(Received for publication, April 18, 1995; and in revised form, May 24, 1995)
From the
In this report we describe a series of experiments designed to
probe the biosynthesis of the bikunin proteins. The bikunin proteins
are serine proteinase inhibitors found in high concentrations in human
plasma. The proteins are composed of two or three polypeptide chains
assembled by a newly identified carbohydrate mediated covalent
inter-chain ``Protein-Glycosaminoglycan-Protein'' (PGP)
cross-link (Enghild, J. J., Salvesen, G., Hefta, S. A.,
Th, I. B., Rutherfurd, S., and Pizzo, S. V.(1991) J.
Biol. Chem. 266, 747-751). In this study we show that
transformed hepatocyte cell lines, exemplified by HepG2 cells, have
lost the ability to produce these proteins. In contrast, primary human
hepatocytes produce bikunin proteins identical to the proteins
identified in human plasma. Pulse-chase analysis demonstrate that the
PGP-mediated cross-linking of the polypeptide chains occurs late in the
secretary pathway. Moreover, the mechanism responsible for the
formation of the PGP cross-link is divided in two steps involving a
proteolytic cleavage followed by carbohydrate attachment. The results
indicate that normal hepatocytes contain the biosynthetic machinery
required for correct synthesis and processing. However, transformed
cell lines are defective in several aspects of bikunin biosynthesis
precluding such systems from being used as relevant in vitro models.
Bikunin proteins are members of the pancreatic trypsin inhibitor
(Kunitz) family (Salier, 1990). The term ``bikunin,'' a
contraction of bis Kunitz inhibitor, was
suggested to describe the double headed proteinase inhibitor of human
inter-
Figure 1:
Precursors and components of the three
bikunin proteins found in human plasma. The bikunin proteins are
multichain plasma proteins composed of bikunin and one or two distinct
but homologous heavy chains. The polypeptides constituting the bikunin
proteins are assembled by a GAG chain originating from Ser
Human plasma bikunin is covalently bound to
three homologous heavy chains (HC1, HC2, and HC3) (Enghild et
al., 1989). Three combinations of bikunin and heavy chains have
been identified in human plasma (Fig. 1); (i) I Although the bikunin proteins are composed of
more than one subunit, they resist dissociation in reduced SDS-PAGE.
However, the subunits of I Previous reports have described attempts to investigate the
synthesis of the bikunin proteins in the human carcinoma cell line
HepG2 (Perlmutter et al., 1986; Bourguignon et al.,
1989; , 1992; Heron et al., 1994). These authors
demonstrated, employing a pulse-chase labeling protocol and antisera
that reacted with a number of bikunin proteins, the synthesis of
miscellaneous components of the bikunin proteins. The antisera used by
these investigators were non-selective and did not distinguish between
the different bikunin proteins, precursors, and mature proteins. Due to
the complex composition of these proteins, studying their biosynthesis
is an almost impossible undertaking without specific reagents.
Consequently, limited consensus can be drawn from these reports. In
this study we describe a series of experiments designed to probe the
biosynthesis of the bikunin proteins. In contrast to previous reports,
which only examined the biosynthesis in transformed cell lines, we have
studied the biosynthesis in primary human hepatocytes. These studies
demonstrate that bikunin proteins are processed correctly in primary
human hepatocytes while HepG2 cells and other transformed cell lines do
not assemble the complete bikunin proteins. Although HepG2 cells failed
to produce authentic bikunin proteins the investigations of the
defective processing events in these cells provided important clues
concerning the mechanism of PGP-mediated chain assembly.
Figure 2:
HepG2
cells are not producing the mature bikunin proteins. Aliquots of human
plasma and HepG2 serum-free medium were run in 5-15% reduced
SDS-PAGE. The samples were analyzed before(-) and after (+)
treatment with 50 mM NaOH, a procedure known to dissociate the
PGP cross-links (Enghild et al., 1991, 1993). Following
electrophoresis proteins were transferred to polyvinylidene difluoride
membranes for immunoblotting. The blots were cut in strips and
developed using antiserum as indicated. Lanes containing molecular
weight markers were removed and stained with Coomassie Blue. Panel
A, the bikunin proteins present in normal human plasma are shown.
