Biochemical characterization of netrin-synergizing activity.

The netrin-1 protein elicits spinal commissural axon outgrowth and turning in vitro and has been shown to be required for commissural axon guidance in vivo in the developing spinal cord. Biochemical observations made during the purification of netrin-1 suggest that this ligand and its receptor, DCC, may not function alone in directing commissural axon guidance. Recombinant netrin-1 protein is approximately 10 times more active in eliciting axon outgrowth from embryonic day (E) 13 rat dorsal spinal cord explants than from E11 rat dorsal spinal cord explants (Serafini, T., Kennedy, T. E., Galko, M. J., Mirzayan, C., Jessell, T. M., and Tessier-Lavigne, M. (1994) Cell 78, 409-424) even though the starting material for the netrin purification, a high salt extract of E10 chicken brain membranes, is equally active on E13 and E11 explants. We previously reported an activity termed netrin-synergizing activity (NSA) that can potentiate the outgrowth-promoting activity of netrin-1 on E11 explants (Serafini et al.). Here we report a biochemical characterization of NSA in netrin-depleted high salt extracts of E10 chicken brain membranes. We provide evidence that NSA is composed of a denaturation-resistant basic protein(s) in the 25-35-kDa size range. We also provide evidence that the activity may be heterogeneous, splitting into three species that may be distinct or related. The results reported here should facilitate purification of this activity from a more abundant source or identification of the activity based on similarity to known proteins that share its distinctive biochemical properties.

The faithful guidance of axons to their targets during development of the embryonic nervous system is believed to occur through the action of both positive and negative guidance factors (1,2). These factors may act either locally or at a distance within the terrain of the developing embryo. A large body of experimental evidence has implicated the phylogenetically conserved netrin gene family in directing the long-range attraction of circumferentially migrating neurons in Caenorhabditis elegans, Drosophila, and vertebrates (reviewed in Ref. 1). In vertebrates, the netrin-1 mRNA is expressed in the floor plate, the ventral midline intermediate target of migrating commissural neurons, before and during the migration of these neurons toward this structure (3). The netrin-1 receptor on migrating commissural neurons is the netrin-binding DCC (deleted in colorectal cancer) protein, which is expressed on commissural axons as they migrate toward the floor plate (4). Mutant mice deficient for either the netrin-1 gene product or DCC exhibit defects in commissural axon migration from their birthplace in the dorsal spinal cord to the floor plate, demonstrating that their proper functioning is necessary for commissural axon guidance (5,6).
The vertebrate netrin-1 protein was originally purified from a high salt extract of embryonic day (E) 1 10 chicken brain membranes on the basis of its ability to mimic the axon outgrowth-promoting effect of floor plate extracts from dorsal spinal cord explants cultured in vitro within three-dimensional collagen matrices (7). During this purification, an activity was discovered that collaborates with netrin-1 to promote outgrowth. Although recombinant netrin-1 protein is ϳ10 times more active in eliciting axon outgrowth from E13 rat dorsal spinal cord explants than from E11 rat dorsal spinal cord explants, a high salt extract of E10 chicken brain membranes is equally active on E13 and E11 explants. We show that the difference is due to the presence in brain extracts of a distinct activity, netrin-synergizing activity (NSA), that is capable of potentiating the axon outgrowth-promoting effects of netrin-1 from E11 explants (7). Because of its dramatic potentiation of netrinmediated axon outgrowth in vitro, it seems likely that this activity may play an important role in commissural axon guidance in vivo. As a first step toward identifying NSA and determining its role in commissural axon guidance in vivo, we report here the results of a biochemical characterization of NSA from a netrindepleted high salt extract of E10 chicken brain membranes.

EXPERIMENTAL PROCEDURES
Synergy Assay and Culture of E11 Dorsal Spinal Cord Explants-E11 rat dorsal spinal cord explants (E0 ϭ day of vaginal plug) were dissected as described (8) with the protease digestion protocol described previously (7). Explants were cultured for 40 h at 37°C and 5% CO 2 in 50% nutrient mixture F-12 and 38.25% Opti-MEM containing 40 mM glucose, 5% heat-inactivated horse serum, 1% penicillin/streptomycin, and 1% Glutamax-1 additive (Life Technologies, Inc.). For most assays, a concentrated high salt extract of stably transfected netrin-expressing cells (9) was diluted 133-fold into the above culture medium to give a final netrin-1 concentration of ϳ50 ng/ml in the culture medium. Control explants without netrin-1 were cultured with an equivalent dilution of 1.0 M NaCl and 20 mM Na 2 HPO 4 (pH 7.0).
