Commercial Recombinant Human β‐Nerve Growth Factor and Adult Rat Dorsal Root Ganglia Contain an Identical Molecular Species of Nerve Growth Factor Prohormone
2000; Wiley; Volume: 74; Issue: 5 Linguagem: Inglês
10.1046/j.1471-4159.2000.0742127.x
ISSN1471-4159
AutoresMax Reinshagen, Irmlind Geerling, Viktor E. Eysselein, G Adler, Kenneth R. Huff, Gordon Moore, Jayaraman Lakshmanan,
Tópico(s)Neuropeptides and Animal Physiology
ResumoJournal of NeurochemistryVolume 74, Issue 5 p. 2127-2133 Free Access Commercial Recombinant Human β-Nerve Growth Factor and Adult Rat Dorsal Root Ganglia Contain an Identical Molecular Species of Nerve Growth Factor Prohormone Max Reinshagen, Max ReinshagenSearch for more papers by this authorIrmlind Geerling, Irmlind GeerlingSearch for more papers by this authorViktor E. Eysselein, Viktor E. EysseleinSearch for more papers by this authorGuido Adler, Guido AdlerSearch for more papers by this authorKenneth R. Huff, Kenneth R. HuffSearch for more papers by this authorGeoffrey Philip Moore, Geoffrey Philip MooreSearch for more papers by this authorJayaraman Lakshmanan, Jayaraman LakshmananSearch for more papers by this author Max Reinshagen, Max ReinshagenSearch for more papers by this authorIrmlind Geerling, Irmlind GeerlingSearch for more papers by this authorViktor E. Eysselein, Viktor E. EysseleinSearch for more papers by this authorGuido Adler, Guido AdlerSearch for more papers by this authorKenneth R. Huff, Kenneth R. HuffSearch for more papers by this authorGeoffrey Philip Moore, Geoffrey Philip MooreSearch for more papers by this authorJayaraman Lakshmanan, Jayaraman LakshmananSearch for more papers by this author First published: 30 July 2008 https://doi.org/10.1046/j.1471-4159.2000.0742127.xCitations: 42 Address correspondence and reprint requests to Dr. M. Reinshagen at Department of Internal Medicine I, University of Ulm, Robert-Koch-Strasse 8, D-89081, Ulm, Germany. E-mail: max.reinshagen@medizin.uni-ulm.de Lippincott Williams & Wilkins, Inc., Philadelphia Abbreviations used: DRG, dorsal root ganglia; NANase II, N-acetylneuraminidase H; NGF, nerve growth factor; O-glycosidase DS, O-glycosidase disaccharide; PAGE, polyacrylamide gel electrophoresis; PNGase F, peptide N-glycosidase; PVDF, polyvinylidene difluoride; rh-β-NGF, recombinant human β-nerve growth factor; SDS, sodium dodecyl sulfate. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Abstract: Examination of commercial recombinant human β-nerve growth factor (rh-β-NGF) preparations with polyclonal antibodies specific to 13-kDa NGF and pro-NGF-specific domains revealed the presence of high-molecular-mass immunoreactive proteins, including a 60-kDa NGF prohormone. On incubation with a mixture of N- and O-specific glycosidases, the 60-kDa NGF prohormone generated a 32-kDa protein corresponding to the molecular size of NGF precursor predicted by the cloned human NGF cDNA. Highly sensitive chemiluminescence immunoblot analysis of adult rat dorsal root ganglia, spinal cord, and colon tissues with NGF- and pro-NGF domain-specific antibodies also revealed the presence of high-molecular-mass proteins, including the 60-kDa NGF prohormone. Based on the presence of the 60-kDa NGF prohormone in dorsal root ganglia and its efferent tissues, we suggest that proteolytically unprocessed, glycosylated NGF prohormone may mediate interactions between neurons and the tissues they innervate. Nerve growth factor (NGF), a 13-kDa basic protein, was the first biochemically defined member of the neurotrophin family (12; 13). This protein supports the survival and differentiation of specific neurons in the sympathetic and sensory PNSs. The structure of cloned NGF cDNA suggests that NGF is synthesized as part of 32- and 27-kDa precursors (18; 22). Based on the high concentrations of 13-kDa NGF that have been isolated from adult mouse submaxillary gland (1) and studies with cell lines transfected with full-length NGF cDNA, it has been hypothesized that the precursor is processed to the mature 13-kDa form in the trans-Golgi network (7; 2; 19). In contrast, high-molecular-weight NGF proteins (17; 4; 10), including the NGF precursor, have also been reported in adult male mouse submaxillary gland, a known site of NGF gene expression (18; 22). In addition, conditioned media of