As expected I
The pulse-chase experiments of cell lysates and conditioned medium
are shown in Fig. 3. Radiosequence analysis of the 45-kDa
proteins immunoprecipitated from the cell lysate identified the
N-terminal of
Figure 3:
Biosynthesis of bikunin in primary human
hepatocytes using a pulse-chase protocol. The cells were metabolically
labeled with [
Figure 9:
Radiosequence analysis of bikunin proteins
produced by HepG2 cells. This figure summarizes the results of the
radiosequence analysis performed on immunoprecipitations from HepG2
cell lysate and medium. Raw radioactive counts per minute (cpm)
associated with each cycle of Edman degradation is plotted for
[
Figure 10:
Radiosequence analysis of proteins
immunoprecipitated from primary human hepatocytes. Two double label
experiments [
Figure 4:
Pulse-chase kinetic analysis of
Figure 5:
Pulse-chase analysis of bikunin synthesis
in HepG2 cells. The
Figure 6:
Pulse-chase analysis of HC2 synthesis in
HepG2 cells. Panel A, the HC2 antiserum immunoprecipitated a
100-kDa protein band in the cell lysate (left panel). A 100 to
70-kDa shift in the molecular mass was observed 15 min after the onset
of biosynthesis. Both bands were identified by radiosequence analysis
as HC2 alone. Radiosequence analysis of the heterogeneous 225-kDa
protein band showed the presence of bikunin and HC2 (right
panel). Panel B, the HC2 C-terminal extension peptide
antisera immunoprecipitated the same 100-kDa band seen in panel A as determined by radiosequencing. The 70-kDa band observed in panel A did not react with the HC2 C-terminal specific
antiserum and most likely represents non-assembled free mature HC2. The
mature HC2 was secreted into the medium as two tightly spaced bands
probably the result of the addition of N-linked carbohydrate.
The open arrows indicate bands analyzed by
radiosequencing.
Figure 7:
Pulse-chase analysis of HC3 synthesis in
HepG2 cells. Panel A, anti-HC3 immunoprecipitated 100-kDa
protein. This protein was secreted 15 min after the onset of
biosynthesis apparently without undergoing any size altering
post-translational modification. The 43- and 15-kDa proteins
immunoprecipitated from the conditioned medium probably represent
proteolytic fragments of HC3, due to adventitious proteolysis. The
66-kDa protein band seen in this panel reacted with the nonspecific
preimmune antiserum seen in the control lane. Panel B, the
antiserum specific to the C-terminal extension of HC3
immunoprecipitated a 100-kDa protein both from the cell lysate and the
conditioned medium. Radiosequence analysis was employed to identify the
100-kDa band from the cell lysate and the 100-kDa band from the
conditioned medium. Both bands were identified as HC3. This suggests
that HepG2 synthesize and secrete only the precursor of HC3. The open arrows shown in the two panels indicate bands analyzed by
radiosequencing.
In
the cell lysate, a heterogeneous band stretching between the 45- and
66-kDa size markers is apparent (Fig. 3A, left panel,
see bracket). This The data
described above suggest that the liver, in vivo, secrete the
assembled bikunin proteins. The bikunin proteins P
The cleavage of the 30-kDa
C-terminal extension occurs at an Asp-Pro bond within the heavy chain
precursor consensus sequence Asp-Pro-His-Phe-Ile-Ile. Consequently, the
mature heavy chains have a C-terminal Asp. Since the PGP cross-link
involves the The
processing of proteins by mutant cell lines has previously proven
useful for unraveling biosynthetic mechanisms. We therefore decided to
further investigate the defective intracellular processing events of
the bikunin proteins in HepG2 cells. These studies were directed toward
understanding the mechanism of chain processing and PGP-mediated chain
assembly.
