Neurochemical Research (3) 175-183 (1978)

ISOLATION OF H U M A N NERVE GROWTH FACTOR FROM PLACENTAL TISSUE L. D . GOLDSTEIN, C. P. REYNOLDS, AND J. R. PEREZPOLO 1 Department of Human Biological Chemistry and Genetics University of Texas Medical Branch Galveston, Texas 77550

Accepted August 12, 1977

Nerve growth factor (NGF) has been isolated from human placental tissue. Using the chicken embryo dorsal root ganglia assay, we determined levels of NGF activ.ity for the amnion, placental cotyledons, cord serum, fetal serum, and maternal serum. The highest levels of NGF activity were measured in placental cotyledons. After homogenization and centrifugation of the placental cotyledons, the superuatant was sequentially chromatographed, at neutral pH, on Sephadex G- 100, DEAE- 11, and Sephadex G- 150. A high-molecular-weight protein fraction (-150,000), which contained all the biological activity, was isolated in this fashion. Analytical isoelectric focusing of this fraction revealed a basic protein component (pI 9.5) of the high-molecular-weight species. Assays for NGF activity of all protein components separated by analytical isoelectric focusing showed that NGF activity was associated only with the basic protein component. Correspondingly, preparative isoelectric focusing of the high-molecular-weight species yielded a basic protein with very high biological activity (1-3 ng per biological unit) that was immunochemically active against rabbit IgG made against mouse /3-NGF.

INTRODUCTION A number of recent studies suggest that there may be a relationship between altered levels of nerve growth factor activity (NGFA) in human tissues and neurological disorders that have a known genetic component

(1-4). NGF is a protein that is essential to the development and maintenance of adrenergic neurons in peripheral, sensory, and sympathetic ganglia J To whom reprint requests should be addressed.

175 0364-3190178/0400-0175505.00/09 1978PlenumPublishingCorporation

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(5). A partial manifestation of this response is a m a r k e d proliferation and acceleration of neurite outgrowth, which is the basis for an in vitro a s s a y (6,7)= Through the use o f a biological assay, it has b e e n d e m o n s t r a t e d that N G F is present in detectable levels in all v e r t e b r a t e s investigated (8-11). Although N G F is found in low levels in m o s t tissues investigated, t w o exceptions to this finding are the submaxillary gland of sexually mature, r a n d o m - b r e d Swiss W e b s t e r male mice (12) and a n u m b e r of snake v e n o m s (13). All previous information regarding the chemical structure and the nature of N G F is limited to m o u s e or snake N G F . A n u m b e r of purification schemes h a v e b e e n devised for N G F f r o m m o u s e submaxillary gland (14-16) as well as f r o m a variety of other sources (17,18). When m o u s e N G F is isolated at neutral p H under conditions resembling the in vivo situation, it is found to be a 7S protein multimer (14), with a molecular weight of 131,750 (19), m a d e of three dissimilar subunits (a,/3,T) (20) in equilibrium with its c o m p o n e n t subunits (21). The a/3T, 7S N G F , protein complex is stable in the p H range of 5 to 8 (22), with the biological N G F activity residing in the basic /3 subunit. Alternatively, N G F can be purified from snake v e n o m as a partially degraded basic protein (23), similar to the m o u s e / 3 - N G F (17,18), or as a c o m p l e x , one of whose c o m p o n e n t s is a basic protein similar to the m o u s e / 3 - N G F (18). To date, all m e a s u r e m e n t s of N G F levels in h u m a n tissues h a v e used m o u s e N G F and antibodies to m o u s e N G F as standards, yielding contradictory results (3,4). The availability of purified h u m a n N G F will allow the standardization and unequivocal quantitation of m e a s u r e m e n t s of N G F levels in h u m a n tissues and fluids in a n u m b e r of neuropathies. In this communication, we d o c u m e n t the p r e s e n c e of N G F A in h u m a n placenta and describe a purification scheme for h u m a n N G F .