established human germ cell lines and rat germ cells have been noted to contain biologically active, highmolecular-weight NGF proteins (5; 15). Recently, we identified several high-molecular-weight NGF proteins, including a 60-kDa NGF prohormone, in different mouse NGF preparations obtained from commercial sources (16). Here we report the presence of high-molecular-mass NGF proteins, including a 60-kDa proteolytically unprocessed, glycosylated NGF prohormone, in a commercial recombinant human β-NGF (rh-β-NGF) preparation as well as in the adult dorsal root ganglia (DRG) and two of its efferent target tissues. MATERIALS AND METHODS Source of rh-β-NGF rh-β-NGF was purchased from Genzyme (Cambridge, MA, U.S.A.; catalogue no. 80-3938-01, lot no. B 50149), Sigma (St. Louis, MO, U.S.A.; catalogue no. N-4273, lot no. 044H02862), and R & D Systems (Minneapolis, MN, U.S.A.; catalogue no. 256-CF, lot no. HS 188081). Source of antibodies Human and mouse NGF monoclonal antibodies were obtained from Promega (Madison, WI, U.S.A.; catalogue no. G1131, lot no. 38088) and Boehringer-Mannheim, Germany (catalogue no. 1087754, clone 27/12), respectively. Mouse β-NGF antibody was obtained from Pro-Hormone Science, Torrance, CA, U.S.A. (catalogue no. 413, lot no. 4647). Antibodies to prepro-NGF-specific domains corresponding to the larger (catalogue no. 418, lot no. 101) and smaller (catalogue no. 421, lot no. 101) NGF precursors (6) were also obtained from Pro-Hormone Science. All antibodies were used in a 1:1,000 final concentration. In a previous study we examined the specificity of these antibodies by western analysis using different mouse NGF preparations obtained from various commercial sources (16). Source of deglycosidases The deglycosylation kit (catalogue no. 170-6500) containing recombinant N-acetylneuraminidase H (NANase II), peptide N-glycosidase (PNGase F), and O-glycosidase disaccharide (O-glycosidase DS) was purchased from Bio-Rad Laboratories (München, Germany). Electrophoresis and immunoblotting The different rh-β-NGF preparations were dissolved in the electrophoretic sample buffer of Laemmli (9), and concentrations equivalent to 1 μg (per lane) were subjected to electrophoresis on a 12.5% gel under heat-denatured and reducing conditions. The GIBCO 10-kDa protein ladder was used as a marker (catalogue no. 10064-012). The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane by electrotransfer according to the procedure of Towbin et al. (21). The membrane strips were incubated with different antibodies (1:1,000), and the immunoreactive bands were identified with chemiluminescence using the ECL reagent (Amersham). Control experiments using preimmune serum did not result in any significant protein bands. Deglycosylation assay The rh-β-NGF preparation (containing the 60-kDa pro-NGF domain-immunoreactive protein) purchased from Sigma was subjected to deglycosylation assay according to the procedure provided by the manufacturer. In brief, 2.4 μg of rh-β-NGF preparation was incubated with NANase and O-glycosidase DS in a total volume of 20 μl at 37°C for 1 h. After incubation, the pH adjustment buffer and the denaturing solution were added, and the samples were heated for 5 min in a boiling water bath. The samples were cooled on ice, and incubation was continued for 3 h following addition of NP-40 and PNGase F in a total volume of 47 μl of Laemmli's electrophoretic sample buffer (4×). In parallel, the same amount of rh-β-NGF was incubated without deglycosidases. The contents of the vials were then loaded onto 12.5% sodium dodecyl sulfate (SDS)—polyacrylamide gel electrophoresis (PAGE). The deglycosidase enzyme mixture and rh-β-NGF were loaded on separate lanes as a control. After electrophoresis, the gels were subjected to Coomassie Blue protein staining. The gels were then photographed. A separate set of sample incubated with deglycosidases was subjected to immunoblotting analysis with mouse NGF monoclonal antibody obtained from Boehringer-Mannheim (catalogue no. 1087754, clone 27/12). The rh-β-NGF sample incubated without deglycosidases was subjected to immunoblotting analysis in parallel under the same incubation conditions. Tissue processing and immunoblotting analysis of tissue extracts Adult Sprague—Dawley rats (weighing 250-300 g) were killed by