These observations suggest that processing of
Secreted bikunin migrated in SDS-PAGE as a
protein of approximately 43-kDa (Fig. 5, right panel)
similar to the 45-kDa intracellular Biosynthesis of wild type bikunin proteins should result in
the appearance of three polypeptide species of 225, 125, and 43 kDa
representing I
Immunoblot
analysis of HepG2 serum-free medium failed to detect secretion of HC1 (Fig. 2B) and pulse-chase analysis of cell lysates
confirmed that HepG2 cells do not produce HC1 (data not shown). A band
of approximately 100 kDa was detected in cell lysates
immunoprecipitated with HC2 antiserum (Fig. 6A, left
panel). Radiosequence analysis confirmed this band as HC2 (Fig. 9D). We did not detect the putative propeptide by
radiosequence analysis of immunoprecipitates (Fig. 6A,
0 min chase time). The 100-kDa band is 30 kDa larger that mature HC2
(see Fig. 2A) and reacted with the HC2 C-terminal
extension peptide antiserum (Fig. 6B). Consequently,
this band represents the precursor of HC2. Additionally, heterogeneous
high molecular weight material, similar to the diffuse bands seen with
the HepG2 bikunin immunoprecipitates, was observed in the cell lysate
and in the conditioned medium (Fig. 6). Radiosequence analysis
of this material revealed the presence of both bikunin and HC2 (Fig. 9, panels C, D, and E). Apparently, this
material represents assembled bikunin and HC2. NaOH treatment results
in the dissociation of the two components suggesting that a PGP
cross-link was formed (see Fig. 2B). As described
above, this protein is dissimilar to the mature wild type bikunin
proteins identified in vivo and does not represent an
authentic bikunin protein. However, the formation of the PGP cross-link
between bikunin and HC2 appear to have taken place. The assembly of
these aberrant bikunin proteins was observed intracellularly
approximately 15-30 min after the onset of biosynthesis followed
by immediate secretion (Fig. 6A). However, the assembly
was incomplete since the HC2 precursor was observed in the medium (Fig. 6B). The secreted HC2 precursor migrated as two
bands most likely due to N-linked carbohydrate heterogeneity.
Of particular significance, some of the 100-kDa HC2 precursors were
processed to a smaller 70-kDa protein, concomitant with the appearance
of the heterogeneous high molecular weight material (Fig. 6A). The failure of this 70-kDa protein to react
with the HC2 extension antiserum (Fig. 6B) suggests
that the band represents free mature HC2. Following the observed
processing event, no 30-kDa extension was detected and we hypothesize
that the released polypeptide is degraded upon removal from HC2.
Apparently, the C-terminal extension is excised from the HC2 precursor
without the formation of the PGP cross-link. This suggests that a
proteinase cleaves the Asp-Pro peptide bond followed by the addition of
GAG to the now available
To investigate these possibilities we determined if
primary hepatocytes, which are capable of producing fully assembled
bikunin protein, also secrete heavy chain precursors.
Immunoprecipitations of cell lysates and conditioned medium using the
HC3 C-terminal extension antiserum was performed (Fig. 8). A
100-kDa band was detected in the cell lysate. Radiosequence analysis
only identified the mature N-terminal of HC3 and not the propeptide.
The processing of the C-terminal extension 15-30 min after the
onset of biosynthesis coincides with the assembly of P
Figure 8:
Biosynthesis of HC3 precursor in primary
human hepatocytes. The processing of the heavy chain precursors in
primary human hepatocytes was investigated using the HC3 C-terminal
peptide antiserum. The antiserum immunoprecipitated a 100-kDa protein
band that we identified as HC3 by radiosequence analysis. Removal of
the C-terminal extension appeared at the same time as the assembly of
the P
The biosynthesis of bikunin proteins in HepG2 cells may be
summarized as follows: (i) the cells are not making HC1 and they are
therefore unable to produce I Primary human hepatocytes produce
bikunin proteins identical to the proteins detected in plasma (Fig. 3, 8, and 10). GAG is added to Ser
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
SUMMARY
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-inhibitor (I
I) (
)(Gebhard et al.,
1989). The ``bikunin proteins'' refer to the multichain
proteins found in human plasma composed of bikunin and one or two heavy
chains (see Fig. 1). Bikunin is structurally related to the
proteinase inhibitor aprotinin, also called Bovine Pancreatic Trypsin Inhibitor or
Trasylol
. We have extended this nomenclature (Enghild et al., 1990; Gebhard et al., 1990) to encompass the
``monokunins'' A4 amyloid peptide precursor-751 (Tanzi et
al., 1988), collagen type VI
3 chain (Chu et al.,
1990), collagen type VII (Greenspan, 1993), and the
``trikunin'' tissue factor pathway inhibitor (Wun, et
al., 1988). In general, a ``kunin'' is a member of the
pancreatic trypsin inhibitor (Kunitz) family. The relationship between
these proteins has emerged recently as cDNA and protein sequences have
become available, but the functions of most of the kunins remains
unclear. The kunins for which a function is suspected include; (i) rat
mast cell trypstatin, which is identical to the second domain of rat
bikunin and appears to regulate mast cell tryptase activity (Kido et al., 1988; Itoh et al., 1994), (ii) tissue factor
pathway inhibitor, which has been implicated in the regulation of the
extrinsic pathway of coagulation (Broze et al., 1990), and
(iii) bovine bikunin proteins which are thought to be involved in the
stabilization of the extracellular matrix of cells that surround the
developing ovum (Chen et al., 1992; Castillo and Templeton,
1993; Camaioni et al., 1993; Huang et al., 1993; Chen et al., 1994).
