©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Studies on the Structure and Function of Glycosylated and Nonglycosylated neu Differentiation Factors
SIMILARITIES AND DIFFERENCES OF THE alpha AND beta ISOFORMS (*)

(Received for publication, October 17, 1994; and in revised form, December 7, 1994)

Hsieng S. Lu (1)(§) David Chang (1) John S. Philo (1) Ke Zhang (1) Linda O. Narhi (1) Naili Liu (1) Mei Zhang (1) Jilin Sun (1) Jie Wen (1) Donna Yanagihara (1) Devarajan Karunagaran (2) Yosef Yarden (2) Barry Ratzkin (1)

From the  (1)From Amgen Inc., Amgen Center, Thousand Oaks, California 91320 and the (2)Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Comparative analyses of both glycosylated and nonglycosylated neu differentiation factor (NDF) isoforms revealed significant similarities and differences of their overall structures and functions. Biophysical analyses confirmed that all NDF isoforms are monomeric, but have an extended ellipsoidal shape in solution. All full-length NDFs are similar in secondary and tertiary structures and they contain no alpha-helix but are abundant in beta-strand structures. A small NDF fragment containing only the epidermal growth factor domain is also rich in beta-strand structures, but exhibits tertiary structure different from the long NDF forms. Monoclonal antibodies that selectively recognize epidermal growth factor domains of human NDF-alpha or NDF-beta can specifically bind the respective NDF-alpha and -beta isoforms independent of NDF origins. Western blot analysis and quantitative binding assays further identify that an NDF preparation produced naturally from Rat1-EJ cells contains both alpha and beta isoforms in a 3 to 2 ratio. In receptor-binding competition experiments, human and rat NDF-beta isoforms have higher affinity than NDF-alpha isoforms. NDF-beta isoforms can dramatically enhance the stimulation of DNA synthesis for transfected NIH3T3 cells that overexpress HER-3 and HER-4 receptors, while NDF-alpha isoforms can only stimulate proliferation of HER-4-transfected cells with lower activity. Taken together, NDF-alpha and -beta isoforms share similar gross protein conformations but are biologically distinct.


INTRODUCTION

Neu differentiation factors (NDF) (^1)or heregulins, which stimulate autophosphorylation of mammary carcinoma cell lines expressing EGF receptor-like receptors, are soluble, secreted proteins that are processed from membrane-associated precursors, pro-NDFs(1, 2, 3, 4) . Pro-NDFs exist in multiple isoforms which exhibit significant sequence differences in the third disulfide loop of the EGF domain, the juxtamembrane region, and the C-terminal cytoplasmic tail(5) . These isoforms together with two other distinct molecules, glial cell growth factor (6) and acetylcholine receptor inducing activity (7) are now known to belong to a related gene family, termed as neuregulin(5) . The molecular diversity of neuregulin has raised interesting questions on aspects of structural and functional multiplicity of various isoforms. It is still unknown whether each molecular species within the family can display distinct biological actions in its membrane-bound or secreted form. Little is known about the contribution of the N-terminal Ig unit to the biological function of NDFs. There has also been no detailed analysis on structural characteristics of glycosylated NDF. Recent studies have shown that NDF or heregulins induce tyrosine phosphorylation of HER-4 (8) and that NDF isoforms bind to both HER-3 and HER-4 receptors, (9) suggesting that instead of HER-2, HER-3 and HER-4 may function as direct physiological receptors for neuregulin.

In the preceding paper(10) , we expressed various human and rat pro-NDF isoforms in CHO cells and isolated different secreted isoforms to apparent purity. Purified recombinant NDF-alpha and -beta isoforms can stimulate phosphorylation of mammary carcinoma cell lines and exist as highly glycosylated forms of similar molecular size. The soluble factors are apparently derived from specific proteolytic processing of pro-NDFs at both N and C termini, which leads to the secretion of only two types of isoforms, i.e. alpha and beta(10) . In this report, we investigate further the molecular, immunological, and biological properties of these isolated isoforms and compare some of the properties to the Escherichia coli-derived, nonglycosylated molecules. All full-length NDF molecules, either glycosylated or nonglycosylated, and alpha or beta isoforms, displayed similar structural folding and behaved as though they have a very extended, elongated shape. However, alpha and beta isoforms are immunologically and biologically distinct.


