centrifuged for 10 min at 2000 × g at 4 °C. The proteins in the supernatant were quantified (Coomassie Plus protein assay reagent; Pierce), diluted to a concentration of 6 mg/ml in the homogenization buffer, and stored at -80 °C until used. Subcellular Fractionation—Cerebella of postnatal rats (P1) were explanted, cleaned free of meninges, and subjected to subcellular fractionation using the proteoExtract subcellular proteome extraction kit from Calbiochem, following exactly the manufacturer's instructions. Briefly, 50 mg of tissue were resuspended in 1 ml of extraction Buffer I and incubated for 10 min at 4 °C under gentle agitation. After centrifugation for 10 min at 1000 × g, the supernatant containing the cytosolic fraction was kept apart. Then the pellet was resuspended in 1 ml of extraction Buffer II and incubated for 30 min at 4 °C, and the suspension was centrifuged for 30 min at 6000 × g. The supernatant, containing the membrane protein fraction, was kept apart, and the pellet was incubated with 500 μl of Buffer III containing 375 units of Benzonase (Calbiochem) for 10 min at 4 °C under gentle agitation. Another centrifugation at 10,000 × g allowed the separation of nuclear protein remaining in the supernatant. The pellet, dissolved in 500 μl of Buffer IV, allowed the recovery of the cytoskeletal fraction. All buffers contained 5 μl of inhibitor mixture. Antibodies Used and Western Blot Analysis—The site-specific antibodies to CRMP2 were obtained as previously described (21Bretin S. Reibel S. Charrier E. Maus-Moati M. Auvergnon N. Thevenoux A. Glowinski J. Rogemond V. Premont J. Honnorat J. Gauchy C. J. Comp. Neurol. 2005; 486: 1-17Crossref PubMed Scopus (95) Google Scholar, 26Quach T.T. Duchemin A.M. Rogemond V. Aguera M. Honnorat J. Belin M.F. Kolattukudy P.E. Mol. Cell Neurol. 2004; 25: 433-443Crossref PubMed Scopus (71) Google Scholar). The peptide sequences used to generate specific antisera were 557IVAPPGGRANITSLG572 targeting the CRMP2 C-terminal region (C-ter), and 454LEDGTLHVTEGS465 (pep4). Peptides were conjugated to keyhole limpet hemocyanin and used to immunize rabbits. The anti-peptide antibodies (IgG) were purified by affinity chromatography on the corresponding immobilized peptide. Extracts from brain or transfected cells (30 μg) were diluted in Laemmli sample buffer, resolved by SDS-PAGE (8 or 10% polyacrylamide gels), transferred onto a polyvinylidene difluoride membrane, and incubated with different antibodies as described (25Bretin S. Rogemond V. Marin P Maus M Torrens Y. Honnorat J. Glowinski J. Prémont J. Gauchy C. J. Neurochem. 2006; 98: 1252-1265Crossref PubMed Scopus (66) Google Scholar). The target protein was detected using diaminobenzidine as peroxidase substrate or an enhanced chemiluminescence (ECL) detection system (Covalab, Lyon, France) and x-ray film. Densitometric quantification of the immunoblot bands was performed using ImageQuant (Amersham Biosciences). Protein Preparation, In-gel Enzymatic Proteolysis, and Mass Spectrometry—A 300-μl aliquot of the cytosolic fraction from mouse brain cortex was dialyzed against 25 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm dithiothreitol, 5 mm MgCl2, and 10 mm PPi and applied to 1 ml of Q-Sepharose columns (Hi-trap Q; GE Healthcare). After elution with 0.5 m NaCl, in the same buffer, CRMP2-enriched fractions were pooled and concentrated on an Ultrafree Biomax membrane (Millipore), dialyzed, and then subjected to SDS-PAGE (8% polyacrylamide). Protein bands were stained with Coomassie Blue, according to standard protocols, the bands of interest were excised from the gel, and in-gel digestion with trypsin was carried out as described by Shevchenko et al. (27Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7831) Google Scholar) with minor modifications. Tryptic peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, using a Voyager DE-PRO work station (Applied Biosystems, Courtaboeuf, France). Spectra were recorded in the mass range 700-5000 Da. A 200-μl solution of 0.5% α-cyano-4-hydroxycinnamic acid (LaserBioLab, Sophia-Antipolis, France) in 50% acetonitrile, 50% H2O, and 0.1% trifluoroacetic acid was used as matrix. The instrument was calibrated using trypsin autolysis fragments at m/z 842.5100 and 2211.1046 Da. Peptide mass fingerprinting was compared with the theoretical masses from the SwissProt sequence data base using Protein Prospector MS-Fit software. Typical search parameters were as follows: ±40 ppm of mass tolerance, carbamidomethylation of cysteine residues, methionine considered in oxidized form, one missed enzymatic cleavage for trypsin. A minimum of five peptide mass hits was required for a match, and a protein mass range from 5 to 100 kDa was permitted. Probability-based molecular weight search scores greater than 1000 were considered as significant. Data Base and Structure Analysis—Sequences were aligned using the Clustal program (28Higgins D.G. Sharp D. Gene (Amst.). 