of bikunin and one or two heavy chains covalently bound to this
carbohydrate chain. Bikunin is encoded by a tandem
m-bikunin mRNA (top). The cDNAs of the heavy
chains encodes a signal peptide followed by a propeptide that is not
present in the mature proteins purified from plasma. The sequence also
predicts a putative 30-kDa C-terminal extension not found in the mature
plasma proteins (middle panel) The complex composition of
bikunin proteins, cross-linking, precursors, and products, predict
several processing events required for the formation of the mature
bikunin protein. These include the usual reactions that most proteins
undergo such as cleavage of the signal peptide, addition of
carbohydrate (♦) and proteolytic processing of the precursors. In
addition, the bikunin proteins undergo a unique post-translational
modification: the formation of the PGP cross-link. The mature bikunin
proteins are shown in the three inserts (bottom). The
molecular mass of the various unglycosylated precursors and
glycosylated mature bikunin proteins are indicated in
kDa.
I, composed
of HC1, HC2, and bikunin, (ii) pre-
-inhibitor (P
I), composed
of HC3 and bikunin, and (iii) HC2/bikunin, composed of HC2 and bikunin
(Enghild et al., 1989). The complete cDNA sequences encoding
HC1, HC2, HC3, and bikunin have been determined (Kaumeyer et
al., 1986; Gebhard et al., 1988, 1989; Diarra-Mehrpour et al., 1992; Bourguignon et al., 1993). The three
heavy chains are homologous to another recently discovered plasma
protein called inter-
-trypsin inhibitor family heavy chain related
protein (IHRP). Interestingly, IHRP is not bound to bikunin (Saguchi et al., 1995; Choi-Miura et al., 1995). The cDNAs of
all the heavy chains encode 30-kDa C-terminal extensions, not present
in the mature proteins (except IHRP), as well as putative N-terminal
pro-peptides (see Fig. 1). The bikunin cDNA encode two tandemly
arranged proteins, namely,
-microglobulin
(
m) and bikunin. A short dibasic connecting peptide
separates the two proteins. Proteolysis of the connecting peptide
release
m and bikunin (Kaumeyer et al., 1986;
Barr, 1991; Bratt et al., 1993, 1994). In humans,
m is found in plasma complexed with IgA and albumin
(Tejler and Grubb, 1976). In the rat,
m is associated
with
-inhibitor 3 and fibronectin (Falkenberg et
al., 1990, 1994).
I (Jessen et al., 1988; Enghild et al., 1989), P
I, and HC2/bikunin (Enghild et
al., 1989) can be dissociated by treatment with chondroitin
sulfate degrading enzymes. Indeed, we have recently determined that the
unusual stability of these proteins are the result of a novel protein
cross-link in which polypeptide chains are joined through a
carbohydrate chain. The subunits are assembled by a
chondroitin-4-sulfate chain that originates from Ser
of
bikunin. The heavy chains are covalently bound to the
chondroitin-4-sulfate chain via an ester bond between the
-carbon
of their C-terminal Asp residues and carbon-6 of an internal N-acetylgalactosamine of the chondroitin-4-sulfate chain. We
call this new structure a Protein Glycosaminoglycan Protein (PGP) cross-link (Enghild et al., 1991,
1993).