MATERIALS AND METHODS

Expression of NDF Molecules in E. coli and CHO Cells

Recombinant human and rat NDF molecules and the NDF EGF domains were produced in E. coli and purified according to previously described procedures(4) . Extra methionine residues were added at the N terminus of NDF as initiation sites for protein translation. To conserve structural differences near the juxtamembrane region among isoforms, the following human and rat NDF isoforms containing varying sequence lengths were prepared: human NDF-alpha2, human NDF-beta1, human NDF-beta2, rat NDF-alpha2, and rat NDF-beta4. Two EGF domains for human NDF-alpha2 and NDF-beta1 were also prepared using similar procedures. Unless stated, these molecules are described later using abbreviated names such as human NDF-alpha2, rat NDF-beta4, etc. Production of CHO cell-derived human and rat NDF isoforms and preparation of rat NDF from Rat1-EJ cells were described previously(1) . The protein concentration for all preparations were determined by absorbance at 280 nm and by quantitative amino acid analysis.

Analytical Gel Filtration

Gel filtration of NDF was performed in an FPLC Superdex 75 column (Pharmacia Biotech Inc.) pre-equilibrated with 10 mM phosphate buffer (pH 7.2) containing 0.1 to 1.0 M NaCl depending on experimental conditions. Samples were analyzed by an HP-1050 Ti system (Hewlett Packard) equipped with an automatic sampler, a multiple wavelength detector, and a ChemStation for data collection and processing. The experiments were performed at a flow rate of 1 ml/min with detection at 225 nm. The column was calibrated using proteins of known molecular weight.

Light Scattering

Molecular weights were also determined using on-line laser light scattering, refractive index, and absorbance detectors in series with a size-exclusion chromatography system (see above). As described previously(11, 12) , by combining the outputs of all three detectors, and using the extinction coefficients calculated for the polypeptide, it is possible to determine the molecular weight of the polypeptide component of a glycoprotein, since by this procedure the carbohydrate contributions to the scattering and refractive index signals are canceled.

Analytical Ultracentrifugation

Sedimentation equilibrium and sedimentation velocity experiments were carried out in a Beckman Optima XL-A ultracentrifuge with measurement at 280 nm. Equilibrium experiments were done at 25 °C and rotor speeds of 15,000 and 21,000 rpm with loading concentrations of 500, 250, and 125 µg/ml in phosphate-buffered saline (pH 7.1). Data for all three concentrations and 2 rotor speeds were simultaneously analyzed using non-linear least squares techniques similar to those in NONLIN(13) . Conversion of the measured buoyant molecular weight, M(b), to the true molecular weight requires a value for the partial specific volume, V, which cannot be estimated accurately for a glycoprotein of unknown carbohydrate content. Therefore, we use a self-consistent approach in which the total mass and the V calculated from its amino acid composition(14) , while the carbohydrate is assumed to have a V of 0.63 ± 0.01 ml/g(15) . It is then possible to uniquely solve for the total molecular weight from the measured M(b). The error in this calculated value is estimated by propagation of the error estimates for M(b) and the V values of polypeptide and carbohydrate.

Sedimentation velocity experiments were performed at 20 °C and 60,000 rpm and the sedimentation coefficient determined as described previously(16) . Conversion to standard conditions, calculation of Stoke's radii hydration, and axial ratios followed procedures described by Laue et al.(14) , using the V and molecular weight determined by sedimentation equilibrium, and assuming that the hydration of the carbohydrate is 0.43 g/g, as calculated for the polypeptide.

Circular Dichroism (CD) and Infrared Spectroscopic Analyses

CD measurements were obtained using a JASCO J-720 spectropolarimeter and cuvettes with a 0.02 cm (for far-UV CD) or 1 cm (for near-UV CD) path length. Thermal stability was determined using a Pelletier temperature control unit, a rectangular cuvette with a 0.1-cm path length, and a heating rate of 20 °C/h. Changes in ellipticity at 215 nm were measured every 0.5 °C, while spectra from 240 to 200 nm were determined every 2 °C. Protein samples were clarified through sterile filtration using a 0.2-µm membrane, and all analyses were carried out in Dulbecco's phosphate-buffered saline buffer (pH 7.1).

NDF was prepared for infrared spectroscopy by dialyzing against pure water, lyophilizing, and dissolving the lyophilized powder in a 20 mM sodium phosphate, 100 mM NaCl buffer prepared in D(2)O (Sigma, 99.9% isotopic purity), with pD = 7.0. Protein concentrations were approximately 1.5-2.0% (w/v) for the E. coli-derived and CHO cell-derived proteins, respectively. Solutions were placed in infrared cells with CaF(2) windows and 50-µg Teflon spacers. All spectra were collected using a Mattson Research Series FTIR spectrometer using a liquid nitrogen-cooled MCT detector. Four thousand scans (15 min collection time) were co-added for each spectrum; resolution was set at 4 cm. Analysis of infrared spectra was done as described previously(17, 18, 19) .