Immunostaining-Explants were fixed after culture for 1 h with 4% paraformaldehyde at room temperature, washed with phosphate-buffered saline, and then blocked with phosphate-buffered saline containing 1% heat-inactivated normal goat serum and 0.1% Triton X-100. Analysis of TAG-1 expression was performed with a 1:100 dilution of monoclonal antibody 4D7 (10), followed by horseradish peroxidase/ diaminobenzidine detection. Analysis of DCC expression was performed with a 1:100 dilution of monoclonal antibody AF5 (Oncogene Science Inc.), followed by horseradish peroxidase/enhanced diaminobenzidine detection (Sigma). The difference in the detection reagents accounts for the difference in the color of labeled axons in Fig. 1 (E and F).
Quantification of Axon Outgrowth-Axon fascicles emerging from each explant were measured and added together to determine the total bundle length of these fascicles per explant. Four to six explants from each condition in three independent experiments were counted. Thin single neurons, which were rare, were not counted.
Testing of Known Factors in the Synergy Assay-Factors were tested in one or more of the following three ways: 1) as pure protein serially diluted in assay medium (generally 2-fold dilutions from 1 g/ml to 0.5 ng/ml), 2) as conditioned medium harvested from cells 48 h after transfection with an expression plasmid for the factor of interest, and 3) from clumps of cells transfected with an expression plasmid for the factor of interest and positioned opposite E11 dorsal spinal cord explants grown in three-dimensional collagen gels and in the presence of ϳ50 ng/ml netrin-1.
Electrophoresis and Dialysis-Electrophoretic and silver stain analyses of proteins present in fractions from nonreducing SDS-PAGE, native acid/urea electrophoresis, and reverse-phase chromatography were carried out by standard techniques. All dialyses, unless otherwise specified, were performed with Spectrapor-2 12-14-kDa molecular mass cutoff membranes at 4°C with vigorous stirring.
Preparation of NSA-containing Heparin Flow-through-An NSAcontaining heparin flow-through fraction was prepared from E10 chicken brains as described (7) except that some preparations were homogenized with a Yamato automatic homogenizer using the following scheme: one pass at 100 rpm and two passes at 110 rpm. The low-speed and mid-speed pellets were rehomogenized with one pass at 40 rpm. Salt extraction of membranes and collection of the heparin flow-through fraction on heparin-Sepharose CL-6B were as described previously (7).
Ion-exchange/Heparin Affinity Chromatography-The heparin flowthrough fraction (see above) was prepared for ion-exchange/heparin affinity chromatography in the following manner. 200-ml batches of the heparin flow-through fraction (ϳ2000 brains' worth of material) were dialyzed at 4°C with vigorous stirring against 20 liters of equilibration buffer A (2 M urea, 0.3% CHAPS, 150 mM NaCl, and 20 mM Tris-Cl (pH 8.0)). The dialysate was spun in a Ti-50.2 rotor (Beckman Instruments) at 100,000 ϫ g for 1 h at 4°C to remove precipitated proteins and nucleic acids. The supernatant was loaded at 2.5 ml/min onto a 5-ml HiTrap Q column (Amersham Pharmacia Biotech; to which the activity did not bind) in line before a HiTrap heparin column (to which the activity did bind). Both columns were equilibrated with buffer A. The column was developed with a 10-column volume gradient from 0 to 100% buffer B (buffer A with 1 M NaCl). 2-min fractions (5 ml) were collected. All fractions were assayed by serial dilution in the synergy assay. The peak activity fractions, 7-10, corresponding to ϳ500 -800 mM NaCl, were pooled and dialyzed overnight at 4°C against 10 liters of 1 M NaCl and 20 mM Na 2 HPO 4 (pH 7.0), aliquoted, frozen in liquid nitrogen, and stored until further biochemical analysis.
