(Received for publication, October 17, 1994; and in revised form, December 7, 1994)
From the
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 ( and
) 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.
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 p185
expression 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(
)-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) .
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.
Dialyzed material was
then loaded onto a DEAE-Sepharose 6B (fast flow) column (1.2 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 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) .
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.
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.
Table 1summarizes the isolation
procedure and yield of NDF obtained from each isolation step using rat
NDF-2 as an example. Fig. 2A shows the three
chromatographic separation steps used in the isolation of rat
NDF-
2. 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-2. Panels 1-3,
heparin-Sepharose, DEAE-Sepharose, and phenyl-Sepharose
chromatographies, respectively. B, SDS-PAGE (Tricine gels) of
column fractions containing rat NDF-
2. 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-2 purification has been successfully used in
the purification of other recombinant NDFs including rat NDF-
4,
and human NDF-
1, -
2, -
1, and -
2 isoforms.
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-2 and -
4; lanes 3-7, human NDF-
1, -
2, -
1, and
-
2, respectively; lane 8, Rat1-EJ NDF; and lane
9, bacterially derived human NDF-
2
.
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-
1 EGF domain, 1 ng/ml; B, control, no factor added; C and D, rat NDF-
2 and rat NDF-
4; E-H, human NDF-
1, -
2, -
1, and -
2.
Each group contains four concentrations, 0.5, 1, 2, and 4 ng/ml (from
the left).
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- as well as
forms.
In comparison with recombinant rat NDF-
2 and -
4, Lys, Gly,
and Arg values are low in Rat1-EJ NDF, which is consistent with the
deletion of its N-terminal nanopeptide, KEGRGKGKG (16) .
Figure 4:
Reversed phase HPLC peptide map analyses
of reduced and carboxymethylated NDF isoforms. Panels A-D,
human NDF-1, rat NDF-
2, human NDF-
1, and rat NDF-
4
(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-1 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-
1 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-
1 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-2, rat NDF-
2, and rat NDF-
4 can also be isolated and
structurally elucidated (Table 3). Human NDF-
2 and rat
NDF-
4 are also processed at the nearby sites found for NDF-
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).
Fig. 5A illustrates the HPLC separation of
rat-NDF-2 glycopeptide after digestion with both endoproteinase
Glu-C and trypsin, and Fig. 5B is the profile for rat
NDF-
4 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-
2 and two fractions (S-1 and S-2)
from glycopeptide fraction of NDF-
4. 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-2
glycopeptide (see Fig. 4). B, HPLC separation of
peptides derived from endoproteinase Glu-C digestion of a rat
NDF-
4 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-
2 and -
4.
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:
MSER
K
EGRGKGKGKKK-(17, 19, 21) .
In CHO cells all pro-NDF isoforms are invariably processed at the
Arg
-Lys
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
-Ser
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
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-1, -
2, -
1, -
2, -
3,
and -
4 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-
isoforms,
and the Lys
-Val
bond to be the minor
cleavage site (Fig. 1). Pro-NDF-
isoforms were also
processed at sites similar to those found in the
isoforms. Human
NDF-
1 and -
2 were secreted by cleavage at three sites,
Met
-Ala
,
Ala
-Ser
, and
Phe
-Tyr
, and rat pro-NDF-
4 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 (
and
) 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 and
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-
and -
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-
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-
and -
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- or
-
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 and
isoforms after
processing. Therefore, it is reasonable to only use
and
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-
and -
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.