Materials
Tissue culture medium, fetal calf
serum, and other tissue culture supplies were obtained from Life
Technologies, Inc. Rat tail collagen, type 1, for coated tissue culture
dishes was from Collaborative Research. Protein G-Sepharose Fast Flow
was from Pharmacia, m-maleimidobenzoic
acid-N-hydroxysuccinimide ester was from Pierce and
[S]methionine (1220 Ci/mM),
[
H]leucine (179 Ci/mM),
[
H]isoleucine (111 Ci/mM), and
[
H]valine (70 Ci/mM) were from Du Pont
NEN. 1,10-Phenanthroline was from Sigma. The general serine proteinase
inhibitor 3,4-dichloroisocoumarin and the general cysteine proteinase
inhibitor N-[[[N-[(-3-trans-carboxy-oxiran-2-yl)-carbonyl]-L-leucyl]amino]-butyl]-guanidine
(E-64) were from Boehringer Mannheim. I
I, P
I, and HC2/bikunin
were purified as described previously (Enghild et al., 1989).
HepG2, Hep3B, SK-Hep1, and Chang liver cells were obtained from the
American Type Culture Collection (Rockville, MD). Primary human
hepatocytes were a gift from Drs. Randy L. Jirtle and Herbert
Reisenbichler, Duke University Medical Center (Durham, NC.)
Production of Specific Antisera
Purified II,
P
I, and HC2/bikunin were treated with 50 mM NaOH as
described before (Enghild et al., 1991). The dissociated
polypeptides were separated by SDS-PAGE and the specific polypeptide
chains HC1, HC2, HC3, and bikunin were then electroeluted according to
Hunkapiller et al.(1983). Antisera to the purified heavy
chains and bikunin were raised commercially in rabbits using a standard
protocol (Pel-Freez). Following bleed out the IgG fraction of the serum
was recovered by affinity chromatography on a protein G-Sepharose Fast
Flow column (Pharmacia). To eliminate cross-reactivity the antisera
were further purified by immunoadsorption on HC1-, HC2-, and
HC3-Sepharose columns. The specificity of the individual antisera was
investigated by immunoblotting of human plasma and purified antigens.
Peptide Antiserum
Peptides, DGAYTDYIVPDIF (HC1),
CFVPQLYSFLKRP (HC2), and GVHTDYIVPNLF (HC3), corresponding to the
putative C-terminal extensions of heavy chain 1, 2, and 3 were
synthesized on an Applied Biosystems 430A peptide synthesizer. The
structure of the peptides was verified by Edman degradation and plasma
desorption mass spectrometry as described before (Rubenstein et
al., 1993). The peptide was coupled to ovalbumin by using m-maleimidobenzoic acid-N-hydroxysuccinimide ester
(Kitagawa and Aikawa, 1976) and glutaraldehyde (Kagen and Glick, 1979).
Rabbit antisera to the peptide-ovalbumin conjugates were raised
commercially (Pel-Freez). The specificity of the different antisera
were examined by immunoblotting of purified proteins, plasma samples,
and peptide-bovine serum albumin conjugates.Metabolic Labeling and Pulse-Chase Analysis
HepG2
cells and primary human hepatocytes were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum
at 37 °C in 5% CO atmosphere. Primary human hepatocytes
were cultured using the same medium; however, rat tail collagen type
1-coated tissue culture dishes were employed to facilitate cell
attachment. For standard biosynthetic radiolabeling, cells were grown
in 50-mm tissue culture plates until 80% confluent. The cells were
washed twice with RPMI 1640 salt solution, and then incubated with RPMI
1640 salt solution without fetal bovine serum and lacking the amino
acids that we intended to subsequently use for metabolic labeling.
