©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Post-translational Processing of Membrane-associated neu Differentiation Factor Proisoforms Expressed in Mammalian Cells (*)

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

Hsieng S. Lu (§) Shinichi Hara Lisa W.-I. Wong Michael D. Jones Viswanatham Katta Geri Trail Aihua Zou David Brankow Sean Cole Sylvia Hu Duanzhi Wen

From the From Amgen Inc., Amgen Center, Thousand Oaks, California 91320

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Expression vectors constructed from human and rat pro-neu differentiation factor (NDF) cDNAs were transfected in Chinese hamster ovary cells for expression of recombinant NDF molecules. Soluble NDF forms were released into culture medium after post-translational processing of the membrane-bound pro-NDF forms. Different human and rat NDF isoforms, after being purified from the culture medium, were subjected to structural and biochemical characterizations. The isolated human and rat NDF isoforms have been proteolytically processed at a specific site at the N terminus, which is different from that observed for the processing of rat or human NDF molecule prepared from natural origins. The processing of each recombinant NDF isoform at its C terminus was heterogeneous but consistently occurred at nearby peptide bonds. Specific N- and C-terminal processing by Chinese hamster ovary cells has resulted in the production of two types (alpha and beta) of recombinant NDFs containing 222-225 amino acid residues. Both human and rat NDF molecules are heavily glycosylated at two of the three potential Asn-linked glycosylation sites and contain O-linked sugars at 11 of the Thr/Ser sites. Glycosylation occurs at a short, Ser/Thr-rich spacer region that connects the N-terminal immunoglobulin homology unit to the epidermal growth factor domain. Cellular phosphorylation assay indicated that these secreted forms contain similar biological activity in receptor tyrosine autophosphorylation of mammary tumor cells.


INTRODUCTION

The neu proto-oncogene (also known as HER-2 or c-erbB-2) encodes a 185-kDa transmembrane receptor tyrosine kinase (p185). The protein products are found in many epithelial and neuronal tissues(1, 2, 3, 4, 5, 6, 7, 8) . The levels of p185expression are frequently elevated in certain human neoplasm. Overexpression of HER-2 occurs in approximately 20% of various carcinomas (9) and is associated with poor prognosis for breast and ovarian cancers(10) . HER-2 is highly homologous to, but distinct from, epidermal growth factor receptor(11, 12) . Numerous studies have demonstrated that p185 glycoprotein can be activated and phosphorylated on tyrosine residues by isolated proteins obtained from various sources(13, 14, 15, 16, 17, 18) .

Purification of rat or human protein that activates p185 led to the isolation of cDNAs encoding EGF(^1)-related molecules, termed neu differentiation factors (NDF) (19) or heregulins (17) . Elucidation of cloned cDNA sequences predicted the existence of pro-NDF mRNAs encoding multiple transmembrane glycoprotein precursors, with an extracellular domain containing an EGF-like domain and an immunoglobulin homology unit, and a cytoplasmic domain(19, 20) . Identification of distinct rat and human pro-NDF genes expands the NDF/heregulin family(21) , referred to as neuregulin family(20) , which now includes glial cell growth factor (22) and chicken acetylcholine receptor inducing activity(23) .

The major structural difference among pro-NDF isoforms resides at the C terminus of the extracellular domain that includes the last disulfide loop of EGF domain and the juxtamembrane region, and at the C-terminal cytoplasmic tail(17, 19, 20, 21) . Some structural and functional aspects of the multiplicity of NDF genes have recently been investigated, which compared bacterially derived recombinant rat NDF soluble isoforms(24) . These isoforms were prepared in different polypeptide lengths including juxtamembrane sequences that diverge in each isoform. Since the natural NDF molecules can only be obtained in minute quantities(16) , it has been impossible to study the processing of pro-NDF isoforms and the structural complexity of naturally occurring soluble NDF forms in relation to their possible functional multiplicity. To overcome this, we started to study the expression of NDF in CHO cells which may process and secrete NDF isoforms similar to those from natural sources. In this report, we express several human and rat NDF isoforms in engineered CHO cells containing respective pro-NDF clones and isolate the secreted recombinant molecules to apparent purity for biological and structural characterizations. The secreted, biologically active NDF isoforms appear to be heavily glycosylated and processed at both N- and C-terminal ends. Multiple N- and O-linked glycosylations consistently take place at the NDF spacer domain. The glycosylated NDF forms can stimulate receptor autophosphorylation with similar potency. In a subsequent paper, molecular behavior and biological characteristics of these NDF glycoforms are compared(25) .