Production of Monoclonal Antibodies and Western Blot Analyses

To raise antibodies against E. coli-derived recombinant human NDF-alpha2 and NDF-beta1 EGF, the following procedures were followed. An NDF-alpha2 specific monoclonal antibody (mAb), 1H7A3, was produced by immunization of BALB/c mouse with human NDF-alpha2, while an NDF-beta specific mAb, 10-125A, was generated by immunization with NDF-beta1 EGF domain conjugated to Keyhole limpet hemocyanin (Calbiochem). After several booster injections, the spleen cells were fused with sp2/0 mouse myeloma according to the procedures described by Nowinski et al.(20) . Screening and cloning of the hybridomas were done by enzyme-linked immunosorbent assay. The specificities of mAb were confirmed by enzyme-linked immunosorbent assay and Western blot with all isoforms of NDF.

Immunodetection of NDFs in Western blot analysis was done with mAb at a concentration of 5 µg/ml followed by a 1:1000 dilution of peroxidase-labeled goat anti-mouse IgG. The blot was then developed and visualized according to ECL instruction (Amersham, United Kingdom). The real-time biospecific interaction analysis (BIA) was performed using a BlAcore instrument (Pharmacia Biosensor AB, Uppsala, Sweden) as described(20) . Biospecific affinity assays using BIAcore (Pharmacia) were employed to quantitate NDF that binds to specific antibodies as described(21) .

Binding of NDF Isoforms to T47D Human Mammary Carcinoma Cells

The ligand binding analyses were essentially identical to a previously described procedure(4) .

Transfection of NIH3T3 Cells with HER-3 and HER-4 and the Tritium Thymidine Uptake Assays

Construction of both HER-3 and HER-4 plasmids from the respective cDNA libraries of human breast carcinoma cell line SKBR-3 and fetal brain was performed using procedures as described(8, 9) . NIH3T3 cells were then transfected with a plasmid DNA (500 ng) carrying human HER-3 or HER-4 full-length cDNA by a standard protocol using the calcium phosphate method. Transfected cells were selected by growing in medium containing 0.5 mg/ml gentamicin (G) for 3 weeks. Cells resistant to gentamicin were collected and used for assays. Cell lines carrying HER-3 or HER-4 were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 0.5 mg/ml gentamicin.

HER-3 or HER-4 transfected cells were plated in 6-well plates with a cell density of 10^5 cells/plate. After 24 h, cells were starved in serum-free medium for 6 h and then changed to medium containing the indicated concentrations of human or rat NDF isoforms. After 12 h incubation, the medium was pulse-labeled by [^3H]thymidine (2 µCi/ml) for 2 h. The medium was then removed and the residual radioactivity washed by phosphate-buffered saline. Cells on the plate were harvested by cell scraper and counted for radioactivity.


RESULTS

Determination of NDF Molecular Sizes in Solution

When glycosylated and nonglycosylated NDF isoforms were analyzed by gel filtration using a 10 mM sodium phosphate buffer + 0.1 M NaCl eluant, they consistently exhibited abnormal elution profiles which include extremely low peak height, broadness of peaks, and peak tailing (data not shown). This anomalous behavior of NDFs in the column may be due to ionic interaction at lower ionic strength and not an aberrant conformation in 0.1 M NaCl, as this phenomena can be corrected by increasing the NaCl concentration (optimum at 0.5-1 M concentration). Under high salt conditions, each NDF isoform eluted as a symmetrical, sharp peak with increased peak height when detected at 225 nm. After calibrating with proteins of known molecular mass, the molecular mass for CHO cell-derived NDF forms calculated from elution positions ranged from 118,000 ± 4,000 to 114,000 ± 3,000 kDa (Table 1). These molecular sizes are significantly larger than those determined by SDS-polyacrylamide gel electrophoresis. Gel filtration analysis of E. coli-derived, nonglycosylated NDF isoforms gave molecular masses of 53,000-55,000 kDa which is also significantly larger than expected (approximately 25,000). These results suggest that NDFs may be dimeric in solution. However, in experiments using gel filtration in conjunction with light scattering detection, the polypeptide molecular weights of all human and rat NDF isoforms, nonglycosylated or glycosylated, were determined to be in the range between 21,000 and 26,000 (±3,000) (Table 1).



Recombinant CHO-derived human NDF-alpha1 and rat NDF-alpha2 were further analyzed by sedimentation equilibrium. The molecular weight determined by this technique is 39,100 ± 1000 (38.5 ± 0.5% carbohydrate) for rat NDF-alpha2 and 37,200 ± 900 (35.6 ± 0.5% carbohydrate) for the human NDF-alpha1, with most of the uncertainty arising from that in the partial specific volume of the carbohydrate. Both data obtained from light scattering and sedimentation equilibrium experiments appear to indicate that the NDF molecule is monomeric in solution.