After the addition of [
S]Met (100 µCi/ml),
the cells were incubated for 5 min (pulse period). If the
immunoprecipitated proteins were destined for radiosequence analysis
250 µCi/ml [
S]Met was added together with
[
H]Ile (400 µCi/ml),
[
H]Leu (400 µCi/ml), or
[
H]Val (400 µCi/ml). At the end of the
labeling period, cells were promptly rinsed twice with serum-free
Dulbecco's modified Eagle's medium and chased with
``cold'' complete medium for various periods of time.
Lysis and Immunoprecipitation
The conditioned
medium was collected and frozen. Cell lysates were prepared by three
rapid freeze-thaw cycles in 1 ml of 25 mM Tris-Cl, pH 7.5, 500
mM NaCl, 5 mM EDTA, 0.5% Triton X-100 (lysis buffer)
containing 200 mM 3,4-dichloroisocoumarin, 40 µM E64, and 4 mM 1,10-phenanthroline (inhibitor mixture).
Prior to immunoprecipitation, the samples of lysates and conditioned
medium were cleared by the addition of 10 µl of a preimmune serum
for 2 h followed by the addition of 30-50 µl of protein
G-Sepharose 4 FF (Pharmacia) for 2 h. The supernatants were incubated
overnight at 4 °C with 10 µl of a relevant specific antiserum.
The next day 30-50 µl of protein G-Sepharose 4 FF (Pharmacia)
was added and incubated for 2 h before the immunoprecipitates were
collected by gentle centrifugation. The immunoprecipitates were then
washed several times with 50 mM Tris-Cl, pH 7.5, 1 M
NaCl, 5 mM EDTA, 0.5% Triton-100, and 10 mM Tris-Cl,
pH 8, 1 mM EDTA. The immunoprecipitates were released from the
protein G-Sepharose 4 FF (Pharmacia) by boiling in SDS sample buffer or
by 100 mM glycine-HCl, pH 2.7.Radiosequence Analysis
Radiosequence analysis was
performed essentially as described previously (Salvesen and Enghild,
1990). Briefly, following immunoprecipitation and SDS-PAGE the S and
H double labeled proteins were
electrotransferred to Immobilon
membranes. The proteins
were identified by autoradiography and bands of interest were excised
and analyzed by automated Edman degradation in an Applied Biosystems
477A sequencer. The anilinothiazolinone-amino acids released after each
cycle of Edman degradation were collected and counted for
S and
H radioactivity. In the experiments
destined for radiosequence analysis the metabolic labelings were
performed using appropriate radioactive amino acids found within the
first 20 N-terminal residues of the mature proteins. Subsequent
radiosequence analysis of the bands and release of radioactive
anilinothiazolinone-amino acid in the expected cycle of Edman
degradation provided unequivocal identification of the protein band.
Polyacrylamide Gel Electrophoresis
The
supernatants from SDS-treated immunoprecipitates were recovered by
centrifugation and run in SDS-PAGE in 5-15% gradient gels (10 cm
10 cm
1.5 mm) using the glycine,
2-amino-2-methyl-1,3-propanediol/HCl system described by Bury(1981).
Gels were stained, destained, equilibrated in 1 M sodium
salicylate (Chamberlain, 1979), dried, and fluorographed for 1-3
days at -70 °C. Other gels were stained, dried, and subjected
to imaging on a PhosphorImager 410A (Molecular Dynamics).
Immunoprecipitates for radiosequence analysis were transferred to
Problott
membranes (Matsudaira, 1987). Following
electrophoresis, the Problott
membranes were dried and
exposed directly to x-ray film overnight at -70 °C.
Other Techniques
NaOH treatment of
immunoprecipitates of purified proteins was employed to dissociate the
PGP cross-link (Enghild et al., 1991). Western blotting was
performed as described by Enghild et al.(1989).