EXPERIMENTAL PROCEDURES

Materials

Full-length rat and human NDFs (including Ig, spacer, and EGF domains) and NDF-EGF forms (including only EGF domain) derived from Escherichia coli were produced as described in previous papers(21, 24) . NDF isolated from Rat1-EJ cells was prepared according to a previous report(16) . Heparin-Sepharose, DEAE-Sepharose (fast-flow), and phenyl-Sepharose gel media used for purification are products of Pharmacia Biotech Inc. All of the HPLC solvents are from Burdick and Jackson. Sequencing reagents and solvents were from Applied Biosystems, Inc. (Foster City, CA) or Hewlett Packard (Mountain View, CA). Proteases (endoproteinases Glu-C and Lys-C, and trypsin) were products of Boehringer Mannheim.

Rat and Human Pro-NDF Clones

A number of rat and human pro-NDF cDNA clones (i.e. NDF-alpha1, alpha2, alpha3, beta1, beta2, beta3, and beta4, etc.) have been isolated in our laboratory(19, 21) . Several human NDF cDNA clones were also isolated and characterized by Holmes et al.(17) . As shown in Fig. 1, pro-NDF clones, i.e. human NDF-alpha1, -alpha2, -beta1, and -beta2, rat NDF-alpha2 and -beta4, were selected for mammalian expression. The coding sequences of alpha and beta forms (both human and rat species) differ in the C-terminal EGF domain which extends to the membrane and the cytoplasmic domains(21) . Their sequence divergence starts from the last disulfide loop of the EGF domain as indicated in Fig. 1.


Figure 1: Predicted partial amino acid sequences (in single letter code) of several human and rat pro-NDF isoforms at the N-terminal and juxtamembrane regions. A, N-terminal region: large arrows illustrate where the human and rat pro-NDFs are processed in CHO cells and small arrows are the assigned cleavage sites for natural human heregulin (17) and natural rat NDF(19) . B, juxtamembrane region: large sequence variations among different pro-NDF forms are indicated from the last disulfide loop of the EGF domain in which the boldface letters designate homology. The transmembrane domain is underlined and several pro-NDF forms have longer sequences in this region where the dashed lines are used for maximal sequence alignment. CHO cells process pro-NDF isoforms at several major (large arrows) and minor (small arrows) C-terminal processing sites.



Expression of Recombinant NDF in Mammalian Cells

Construction of Expression Plasmids

Similar approaches were used to construct various pro-NDF expression plasmids for rat pro-NDF-alpha2 and beta4 and human pro-NDF-alpha2. The following is an example for construction of rat pro-NDF-alpha2 expression vector. Rat pro-NDF-alpha2 cDNA encoding 632 amino acids was subjected to polymerase chain reaction using two primers. The 37-base oligonucleotide sense primer has the sequence: 5`-CGG TCT AGA AGC TTC CAC CAT GTC TGA GCG CAA AGA A-3`. It included 5` XbaI and HindIII restriction cloning sites followed by the Kozak consensus sequences CCACC, as well as the initial 18 bases of the rat NDF coding sequences. The 30-base oligonucleotide antisense primer had the sequence: 5`-GCC GTC GAC CTA TTA CCA TTC GCT ATG AGG-3`. It included a SalI site, two tandem translation stop codons, and 15 bases of the carboxyl end of the rat NDF coding sequences. The polymerase chain reaction product was digested with XbaI and SalI to generate a 1.4-kilobase DNA fragment containing the entire coding sequence of rat pro-NDF-alpha2. This fragment was then subcloned into the expression vector pDSRalpha2 (see European patent application A20398753) that had been cut with XbaI and SalI. The sequence of the entire insert was verified through DNA sequencing. The final plasmid was designated at pDSRalpha2/rNDF-alpha2.

Chimeric Human-Rat NDF-alpha1, NDF-beta1, and NDF-beta2 Expression Vectors

Chimeric pro-NDF molecules were designed in which the 197-amino acid cytoplasmic domain of the human NDF-alpha1, NDF-beta1, or NDF-beta2 (coding for NDF of 542, 645 and 637 amino acids, respectively) was substituted with the 157-amino acid cytoplasmic domain of the rat NDF-alpha2.

Production of Human and Rat NDFs in CHO Cells

A Chinese hamster ovary cell line (CHO D) deficient in dihydrofolate reductase was transfected with the above mentioned plasmid DNA by calcium phosphate precipitation(21) . The transfected clones that express the highest NDF level were selected for generating conditioned media as described(21) .

