Proc. Nati. Acad. Sci. USAVol. 91, pp. 9593-9596, September 1994Neurobiology

Identification of prosaposin as a neurotrophic factorJOHN S. O'BRIEN*t, GEOFFREY S. CARSON*, HEE-CHAN SEO*, MASAO HIRAIWAf, AND YASuo KISHIMOTO**Department of Neurosciences, Center for Molecular Genetics, University of California at San Diego, School of Medicine, La Jolla, CA 92093-0634; andtDepartment of Health Chemistry, Niigata College of Pharmacy, Niigata, Japan

Communicated by Morris E. Friedkin, June 15, 1994

ABSTRACT Prosaposin was identified as a neurotrophicfactor stimulating neurite outgrowth in murine neuroblastoma(NS20Y) cells and choline acetyltranferase (ChAT) activity inhuman neuroblastoma (SK-N-MC) cells. The four naturallyoccurring saposs, which are derived by proteolytic processingof prosaposin, were tested for activity. Saposin C was found tobe active, whereas saposns A, B, and D were inactive asneurotrophic factors. Dose-response curves demonstrated thatnanomolar concentrations of prosaposin and saposin C stim-ulated neurite outgrowth and increased ChAT activity. Prosa-posin and saposin C exerted activity by a mechanlsm indepen-dent of nerve growth factor, brain-derived neurotrophic fac-tor, and neurotrophin 3. Binding assays uing saposin C asa ilgand gave two saturable binding constants, a hig-affnity(Kd = 19 pM) and a low-affinity (Kd = 1 uM) constant, with2000 and 15,000 sites per NS20Y cell, respectively. Phosphor-ylation stimulation experiments demonstrated that brief treat-ment with prosaposin or saposin C enhanced phospholationof a variety of proteins, some of which contained phosphory-lated tyrosine(s). Since both cell lines were also stimulated byciary neurotrophic factor (CNTF) as well as prosaposin,inhibition was tested by utilizing an anti-gpl30 monoclonalantibody, which s lly inhibited CNTF stimulation; thlsantibody did not inhibit prosaposin or saposi C smulation.These results indicate that prosaposin and saposin C areneurotrophic factors which initiate signal transduction bybinding to a high-afinity receptor that induces protein phos-phorylation.

Prosaposin is the precursor of the lysosomal saposin pro-teins, which are required for hydrolysis ofglycosphingolipidsby lysosomal hydrolases (1, 2). In addition to its role as alysosomal precursor, prosaposin is presumed to have addi-tional functions, since it exists as a secretory protein inhuman milk, cerebrospinal fluid, and seminal plasma (3-5); itis present in unprocessed form in high concentrations inhuman (6) and rat (7) brain; its mRNA is abundant in brainand dorsal root ganglia during embryonic development (8); itis present predominantly in neurons after immunostaining(9); and it occurs as an integral membrane component (10).

Recently we demonstrated that prosaposin binds ganglio-sides with high affinity and facilitates their transfer frommicelles to membranes (11). Since gangliosides have beenshown to promote neurite outgrowth in cultured neuronalcells (12-14), we investigated whether prosaposin was alsoactive. In this report we identify prosaposin as a potentneurotrophic factor and locate the active region to thesaposin C domain.

MATERIALS AND METHODSCels. Murine neuroblastoma cells (NS20Y), which extend

neurites in response to gangliosides (15), were from T.Taketomi. Human neuroblastoma cells (SK-N-MC) were

obtained from the American Type Culture Collection. Bothlines were maintained in Dulbecco's modified Eagle's me-dium (DMEM)/10%o fetal bovine serum (FBS) plus penicillinand streptomycin. PC12M cells are a subclone of the originalPC12 cell line (16) selected in M. Montminy's laboratory(Salk Institute, La Jolla, CA) for their enhanced attachmentto plasticware.