With the true molecular weight of NDF known from light scattering and sedimentation equilibrium, we decided to confirm its molecular shape using sedimentation velocity. For CHO cell-derived human NDF-alpha1, the measured s(w) of 2.65 S implies a Stokes radius of 3.81 nm, which is 74% larger than expected for a spherical molecule of this molecular weight, and which would be consistent with a hydrated prolate ellipsoid with an axial ratio of 8. The 1.90 S sedimentation coefficient of recombinant human NDF-alpha2 produced in E. coli implies that it is also elongated, with an axial ratio similar to that of CHO cell-derived NDF.

CD and FTIR Analyses

Fig. 1A shows the near UV CD spectra of the CHO cell-derived NDF-alpha and -beta isoforms. The spectra of the NDF-alpha and -beta species are very similar, with fine structure in the 290 nm (Trp), 270-280 nm (Tyr), and 220-250 nm regions, indicating that these molecules display very similar structural folding in solution, and that the conserved Trp is in a similar environment in both NDF isoforms. A few differences are apparent, however. The human and rat NDF-beta species all have stronger and better defined signals in the 270-280 nm region, indicating that there are differences in the environment(s) of Tyr residue(s). The CHO cell-derived NDF-beta isoforms, regardless of species of origin, have one more tyrosine than the alpha-isoforms at the C-terminal tail. It therefore appears likely that this particular tyrosine is located in an asymmetric structural environment, and that its contribution to the spectral signal is stronger than that of the 5 tyrosines conserved in all NDF molecules.


Figure 1: Near UV and far UV CD analysis (A and B, respectively) of CHO cell-derived NDF isoforms. Spectra a-d, rat NDF-alpha2, human NDF-alpha1, rat NDF-beta2, and human NDF-beta1, respectively.



The far UV CD analysis of both CHO cell-derived human and rat alpha and beta isoforms is illustrated in Fig. 1B. Both NDF-alpha and -beta isoforms displayed a negative CD spectrum at 195 nm. All NDF species also appear to have a small positive peak in the 220-230-nm region. This structural characteristic appears in other proteins as well and is believed to arise from ring stacking of aromatic amino acids, probably Tyr, in beta-sheet containing proteins(22, 23) . The rest of the far UV CD spectra for all NDF isoforms are consistent with a protein containing beta-sheets and unordered structures.

CD spectra of E. coli-derived NDF isoforms and NDF-EGF domains were also measured and shown in Fig. 2, A and B. In the near UV CD region, these nonglycosylated isoforms also display structural features similar to glycosylated NDFs produced in CHO cells (Fig. 1A). The NDF-beta species (Fig. 2A, spectra c and d) also have stronger and better defined spectra at 270-280 nm, which again may be due to the two extra tyrosines at the C termini of both human NDF-beta1 and -beta2 isoforms. There is an obvious difference between the spectra of the NDF species derived from E. coli and the spectra of the NDF's derived from CHO cells in the region near 245-240 nm. Whether human or rat NDF, alpha or beta isoform, all CHO-derived NDF proteins have spectra which are characterized by an increase in ellipticity in this region, while the E. coli-derived molecules all have spectra which continue to be negative.


Figure 2: Near UV and far UV CD analysis (A and B, respectively) of E. coli-derived NDF isoforms and NDF EGF domains. Spectra a-d, human NDF-alpha2, rat NDF-beta2, human NDF-beta1, and rat NDF-beta4, respectively. Spectrum e, human NDF-beta1 EGF domain.



The far UV CD spectra of E. coli-derived human NDF-alpha and -beta isoforms and beta-NDF EGF domain are shown in Fig. 2B. A strong negative CD band near 193-196 nm found in CHO cell-derived NDFs (Fig. 1B) is also common in E. coli-derived NDF isoforms (Fig. 2B, spectra a-d). However, there is an obvious spectral difference around 220-235 nm. The CHO proteins all display broad but distinct CD spectra in this region, which are missing from the E. coli-derived proteins. Nonetheless, the far UV CD spectra of NDF isoforms as described above appear to indicate that these molecules are not typical alpha helical proteins. The spectra of rat NDF-alpha2 in 1 M NaCl, 10 mM phosphate buffer (pH 7.1) are identical to those obtained in phosphate-buffered saline, indicating that salt does not effect the conformation in solution.