Isolation of Recombinant Rat and Human NDF Molecules

Concentration

Unless mentioned, all of the isolation steps described below were performed in the cold room at 5 °C. Pooled and filtered serum-free conditioned medium (20-100 liters) derived from engineered CHO cells was concentrated to 1-2 liters in phosphate-buffered saline (PBS; pH 7.0) with a Pellicon diafiltration system (Millipore, MA), using a membrane with a 10-kDa molecular size cutoff. Total NDF content in the concentrated retente was determined by Western blot analysis (21) and tyrosine kinase stimulatory activity. The total protein concentration was estimated by UV absorption at 280 nm or by a Coomassie Brilliant Blue binding assay supplied by the vendor (Bio-Rad).

Column Chromatography

Three chromatographic steps, i.e. heparin-Sepharose, DEAE-Sepharose, and phenyl-Sepharose chromatographies, were applied to isolate various NDF isoforms. The clarified and concentrated material (1-2 liters) as described above was loaded on a 80-ml heparin-Sepharose column (1.5 times 15 cm), pre-equilibrated with PBS buffer (pH 7.2), at a flow rate of 0.3 liter/h. Conditions for washing the column and eluting NDF from the column were described previously(16) . NDF-containing fractions were pooled and dialyzed against PBS buffer.

Dialyzed material was then loaded onto a DEAE-Sepharose 6B (fast flow) column (1.2 times 20 cm) equilibrated with the same PBS buffer as described. After washing the column with PBS buffer, it was then developed with a 300-ml gradient of 0.02 M to 0.5 M NaCl in PBS (pH 7.2) using a flow rate of 50 ml/h. Eluates were monitored at 280 nm; and fractions containing NDF activity or exhibiting NDF protein band were pooled for subsequent purification.

Ammonium sulfate was added to the pooled NDF fraction to achieve a concentration of 1.5 M. The material was immediately loaded on a phenyl-Sepharose 6B column (1.2 times 20 cm) pre-equilibrated with the initial buffer which contains PBS buffer and 1.5 M ammonium sulfate. After loading, the column was washed with the initial buffer to remove any unbound non-NDF proteins. The NDF protein was eluted with a 300-ml gradient of 1.5 to 0 M ammonium sulfate in PBS buffer (pH 7.2) at a flow rate of 1-1.5 ml/min. Fractions in 2-ml volume were collected, and protein peaks were measured on-line at 280 nm. Fractions containing a 40-44-kDa protein band were pooled, dialyzed against PBS buffer, concentrated, and then sterile-filtered by a 0.2-µm membrane and sample aliquots were transferred to vials and stored at -80 °C. Overall NDF yield was obtained from biospecific affinity assays as described(26) .

SDS-PAGE and Assays for Kinase Stimulatory Activity

SDS-PAGE of column fractions under reducing conditions was performed using 14% Novex Tricine precast gels. Electrophoresis was run using Novex Tricine buffer containing 0.1% SDS and a fixed voltage at 100 V for 1.5-2 h. The final purified NDF preparations were run on 14% gels using a Laemmli system as described previously(16) .

Assays for tyrosine phosphorylation activity were performed in MDA-MB453 human cancer cells according to previously described procedures(16) . Phosphorylation of membrane-associated receptor was monitored by phosphotyrosine antibody coupled with chemiluminescence detection after SDS-PAGE and electroblotting of the isolated membrane fractions.

Amino Acid Analysis and Sequence Determination

Acid hydrolysis of purified NDF samples (1-3 nmol) using 6 N HCl was performed as described previously(27) . The hydrolysates were dried, reconstituted, and injected into the Beckman 6300 amino acid analyzer for compositional analysis. Analysis of Rat1-EJ cell-derived NDF was performed with an automated PTC-derivative analyzer Model 420 (Applied Biosystems) with procedures according to the manufacturer's recommendation.

Sequential Edman degradations (28) were performed either with ABI Model 477 and 473 sequencers (Applied Biosystems, Foster City, CA) or with HP1000 sequencers (Hewlett Packard) using sequencing programs recommended by the manufacturers.

Endoproteinase Lys-C Digestion of NDFs

CHO cell-derived rat NDF-alpha2 and -beta4, as well as human NDF-alpha1 and -beta2 (200-300 µg) were dried by Speed Vac and reconstituted in 300 µl of 0.3 M Tris-HCl containing 6 M guanidine HCl (pH 8.4). Samples were then reduced with dithiothreitol and alkylated with iodoacetate as described(29) . Alkylated NDF samples at 0.5 mg/ml in 0.1 M Tris-HCl (pH 7.2) containing 2 M urea were digested by endoproteinase Lys-C at 37 °C for 18-24 h at an enzyme-to-substrate ratio of 1:100. The peptide mixture was separated by reverse-phase HPLC and monitored at 215 nm using a Vydac C4 column (4.6-mm inner diameter times 25 cm, 300 Å) and an HP 1090 liquid chromatography system equipped with a diode-array detector and a workstation. The column was equilibrated with mobile phase A (0.1% trifluoroacetic acid). The peptides were eluted with a linear gradient from 3 to 35% mobile phase B (90% acetonitrile in 0.1% trifluoroacetic acid) for 70 min, then to 50% B for 10 min and washed with 95% B. HPLC analysis was conducted at ambient temperature with a flow rate of 0.7 ml/min. The peptide peaks were collected manually and dried immediately after collection. Dried samples were then reconstituted in 0.1% trifluoroacetic acid prior to further analysis.