1988; 73: 237-244Crossref PubMed Scopus (2885) Google Scholar). The prediction programs PROSITE (available on the World Wide Web) and PSORT II (available on the World Wide Web) were used to identify putative NLS. The online prediction program (available on the World Wide Web) was used to identify the intrinsically unstructured protein-related structure of the C-terminal part of CRMP2. The structure of CRMP2 was modeled, based on the coordinates available for CRMP2 chain D (protein Data Bank entry 2GSE), using Viewerlite version 4.2 (Accelrys). Expression Constructs, Cell Culture, and Transfection—Full-length CRMP2 or the C-terminally truncated ΔC503 was amplified by PCR and inserted directionally into the pCMV2-FLAG vector (Sigma), which generated a protein with a FLAG tag at its N terminus. A two-step PCR procedure was used for the preparation of CRMP2 ΔC503 mutants. First, C-terminal fragments were generated using ΔC503 reverse primers introducing an EcoRI site at the 3′-end and forward primers with substituted codons as follows: mutant 1, Arg471 (CGG) and Lys472 (AAG), both substituted with Ala (GCG); mutant 2, Lys480 (AAA) and Arg481 (CGC), substituted with Ala (GCA) and Ala (GCC) respectively; mutant 3, Lys483 (AAG), Arg485 (AGG), and Arg487 (AGG) each substituted with Ala (GCG). Next, the three mutated fragments were used as reverse primers in the PCR with a wild type forward primer introducing a HindIII site at the 5′-end. For the full-length mutant Arg471-Lys472, two overlapping PCR products bearing the mutated codons (nucleotides 1-1506 and 1387-1716) were first obtained by separated reaction using wild-type CRMP2 as a template. Fragment 1-1506 corresponded to ΔC503 mutant 1. Fragment 1387-1716 was generated using a mutagenic forward primer introducing the above mentioned substituted codons and a wild-type reverse primer. Both fragments were mixed and then allowed to hybridize (5 min at 50 °C) and elongate (10 min at 70 °C) for the generation of the mutated template. Finally, this template was used in a last PCR step to generate full-length mutated CRMP2, using the above described forward and reverse CRMP2 primers introducing HindIII and EcoRI sites. The correct DNA sequences of all constructs were verified. The final PCR product was cloned into the HindIII and EcoRI sites of the pCMV2-FLAG vector. PC12 cells were transfected by 2 μg of purified plasmid using the Amaxa nucleofector reagent and electroporation, following precisely the manufacturer's protocol. N1E-115 cells were seeded in 10% fetal bovine serum-containing medium and cultured overnight. The cells were transiently transfected, in the absence of serum, using Lipofectamine LTX (Invitrogen) essentially as described (4Fukata Y. Itoh T.J. Kimura T. Ménager C. Nishimura T. Shiromizu T. Watanabe H. Inagaki N. Iwamatsu A. Hotani H. Kaibuchi K. Nat. Cell Biol. 2002; 4: 583-591Crossref PubMed Scopus (637) Google Scholar, 5Suzuki Y. Nakagomi S. Namikawa K. Kiryu-Seo S. Inagaki N. Kaibuchi K. Aizawa H. Kibuchi K. Kiyama H. J. Neurochem. 2003; 86: 1042-1050Crossref PubMed Scopus (73) Google Scholar). Twenty-four hours post-transfection, cells were cultured in 5% serum-containing medium for 24 h and were treated for immunohistochemistry analysis. Cortical neurons from E15 mouse embryos were prepared as described (29Bouillot C. Prochiantz A. Rougon G. Allinquant B. J. Biol. Chem. 1996; 271: 7640-7644Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Dissociated cells were seeded on plastic dishes coated overnight with 1.5 μg/ml poly-d-ornithine in chemically defined Dulbecco's modified Eagle's medium/F-12 medium free of serum with 2 mm glutamine, 9 mm NaHCO3, 10 mm HEPES, and 33 mm glucose. The medium was supplemented with hormones and proteins (100 μg/ml transferin, 25 μg/ml insulin, 20 nm progesterone, 60 μm putrescine, 0.1% ovalbumin, and 30 nm sodium selenite). After 3 days of in vitro culture, neurons were transfected using Lipofectamine LTX (Invitrogen) in medium without hormones and proteins. Twenty-four hours post-transfection, neurons were cultured in the presence of hormones and proteins for a further 48 h. Immunofluorescence Study and Microscopic Observation—The PC12 cells were observed 48 h after transfection using a laser-scanning confocal system (Leica TCF SP2) imaging platform. The cells were fixed in 4% paraformaldehyde for 20 min, washed in phosphate-buffered saline, and blocked with phosphate-buffered saline containing 2% gelatin and 