Materials. Human saposins A, B, C, and D, milk prosa-posin, and recombinant prosaposin (prepared by insect cell/baculovirus expression) were prepared as described (5, 17).Ciliary neurotrophic factor (CNTF), brain-derived neuro-trophic factor (BDNF), and neurotrophin 3 (NT-3) were fromCephalon. Nerve growth factor (NGF) and 5-methylthioad-enosine were from Sigma. AM64, a mouse monoclonal an-tibody to the human membrane glycoprotein gpl30, a signaltransducer, was the gift of T. Taga (Osaka University) andhas been characterized previously (18).

Neurite Outgrowth. Neurite outgrowth in NS20Y cells wasassayed by utilizing a published method (15). In brief, aftertrypsinization 2 x 104 NS20Y cells were plated onto glasscoverslips in 30-mm Petri dishes. After 20-24 hr, the mediumwas replaced with 2 ml of DMEM/0.5% FBS plus effectors.Cells were cultured for an additional 24 hr, washed withphosphate-buffered saline (PBS), and fixed in Bouins solu-tion (30 min). After the fixative had been removed, neuriteoutgrowth was scored under a phase-contrast microscope.Cells bearing neurites longer than 1 cell diameter were scoredas positive, and 100 cells were counted in triplicate fromdifferent portions ofeach dish. Each assay was carried out induplicate dishes. The average error of duplicates (40 assays)was ±3.6%.Enzyme Assays. Assays of choline acetyltransferase

(ChAT) activity in the human neuroblastoma cell line SK-N-MC were carried out as described (19). In brief, cells werewashed three times in PBS, scraped from 60-mm Petri dishes,pelleted, and sonicated in 20 mM Tris HCl (pH 8.6) contain-ing 0.1% Triton X-100. Extracts were assayed for 15 min asdescribed. Two assays were carried out in duplicate dishesfor each determination. Protein was estimated by the bicin-choninic acid method (Sigma).

lodinatin. Human saposin C was iodinated with Na'ZI(Amersham IMS.30, carrier-free) by using Iodo-Beads(Pierce) and following the manufacturer's instructions. ThreeIodo-Beads were used to iodinate 5.2 pg of saposin C insodium phosphate buffer (100 mM sodium phosphate/100mM NaCl, pH 7.0) with 1.2 mCi (1 mCi = 37 MBq) of Na125I.The reaction mixture (total volume 450 p1) was gently shakenfor 6 min, 500 pg ofbovine serum albumin (Sigma no. A0281)was added, and free iodide was removed from the solution onan Excellulose column (Pierce). Radiolabeled saposin C(specific activity 80 cpm/pg) was stored at 40C and usedwithin 3 weeks of preparation. Nonradioactive iodide wasused to label saposin C by the same method to assess the

Abbreviations: FBS, fetal bovine serum; CNTF, ciliary neurotrophicfactor; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin3; NGF, nerve growth factor; ChAT, choline acetyltransferase.tTo whom reprint requests should be addressed.

9593

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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biological activity of radiolabeled saposin C. Iodinated sa-posin C was found to retain full biological activity comparedwith native saposin C in the neurite outgrowth assay.

Binding Studies. Nearly confluent NS20Y cells were usedfor the binding studies. Cells were rinsed twice with warmHanks' balanced saline solution (HBSS; GIBCO) and disso-ciated from the culture plate by trituration in HBSS. The cellsuspension (5-7 x 105 cells per ml) containing bovine serumalbumin (fatty acid- and globulin-free; Sigma A0281) at 100,gg/ml was used for each concentration in a 1.5-ml Eppendorftube (final volume, 1 ml) in the presence of 125I-labeledsaposin C (125I-saposin C). After incubation for 30 min at 37TCeach tube was centrifuged in a Sorvall microcentrifuge (Mi-crospin 12) for 20 sec. An aliquot (100 std) was taken from thesupernatant for determination of free ligand concentration,and the pellet was rinsed once with cold HBSS. The bottomof the tube, containing the pellet, was cut with a toenailclipper and the radioactivity was measured in a Beckman 'ycounter (model 5500). Specific binding was determined astotal binding minus nonspecific binding. Nonspecific bindingwas determined by inclusion of a 100-fold excess of unlabelediodinated (nonradioactive) saposin C for each concentrationpoint to 60 pM (below 60 pM, 6 pmol of unlabeled ligand wasused regardless of concentration). Nonspecific binding wasfound to increase with increasing concentrations of 125I-saposin C and typically was 30-50%6 at higher concentrationsof ligand and 5-20% at lower concentrations. Each experi-ment was performed in quadruplicate and Scatchard analysiswas performed with the computer program MAcLIGAND(Robert E. Williams, University of California, Los Angeles).We used fatty acid- and globulin-free albumin in low con-centrations because high concentrations inhibited saposin Cbinding. Less purified bovine serum albumin inhibited sa-posin C binding even at low concentrations, and higherconcentrations (1 mg/ml) gave very high nonspecific binding(80-150%o of specific binding).