CO2 narcosis. Sensory ganglia and spinal cord from L3 to S3 were dissected under a microscope. Colon tissues were cleaned of their contents with cold saline. Pooled ganglia (10-20) were homogenized in 300 μl of buffer (20 mM TrisHCl, 10 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 0.1% SDS) containing protease inhibitors (5 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 5 μg/ml pepstatin, and 5 μg/ml aprotinin) and phosphatase inhibitors (0.5 mM orthovanadate). Spinal cord and colon tissues (50-100 mg) were homogenized in 1 ml of buffer using a Tekmar tissue homogenizer for 12 s. The homogenates were centrifuged at 12,000 g for 3 min, and aliquots of the supernatants were assayed for protein content (Bio-Rad Laboratories). The homogenates were diluted in Laemmli's electrophoretic sample buffer, heated in a boiling water bath for 8 min, cooled, and centrifuged. Aliquots were subjected to electrophoresis on an SDS polyacrylamide gel (12.5% resolving gel and 4.5% stacking gel), and proteins were transferred to PVDF membranes according to the method of Towbin et al. (21). The GIBCO 10-kDa protein ladder (range, 200-10 kDa) was used as a reference molecular mass marker. PVDF membrane strips were incubated with the different antibodies, and the immunoreactive bands were identified using the ECL reagent (Amersham). RESULTS Figure 1A shows the results of immunoblotting analysis of three commercial rh-β-NGF preparations with a rabbit polyclonal antibody to mouse β-NGF. All three preparations contained an intense 13-kDa NGF band, as expected. The rh-β-NGF preparations obtained from Sigma (lane 2) and R & D Systems (lane 3) contained a 27-kDa immunoreactive protein. An intensely immunoreactive NGF protein corresponding to the molecular mass of 53 kDa was observed in the rh-β-NGF preparation obtained from Sigma (lane 2). This preparation also contained a less intensely immunoreactive protein with a molecular mass of 42 kDa. The rh-β-NGF preparations obtained from Genzyme (lane 1) and R & D Systems (lane 3) contained a faintly immunoreactive 75-kDa protein that was barely detectable. Figure 1Open in figure viewerPowerPoint rh-β-NGF (1 μg per lane) obtained from three different commercial sources were subjected to electrophoresis and immunoblotting as stated in Materials and Methods. A: Lanes were incubated with a rabbit polyclonal mouse β-NGF antibody. B: Lanes were incubated with antibody to prepro-NGF domain (-144 to -163). C: Lanes were incubated with antibody to prepro-NGF (-60 to -91). These domains correspond to the larger and smaller NGF precursors as predicted by Edwards et al. (6). Lanes were incubated with the respective antibodies at a 1:1,000 dilution. See text for further details. Lane 1, rh-β-NGF from Genzyme; lane 2, rh-β-NGF from Sigma; and lane 3, rh-β-NGF from R & D Systems. Immunoreactive bands were detected using the ECL detection system (Amersham). FIG. 1. Figure 1B shows the results of an immunoblotting analysis of rh-β-NGF preparations with rabbit polyclonal antibody to the prepro-NGF domain (-144 to -163), corresponding to the larger NGF precursor predicted by Edwards et al. (6). This antibody identified a 53-kDa immunoreactive protein in the rh-β-NGF sample obtained from Sigma (lane 2) and a 60-kDa immunoreactive protein in the preparation obtained from R & D Systems (lane 3). All three rh-β-NGF preparations contained a 13-kDa immunoreactive protein. Figure 1C shows the results of an immunoblotting analysis of rh-β-NGF preparations with rabbit polyclonal antibody to the prepro-NGF domain (-60 to -91) corresponding to the smaller NGF precursor predicted by Edwards et al. (6). A broad immunoreactive protein comigrating with 60-kDa albumin was observed in the recombinant preparation obtained from Sigma (lane 2). A faintly immunoreactive protein corresponding to 60 kDa in size was observed the rh-β-NGF preparations obtained from Genzyme (lane 1) and R & D Systems (lane 3). All three preparations also contained a 13-kDa immunoreactive protein. Figure 2 shows the results of Coomassie protein staining of the rh-β-NGF preparations subjected to deglycosylation. The specific rh-β-NGF preparation used for the enzyme assay was obtained from Sigma. This preparation contained human serum albumin as a carrier. The protein staining