Analysis of Isolated Glycopeptides after Endoproteinase Glu-C and Trypsin Digestion

Glycopeptide fractions obtained from previously mentioned peptide mapping were pooled, dried, and reconstituted in 0.1 M ammonium bicarbonate buffer (pH 7.9). The reconstituted sample was digested with endoproteinase Glu-C at 25 °C for 24 h at an enzyme-to-substrate ratio of 1:10. The digest mixture was further incubated with trypsin at 37 °C for 4 h at an enzyme-to-substrate rate of 1:5. The resulting peptide mixture was separated by reverse-phase HPLC as described above. The elution was performed with a linear gradient from 3 to 15% B for 20 min, to 25% B for 40 min, and then to 50% B for 40 min, followed by a wash with 95% B with the flow rate of 0.5 ml/min at 25 °C.

Mass Spectrometry

The molecular mass of the purified peptides was determined by mass spectrometry using a Sciex APIII electrospray mass spectrometer as described (29) or a Krato MALDI matrix-assisted laser desorption mass spectrometer. Samples analyzed by laser desorption mass spectrometry were first spotted on a metal probe surface in a 0.5-µl volume and dried. After the probe surface was further coated with 0.5 µl of sinapic acid or 4-cyanohydroxyl sinapic acid at 5-10 mg/ml concentration and dried to completeness, samples were ready for laser desorption mass spectrometry analysis.


RESULTS

Expression and Isolation of Rat and Human NDFs

We have constructed various expression plasmids containing rat, human, and human-rat chimeric pro-NDF DNAs. These plasmids were prepared for transfection in CHO cells to express different human and rat NDF forms. Stable clones expressing higher levels of NDFs were subcloned and selected for large scale production and for subsequent isolation of biologically active NDFs. For example, secretion of rat NDFs ranges from approximately 300 to 700 ng/ml of culture medium. Human NDFs were expressed at much lower levels (<50 ng/ml) when full-length human pro-NDF (such as human NDF-alpha2) genes were constructed into the expression vector. However, we found that levels of NDF mRNA and the respective membrane-bound pro-NDF protein expressed by the human NDF clones are comparable to those found in the rat pro-NDF clones. Therefore, it is likely that inefficient processing of membrane-bound pro-NDFs may be partly related to the low expression of human NDFs. We also found that expression of soluble human NDFs can be increased to the level of rat NDF forms using human-rat chimeric pro-NDF constructs (i.e. human NDF-alpha1, -beta1, and -beta2) where the cytoplasmic domain of each human NDF isoform was replaced by the same domain for rat NDF-alpha2. These observations suggest that cytoplasmic tails of human pro-NDF genes may be responsible for the low secretion levels of human NDF by CHO cells, possibly due to the effect of the isoforms-specific rate of pro-NDF processing(21) .

Table 1summarizes the isolation procedure and yield of NDF obtained from each isolation step using rat NDF-alpha2 as an example. Fig. 2A shows the three chromatographic separation steps used in the isolation of rat NDF-alpha2. Following heparin, DEAE, and phenyl-Sepharose chromatographies, recombinant NDF can be prepared to homogeneity with an overall yield of 36.5% (Table 1). Fig. 2B (panels 1-3) illustrates typical SDS-PAGE profiles of the eluting fractions in reducing conditions (using Tricine gels as described) for the detection of 40-44-kDa NDF bands for samples pooled at different purification steps.




Figure 2: A, chromatographic separation of recombinant rat NDF-alpha2. Panels 1-3, heparin-Sepharose, DEAE-Sepharose, and phenyl-Sepharose chromatographies, respectively. B, SDS-PAGE (Tricine gels) of column fractions containing rat NDF-alpha2. Panel 1, fractions (25 µl each) from heparin-Sepharose chromatography. Panel 2, fractions (25 µl each) from DEAE chromatography in A. Panel 3, NDF samples (25-40 µl each) pooled at each separation step. Conc, concentrate; FT, unbound fraction from heparin column; Hep., NDF pool from heparin column; DE, NDF pool from DEAE column; and Phe, NDF preparation from phenyl-Sepharose column.