EXPERIMENTAL PROCEDURE

Placental Tissue Analysis Fresh human placentas were processed the same day they were obtained. Blood was flushed from the placenta with cold (4~ 0.05 M Tris-HCl, pH 7.4, buffer, and the serum after centrifugation was lyophilized. The amnion was separated from the placenta and frozen for later analysis. The placenta was then flushed with 0.9% saline solution to purge any residual blood, and then the cotyledons were dissected, freed of vesicles, blotted dry, and frozen. Fetal serum and maternal serum lyophilizates were resuspended in 0.05 M TrisC1, pH 7.4, buffer and aliquots were bioassayed for NGF activity using the embryonic chick dorsal root ganglia assay. Cotyledons and amnion were homogenized in a Sorvall Omnimixer for 3 min at high speed with 0.05 M Tris-C1, pH 7.4, buffer. The homogenate was centrifuged for 30 min at 10,400g, and the supernatants were tested for NGF activity.

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Bioassay The bioassay, which uses 8-9-day-old chick embryo dorsal root ganglia, is capable of detecting as little as 1-10 ng/ml of NGF activity. This bioassay was done according to standard methods, using threefold serial dilution of samples tested (5,6). The biological unit (BU) is defined as that concentration of NGF giving optimal fiber outgrowth as scored on a 1 to 4 scale; a score of 4 corresponds to a dense halo of neurites extending from the explant. Such a solution is said to have 1 BU/ml. Protein was determined by the method of Bradford (24), using bovine serum albumin as a standard.

NGF Purification The isolation procedure was based on the method of Varon et al. (17) developed for mouse NGF. The cotyledonary tissue of a single placenta was homogenized with cold (4~ distilled-deionized water for 1-3 min at high speed in a Sorvall Omnimixer. The homogenate was centrifuged at 10,000g in a preparative ultracentrifuge for 25 rain. The supernatant was then lyophilized and resuspended in 0.05 M Tris-C1, pH 7.4, buffer. The suspension was next chromatographed on a column of Sephadex G-100 (5 • 100 cm), and the protein was eluted with 0.05 M Tris-HCl, pH 7.5, buffer at a rate of 0.25 ml/cm2/h. The fractions containing NGF activity were pooled and then chromatographed on a DEAE cellulose (DE-11) column (5 • 15 cm). The NGF activity is eluted by using successive solutions of increasing salt concentration (1 liter each of 0.01, 0.08, and 2.0 M NaCI, all in 0.05 M Tris-C1, pH 7.4, buffer). The NGF activity was eluted by the 0.08 M NaCI. This fraction was concentrated to a small volume by pressure dialysis and then applied to a column (2 • 90 cm) of Sephadex G-150. The NGF activity was then eluted by 0.05 M K+,Na+-phosphate, pH 6.8, buffer.

Analytical Isoelectric Focusing An aliquot of the NGF fraction isolated by the Sephadex G-150 step was subjected to thin-layer isoelectric focusing in polyacrylamide gel (TL1EF). The gel mixture, having a final concentration of T = 5% (w/v) and C = 3% (w/v) (20), 2% ampholine (3.5-10, 40% (w/v), and 0.0004% riboflavin 5'-phosphate, was degassed, and a 1-mm-thick gel was cast. Electrofocusing was conducted as described by the LKB protocol (LKB, Bromma, Sweden, application note #75 of LKB instructions). The gel was sectioned into 5-mm pieces (50 mm 2) and eluted in a small volume (500 tzl) of Gey's Balanced Salt Solution overnight at 4~ Eluted fractions were then assayed for NGF activity. Double immunodiffusion was performed according to the Ouchterloney technique (25), using rabbit IgG made against mouse/3-NGF.

Basic Subunit Isolation Preparative isoelectric focusing was used to isolate the basic subunit in human NGF (so designated because the purification protocol used for human NGF parallels that of mouse NGF in which a basic biologically active subunit, mouse fl-NGF, is demonstrated), hereafter called human/3-NGF. An aliquot of the G-150 pool was dialyzed for 4 h vs. two 4-liter changes of 1% glycine; The dialysate (2.7 ml containing 33 nag of protein) was subjected to sucrose (0-47%) density gradient isoelectric focusing using an LKB 8102 Column (440-ml capacity). The column, containing 2% ampholine (pH 3.5-10, 40% w/v), was prefocused

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for 19 h at an average constant wattage of 7.5 W and a voltage limit of 1200 V. A volume equal to that of the sample and corresponding to a pH of 3.9 was withdrawn from the column. The sample, which was made isodense with respect to the withdrawn aliquot, was introduced into the column in substitution. Electrofocusing was conducted at 4~ using the constant wattage procedure outlined by LKB application note #194. Constant 1.5-ml fractions were collected at a constant flow rate of 4.0 ml/min by using a Varioperpex II pump in tandem with a Redirac fraction collector (LKB). Each fraction was monitored for UV absorbance at 280 nm and for pH, which was determined at 4~