identified a strong 60-kDa serum albumin band and a 13-kDa NGF band in the rh-β-NGF preparation (lanes 1 and 3). In addition, the preparation contained minor bands with molecular masses of 53, 36, and 32 kDa (lane 1). Protein staining also identified a 36-kDa band in the deglycosidase enzyme mixture (lane 2). The 32-kDa protein band was observed only in the rh-β-NGF sample incubated with deglycosidases (lane 1). Figure 2Open in figure viewerPowerPoint rh-β-NGF obtained from Sigma was subjected to deglycosylation as stated in Materials and Methods. Protein staining was performed with Coomassie Blue. Lane 1, rh-β-NGF preparation incubated with deglycosidases. Lane 2, deglycosidase enzyme control. Lane 3, rh-β-NGF control. Note the presence of the 32-kDa protein band only in the rh-β-NGF sample subjected to enzymatic digestion (lane 1). Lane 4, immunoblot of rh-β-NGF probed with mouse NGF monoclonal antibody (Boehringer-Mannheim) after incubation with deglycosidases. Note the presence of intense staining for the 32-kDa immunoreactive protein and the absence of the 13-kDa β-NGF band. Lane 5, immunoblot of rh-β-NGF probed with mouse NGF monoclonal antibody (Boehringer-Mannheim). The sample was incubated without deglysidases. Note the presence of intense staining for the 13-kDa β-NGF band. Immunoreactive bands at 60 and 28 kDa were also present. FIG. 2. The result of immunoblotting analysis of rh-β-NGF preparations subjected to deglycosylation is shown in lane 4. The mouse 2.5S NGF monoclonal antibody (Boehringer-Mannheim) identified a strongly immunoreactive 32-kDa band in the rh-β-NGF subjected to deglycosylation (lane 4). This protein was not observed in the rh-β-NGF incubated without deglycosidases (lane 5). The monoclonal antibody identified the 13-kDa rh-β-NGF band in the sample incubated without deglycosidases (lane 5). This band was not observed in the sample incubated with deglycosidases (lane 4). The mouse NGF monoclonal antibody identified a sharp 60-kDa immunoreactive band in the rh-β-NGF sample incubated with (lane 4) and without (lane 5) deglycosidases. An intensely immunoreactive band with a molecular mass of 28 kDa was noted in the rh-β-NGF sample incubated without the enzymes (lane 5). A similar immunoreactive band with less intensity was observed in the sample incubated with deglycosidases (lane 4). Figure 3 shows the immunoblotting analysis of adult rat DRG with NGF polyclonal and monoclonal antibodies. The mouse β-NGF polyclonal antibody identified 73- and 60-kDa immunoreactive proteins with strong intensity (Fig. 3, lane A). This antibody immunoreacted less strongly with a 53-kDa protein. In addition, the polyclonal antibody weakly immunoreacted with a 170-kDa protein. Both the human and mouse NGF monoclonal antibodies reacted strongly with 170-, 60-, and 53-kDa proteins (Fig. 3, lanes B and C). The latter molecule appeared as a doublet. The monoclonal antibodies identified faintly immunoreactive bands of ∼40 kDa. These antibodies less consistently identified a faint 13-kDa NGF band. Figure 3Open in figure viewerPowerPoint Western analysis of adult rat DRG with polyclonal and monoclonal antibodies to NGF. DRG extracts equivalent to 20 μg of protein were subjected to electrophoresis on a 12.5% SDS-PAGE gel. Proteins were transferred to a PVDF membrane, and membrane strips were incubated with polyclonal NGF antibody (lane A), human NGF monoclonal antibody (lane B), and mouse NGF monoclonal antibody (lane C). Immunoreactive bands were detected using the ECL detection system (Amersham). FIG. 3. Figure 4 shows the immunoblotting analysis of rat DRG with two prodomain NGF precursor-specific antibodies. The prepro-NGF domain (-144 to -163) antibody, corresponding to the larger mouse and human NGF precursors predicted by Edwards et al. (6), strongly immunostained the 60-kDa protein band (Fig. 4A). Similarly, the prepro-NGF domain (-60 to -91) antibody, corresponding to the smaller mouse and human NGF precursors predicted by Edwards et al. (6), also stained the 60-kDa protein band (Fig. 4B). Both predomain NGF precursor antibodies immunostained the 73- and 32-kDa proteins with lesser intensity. Figure 4Open in figure viewerPowerPoint Western analysis of adult rat DRG with two NGF prodomain-specific antibodies. DRG extracts equivalent