Stability Experiments-For the water and 6 M urea conditions, the NSA-containing heparin eluate was dialyzed for 2 h against a 1000-fold volume excess of these solutions. The dialysate was spun at 100,000 ϫ g for 1 h at 4°C in a Sorvall S120-AT2 mini-ultracentrifuge rotor to remove precipitated protein. The supernatant was then dialyzed against nutrient mixture F-12 for subsequent assay of serial dilutions in the synergy assay. Activity recoveries were determined by comparing the actual dilution of a fraction that gave maximal activity with the dilution that would have given maximal activity if 100% of the starting activity had been recovered. For testing of SDS and trifluoroacetic acid, the NSA-containing heparin eluate was first dialyzed against water and centrifuged as described above. Trifluoroacetic acid was added to 0.1% (pH 1.6), and the solution was allowed to stand for 1 h at room temperature before neutralization to pH 7.0 with 5 M NaOH. This material was dialyzed against nutrient mixture F-12 for assay, and serial dilutions of trifluoroacetic acid-exposed sample were compared with serial dilutions of sample in water that had stood at room temperature for 1 h. No loss of activity was observed compared with the water-only standard. For testing SDS stability, SDS was diluted from a 10% stock to a final concentration of 0.5% into an NSA-containing sample in water. The sample was allowed to stand for 1 h at room temperature along with a mock control that did not receive detergent. Both samples were then dialyzed for 2 h against 2 liters of 6 M urea and 50 mM Tris-Cl (pH 7.8) at room temperature. The dialysates were loaded onto AG 1-X2 resin (Bio-Rad) that had been equilibrated with dialysis buffer to remove SDS according to the protocol of Weber and Kuter (11). The resin was rotated for 0.5 h at room temperature and then pelleted in a microcentrifuge at 16,000 ϫ g. The SDS-depleted supernatant was then dialyzed overnight against 1 M NaCl and 20 mM Na 2 HPO 4 (pH 7.0) to stabilize NSA and to remove the 6 M urea. The following morning, these dialysates were dialyzed against nutrient mixture F-12 for subsequent assay. The recovery of material in the mock sample (45%) did not differ from that of the detergent sample, so it can be assumed that the losses were due to the lengthy SDS removal protocol and not to the presence of SDS. For heating, the NSA-containing heparin eluate in 1 M NaCl and 20 mM Na 2 HPO 4 (pH 7.0) was heated for 15 min at 95°C, followed by 10 min on ice. A control sample was left at room temperature for 15 min, followed by 10 min on ice. The mock and heated samples were centrifuged in an Eppendorf microcentrifuge at 16,000 ϫ g for 15 min at 4°C to remove the precipitate (present only in the heated sample). The supernatant was then dialyzed against nutrient mixture F-12 for subsequent assay. To test protease stability, 600 l of NSA-containing heparin eluate in 1.0 M NaCl and 20 mM Na 2 HPO 4 (pH 7.0) was added to trypsin-or bovine serum albumin-agarose beads (Sigma) equilibrated with the same buffer. The beads with sample were rotated overnight at room temperature prior to dialysis of each sample against nutrient mixture F-12 for assay. Recovery of total protein (by the Bio-Rad Bradford protein assay) was 82% on bovine serum albumin and 20% on trypsin. Recovery of activity was ϳ100% on bovine serum albumin and Ͻ12% on trypsin.
Nonreducing SDS Electrophoresis-3.2 ml of NSA-containing heparin eluate was prepared for electrophoresis by dialysis against 4 liters of unbuffered water for 2 h. The dialysate contained a precipitate that was removed by centrifugation at 100,000 ϫ g for 1 h at 4°C in a Sorvall S120-AT2 mini-ultracentrifuge rotor. The sample was then concentrated ϳ40-fold by spinning in Centricon-30 2-ml concentrators (Amicon, Inc.) to a final volume of 80 l. This concentrated sample was diluted with 2ϫ nonreducing sample buffer containing 1.25% SDS, 100 mM Tris-Cl (pH 6.8), 40% glycerol, and 0.025% bromphenol blue. 150 l of the sample was loaded into the middle well of a five-well comb on a 1.5-mm thick ϫ 5-cm high 10% SDS-polyacrylamide minigel covered with a standard stacking gel. Adjacent lanes contained an equivalent amount of 1ϫ loading buffer. The gel was run at room temperature at 60 V through the stack and at 120 V through the separating gel. Once the dye front ran off the gel, the middle lane was cut up into 11 ϳ4-mm slices with clean razor blades. Each slice was then minced with a clean blade, and the pieces from each were transferred to individual wells of a four-well tissue culture dish (Nunc) containing 400 l of 6 M urea and 50 mM Tris-Cl (pH 7.8) and 1 mg/ml ovomucoid carrier protein. The dishes were rocked at room temperature overnight to elute protein from the minced slices. The supernatant was then removed from each well, brought back up to a 400-l volume with the same buffer, and taken through the SDS removal protocol described above for the SDS stability experiment (11). The supernatant from each fraction was dialyzed against 5.4 liters of nutrient mixture F-12 for 4 h at 4°C. Fractions were then analyzed both by serial dilution in the synergy assay and by SDS-PAGE on a 12% gel (the ovomucoid carrier protein binds to the AG 1-X2 resin during SDS removal and so does not interfere with the SDS-PAGE analysis).