HepG2 Cells Do not Secrete Mature Bikunin
Proteins
Initial investigations into the biosynthesis of the
bikunin proteins were targeted toward the examination of HepG2 cell
secretory products. Cultured cells were maintained in serum-free medium
to ensure that the detected polypeptides were secreted by the HepG2
cells and not exogenous constituents of added fetal bovine serum. HepG2
cell conditioned medium was subjected to SDS-PAGE and the bikunin
proteins were visualized by immunoblot analysis using antisera specific
to the individual components. This allowed us to discern and identify
the specific polypeptide chains HC1, HC2, HC3, bikunin,
m, and the precursors of these proteins (see Fig. 1). The presence of the PGP cross-link was probed by gentle
NaOH induced dissociation (Enghild et al., 1991). This
property was employed to probe for PGP-mediated chain assembly of the
bikunin proteins. Human plasma (Fig. 2, panel A) and
HepG2 cell serum-free medium (Fig. 2, panel B) were
analyzed and a comparison of the immunoreactivity and the NaOH induced
dissociation pattern demonstrate that HepG2 cells do not correctly
assemble the bikunin proteins I
I and P
I. HepG2 cells do not
produce HC1, a component of I
I; however, they do secrete
m, bikunin, HC2 precursor, HC3 precursor, and a
heterogeneous high molecular weight protein with reactivity toward HC2
and bikunin (Fig. 2, panel B, see bracket).
These results suggest that HepG2 cells are incapable of synthesizing
the bikunin proteins found in plasma.
I reacts with anti-HC1, anti-HC2, and bikunin
antisera. Similarly, HC3 and bikunin antiserum reacted with P
I.
The I
I antiserum recognized HC1, HC2, and bikunin. Since P
I
contain bikunin this protein is also recognized by the I
I
antiserum. The C-terminal extension antisera did not identify any
plasma proteins. The expected dissociation of I
I and P
I
following NaOH treatment is observed. Panel B, an examination
of the immunoblots suggest that HepG2 cells are not producing the
bikunin proteins found in human plasma. We did not find evidence for
the production of HC1, thus I
I cannot be produced (see Fig. 1). Moreover, the cells appear to secrete precursors of HC2
and HC3. Some high molecular weight protein material with reactivity
toward bikunin and HC2 was observed. This heterogeneous protein is not
found in normal human plasma.
Pulse-Chase Analysis of Bikunin Portein
Biosynthesis
In an attempt to identify a cell line that secreted
normal bikunin proteins we examined other transformed human liver cell
lines. However, Hep3B, SK-Hep1, and Chang liver cell lines did not
secrete normal bikunin proteins either (data not shown). The failure of
authentic assembly by transformed cell lines prompted us to investigate
the biosynthesis in primary human hepatocytes employing a pulse-chase
protocol. This allowed us to analyze transient intracellular
biosynthetic processing events including (i) the processing of the
m-bikunin tandem protein, (ii) processing of the
putative heavy chain N-terminal propeptide, (iii) cleavage of the
C-terminal extensions of the heavy chain precursors, and (iv) assembly
of heavy chains and addition of carbohydrate. N-terminal radiosequence
analysis (Salvesen and Enghild, 1990) of intracellular polypeptides
made it feasible to positively identify processing intermediates.
Furthermore, the technique provided unequivocal evidence that the
identified polypeptides are indeed components of the bikunin protein
and not immunologic cross-reactive species.
Primary Hepatocytes Produces the Bikunin Proteins Found
in Plasma
Since the transformed hepatocyte cell lines failed to
produce the authentic bikunin proteins we investigated the biosynthesis
in primary human hepatocytes. Due to the limited availability of viable
cells we used the bikunin antiserum for most of the
immunoprecipitations, since bikunin is a component of all the proteins
of interest (see Fig. 1). The properties of the
immunoprecipitated proteins could subsequently be established by
SDS-PAGE, stability to 50 mM NaOH, and radiosequence analysis. m, suggesting that the band represents
the
m-bikunin tandem protein (Fig. 3A, left
panel). Radiosequence analysis of the 43-kDa band observed in the
conditioned medium identified the N-terminal of bikunin (Fig. 9, panel C). Apparently, the primary human hepatocytes secreted
large amounts of glycosylated free bikunin (Fig. 3A, right
panel). This has also been observed with primary rat hepatocytes
(Sjberg and Fries, 1992). The appearance and size
of P
I (125 kDa) and I
I (225 kDa) in SDS-PAGE (Fig. 3A, right panel) and in SDS-PAGE following
treatment with 50 mM NaOH (Fig. 3B, right
panel) mirrored the behavior of the bikunin proteins found in
plasma (Fig. 2A). Furthermore, radiosequence analysis
of the two bands identified the expected sequences of I
I and
P
I (Fig. 10, panels A, B and C, D).