to 20 μg of protein were subjected to electrophoresis and western analysis as stated in the legend of Fig. 3. Lanes were incubated with polyclonal antibodies specific to the prepro-NGF domain (-144 to -163) (A) and the prepro-NGF domain (-60 to -91) (B). FIG. 4. Figure 5 shows the results of immunoblotting analysis obtained with colon extracts with two prodomain NGF precursor antibodies. Both the prepro-NGF domain (-144 to -163) (Fig. 5A) and prepro-NGF (-60 to -91) antibodies (Fig. 5B) immunoreacted strongly with the 60-kDa protein. These antibodies also immunostained the 170-, 73-, and 42-kDa immunoreactive proteins with lesser intensity. Figure 5Open in figure viewerPowerPoint Western analysis of adult rat colon extracts with two NGF prodomain-specific antibodies. Colon extracts equivalent to 20 μg of protein were subjected to electrophoresis and immunoblotting as stated for the analysis of DRG extracts in the legend of Fig. 3. A: Lanes were incubated with prepro-NGF domain (-144 to -163) antibody. B: Lanes were incubated with prepro-NGF domain (-60 to -91) antibody. FIG. 5. Figure 6 shows the results of the immunoblotting analysis obtained with spinal cord extracts using two prodomain NGF precursor antibodies. Both the prepro-NGF domain (-144 to -163) (lane A) and prepro-NGF (-60 to -91) (lane B) antibodies corresponding to the larger and smaller mouse and human NGF precursors immunoreacted strongly with 73-, 60-, and 53-kDa protein bands. Both antibodies identified a faintly immunoreactive 32-kDa band. The high-molecular-weight proteins that remained near the origin also weakly immunoreacted with both antibodies. Figure 6Open in figure viewerPowerPoint Western analysis of adult rat spinal cord with two NGF prodomain-specific antibodies. Spinal cord extracts equivalent to 20 μg of protein were subjected to electrophoresis and immunoblotting as stated for the analysis of DRG extracts in the legend of Fig. 3. Lane A, incubated with prepro-NGF domain (-144 to -163) antibody. Lane B, incubated with prepro-NGF domain (-60 to -91) antibody. FIG. 6. DISCUSSION In the present study, we examined commercially available rh-β-NGF preparations, adult rat DRG, and two of its efferent tissues for the presence of high-molecular-weight NGFs by western analysis. For the immunodetection, we used antibodies specific for mature β-NGF and prepro-NGF-specific domains conserved in mouse and human NGF precursors as predicted from the structure of the respective cDNAs (18; 22). Of the three commercial rh-β-NGF preparations tested, two of them (Sigma and R & D Systems) were expressed in a mouse myeloma cell line (NSO) transfected with NGF cDNA encoding the human β-NGF precursor. The third preparation, according to the manufacturer (Genzyme), was expressed in an Escherichia coli system using a synthetic gene. Among the three rh-β-NGF preparations, the sample obtained from Sigma contained human serum albumin as carrier. The mouse β-NGF antibody identified the 13-kDa β-NGF band in all three samples, as expected. The preparation obtained from Sigma contained 53-, 42-, and 27-kDa NGF immunoreactive proteins. The presence of 53- and 42-kDa NGF immunoreactive proteins in the recombinant preparation is interesting in light of the fact that Seidah et al. (19) reported expression of a 48.5-kDa glycosylated NGF precursor in a human colonic carcinoma cell line transfected with full-length human NGF cDNA. Based on the close similarity in molecular masses, we infer that the 53- and 42-kDa NGF immunoreactive proteins likely represent the glycosylated NGF precursors. This inference is further supported by the fact that the structure of the NGF precursor(s) predicted by the cloned mouse and human NGF cDNAs contains three distinct glycosylation sites (18; 22). The unglycosylated NGF precursors are predicted to be either 32 or 27 kDa in size, depending on the initiation sites. The rh-β-NGF preparations obtained from Sigma and R & D Systems also contained a 27-kDa NGF-immunoreactive protein. It is unlikely that the 27-kDa NGF protein is a dimer of β-NGF because the recombinant preparations were subjected to electrophoresis under heat-denatured and reducing conditions. Another possibility is that the 27-kDa protein corresponds to the smaller NGF precursor predicted from the structure