The main feature of the spectra of human NDF-beta1 EGF domain consists of a trough from 320 to 250 nm (Fig. 2A, spectrum e), possibly arising from major contributions of the three disulfide bridges, with some fine structure from Tyr and Phe superimposed on it. This structural characteristic is thus very different from the spectra of the longer isoforms of glycosylated or nonglycosylated NDFs as studied above. Human NDF-beta1 EGF domain also displays significantly different far UV CD spectrum than the full-length NDF's. There is a positive and broad 220-235-nm CD band and a negative CD band around 197 nm (Fig. 2B, spectrum e). Human NDF-alpha EGF domain also displays CD spectra similar to those of the type-beta EGF domain (data not shown).

The conformational stability of several NDF species was studied using thermal stability as a probe. This was determined by following changes in the far UV CD region upon heating as indicators for loss of secondary structure. As listed in Table 2, NDF-beta EGF domain, like EGF itself, does not show a single cooperative transition upon melting and therefore its T(m) (the transition midpoint) could not be determined. The T(m) of various full-length NDF species were determined from changes in the ellipticity at 208 nm. E. coli-derived NDF isoforms have a T(m) between 44 and 50 °C. There appears to be a slight increase in T(m) (=52 and 53 °C) for CHO-derived NDF isoforms. The thermal denaturation of NDFs is irreversible, so a thorough thermodynamic analysis was not possible. Most of the beta-sheet was recovered after cooling, but the 230-nm feature was never fully regained. However, all of the full-length NDF forms melted with a single cooperative transition which ranged over 10 °C.



The second derivative infrared spectra in the amide I region (1700-1620 cm) of E. coli-derived NDF and CHO cell-derived NDF isoforms are shown in Fig. 3. These spectra are related to the polypeptide backbone conformation (24, 25) and are essentially identical for both alpha and beta isoforms, as well as for both nonglycosylated and glycosylated isoforms (spectra a-d). Human NDF-alpha2 EGF domain also displays a spectrum similar to the full-length NDF molecules (spectrum e). These spectra contain a strong band at 1631-1634 cm and a clear 1673-1678 cm band. Curve fitting of the deconvoluted infrared spectra was used to provide quantitative estimates of secondary structures, and the resulting band assignments for various isoforms are listed in Table 3. Band assignments are based on previous literature reports(24, 25, 26) . Thus, by infrared studies, it is estimated that the conformations of the recombinant NDFs are essentially identical and consist of no alpha-helix, 42-52% beta-sheet, a moderate amount of reverse turns, and the remaining disordered structures.


Figure 3: FTIR analysis of NDF isoforms. Spectra a-e, E. coli-derived human NDF-alpha2, CHO cell-derived rat NDF-alpha2, E. coli-derived human NDF-beta1, CHO cell-derived human NDF-beta1, and E. coli-derived human NDF-alpha2 EGF domain.





Immunological and Functional Analysis of Bacteria and CHO Cell-derived NDF Isoforms

Two specific monoclonal antibodies against recombinant human NDF-alpha2 (clone 1H7A3) and human NDF-beta1 (clone 10-125A) were prepared and used to identify different NDF isoforms. The epitope region in NDF for clone 1H7A3 resides at the C-terminal disulfide loop in the EGF domain of human NDF-alpha2, while the epitope for the 10-125A clone produced against the NDF-beta1 EGF domain also locates at the same region within the beta isoform. (^2)The recognition of mammalian cell-derived human and rat NDF isoforms was investigated by Western blot analysis using these two antibodies. As shown in Fig. 4A, at 50 ng of NDF loading on the gel, clone 1H7A3 can only recognize the 25-kDa E. coli-derived NDF-alpha2 (lane 1) and the 6.6-kDa NDF-alpha2 (an EGF domain) (lane 2), as well as the 44-kDa CHO cell-derived human NDF-alpha1 and rat NDF-alpha2 (lanes 5 and 6). However, all of the beta isoforms, either from bacterial or mammalian origin, full-length or EGF domain, are not recognized by the NDF-alpha-specific monoclonal antibody (Fig. 4A, lanes 3, 4, 8, and 9). Likewise, clone 10-125A can strongly recognize all E. coli- and CHO cell-derived NDF-beta isoforms (25- and 44-kDa bands, respectively) and the 6.5-kDa beta-EGF domain (data not shown), but did not bind to all human and rat alpha isoforms (Fig. 4B).