Native Acid/Urea Electrophoresis-For native acid/urea electrophoresis, we used a protocol similar to that reported by Panyim and Chalkley (12) with the sample buffer employed by Barde et al. (13). 3.8 ml of NSA-containing heparin eluate was prepared for acid/urea electrophoresis in the following manner. First, the sample was heated as described above for the stability experiment. Second, the supernatant was collected and dialyzed against 4 liters of unbuffered water (to remove salt and to remove precipitated proteins) for 2 h. Third, the dialysate was then spun at 100,000 ϫ g for 1 h at 4°C in an S100-AT5 rotor in a Sorvall RC-M120EX mini-ultracentrifuge to remove precipitated protein. Fourth, the supernatant from this spin was then dialyzed for 2 h at room temperature against 4 liters of acid/urea sample loading buffer containing 6 M urea and 250 mM sodium acetate (pH 5.8). Finally, this second dialysate was concentrated 38.5-fold in a Centricon-30 microconcentrator. A 1.5-mm thick 10% acrylamide minigel was poured at the beginning of the sample preparation protocol. The gel contained no SDS, but did contain 6 M urea and 0.9 M acetic acid (pH 2.75). To aid polymerization of the acrylamide under acidic conditions, a 1:100 dilution of freshly made 300 mM sodium sulfite and a 1:133 dilution of freshly made 10% ammonium persulfate were included in the gel mixture. A 1:2000 dilution of TEMED was used to start the polymerization. A five-well comb was inserted above the gel; there was no stacking gel. Prior to loading the sample, the gel was prerun at room temperature for 1 h at 150 V of constant voltage to remove charged species from the gel. The concentrated sample was loaded onto one lane and was flanked by lanes containing cytochrome c, a red-colored highly basic protein of 12.4 kDa (Sigma), to help visualize the progress of the electrophoresis. The sample was run on the gel at 20 mA of constant current until the cytochrome c marker was at the bottom of the gel. The sample lane was cut into 3-mm strips with clean razor blades starting 1.5 cm in from the bottom of the gel (previously shown not to contain any activity; data not shown). Each strip was minced with a clean razor and transferred into 400 l of 6 M urea in one well of a four-well tissue culture dish precoated with 10% serum for several hours to block the protein-binding capability of the dish. The dishes were shaken overnight at room temperature to elute protein from the minced slices. The following morning, the elution buffer was recovered from each well, and a portion of each fraction was analyzed by SDS-PAGE. The rest of each fraction was dialyzed for several hours against 5.4 liters of nutrient mixture F-12 for subsequent analysis in the synergy assay.
Reverse-phase Chromatography-7 ml of NSA-containing heparin eluate was first prepared for chromatography by heating as described above. The supernatant was then dialyzed for 2 h against a 1000-fold volume excess of unbuffered water. The dialysate was spun in a miniultracentrifuge (Sorvall S100-AT5 rotor) at 100,000 ϫ g for 30 min at 4°C. The supernatant was adjusted to 0.1% trifluoroacetic acid and 25% acetonitrile at room temperature and then injected onto a 250 ϫ 4.6-mm Vydac C4 reverse-phase column equilibrated with 25% acetonitrile and 0.1% trifluoroacetic acid. The column was developed with a 65-min linear gradient from 25 to 100% acetonitrile at room temperature. The flow rate was 1 ml/min, and 1-ml fractions were collected. 75% of each fraction was adjusted to 1 mg/ml ovomucoid carrier protein, dried down in a SpeedVac, resuspended in 150 mM NaCl and 100 mM Tris-Cl (pH 8.0) to neutralize residual acid, dialyzed against nutrient mixture F-12 and serially diluted into explant medium for assay. The remaining 25% of each fraction was dried down without carrier protein and used for the Bio-Rad/Bradford protein assay and analysis of the fractions by SDS-PAGE.

RESULTS
Initial Characterization of NSA-We previously demonstrated that netrin-depleted high salt extracts of E10 chick brain membranes contain an activity (NSA) that can potentiate netrin-mediated axon outgrowth from explants of E11 rat dorsal spinal cord (7). In those experiments, the addition of brain extracts caused necrosis within the explants, resulting in variability of responses (data not shown). We found, however, that reproducible non-necrotic growth of E11 dorsal spinal cord in the presence of complex protein fractions from chicken brain was obtained if the cultures were performed in a mixture of nutrient mixture F-12/Opti-MEM supplemented with a stable glutamine derivative, Glutamax-1 (Fig. 1, A-D). E11 explants grown alone, in the presence of ϳ50 ng/ml recombinant netrin-1 protein, or in the presence of a heparin flow-through fraction containing NSA exhibited little or no commissural axon outgrowth into the surrounding collagen (Fig. 1, A-C). Explants grown in the presence of both ϳ50 ng/ml netrin-1 protein and NSA exhibited robust axon outgrowth primarily but not exclusively from the ventral cut edge of the dorsal spinal cord explants (Fig. 1, D-F).
The rat dorsal spinal cord explants employed in the synergy assay contain both commissural neurons and association neurons. Commissural neurons are distinguishable by virtue of their expression of the axonal glycoprotein TAG-1 (14) and the netrin receptor, DCC (4). Most or all of the responsive axons in the synergy assay are commissural neurons since they were labeled by antibodies directed against the axonal glycoprotein TAG-1 (Fig. 1E) and the netrin receptor, DCC (Fig. 1F).
The commissural axons present in the E11 explants responded to the NSA-containing heparin flow-through fraction in a dose-dependent fashion in the presence of a constant amount (ϳ50 ng/ml) of recombinant netrin-1 protein. NSA exhibited a maximal outgrowth response at 0.26 mg/ml heparin flow-through fraction. The dose-response curve for NSA was also very steep, showing minimal commissural axon outgrowth at one-quarter the peak concentration as well as a diminished response beyond the peak concentration (Fig. 1G).