of cloned human and mouse NGF cDNA (6) or a protein derived from high-molecular-weight NGF proteins by proteolysis. The prepro-NGF domain (-144 to -163)-specific antibody, corresponding to the larger NGF precursor predicted by Edwards et al. (6), identified a 53-kDa and a 60-kDa immunoreactive protein in the rh-β-NGF samples obtained from Sigma and R & D Systems, respectively (Fig. 1B). This is interesting in light of the fact that this antibody also identified the presence of a 60-kDa immunoreactive protein in different NGF preparations isolated from mouse salivary glands (16). Also, in our previous study we observed the presence of 53- and 60-kDa NGF immunoreactive proteins in human and mouse NGF preparations using human and mouse NGF-specific monoclonal antibodies 16). In the present study, the prepro-NGF domain (-144 to -163) antibody also identified a 13-kDa immunoreactive protein in all three rh-β-NGF preparations. The prepro-NGF domain (-60 to -91)-specific antibody, corresponding to the smaller NGF precursor predicted by Edwards et al. (6), identified a strong immunoreactive band spanning between 53 and 60 kDa (Fig. 1C). It is possible that this broad band may represent two closely migrating NGF precursors. This prediction is based on the fact that the prepro-NGF domain (-144 to -163) identified a 53- and a 60-kDa immunoreactive protein in the rh-β-NGF preparations obtained from Sigma and R & D Systems, respectively. As stated earlier, mouse and human NGF monoclonal antibodies also identified a broad 60-kDa band in the rh-β-NGF preparation obtained from Sigma (16). The prepro-NGF domain (-60 to -91)-specific antibody identified a faintly immunoreactive 60-kDa protein in the rh-β-NGF preparations obtained from both Genzyme (Fig. 1C, lane 1) and R & D Systems (Fig. 1C, lane 3). In the present study, the prepro-NGF antibody also recognized a distinct 13-kDa immunoreactive protein in all three rh-β-NGF preparations. Both prodomain NGF antibodies identified the 13-kDa immunoreactive protein in all three rh-β-NGF samples. This finding is interesting in light of the fact that the proregion of the human NGF precursor (22) contains two dibasic cleavage sites. The presence of the proregion-derived immunoreactive protein with a similar molecular size as 13-kDa β-NGF is also consistent with the results of Dicou (3), who detected a 13-kDa proregion-derived peptide by digestion of the mouse NGF precursor synthesized in wheat germ extract using a distinct prepro-NGF (-71 to -45) domain antibody. Incubation of an rh-β-NGF sample (containing the 60-kDa NGF prohormone) with glycosidases resulted in the generation of a 32-kDa protein. The molecular mass of this protein corresponded to the size predicted for the translation product of the larger NGF mRNA (6). Mouse NGF monoclonal antibody strongly immunoreacted with 32-kDa protein generated after incubation with glycosidases. It should be noted here that other investigators reported isolation of a biologically active 32-kDa NGF precursor from adult mouse salivary glands using a polyclonal antibody to mouse 2.5S NGF (17). Our attempts to confirm the identity of 32-kDa protein as unglycosylated NGF precursor with both pro-NGF-specific domain antibodies were not successful because both antibodies failed to immunoreact with 32-kDa protein (data not shown). The reason for this is not clear. This is also in contrast with our previous investigation in that we identified a 32-kDa NGF precursor with both prodomain antibodies in hippocampal extracts of adult mouse and rat brains (11). In the present study the mouse NGF monoclonal antibody failed to immunoreact with 13-kDa rh-β-NGF after incubation with deglycosidases (Fig. 2, lane 4). This does not appear to be due to degradation of rh-β-NGF because the Coomassie Blue staining indicated the presence of β-NGF in the sample incubated with deglycosidases (Fig. 2, lanes 1 and 3). This clearly suggests that the deglycosidases used in the present study do not exhibit any proteolytic activity. Based on the sensitivity to deglycosidases, we suggest that the rh-β-NGF contains glycosyl residues on its backbone. It is interesting to note that all three forms of NGF (7S NGF, β-NGF, and 2.5S NGF) isolated from mouse salivary gland were reported to contain a small amount