Figure 4: Western blot analysis of NDF isoforms by specific monoclonal antibodies against E. coli-derived human NDF-alpha2 (A) and human NDF-beta1 EGF domain (B). Unless mentioned, each sample containing 50 ng of NDF was loaded onto the gels. A, lanes 1-4: E. coli-derived human NDF-alpha2, human NDF-alpha2 EGF domain, human NDF-beta1, and human NDF-beta1 EGF domain, respectively. Lanes 5-6 and 8-9: CHO cell-derived human NDF-alpha1, rat NDF-alpha2, human NDF-beta1, and rat NDF-beta4, respectively. Lane 7: Rat1-EJ cell-derived NDF. B, Lane 1: low molecular weight standards; lanes 2-3: E. coli derived human NDF-beta1 and NDF-alpha2, respectively; lanes 4-7: CHO cell-derived human NDF-alpha1, human NDF-alpha2, human NDF-beta1, and rat NDF-beta4, respectively; and lanes 8-10: Rat1-EJ cell-derived NDF at 50, 100, and 200 ng loading, respectively.



Western blot analysis of Rat 1-EJ cell-derived NDF with the monoclonal antibodies was also studied. Both 1H7A3 and 10-125A antibodies can recognize this natural rat NDF preparation; however, stronger binding was found with mAb 1H7A3 than with 10-125A at the same sample loading (Fig. 4A, lane 7, and B, lane 8). Clone 10-125A can also clearly recognize Rat1-EJ cell-derived NDF at higher NDF sample loading (Fig. 5B, lanes 9 and 10). Based upon this qualitative comparison of the intensity of blotted bands, we conclude that natural rat NDF contains both alpha and beta isoforms in an approximately 3:2 ratio. When the presence of alpha and beta isoforms was quantified by the bioaffinity technique (21) using these monoclonal antibodies, a 38-43% beta isoform was estimated in the natural rat NDF preparation with sample loading at concentrations of 100-250 ng/ml.


Figure 5: Binding of NDF isoforms to T47D human mammary carcinoma cells. The ability of various NDF proteins to displace radiolabeled NDF-beta1 was analyzed on monolayers of T47D human breast cancer cells. Binding reactions were carried out with 5 ng of I-NDF-beta1 per ml for 2 h at 4 °C. This was followed by extensive washing of the cell monolayers and determination of bound radioactivity. The amount of bound NDF-beta1 is expressed relative to ligand binding in the absence of competitor unlabeled protein. A, the following unlabeled proteins were used to displace cell-bound human NDFbeta1; bacterially made human NDF-beta1 (), and two rat NDF isoforms, alpha2 (bullet) and beta4 (), expressed in CHO cells. B, the following human NDF isoforms expressed in CHO cells were used as unlabeled competitors to bacterially made human NDF-beta1 (): alpha1 (circle), alpha2 (bullet), beta1 (box), and beta2 (). Averages of duplicate determinations and the corresponding range (bars) are shown. Each experiment was repeated three times.



To quantitatively compare receptor binding characteristics of different NDF isoforms, the purified NDF preparations were analyzed for their binding to T47D human mammary carcinoma cells. Ligand displacement analysis was performed with radiolabeled human NDF-beta1. The latter differs from the corresponding rat protein in only 1 amino acid. This EGF-like domain displayed an apparent dissociation constant of 200-400 pM (Fig. 5A). Comparative ligand displacement analysis confirmed that CHO cell-derived rat NDF-alpha2, as well as human NDF-alpha1 and -alpha2, have lower binding affinities by a factor of 8-10 (Fig. 5, A and B). Recombinant CHO rat NDF-beta4 displayed ligand displacement activity that is three times higher than rat NDF-alpha2 (Fig. 5A), but is less active than human NDF-beta1 or -beta2 (Fig. 5B). Human NDF-beta1 or -beta2 can best compete with labeled human NDF-beta1 EGF domain in ligand displacement analysis.

We also performed tritiated thymidine uptake experiments to examine NDF-dependent stimulation of DNA synthesis in NIH3T3 cells transfected with plasmids carrying human HER-3 or HER-4 receptor genes. Expression of HER-3 or HER-4 receptor proteins was examined by immunoprecipitation using specific antibodies against either receptor molecule (data not shown). As shown in Fig. 6A, CHO cell-derived NDF-beta isoforms (curve 3: rat NDF-beta4; curve 4, human NDF-beta1) can induce more than a 16-fold stimulation of DNA synthesis in cells transfected with HER-3 receptor in a dose-dependent manner. Rat NDF prepared from medium conditioned by Rat1-EJ cells also exhibited a similar stimulation profile (Fig. 6A). The 50% effective concentration for stimulation of DNA synthesis by these samples was approximately 3-4 ng/ml or 70-100 pM. In contrast, the CHO cell-derived NDF-alpha isoforms appeared to have little stimulatory effect on cells expressing HER-3 when compared to the stimulation on cells without transfection of receptor.