0.1% Triton X-100 before incubation with different antibodies. The cells were double-stained with polyclonal anti-FLAG (F 7425; Sigma) and anti-rabbit Alexa Fluo 488 (Invitrogen) antibodies and with monoclonal anti-α-tubulin (Sigma) and anti-mouse Cy3 (Jackson) antibodies. For some experiments, anti-rabbit Alexa Fluo 546 (Invitrogen) was used. Nuclei were visualized by DAPI staining, and Alexa Fluo 546-phalloidin (Invitrogen) was used for F-actin staining. For morphometric analysis, both N1E-115 cells and mouse embryo cortical neurons transfected with FLAG-CRMP2 (full-length, Δ503, and mutant 1) were viewed using the Axioplan II fluorescence microscope (Carl Zeiss). N1E-115 cells were fixed and stained with polyclonal anti-FLAG and anti-rabbit Alexa Fluo 488 antibodies. The percentage of cells bearing neurites (length >20 μm from the cell body) among the transfected cells was measured. At least 300 cells for each expressed protein from three different experiments were examined. Cortical neurons were fixed and visualized by immunostaining with anti-FLAG antibody. Non-transfected cortical neurons, used as control cells, were stained by anti-pep4 and anti-rabbit Alexa Fluo 488 antibodies. The length of the longest neurite was measured as that of an axon and compared with the axon length of control neurons. At least 40 cells for each expressed protein were examined in one experiment. The axon length from three different experiments was examined. Identification of Different Isoforms of CRMP2 during Brain Development—To study different isoforms of CRMP2, a site-specific antibody recognizing residues 454-465 of CRMP2 was used (anti-pep4; Fig. 1A). The specificity of this antibody toward CRMP2, but not CRMP1, -3, -4, and -5, had previously been confirmed (26Quach T.T. Duchemin A.M. Rogemond V. Aguera M. Honnorat J. Belin M.F. Kolattukudy P.E. Mol. Cell Neurol. 2004; 25: 433-443Crossref PubMed Scopus (71) Google Scholar). Besides the well known and abundant 64-kDa CRMP2, other isoforms of CRMP2 migrating as 62 and 66 kDa could be detected in embryonic brain extract as faint bands (Fig. 1C, anti-pep4), their intensity increasing at the postnatal stages. The presence of such isoforms was reported in previous studies (19Gu Y. Hamajima N. Ihara Y. Biochemistry. 2000; 39: 4267-4275Crossref PubMed Scopus (158) Google Scholar). However, in embryonic brain cortex, an additional isoform presenting an apparent mass of 58 kDa was recognized by the anti-pep4 antibody (Fig. 1C). To determine whether the presence of this short isoform of CRMP2 is development-dependent, it was searched for in brain extracts at embryonic and early postnatal stages. As an internal loading control, the extent of actin was checked in all lanes using anti-actin antibody (data not shown). Anti-pep4 antibody clearly recognized a band migrating at 58 kDa in the cortex extract from embryo (stages E14, E16, and E19) as well as in the extract of early postnatal P1-P15 brain (Fig. 1C, anti-pep4). The intensity of this band decreased gradually during development, since only a faint band was recognized by the anti-pep4 antibody at stage P15 (Fig. 1, C and D), whereas other isoforms (62-66 kDa) of CRMP2B were clearly detected (Fig. 1C). As shown in Fig. 1D, the ratio of the 58 kDa band to 64 kDa decreased at late stages of brain development. This 58 kDa band was also detected by anti-pep4 antibody in an extract from primary cortical neuron cultured at stage E15 (Fig. 1B). In addition, a high molecular mass band (75 kDa) belonging to CRMP2A could be visualized from the P1 stage onward (Fig. 1C). On the other hand, in the adult brain, the 58 kDa band was almost absent, since a very faint band, presenting very low intensity, could be detected by the anti-pep4 antibody. However, significant intensity was detected for the 64-kDa isoform (Fig. 1, C and D). The appearance of t
Specific antibodies are essential tools for studying proteins as well as for diagnostic research in biomedicine. The egg yolk of immunized chicken is an inexpensive source of high-quality polyclonal antibodies. The 12-kDa Parietaria judaica 2 allergen was expressed as a fusion protein and was used to immunize Leghorn chickens. In this paper, we show, using 2-dimensional gel electrophoresis and immunoblotting, that chicken antibodies raised against a recombinant allergen can be used to recognize similar proteins from a pollen raw extract. Allergen identity was confirmed by nanoLC-nanospray-tandem mass spectrometry analysis. Our data demonstrate for the first time that a synergistic combination of molecular biology, 2-dimensional PAGE, and use of nonmammalian antibodies represents a powerful tool for reliable identification of allergens.