The assay described above provides a useful tool for a possible biochemical purification of NSA. In this synergy assay, E11 explants are grown within three-dimensional collagen matrices in the presence of ϳ50 ng/ml netrin-1 protein, a netrin-1 concentration that elicits minimal axonal outgrowth from these explants. The NSA-containing heparin flow-through starting material is then fractionated. Serial dilutions of the generated fractions are dialyzed against nutrient mixture F-12 and added to the explants with netrin-1, cultured for 40 h in three-dimensional collagen matrices, and then scored for the outgrowth of axonal fascicles. For the quantification of the biochemical fractionation experiments detailed below, we did not routinely perform the laborious axonal length measurements presented in Fig. 1G. Rather, we defined a unit of activity as the lowest dilution of any given fraction dissolved in 1 ml of assay medium that would give a maximal response (similar to that shown in Fig. 1, D-F) in the synergy assay.

NSA Is Not Encoded by a Variety of Known Extracellular Factors and, Like Netrin-1, Is a Heparin-binding Protein-
Before embarking on a potentially laborious biochemical purification of NSA, we tested a number of known factors to determine whether they might encode NSA-like activity. These factors were chosen on the basis of one or more of the following criteria: 1) known effects on the in vitro growth of other classes of neurons, 2) expression in the embryonic spinal cord, and 3) similarity to the biochemical properties of NSA (see below). Candidates tested (Table I) included neurotrophic factors, extracellular matrix components, growth factors, extracellular proteases, and protease inhibitors. None of the proteinaceous factors tested to date exhibited any NSA-like activity.
The addition of 2 M urea and 0.3% CHAPS detergent was required to keep NSA soluble and stable in low salt solutions (Ͻ300 mM NaCl; data not shown). In the presence of these additives and 150 mM NaCl, NSA did not bind to anion exchangers (Q-Sepharose) and did bind to cation exchangers (Sand CM-Sepharose) and to heparin-Sepharose, a cationic affinity resin. The majority of NSA in our starting material, when depleted of anionic proteins on Q-Sepharose, bound to heparin-Sepharose in the presence of 2 M urea, 0.3% CHAPS, 150 mM NaCl, and 20 mM Tris-Cl (pH 8.0) and eluted from this resin in a broad peak from ϳ500 to 800 mM NaCl (Fig. 2). With some batches of material (the run shown in Fig. 2 is an example of this), the activity peak was broader than this, with up to one-third of the activity eluting in lower salt fractions. Even when the activity eluted more broadly, we consistently pooled only fractions 7-10 for further biochemical analysis in order to avoid abundant contaminant proteins present in the lower salt fractions. Pooled active fractions were dialyzed against 1 M NaCl and 20 mM Na 2 HPO 4 (pH 7.0) to stabilize NSA and were stored at Ϫ80°C until further biochemical analysis. We sometimes saw, as in Fig. 2, what looked to be two peaks of activity eluting from the heparin column. These results indicate that NSA, like netrin-1, is also a heparin-binding protein(s), although it elutes at lower salt than netrin-1 (500 -800 mM NaCl for NSA compared with ϳ1.2 M NaCl for netrin-1). Note that NSA is present in the flow-through fraction from the heparin column in the netrin purification (7) because that column is loaded at 0.9 M NaCl.
NSA Is a Protein(s) Extremely Resistant to Denaturation-Pilot experiments with a number of chromatographic techniques (lectin affinity chromatography, metal affinity chromatography, high resolution ion-exchange, hydrophobic chromatography, dye chromatography, hydroxylapatite chromatography, and netrin affinity chromatography) demonstrated these techniques to be ineffective in resolving NSA in our partially purified heparin eluate fraction from the bulk of the remaining protein (Ͻ2.5-fold purification was obtained with any of the above techniques), indicating that purification of NSA from our limited source material might not be feasible (data not shown). Further characterization of NSA was facilitated, however, by the observation that NSA is particularly resistant to denaturing conditions. NSA present in the partially purified heparin eluate survived exposure to SDS, trifluoroacetic acid, heat (95°C), and 6 M urea (Table II). In addition, NSA present in the pooled heparin eluate fractions was stable FIG. 2. Ion-exchange/heparin affinity chromatography of the NSA-containing heparin flow-through fraction. The NSA-containing heparin flow-through fraction was dialyzed against equilibration buffer and then centrifuged to remove precipitated protein and nucleic acid. The material was loaded onto 5-ml Q and heparin columns linked in tandem (with the Q column in first) on a fast protein liquid chromatography system. After loading, the Q column was removed, and the bound protein was eluted from the heparin column with a 50-ml linear gradient from 150 mM to 1.0 M NaCl in the presence of 2 M urea, 0.3% CHAPS, and 20 mM Tris-Cl (pH 8.0). 5-ml fractions were collected, and the protein concentration (blue line; mg/ml)), conductivity (red line; millisiemens (mS/cm)), and netrin-synergizing activity (black bars; units/ml) were determined for each fraction. The data represent the results from one such run in which NSA eluted broadly from the heparin column (see "Results"). Fractions 7-10 from this and other runs were pooled for further biochemical analysis. to dialysis against unbuffered water and other low salt-containing solutions despite the presence of a precipitate on dialysis to these conditions (see "Experimental Procedures"), indicating that the solubility problems of NSA in our starting material are more likely the result of coprecipitation with contaminant proteins rather than some intrinsic property of NSA. Finally, it should be noted that despite the remarkable stability of NSA to highly denaturing conditions, the one manipulation that seriously impairs its activity is exposure to the protease trypsin, indicating that NSA requires a necessary protein component.