Figure 6: Effect of NDF on stimulation of thymidine uptake in HER-3 or HER-4 transfected NIH3T3. A, HER-3 transfected cells: CHO cell-derived human NDF-alpha1 (bullet), rat NDF-alpha2 (), rat NDF-beta4 (circle), human NDF-beta1 (box), and rat1-EJ cell-derived NDF (Delta). B, HER-4 transfected cells: CHO cell-derived human NDF-alpha1 (), rat NDF-alpha2 (bullet), human NDF-beta1 (box), rat NDF-beta4 (Delta), rat1-EJ cell-derived NDF (circle), and control with no addition of NDF (). C, effect of E. coli-derived NDFs at 2 µg/ml concentration.



The stimulatory effect on cells transfected with HER-4 receptor is also shown in Fig. 6B. Both CHO cell-derived NDF-alpha and -beta isoforms exhibit very strong stimulation of thymidine uptake by transfected NIH3T3 cells, ranging from approximately 8-16-fold stimulation (Fig. 6B), as compared to the control with no addition of NDF. The concentration required for half-maximal stimulation is approximately 15-100 pM for different isoform preparations. NDF-alpha isoforms exhibited weaker stimulation effect than the beta isoforms. Rat1-EJ cell-derived NDF also displays strong stimulation.

Similar experiments were also performed by using E. coli-derived NDF isoforms including full-length human and rat NDFs, as well as NDF EGF domains. Consistent with CHO cell-derived beta isoforms, all human and rat NDF-beta isoforms and EGF domain isoform exhibited strong stimulation on cells transfected with HER-3 or HER-4, while alpha isoforms only display weaker stimulation on HER-4 transfected cells (Fig. 6C).


DISCUSSION

By gel filtration analysis, the determined molecular weight of NDFs appears to be greater than 100,000 in all CHO cell-derived NDF isoforms and is also greater than 50,000 in E. coli-derived NDF isoforms. However, the molecular weights of NDFs determined by gel filtration in conjunction with light scattering are consistent with a monomeric NDF molecule predicted from their primary sequences (Table 1). These data suggest that NDFs are monomeric, but have an atypical shape in solution.

The molecular properties of NDFs were further confirmed by sedimentation velocity experiments in which the measured s(w) is consistent with a hydrated prolate ellipsoid with an axial ratio of 8, i.e. a rod-like shape. Most of this asymmetry appears to be due to the polypeptide rather than the carbohydrate because a similar axial ratio is found for nonglycosylated NDF molecules derived from E. coli. Other experiments by sedimentation equilibrium indicated that glycosylated NDF isoforms have a carbohydrate content of 35-39%. The unique rod-shaped structure deduced from physical analysis is consistent with the proposed NDF domain structures (4, 10) where the Ig-like homology unit and the glycosylated spacer region may be very extended and separated away from EGF domain.

Since multiple pro-NDF transcripts do exist in Rat1-EJ cells, it is reasonable to predict that rat1-EJ cells may express different isoforms (14) . However, we were unable to obtain enough quantities of Rat1-EJ cell-derived NDF for comparative C-terminal determination and for verification of isoforms. In this report, Western blot analyses using specific monoclonal antibodies against human NDF-alpha and -beta isoforms confirm that the natural rat NDF preparation contains a mixture of both isoforms at a ratio of 3 to 2. When the amino acid composition of rat 1-EJ NDF was used to fit the amino acid composition data for the mixture (see the preceding paper, (10) ), there is sufficient agreement in the data to suggest that Rat1-EJ NDF may share identical C-terminal processing sites with CHO cell-derived rat NDF-alpha2 and -beta4. Other evidence to support a similar processing is that the Rat1-EJ NDF shows a similar 40-44-kDa molecular size upon SDS-polyacrylamide gel electrophoresis.

Glycosylated and nonglycosylated NDF-alpha and -beta isoforms share similar secondary structures which are rich in beta-sheet structure with no detectable helical structure. We believe that the beta-sheet structures may spread over the whole molecule including the Ig domain, spacer region, and EGF domain in order to account for the high proportion of beta structure (see Table 3). The EGF domain alone, either alpha or beta isoform, also contains approximately 50% beta-sheet structure, which is consistent with that observed for the EGF and TGF-alpha molecules(27, 28) . This observation is also consistent with the solution structure of heregulin-alpha EGF domain (29) and NDF-beta1 EGF domain (^3)elucidated by two-dimensional NMR analysis.