A similar process for recombinant rat NDF-alpha2 purification has been successfully used in the purification of other recombinant NDFs including rat NDF-beta4, and human NDF-alpha1, -alpha2, -beta1, and -beta2 isoforms.

Purity and Molecular Weight of NDF Isoforms

Fig. 3(top panel) shows the SDS-PAGE of the final purified recombinant preparations including rat NDF-alpha2 and -beta4 (lanes 2 and 3), as well as human NDF-alpha1, -beta1, and -beta2 (lanes 4, 6, and 7, respectively) after Coomassie Blue staining (using Laemmli gels as described). Human NDF-alpha2 expressed by a human pro-NDF clone also exhibited a similar molecular size (lane 5). Every NDF isoform together with Rat1-EJ cell-derived NDF (lane 8) exhibits a single diffused band at 40-44 kDa with indistinguishable electrophoretic mobility from each other. The diffused bands shown in all samples are indicative of being glycosylated in all NDF preparations. As a comparison, bacterially derived human NDF-alpha exhibited a sharp band with an estimated molecular mass (30 kDa) slightly larger than the expected value (25 kDa).


Figure 3: Top, SDS-PAGE of NDF isoforms (Laemmli gels and Coomassie staining). Lane 1, protein standards (5 µg each) of known molecular size (90, 65, 45, 31, 20, and 14 kDa, from the top); lanes 2 and 3, CHO cell-derived rat NDF-alpha2 and -beta4; lanes 3-7, human NDF-alpha1, -alpha2, -beta1, and -beta2, respectively; lane 8, Rat1-EJ NDF; and lane 9, bacterially derived human NDF-alpha2. Sample loading is approximately 3-4 µg on each lane. Bottom, chemiluminescence detection of receptor phosphorylation in cells stimulated by NDF isoforms. A, human NDF-beta1 EGF domain, 1 ng/ml; B, control, no factor added; C and D, rat NDF-alpha2 and rat NDF-beta4; E-H, human NDF-alpha1, -alpha2, -beta1, and -beta2. Each group contains four concentrations, 0.5, 1, 2, and 4 ng/ml (from the left).



N-terminal Sequence and Amino Acid Composition Analyses

Sequence analyses revealed that purified rat NDF-alpha2 and -beta4 have the same N-terminal sequence: KEGRGKGKGKKKDRGSR-, while the N-terminal sequence of human NDF-alpha1, -alpha2, -beta1, or -beta2 was also determined to be identical: KEGRGKGKGKKKERGS-. These sequences are 4 amino acids shorter than the N terminus predicted from pro-NDF genes (17, 19, 21) . A minor sequence (<5%) starting with GRGKGKG- was also detected in every isolated isoform. This minor form is a shorter NDF with two amino acid truncation from the N terminus of the main processed form as described above.

Table 2lists the amino acid composition data obtained from analysis of natural NDF isolated from Rat1-EJ cells and different recombinant human and rat NDF isoforms. Protein theoretical numbers of residues provided in the table were based on the assigned N and C terminus for each NDF isoform as described above and later. Amino acid composition data for different recombinant NDF isoforms are in general agreement with the theoretical values predicted from the DNA sequences of different secreted human and rat NDF-alpha as well as beta forms. In comparison with recombinant rat NDF-alpha2 and -beta4, Lys, Gly, and Arg values are low in Rat1-EJ NDF, which is consistent with the deletion of its N-terminal nanopeptide, KEGRGKGKG (16) .



Assignment of C Terminus for NDF Isoforms

Fig. 4shows four comparative peptide maps derived from endoproteinase Lys-C digestion of the reduced and alkylated human NDF-alpha1, rat NDF-alpha2, human NDF-beta1, and rat NDF-beta4. The map derived from human NDF-alpha2 expressed by a human pro-NDF clone is also identical to the map of human NDF-alpha1 (data not shown). Several peaks as indicated eluted at retention times that are identical in four of the maps. Their sequences are confirmed to be identical by structural analysis in all cases.


Figure 4: Reversed phase HPLC peptide map analyses of reduced and carboxymethylated NDF isoforms. Panels A-D, human NDF-alpha1, rat NDF-alpha2, human NDF-beta1, and rat NDF-beta4 (50 µg of each digest was injected).