Recovery of NSA from Nonreducing SDS-Polyacrylamide Gels Indicates That NSA Is a Minor Protein Species That Is
Most Likely in the 25-35-kDa Size Range-We took advantage of the SDS stability of NSA to fractionate the proteins present in our partially purified heparin eluate by nonreducing SDS electrophoresis, reasoning that the dissociative properties of SDS would give us a more accurate size estimate than conventional gel filtration. The NSA-containing heparin eluate was prepared for electrophoresis as described under "Experimental Procedures." The proteins were then separated on a 10% polyacrylamide gel. The gel was cut into strips (fractions were numbered from the bottom up) with a razor blade, and the proteins were eluted from the minced strips by passive diffusion into 6 M urea. After removal of SDS by a modification of the technique of Weber and Kuter (11), the fractions were split into portions that were either rerun on a reducing SDS-polyacrylamide gel that was subsequently silver-stained to assess protein complexity or assayed in the synergy assay to detect NSA. The results of this analysis on one such gel are shown in Fig. 3. All of the activity was found to reside in three fractions that contained proteins centered around 30 kDa. Lower molecular mass proteins present in fraction 1 were inactive, as were higher molecular mass fractions, only some of which are shown. Furthermore, comparison of the intensity of visible protein bands in the active and inactive fractions and particularly between fractions 2 and 3, which possess equivalent activity, demonstrated that NSA is not encoded by any of the major protein species in these fractions. Due to a very low recovery of NSA and gel-to-gel variability in the separation of NSA with respect to the abundant surrounding contaminant bands, it was not feasible, given our limited starting material, to employ this technique for purification of NSA. Nevertheless, our data strongly suggest that NSA resides in a minor protein(s) in the size range of 25-35 kDa (although we cannot exclude that some activity may also be FIG. 3. Recovery of activity from nonreducing SDS-polyacrylamide gels demonstrates that NSA is most likely between 25 and 35 kDa and is encoded by a minor protein species. The NSAcontaining heparin eluate was prepared for nonreducing SDS-PAGE as described under "Experimental Procedures." Portions of each fraction from the nonreducing gel were used for serial dilution in the synergy assay to quantify netrin-synergizing activity (bar graph) and for reducing SDS-PAGE and silver staining to visualize the protein complexity of each fraction (gel). The numbers below the gel indicate the fraction numbers. Molecular mass markers (in kilodaltons) are indicated to the left of the gel. Activity was centered on fractions 2 and 3, with some activity also in fraction 4. These fractions contain proteins in the 25-35-kDa size range. Examination of these proteins and, in particular, comparison of fractions 2 and 3 showed that none of these major proteins were distributed among the fractions in a manner that is consistent with their encoding the activity indicated by the graph. Furthermore, no activity resided with the lower molecular mass proteins in fraction 1 or in the higher molecular mass fractions 5 and 6.
FIG. 4. Native acid/urea electrophoresis of the NSA-containing heparin eluate. The NSA-containing heparin eluate was heated, centrifuged, dialyzed against water, recentrifuged, concentrated, and diluted into native electrophoresis sample buffer as described under "Experimental Procedures." After electrophoresis on the native acid/urea gel (see "Experimental Procedures"), the gel was cut into slices, which were numbered from the bottom of the gel up, and protein was eluted from each slice. A portion of each fraction was analyzed by reducing SDS-PAGE and silver staining to assess the protein complexity of each fraction (gel), and another portion was serially diluted in the synergy assay to assess the amount of netrin-synergizing activity in each fraction (bar graph). Activity was observed in fractions 4 and 5. Numbers below the gel indicate the fraction numbers; Hep refers to the starting heparin eluate fraction, and L refers to this fraction after heating and dialysis against water in preparation for electrophoresis. Molecular mass markers (in kilodaltons) are indicated to the left of the gel. The band present in all fractions between 45 and 66 kDa is a carrier protein added during elution of protein from each of the gel slices.