Protein Phosphorylatlon. Protein phosphorylation and al-kaline hydrolysis were performed by using previously pub-lished methods (20, 21) with minor modifications. Briefly,NS20Y cells were incubated for 1-3 hr in phosphate-freeHBSS containing actinomycin D at 2.5 ,ug/ml, after whichcarrier-free sodium [32P]orthophosphate (New England Nu-clear) (80-100 ,Ci/ml) and effector (0.5-1 ,g/ml) wereadded and the cells were incubated for 10-15 min at roomtemperature. Cells were solubilized in 1x SDS/PAGE sam-ple buffer, and the proteins were analyzed by SDS/PAGEand autoradiographed on Fuji RX film. Gels were also treatedwith NaOH [1 M NaOH for 1 hr at 55°C, then washed in 10%acetic acid/30%o methanol (vol/vol) for 30 min at roomtemperature to neutralize] to hydrolyze phosphate esterlinkages to serine and threonine residues before autoradiog-raphy.

Phosphotyrosine Immunobbfflng. Cells grown in 10-cmplates were labeled and stimulated with effectors as before.The cells were lysed in 20 mM Tris-buffered 150 mM saline(pH 7.8) containing 1% Nonidet P-40, 1% sodium deoxycho-late, and 0.1% SDS (TBS-NDS). Protein concentration wasdetermined, and equal amounts of lysate were immunopre-cipitated by using agarose-conjugated anti-phosphotyrosinemonoclonal antibody 4G10 (anti-ptyr 4G10, Upstate Biotech-nology, Lake Placid, NY) for 2 hr at 4°C, then resolved bySDS/6% PAGE. Nitrocellulose blots were initially probedwith unconjugated anti-phosphotyrosine monoclonal anti-body, using ECL (Amersham) detection, and subsequentlyautoradiographed.

RESULTSA dose-response curve (Fig. 1A) demonstrated that bothnative and recombinant prosaposin promoted reversible neu-

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FIG. 1. Neurogenic response of NS20Y cells treated with proteineffectors. (A) Neurite outgrowth in response to various saposinproteins, expressed as percentage of cells extending neurites as afunction of dose. prosap-r, recombinant human prosaposin; pro-sap-m, purified human milk prosaposin; sap C and sap A, purifiedhuman saposins C and A. (B) Neurite outgrowth in the presence ofeffectors (100 ng/ml), demonstrating inhibition of CNTF-induced,but not saposin C-induced, neurite outgrowth by anti-gp130 mono-clonal antibody AM64 (1 pl/ml of medium).

rite outgrowth in NS20Y neuroblastoma cells, with the lowestconcentration for activity being 14 pM (10 ng/ml). Whenprosaposin was removed, retraction ofneurite outgrowth wascomplete at 36 hr, demonstrating the requirement for itscontinual presence to sustain neurite outgrowth. Saposins A,B, and D were inactive, whereas saposin C also promotedneurite outgrowth at about 5 times higher molar concentra-tions than prosaposin.To test whether prosaposin-ganglioside complexes stimu-

lated neurite outgrowth, complexes of prosaposin with gan-glioside GM1 (molar ratio of ganglioside GM1 to prosaposin,4:1) were prepared in vitro (11). When tested in the neuriteoutgrowth assay at 1 pg/ml (of prosaposin) the complex hadnegligible activity, and the same result was obtained when aganglioside GM3-saposin C complex was tested. Abolish-ment of activity may be due to the masking of functionalstretches of amino acid residues in prosaposin (and saposinC) by bound ganglioside. The concentrations of gangliosideGM1 (100 pg/ml) previously reported to stimulate neurite