Secondary structure prediction (30) suggests that Ig and EGF domains in NDF-alpha or -beta isoform contain moderate amounts of beta-sheet structures. The observation of beta-sheet structures in the Ig domain suggests that the structure of the Ig domain in NDF is homologous to Ig domains found in a number of proteins, including CD2 and CD8(31) . The Ig unit of NDF belongs to the C2 set of immunoglobulin homology units (2) which is shared by the non-immunological members of this gene superfamily. Included in this family are membrane receptors for antibodies, receptors for growth factors (e.g. platelet-derived growth factor), and lymphokines (e.g. interleukin-1) and cell adhesion molecules (e.g. neural cell adhesion molecule)(32) . It is clear that the Ig unit in NDF does not directly participate in receptor recognition. Instead, its structural folding implies that its functional role may be similar to that attributed to other immunoglobulin units, namely, stabilizing homophilic protein-protein interaction(2) .

Glycosylated human and rat NDF-alpha and -beta isoforms also share similar basic tertiary structural folding. However, the human and rat NDF-beta isoforms contain slightly stronger and more defined near UV CD bands (Fig. 1A). This phenomenon may be attributed to the Tyr residue that occurs at the C terminus in the NDF-beta isoforms that is absent in the NDF-alpha isoforms. A similar observation was also found for the nonglycosylated NDF-beta isoforms which contain two extra tyrosines (Fig. 2A). The tertiary structural folding of the NDF EGF domain is apparently distinct from the other longer NDF isoforms (Fig. 2A). The full-length NDF molecule contains three different domain structures; two of them (Ig and spacer domains) have been removed from the NDF EGF domain.

Despite their structural similarity, it is clear that biological functions of NDF-alpha and -beta isoforms are distinct. In binding competition experiments, glycosylated NDF-alpha isoform showed much weaker competition with NDF-beta EGF domain than the glycosylated beta isoform. The data also suggests that NDF-beta isoforms have higher affinity to the receptor on the cell surface.

The higher affinity of NDF-beta isoforms may indicate that the beta isoform can better stimulate DNA synthesis or cell growth. We have used HER-3 and HER-4-transfected NIH3T3 cells to evaluate the stimulatory effects by NDF-alpha and NDF-beta isoforms, and EGF domains. NDF-alpha isoforms, glycosylated or nonglycosylated or the EGF domain only, did not exert significant stimulation of DNA synthesis of HER-3-transfected cells but could stimulate a moderate amount of mitogenic effect on HER-4-transfected cells. In contrast, all NDF-beta isoforms and the NDF-beta EGF domain exhibited strong stimulation of DNA synthesis of both HER-3 and HER-4 transfected cells. Taken together, these data suggest that NDF-alpha and NDF-beta seem to have different biological functions, which must be related to the unique sequence (i.e. structural) difference in the last disulfide loop in the EGF domain and the C-terminal tail (6 amino acids in length). Earlier studies have shown that the solution structures of EGF and transforming growth factor-alpha analyzed by NMR seems to be homologous and rich in beta-sheets, beta-turns, and loops(9) . These structures are shared by other known sequences that bind the EGF receptor. Significantly, the alpha and beta isoforms of NDF EGF domains have a main chain structural fold similar to the EGF molecule (see above and (27) ), yet do not bind to the EGF receptor. These data imply that the receptor binding domain of NDF molecules resides at the C-terminal region of the EGF domain and is distinct from the EGF molecule in overall structural folding. NMR analysis has revealed distinct structural features of heregulin-alpha (29) and NDFbeta(^4) on the molecular surface for possible receptor recognition. Future mutational analysis, synthesis of chimeric molecules, and further NMR studies should prove useful in unraveling the molecular basis of NDF receptor binding specificity.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Amgen Inc., Amgen Center, 1840 DeHavilland Dr., Thousand Oaks, CA 91320-1789. Tel.: 805-447-3092; Fax: 805-499-7464.

(^1)
The abbreviations used are: NDF, neu differentiation factor; EGF, epidermal growth factor; TM, transmembrane; CHO, Chinese hamster ovary; mAb, monoclonal antibody; HER-2, human EGF-like receptor, type 2; FTIR, Fourier transform infrared spectroscopy; Ig, immunoglobulin.

(^2)
H. S. Lu, D. Chang, J. S. Philo, K. Zhang, L. O. Narhi, N. Liu, M. Zhang, J. Sun, J. Wen, D. Yanagihara, D. Karunagaran, Y. Yarden, and B. Ratzkin, our unpublished results.

(^3)
J. Cheetham, personal communication.

(^4)
J. Cheetham, unpublished data.


ACKNOWLEDGEMENTS

We are indebted to Steve Prestrelski, Michael D. Jones, and Lisa W.-I. Wong for their technical help, to Joan Bennett for her help in typing the manuscript, and to Dr. Duanzhi Wen for helpful comments.


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