Assignment of the C-terminal peptides in different NDF isoforms was carried out by both N-terminal sequence analysis and mass spectrometry of the isolated peptides. This is summarized in Table 3. For example, the map of human NDF-alpha1 contains four C-terminal peptide candidates (Fig. 4A and Table 3). Two C-terminal peptides were confirmed. A major peptide fraction eluted at 38.3 min (sequence: JQPGFTGARJTENVPM, where J is the modified carboxymethylcysteine) and a minor peptide fraction eluted at 32.9 min (sequence: JQPGFTGARJTENVPMK), which contains an extra Lys at the C terminus of the 38.3-min peptide. Mass spectrometric analysis of two early eluting peptides at 28.5 and 32.1 min confirmed that they are the methionine-oxidized peptides respective to peptides at 32.9 and 38.3 min. Since there are no other peptides having sequences that extend beyond the above analyzed peptides, human NDF-alpha1 thus ends at Met or Lys at sequence positions 227 and 228 assigned for pro-NDF (see Fig. 1). The above data concludes that human NDF-alpha1 is processed at both termini to become a soluble protein of 223-224 amino acids.



Using similar approaches, several C-terminal peptides together with the respective Met-oxidized peptides from human NDF-beta2, rat NDF-alpha2, and rat NDF-beta4 can also be isolated and structurally elucidated (Table 3). Human NDF-beta2 and rat NDF-beta4 are also processed at the nearby sites found for NDF-alpha isoforms. These NDF isoforms are processed to contain 222-225 amino acid residues. Oxidation of the methionine residue near the C terminus were observed in all NDF isoforms (Table 3).

Identification of Glycosylation Sites in NDF Molecules

As demonstrated in each of the peptide maps derived from various NDF forms (Fig. 4, panels A-D), one fraction between retention times 37 and 43 min behaves as a broader peak with poor resolution, a typical characteristic for a peptide being glycosylated. One of the C-terminal peptide fractions, which has been analyzed as described, reproducibly eluted within this broad fraction in every peptide map (Fig. 4). N-terminal sequence analysis of the broad fraction isolated from each of the maps elucidated a partial peptide sequence: LGNDX(1)ASAX(2)ITIVESNEFITGMP-, where X(1) and X(2) are unassigned due to complete absence of sequence signals. From cDNA sequence, Ser is the assigned amino acid at X(1) and Asn at X(2) position in all of the NDF isoforms. Digestion of each NDF form with Lys-C protease would consistently generate the largest peptide which contains 64 amino acids and has a sequence identical to the sequence found in this broader peak. This peptide fragment is the only NDF peptide to be glycosylated.

Fig. 5A illustrates the HPLC separation of rat-NDF-alpha2 glycopeptide after digestion with both endoproteinase Glu-C and trypsin, and Fig. 5B is the profile for rat NDF-beta4 glycopeptide after Glu-C endoproteinase digestion. Three peptide fractions (ST-1, ST-2, and ST-3) were obtained from the glycopeptide fraction of rat NDF-alpha2 and two fractions (S-1 and S-2) from glycopeptide fraction of NDF-beta4. The absence in sequence signals at a sequencing cycle in the peptide allowed prediction of a modification that may have occurred at that amino acid residue. Fig. 5C summarizes the assignment of possible glycosylation sites for rat NDF glycopeptides. The results revealed that 2 Asn residues at positions 9 and 47 in this peptide are glycosylated and Asn at position 3 is not glycosylated at all. There are at least 11 sites absent in Ser and Thr signals, including Thr at positions 20, 26, 43, 52, 53, and 59 and Ser at positions 25, 32, 33, 42, and 60. Thr and Ser residues at these sites may be attached with O-linked sugars.


Figure 5: A, HPLC separation of peptides derived from endoproteinase Glu-C and trypsin incubation of a rat NDF-alpha2 glycopeptide (see Fig. 4). B, HPLC separation of peptides derived from endoproteinase Glu-C digestion of a rat NDF-beta4 glycopeptide. Peptides ST-1, ST-2, ST-3, S-1, and S-2 are subfragmented glycopeptides. C, glycosylation sites. Asn, Thr, and Ser (residues marked with asterisks) are assigned as potential glycosylation sites in the glycopeptide sequence for rat NDF-alpha2 and -beta4.



Receptor Autophosphorylation of CHO-derived NDF Isoforms

Fig. 3(bottom panel) illustrates receptor phosphorylation in the MDA-MB453 human breast cancer cells stimulated by different human and rat NDF isoforms. Each NDF at 0.5-4 ng/ml was able to actively stimulate receptor phosphorylation. The data also indicate that every NDF isoform can stimulate tyrosine phosphorylation of receptor in a dose-dependent manner. At equimolar concentration, each CHO cell-derived NDF (4 ng/ml) appears to be as potent as human NDF-beta EGF domain (1 ng/ml), used as a standard.