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FIG. 2. ChAT activity in SK-N-MC cells after treatment withsaposins or CNTF (200 ng/ml) for 24 and 48 hr. ChAT activity isexpressed as cpm/mg of extract protein.

outgrowth in NS20Y cells (15) are 6 x 103 higher than theconcentrations we supplied as complexes.We then determined whether NGF, BDNF, NT-3, and

CNTF stimulated neurite outgrowth in NS20Y cells. NGF,BDNF, and NT-3 did not stimulate neurite outgrowth whenadded at concentrations as high as 5 pg/ml (data not shown).When CNTF was tested, stimulation of neurite outgrowth inNS20Y cells occurred and the extent of neurite outgrowthwas similar to that induced by prosaposin. We then tested an

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125I-saposin C in a concentration-dependent manner. (B) Scatchardanalysis of the binding data, using a two-site model.

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FIG. 4. SDS/PAGE showing 32p incorporation into NS20Y pro-teins after incubation (15 min, 22QC) with saposin proteins (19). Theprotein concentration applied was the same in all lanes. (A) Proteinswere from cells treated as follows: lane 1, control; lane 2, saposin A;lane 3, saposin C; and lane 4, prosaposin. Electrophoresis was in a7.5% acrylamide gel. (B) Same gel treated with alkali (1 hr, 55QC),lanes same as in A. Note the higher 32p incorporation into proteinsof 148, 110, 82, 69, 59, 47, 38, and 34 kDa in the samples treated withprosaposin and saposin C. Note the relative resistance of the 110-,82-, 69-, 38-, and 34-kDa proteins to alkali treatment in B.

antibody to gpl3O (AM64) that has previously been reportedto block stimulation by CNTF (18). AM64 completely inhib-ited neurite outgrowth induced by CNTF but did not inhibitneurite outgrowth induced by prosaposin or saposin C (Fig.1B).A similar neurite outgrowth assay was performed in

PC12M cells, which are responsive to NGF. No neuriteoutgrowth was observed in the presence of prosaposin orsaposin C (2 pg/ml). Moreover, addition of 3 mM 5'-methylthioadenosine, which inhibits neurite outgrowth in-

duced by NGF (22), had no effect on prosaposin-stimulatedoutgrowth in NS20Y cells (data not shown).SK-N-MC cells also extended neurites when treated with

prosaposin or saposin C (not shown). Untreated SK-N-MCcells were found to possess readily measurable ChAT activitywhen these cells were grown in media containing low serum(0.5% FBS). After exposure to prosaposin and saposin C for24 and 48 hr. ChAT activity increased and reached a levelabout twice the untreated control value at 48 hr (Fig. 2). Inthis assay, CNTF was also stimulatory, whereas BDNF andNT-3 were inactive. These results demonstrated that prosa-posin and saposin C induced differentiation of a cholinergicneuronal cell line.

After iodination saposin C was found to stimulate neuriteoutgrowth to the same extent as native saposin C (data notshown). Utilizing 125I-saposin C as a ligand, binding studieswere conducted in NS20Y cells. 'wI-saposin C was found tobind to putative receptors on NS20Y cells in a saturablemanner (Fig. 3A). Scatchard analysis (Fig. 3B) revealed thepresence of two different binding sites, a high-affinity sitewith a Kd of 19 pM (11.5-30 pM) and 2000 (1130-2900) sitesper cell and a low-afflinity site with a Kd of approximately 1nM (0.6-1.7 nM) and 15,000 (11,000-21,500) sites per cell.Binding of saposin C was not influenced by the presence ofa 100-fold excess of unlabeled saposin A (not shown).