TABLE II
Stability of NSA to denaturing conditions The NSA-containing heparin eluate was exposed to the conditions below as described under "Experimental Procedures," and the percent of starting activity recovered was assessed by serial dilution of the treated fractions in the synergy assay. associated with smaller protein species that were run off of this gel or that NSA, due to its basic nature (see below), might migrate at an aberrantly high molecular mass on nonreducing SDS-PAGE).
Fractionation of the NSA-containing Heparin Eluate by Native Acid/Urea Electrophoresis-Despite the low recovery of protein and activity on nonreducing SDS-PAGE, we hoped that another electrophoretic technique run under different conditions might prove more useful in the fractionation of NSA. In particular, we wished to investigate native electrophoresis in the presence of acid and urea, a technique that is ideal for separating basic proteins from one another (12,13). We assumed that the activity was a basic protein(s) based on its behavior on ion-exchange/heparin affinity resins (Fig. 2). The results of one native electrophoresis experiment are illustrated in Fig. 4. The partially purified NSA-containing heparin eluate was prepared for electrophoresis as described under "Experimental Procedures." After separation on the polyacrylamide gel, the gel was cut into strips (which were numbered from the bottom of the gel up and minced with a clean razor blade). Portions of each fraction were either rerun on a reducing SDSpolyacrylamide gel and then silver-stained to assess protein complexity or assayed in the synergy assay to detect NSA. The complexity of these fractions is less than that observed on nonreducing SDS-PAGE in Fig. 3 because the material loaded onto this gel was from a batch of the heparin eluate that could be (and was) heated with only a small loss of activity. Comparison of the protein complexity of each fraction revealed that the proteins on the acid/urea gel were not separated solely as a function of their size (as expected for a technique that fractionates by a complex function of charge and mass). Robust activity was recovered in fractions 4 and 5, which contain a set of proteins centered around 30 kDa. This result demonstrates conclusively that NSA is encoded by a basic protein(s) since upon application of the electric field in the presence of the highly dissociative 6 M urea, only basic proteins should migrate through the acid/urea gel. Unfortunately, this technique is also plagued by the low activity recovery problems we encountered with nonreducing SDS-PAGE; and therefore, its use in a purification scheme was precluded because the number of embryonic chicken brains required to include it as a purification step was prohibitive.
Multiple Peaks of NSA Elute from Reverse-phase Columns-The stability of NSA to trifluoroacetic acid suggested that reverse-phase chromatography might prove useful for the fractionation of this activity. The NSA-containing heparin eluate was prepared for C4 reverse-phase chromatography (as described under "Experimental Procedures"). The column was developed with a gradient of increasing acetonitrile concentration. Samples from each fraction from the C4 column were analyzed in the synergy assay as well as by SDS-PAGE and silver staining to examine the protein complexity of the fractions (Fig. 5). Electrophoresis revealed separation of the remaining proteins that was far better than that observed with other non-electrophoretic chromatographic techniques ( Fig. 5 and data not shown). Synergy assays revealed the presence of FIG. 5. Reverse-phase chromatography of the NSA-containing heparin eluate reveals three distinct peaks of netrin-synergizing activity. The NSA-containing heparin eluate was prepared for reverse-phase chromatography as described under "Experimental Procedures" and loaded onto a C4 reverse-phase column in the presence of 25% acetonitrile and 0.1% trifluoroacetic acid. Bound proteins were eluted with a 65-min linear gradient from 25 to 100% acetonitrile in the presence of 0.1% trifluoroacetic acid. The flow rate was 1 ml/min, and 1-ml fractions were collected. Each fraction was analyzed by both SDS-PAGE and silver staining to assess protein complexity (gel) and by serial dilution in the synergy assay to quantify the amount of netrinsynergizing activity in each fraction (bar graph). The numbers below the gel are the fraction numbers; Hep refers to the heparin eluate starting material, and L refers to this fraction after heating at 95°C and dialysis against water to prepare for chromatography. Molecular mass markers (in kilodaltons) are indicated to the left of the gel. Two major peaks(centered on fractions 22 and 26) and one minor peak (beginning in fraction 28) of NSA were observed, each of which contained largely nonoverlapping sets of proteins visible by silver staining. a Owing to the semiquantitative nature of the quantification method employed (see "Experimental Procedures" and "Results"), all unit measures and values calculated using these measurements are subject to a 2-fold uncertainty.
b Although this fraction has already been significantly enriched from the purification source (embryonic chicken brains), it is the first fraction in which NSA could be measured independently of netrin and therefore was considered the starting material for the purification of NSA.
c For ease of comparison, the amounts of these fractions have been normalized to the amount of material used for the C4 reverse-phase chromatography run shown in Fig. 5 and analyzed here, ϳ750 E10 chicken brains. In practice, the Q/heparin step was run with ϳ2000 brains'-worth of heparin flow-through starting material, as in Fig. 2. d As indicated in Fig. 2, the Q flow-through/heparin eluate consists of pooled fractions 7-10 from this run. The values specified here are for these pooled fractions only.