DISCUSSION

Typical chromatographic procedures were able to isolate recombinant human and rat NDF isoforms to their apparent purity. These soluble NDF forms exhibit 40-44-kDa molecular mass in the reducing gel. All human, rat, or human-rat chimeric pro-NDF genes used for mammalian cell expression encode transmembrane isoforms(19, 20, 21) . Initially membrane-bound, glycosylated forms were expressed(21) , which were then processed and secreted into the medium as soluble NDF forms of similar size.

Except glial cell growth factor which contains a normal signal peptide and a Kringle domain(22) , the sequences of various pro-NDF forms in the neuregulin family are shorter at their N termini and have no classical signal peptide sequence(20) . Excluding acetylcholine receptor inducing activity whose N terminus has an 8-amino acid sequence deletion(23) , both human and rat pro-NDF genes encode precursor protein sequence having an N terminus: M^1SER^4K^5EGRGKGKGKKK-(17, 19, 21) . In CHO cells all pro-NDF isoforms are invariably processed at the Arg^4-Lys^5 bond, giving rise to secretion of major NDF forms 4 amino acids shorter than predicted. This result suggests that the N-terminal processing of CHO cell-derived NDF forms is different from natural rat NDF (19) and human heregulin(17) . Human heregulin was proven to have an N terminally blocked Ser(17) , indicating the processing of Met^1-Ser^2 followed by N-terminal acylation. In contrast, rat NDF isolated from medium conditioned by Rat1-EJ cells has an N terminus 9 amino acids shorter than the CHO cell-derived NDF as a result of Gly-Lys^14 cleavage(19) .

Putative sites for C-terminal cleavages from their respective precursors have been postulated for the release of soluble forms of heregulin(17) , NDF(21) , and acetylcholine receptor inducing activity (23) . Based upon the pro-NDF structure, the processing region can be clearly defined between EGF and transmembrane domains. A putative processing site (Lys-Arg near the start of transmembrane domain) is shared by most of the transmembrane forms of NDF(20, 21) . As a result of processing, various NDF isoforms, i.e. NDF-alpha1, -alpha2, -beta1, -beta2, -beta3, and -beta4 etc. will display distinct structural differences at the C-terminal region. However, additional processing sites may also be present within the juxtamembrane region. Here we have provided evidence that the Lys-Arg bond is not a primary cleavage site for all NDF forms expressed in CHO cells. Met-Lys was found to be the major cleavage site for human and rat pro-NDF-alpha isoforms, and the Lys-Val bond to be the minor cleavage site (Fig. 1). Pro-NDF-beta isoforms were also processed at sites similar to those found in the alpha isoforms. Human NDF-beta1 and -beta2 were secreted by cleavage at three sites, Met-Ala, Ala-Ser, and Phe-Tyr, and rat pro-NDF-beta4 was also processed at Ser-Phe together with a minor species cleaved at the Phe-Tyr bond. Therefore, specific processing of pro-NDFs by CHO cells have resulted in the production of two types (alpha and beta) of recombinant NDFs having 222-225 amino acid residues.

The above observations indicate that CHO cell-expressed NDF isoforms are secreted as a result of specific proteolytic processing at both the N- and C-terminal ends. Both alpha and beta isoforms are processed at similar sites despite the large sequence variations in their juxtamembrane regions (Fig. 1). These observations suggest that specific pro-NDF processing enzymes do exist in CHO cells. However, the specificity of an N-terminal processing enzyme may be cell type-specific as the rat NDF, human heregulin, and the CHO cell-derived NDF isoforms display different N termini, apparently due to difference in the processing of NDF precursors in different cells. In CHO cells, the C-terminal processing enzyme seems to recognize pro-NDF-alpha and -beta cleavage sites in the juxtamembrane region, which are 5 to 8 amino acids further downstream from the carboxyl end of the last EGF disulfide loop. The recognition seems to be universal to all pro-NDF forms and is thus not sequence-specific, suggesting that sequence variations among all the pro-NDF isoforms in the juxtamembrane region do not predetermine sites and efficiency of the cleavage. It is interesting to note that the C-terminal processing site of pro-NDF is similar to that of TGF-alpha precursor (30) and is only 3-4 amino acids apart from the processing sites of EGF or amphiregulin precursors(31, 32) . As no signal peptide sequences were found in all pro-NDF genes, the N- and C-terminal processings may be directed by transmembrane domain which is conserved in sequence for all human and rat pro-NDFs. Specific C-terminal processing of the NDF-alpha and -beta isoforms appear to indicate that the high molecular multiplicity in the neuregulin family may only occur in their membrane-associated pro-NDF forms.