of glycosylated NGF (14). From the above findings we infer that the presence of prosthetic groups on β-NGF may alter the orientation of the molecule on the membrane. Both the immunoblotting and protein staining techniques appear to be essential to identify the molecular nature of the NGF precursor and its derivatives. Examination of DRG, colon, and spinal cord extracts with both pro-NGF domain antibodies also revealed the presence of high-molecular-mass immunoreactive proteins with molecular masses of 120, 73, 60, 42, and 32 kDa. Of these, the 60-kDa protein exhibited strong immunoreaction with both pro-NGF domain antibodies, and this protein also strongly immunoreacted with polyclonal and monoclonal NGF antibodies in the DRG extracts. Based on the immunoreaction to prodomain NGF antibodies and antibodies to mature NGF, we conclude that the 60-kDa protein identified in the rat DRG, spinal cord, and colon likely represents the proteolytically unprocessed, glycosylated NGF precursor corresponding to the larger NGF precursor predicted for the mouse and human NGF species (22; 6). This conclusion is further supported by the finding of a 60-kDa NGF prohormone in a commercial rh-β-NGF preparation (present study) as well as in NGF preparations isolated from mouse salivary glands (16). The present finding also suggests for the first time that the rat expresses an NGF prohormone corresponding to the larger NGF precursor predicted by the cloned mouse and human NGF cDNAs (22; 6). However, it should be pointed out here that the nucleotide sequence of the cloned rat NGF cDNA (23) significantly differs from the nucleotide sequences of the cloned mouse and human NGF cDNAs (18; 22). In particular, the rat NGF precursor predicted by the cloned rat NGF cDNA does not contain the amino acid sequences corresponding to the mouse and human prepro-NGF domain -144 to -161. Nevertheless, the identification of 60-kDa NGF prohormone in rat DRG, spinal cord, and colon extracts and the presence of a similar molecular species of NGF prohormone in commercial rh-β-NGF samples as well as in various mouse NGF preparations (16) provide strong evidence that the rat expresses the larger NGF precursor predicted from the cloned mouse and human NGF cDNAs. In addition, the present findings suggest that the larger NGF precursor undergoes a similar posttranslational processing in all three species. Based on the presence of 60-kDa NGF prohormone in the rat DRG, spinal cord, and colon, we suggest that the prohormone itself may mediate the neuron-target interaction. Purification of 60-kDa NGF prohormone from commercial rh-β-NGF preparations should provide an opportunity to confirm our hypothesis. In the present investigation we failed to identify the 13-kDa NGF in a significant concentration in the DRG, spinal cord, and colon extracts. Recently, we also failed to identify the 13-kDa NGF in a significant concentration in hippocampal extracts from the mouse, rat, and human (11; 8). It is interesting that the hippocampal extracts of all three species contained abundant amounts of NGF prohormone isoforms (11; 8). Other investigators who recently identified the presence of NGF prohormone isoforms in human brain parietal regions also reported the absence of 13 kDa in the brain extracts (20). All four antibodies used in the present study also reacted variably with 170-, 73-, and 42-kDa proteins in the extracts of DRG, spinal cord, and colon. These molecules are likely to represent multiple NGF prohormone species generated by extensive posttranslational modifications. Pulse-chase studies with cell lines transfected with full-length NGF cDNA have clearly documented that the NGF precursor undergoes both N-glycosylation and sulfation (19). Both NGF prodomain antibodies also identified a 32-kDa protein in the spinal cord extracts. The molecular mass of this protein corresponds to the prohormone species generated by deglycosidases from glycosylated 60-kDa rh-β-NGF. We cannot rule out the possibility that a 32-kDa prohormone species is formed during the process of tissue homogenization. In summary, most of the NGF analysis performed to date on DRG and its efferent tissues has been restricted solely to immunohistochemistry and immunoassay techniques. 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