Protein phosphorylation was assayed in NS20Y cells in-cubated in the presence of [32P]orthophosphate and variouseffectors. Prosaposin and saposin C enhanced phosphoryla-tion of proteins of approximately 148, 110, 82, 69, 59, 47, 38,a4d 34 kDa by 3- to 5-fold compared with untreated controlcultures or those treated with saposins A, B, or D (Fig. 4A).Subsequent treatment of the gel depicted in Fig. 4A withalkali to hydrolyze phosphoserine and phosphothreonine

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residues revealed (Fig. 4B) that many of these phosphory-lated proteins were alkali-resistant, consistent with eithertyrosine or threonine (adjacent to proline) phosphorylation.To determine which of these proteins contained phospho-

tyrosine residues, 32P-labeled cells were treated with effec-tors and phosphotyrosine-containing proteins were immuno-precipitated by using the 4G10 monoclonal antibody to phos-photyrosine. The 110-, 82-, 69-, 38-, and 34-kDa proteinswere detected on Western blots probed with the same anti-body and, simultaneously, on autoradiographs of the blots.We conclude that the observed increase in protein phosphor-ylation induced by prosaposin and saposin C is the result oftyrosine phosphorylation offive ofthe eight major phosphor-ylated proteins.

DISCUSSIONThe circumstantial evidence that prosaposin not only is alysosomal precursor ofthe saposin proteins but also has otherfunctions, especially in the nervous system, can be summa-rized as follows: Prosaposin exists as an unprocessed proteinin human (6) and rat (7) brain and in cerebrospinal fluid (5,23). Immunocytochemical studies using a prosaposin-specific antibody demonstrated high concentrations of pro-saposin in human cortical neurons (unpublished data). Asimilar survey in rat brain demonstrated widespread prosa-posin immunoreactivity in neurons (9). Total prosaposindeficiency in humans due to a point mutation in the startcodon of the prosaposin gene (24) resulted in severe neuro-logical deterioration evident at birth with prominent corticalhypomyelination and cerebral atrophy (25). Northern anal-ysis during embryonic development (day 12) demonstratedhigh concentrations ofprosaposin mRNA in mouse brain anddorsal root ganglia indicative of a developmental role (8).The present study demonstrated that prosaposin and sa-

posin C stimulated neurite outgrowth and increased ChATactivity in neuronal cell lines at nanomolar concentrations,concentrations similar to those at which other cytokines elicittheir effects. Prosaposin was effective at a molar concentra-tion one-fifth that of saposin C, perhaps indicating enhancedbinding. Binding assays and Scatchard analysis indicated thepresence of both high- and low-affinity binding sites. Spec-ificity of saposin C binding was high, since saposin A, whichis 40%o identical to saposin C, did not inhibit saposin Cbinding.Rapid protein phosphorylation was evident when cells

were exposed to prosaposin and saposin C, indicative ofsignal transduction through a kinase cascade. The data indi-cated that prosaposin and saposin C did not activate by themechanism utilized by the NGF family of neurotrophins.However, both cell lines we tested also responded to CNTFstimulation. CNTF appears to act by binding to a specificity-conferring a-receptor, inducing heterodimerization of two13-receptor components, gpl30 and LIF,8R (leukemia inhib-itory factor (3-receptor), both of which become phosphory-lated upon ligand binding (26). We found no evidence fortyrosine phosphorylation of a protein the size of gpl30 afterprosaposin or saposin C stimulation, and anti-gp130, whichblocked the CNTF response, did not block the prosaposinresponse, indicating that prosaposin acts through a different

mechanism. Further work is needed to identify the prosa-posin receptor and determine the mechanism ofits action andto identify neuronal cell types which are prosaposin respon-sive.

The authors thank Dr. T. Taketomi and K. Uemura for theneuroblastoma cell line; Dr. T. Taga for AM64; Cephalon, Inc., forgifts of CNTF, BDNF, and NT-3; and Susan O'Brien for assistancein the preparation of this manuscript. The project was funded in partfrom National Institutes of Health grants to J.S.O. (NS08682) andY.K. (NS13559) and a Gould Family Foundation grant to J.S.O.

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