e The C4 reverse-phase total pool is the summed protein and activity of fractions 21-26, 28, and 29 of the run shown in Fig. 5. Peak I consists of fractions 21-23; peak II consists of fractions 25 and 26; and peak III consists of fractions 28 and 29. three distinct peaks of NSA (Fig. 5). The presence of these three peaks was confirmed by multiple runs with different batches of the NSA-containing heparin eluate (data not shown). Comparison of the protein profiles of the active fractions revealed that they contain largely nonoverlapping proteins with the exception of band(s) in the ϳ30-kDa range ( Fig. 5 and see "Discussion"). Table III gives a quantitative assessment of the most useful chromatography steps we have identified to date, including the ion-exchange/heparin step shown in Fig. 2 and the reverse-phase chromatography shown in Fig. 5. The modest -fold purification afforded by these steps is insufficient for purification to homogeneity from our current source, but we hope that future fractionation of NSA(s) present in a more easily obtained starting material will allow molecular identification of these activities, which may be related or distinct molecular species. DISCUSSION Axon guidance cues can be classified into four categories: positive or negative cues that act either locally or at a distance to guide axons within the developing embryo (1,2). The fact that NSA does not have any axon outgrowth-promoting activity on its own suggests that this activity may be acting in a mechanistically distinct manner from that of previously identified positive and negative axon guidance factors, through modulating the activity of a known axon guidance cue. It will be difficult to unravel the mechanism of action and in vivo function of this cue without its molecular identification. The results presented here should aid in that endeavor. Here we describe an improved assay for NSA and the use of this assay to define the biochemical properties of NSA. Fractionation of NSA reveals a number of important observations that should facilitate future molecular identification. First, despite its remarkable stability to a variety of denaturing conditions, this activity contains a necessary protein component for activity since it is abolished by protease treatment. This observation, coupled with the finding that NSA is a basic protein (indicated by the behavior of NSA on ion-exchange resins and on native acid/urea electrophoresis), indicates that NSA is not encoded by a glycosaminoglycan or proteoglycan moiety as was originally hypothesized (7) since brain-derived proteoglycans (but not NSA) bind to anion exchangers at 150 mM NaCl (15).
It appears that the NSA present in our starting material, a netrin-depleted high salt extract of embryonic chick brain membranes, may be heterogeneous, as indicated by the existence of multiple activity peaks that elute from a C4 reversephase chromatography column. All of these peaks must be encoded by basic proteins (since they all elute from heparin at basic pH), and they are likely to all be in the same size range (25-35 kDa) since this is where all NSA-like activity is recovered in nonreducing SDS electrophoresis experiments. One could argue that some of these NSAs are stable to trifluoroacetic acid and not to SDS (a possibility our stability experiments do not address) and that we therefore really know the size of only one of the three NSAs. Although this is possible, we think it is unlikely since resistance to one denaturing condition generally implies a structure that imparts resistance to other denaturing conditions. Examples of this principle include the neuregulins (16) and the tissue inhibitors of metalloproteases (17), both of which survive exposure to a number of denaturing conditions. The most likely interpretation of our nonreducing electrophoresis and reverse-phase chromatography experiments is that each peak observed on reverse-phase chromatography is in the 25-35-kDa size range we defined for NSA by nonreducing SDS-PAGE.
Most of the difficulty in purifying this activity can be attributed to the facts that we have not been able to identify a high enrichment affinity chromatography step specific for this activity (since neither a panel of lectins nor netrin affinity resins bind NSA) and that the steps we have identified so far give only modest enrichment of NSA (Table III). Published biochemical purifications of other axon guidance molecules have all included affinity steps and may not have been possible without them (7,18,19). Through future fractionation experiments, we hope to determine whether the three activity peaks we observed with reverse-phase chromatography represent independent proteins, related but distinct proteins, or a single protein with several different modifications. Our suspicion, based on the results of nonreducing SDS-PAGE of NSA-containing fractions, is that NSA is encoded by minor protein species present in trace amounts in our fractions. Mass spectrometry and Edman degradation sequencing of the most abundant protein species that approximately cofractionate with our observed activity on reverse-phase chromatography (the ϳ30-kDa bands shown in Fig. 5) bear this conclusion out; to date, the proteins we have sequenced from these fractions are all highly basic histone and ribosomal proteins. 2 Although these proteins share biochemical properties with NSA, their intracellular nature makes it unlikely that they actually encode NSA. Due to low recoveries of NSA on reverse-phase chromatography, the lack of an affinity chromatography step, and the fact that NSA in embryonic chicken brain appears to be present in trace amounts, purification of NSA from embryonic chicken brain does not seem currently feasible. Identification of an affinity chromatography step or purification from a more abundant activity source (perhaps adult chicken or bovine brain) may surmount these obstacles to the molecular identification of NSAs.