RESULTS

The following placental tissues were bioassayed for NGF activity: amnion, placental cotyledons, cord, fetal, and maternal serums. Although the amnion had the highest specific NGF activity, the placental cotyledon tissue had the highest total activity and, for that reason, was selected as the starting material for the purification of human NGF (Table I). A typical response of the avian dorsal root ganglia to an optional concentration of human NGF activity is shown in Figure 1. NGF was partially purified from an aqueous extract of placental cotyledons. A summary of the purification scheme is presented in Table II, which shows a remarkable increase in the specific activity for NGF (-60,000) obtained as a high-molecular-weight species (-140,000) from the Sephadex G-150 step. The total recovered biological activity increased as the purification proceeded. This anomalous behavior is caused by the removal of endogenous inhibitors during isolation and has been also reported to take place during the purification of murine (14) and snake venom NGF (18). Addition of purified, active mouse fl-NGF to a homogenate results in a similar inhibition. This could be a result of TABLE i NERVE GROWTH FACTOR ACTIVITY PRESENT IN DIFFERENT HUMAN PLACENTAL TISSUES a

Tissue Placental cotyledons Amnion Fetal serum Maternal serum Cord serum

Protein content (mg)

NGF Specific activity (BU/mg)

NGF Activity content (BU)

3843 41 166 2040 47.2

0.35 4.16 0 0 0.06

2070 170 0 0 3

a For details, see the Experimental Procedure section.

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FIG. 1. Fiber outgrowth from explanted 8-day-old chicken embryo dorsal root ganglion cultured for 24 h in the presence of human fl-nerve growth factor isolated by preparative isoelectric focusing. Phase contrast magnification 100x.

inhibition of the NGF effect or nonspecific toxic effects of the placental homogenate on the embryonic avian dorsal root ganglia. Since this highmolecular-weight species was isolated at neutral pH, it was then of interest to determine whether the human NGF, like the mouse NGF, contained a basic component that possessed NGF biological activity (16). This hypothesis was examined by analyzing an aliquot of the G-150 fraction by TLIEF, followed by sectioning and eluting the gel and assaying the diffusible material for NGF activity. The results of this analysis revealed a basic protein species that demonstrated NGF activity. None

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TABLE II PROTEIN AND NERVE GROWTH FACTOR LEVELS FOR THE VARIOUS POOLED FRACTIONS DERIVED FROM HUMAN PLACENTAL COTYLEDONS DURING THE

ISOLATIONOF HUMANNGF a

Tissue homogenate G-100 Pool DE-11 Pool G-150 Pool

Total Protein

% of starting

Specific activity

(mg)

protein

(BU/mg)

3843 853 149 41

100 22 3.9 1.1

0.54 230 1,300 32,000

a A 4 + rating was only observed for the G-150 pool (for details, see the Experimental Procedure section).

of the eluted gel pieces sectioned from any other part of the pH gradient showed N G F activity. These results further suggested that this basic fraction may be isolated preparatively. For this purpose, an aliquot of the high-molecular-weight fraction containing 33 mg of protein was subjected to high-wattage, rapid isoelectric focusing. The 280-nm absorption profile (Figure 2) of the column fractions revealed a single isolated basic protein peak whose specific biological activity for N G F was found to be in the range of 1-3 ng/BU. Of the 33 mg of protein loaded, 2 mg was recovered as the biologically active basic protein species. This fraction was also tested immunologically and found to be partially cross-reactive with rabbit antimouse/3-NGF IgG by the double immunodiffusion technique. The degree of cross-reaction between human NGF and antibodies against mouse /3-NGF was found to be roughly one-tenth that observed with mouse NGF and antibodies against mouse fl-NGF.

DISCUSSION We have demonstrated that human placental cotyledons are a suitable source for the purification of human NGF. As already pointed out, some of the characteristics of human N G F are similar to those of mouse NGF. This report suggests that the human NGF molecule may be obtained from a unique and readily available source, the placenta, as a protein with a molecular weight of about 140,000 daltons. Dissociation of this highmolecular-weight species by preparative isoelectric focusing yields a basic protein that has biological NGF activity. The dissociation of the

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