Endoproteinase Lys-C digestion of NDF isoforms consistently released a 64-amino acid glycopeptide corresponding to the entire spacer domain of all NDF molecules. Sequence analysis of the isolated glycopeptides revealed that there are 2 Asn residues containing N-linked sugars and at least 11 Thr/Ser residues assigned as potential sites for O-linked sugars (Fig. 5). We predict that the NDF molecule contains high levels of N- and O-linked carbohydrate, as evidenced by SDS-PAGE in this study and sedimentation equilibrium analysis in the subsequent paper (25) and also by deglycosylation experiments reported earlier(21) . The glycosylation pattern of NDF is quite unique and similar in all NDF isoforms.

Taken together, the data describes a schematic drawing common for the soluble NDF-alpha or -beta structures (Fig. 6). The N termini of all soluble NDF isoforms are highly conserved (Fig. 1); and the N-terminal 20 amino acids in all isoforms contain 50% highly charged Lys and Arg. This region seems to display high consensus homology to the sequences that bind heparin in molecules such as fibroblast growth factor, heparin-binding EGF-like growth factor, platelet-derived growth factor, and hepatocyte growth factor(33, 34, 35, 36) . Fibroblast growth factor has been demonstrated to bind to cell surface heparan sulfate proteoglycans which have been determined to be low affinity receptors(37, 38) . These low affinity binding sites exist in the extracellular matrix of cells as well. Soluble NDF isoforms actually exhibit very strong binding to heparin-Sepharose which was used as our first purification step ( Fig. 2and Table 1). The functional role of heparin binding of NDF in vivo remains unknown and awaits further investigation.


Figure 6: Schematic drawing of an extended structure of secreted NDF including a putative N-terminal heparin binding region, an Ig domain, a carbohydrate (or spacer) domain, and an EGF domain. The structure is not in a proportional scale. The open circles represent O-linked sugars and the branched closed circles are N-linked sugars. Four disulfide bonds, one in Ig domain and three in EGF domain, are also shown.



Adjacent to the putative N-terminal heparin binding region is the Ig-like loop which contains approximately 60 amino acids linked by a disulfide bridge. The carbohydrate domain contains two Asn-linked and multiple O-linked sugars which are attached in this unique region that separates Ig from EGF domain (Fig. 6). As all the N- and O-linked sugars are attached around this short spacer, the extended and hydrophilic carbohydrate moieties may extensively cover the whole spacer domain. The functional role of carbohydrates also remains unknown in vivo despite that the E. coli-derived, nonglycosylated NDF forms are biologically active in vitro(21) . A structural feature of NDF that contains Ig-like domain connected to the carbohydrate domain is also common in other molecules such as the extracellular region of CD8, a cell surface marker protein involved in vital cellular immune response (39) .

The discovery of multiple pro-NDF forms has raised important questions on their biological functions. Nonetheless, the secreted NDF forms only exist as alpha and beta isoforms after processing. Therefore, it is reasonable to only use alpha and beta isoforms to evaluate their biological action and potential clinical usage. However, the importance of the membrane-associated pro-NDF forms should also not be ruled out, as they may distribute differently in tissues and exert different functions. As found in earlier studies, NDF isoforms cannot stimulate phosphorylation of HER-2 receptor in NIH3T3 cells transfected with the HER-2 gene(24) . However, both NDF-alpha and -beta isoforms exhibit similar stimulating activity using MDA-MB453 cells in our assays (Fig. 3B). The cancer cell line used in the assay has been known to express HER-2 and HER-4 receptors(24) . It was suggested that the stimulatory activity of NDFs may be associated with NDFs being capable of inducing homo- and/or heterodimerization of these receptors present in the cells(24) . More recent reports have described that heregulin induces tyrosine phosphorylation of HER-4 (40) and that NDFs bind to both HER-3 and HER-4 receptors(41) , suggesting that instead of HER-2, the HER-3 and HER-4 receptors may function as physiological receptors for many of the NDF isoforms. More studies are thus required to determine HER-3/HER-4 and NDF interactions, the subsequent signal transduction and biological function. The studies on the structure and processing of mammalian cell-derived NDF isoforms as described here and in the subsequent paper (25) may shed light on elucidating the functional role of NDF isoforms.


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: EGF, epidermal growth factor; CHO cells, Chinese hamster ovary cells; NDF, neu differentiation factor; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PTC-, phenylthiocarbamyl; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We are indebted to Dr. Yosef Yarden, Department of Chemical Immunology, the Weizmann Institute of Science, for critical reading of and comments on the manuscript, and to Joan Bennett for her help in typing the manuscript.


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