S]Met for 5 min and chased for the
indicated times in radiolabel free medium. Following the chase, bikunin
was immunoprecipitated from cell lysates and conditioned medium using a
specific bikunin antiserum. The products were analyzed by SDS-PAGE
followed by imaging on a PhosphorImager. Control lanes contain
immunoprecipitates performed using a preimmune antiserum. The open
arrows indicate bands analyzed by radiosequencing. Panel
A, the 45-kDa
m-bikunin tandem protein (left
panel) was identified in the cell lysate by radiosequence
analysis. The intracellular assembly of the bikunin proteins are
apparent 15-30 min after the onset of the biosynthesis. Following
assembly the bikunin proteins are secreted. The secreted 225-kDa band
and the 125-kDa band were identified as I
I and P
I,
respectively, by radiosequence analysis. The 43-kDa band was identified
as bikunin. Panel B, the bikunin immunoprecipitates were
treated with 50 mM NaOH, and run in SDS-PAGE. Both the
intracellular and secreted bikunin protein dissociated following NaOH
treatment consistent with the presence of the PGP cross-link. It is
apparent that the bikunin proteins produced by the primary human
hepatocytes are analogous to the proteins present in normal human
plasma.
S]Met (closed bars),
[
H]Leu, and [
H]Ile (open bars). To assist in the analysis of the data the
expected N-terminal amino acid sequence of the mature proteins are
shown in each panel. The analysis were designed to identify only one
protein chain during each analysis. If more than one protein chain was
expected in a particular protein band, we performed several analysis
employing different radioactive amino acid. The results, obtained
during radiosequence analysis of the same protein immunoprecipitated in
different experiments, were very similar and only one example of these
analysis are shown. Panel A,Fig. 3A, left
panel, 45-kDa
m-bikunin band; Fig. 4, left panel, 45-kDa
m-bikunin band; Fig. 5, left panel, 45-kDa
m-bikunin
band. Panel B,Fig. 4, right panel, 32-kDa
free
m band. Panel C,Fig. 3A,
right panel, 43-kDa bikunin band; Fig. 5, right
panel, 43-kDa bikunin band and diffuse 200-kDa HC2/bikunin band. Panel D, Fig. 6A, left panel, 100-kDa HC2
precursor and 75-kDa C-terminal processed HC2. Panel E, Fig. 5, right panel, diffuse 200-kDa HC2/bikunin band;
and Fig. 6A, right panel, diffuse 200-kDa HC2/bikunin
band. Fig. 6A, right panel, 100-kDa HC2 precursor and
75-kDa C-terminal processed HC2. Panel F, Fig. 7, A and B, left panels, 100-kDa HC3 precursor band. Panel
G, Fig. 7, A and B, right panels, 100-kDa
HC3 precursor band.
S]Met/[
H]Leu
and [
S]Met/[
H]Val were
performed to identify the three components of I
I. Similarly, as
shown in the two lower panels, we performed two double label
experiments to identify the two components of P
I. The radioactive
amino acids used for the metabolic labeling are shown and the
N-terminal protein sequence of the mature bikunin proteins purified
from plasma are indicated in each panel. In the two upper panels the sequences from top to bottom are bikunin, HC1, and HC2. In the two bottom panels the sequence of bikunin (top) and
the HC3 (bottom) are shown. Radioactive counts per minute
(cpm) associated with each cycle of Edman degradation is plotted for
[
S]Met (closed bars),
[
H]Leu, and [
H]Ile (open bars). The amino acids employed for each particular
radioactive labeling are indicated in the panels. To assist in the
analysis of the data, the expected N-terminal amino acid sequence of
the mature proteins are shown in each panel. Panels A and B,Fig. 3A, right panel, 225-kDa I
I band. Panels C and D, Fig. 3A, right panel, 125-kDa P
I band.
m in HepG2 cells. The 45-kDa
m-bikunin tandem protein was detected in the lysates
and identified by radiosequence analysis. The diffuse band in the left panel (see bracket) incorporated radioactivity
during metabolic sodium [
S]sulfate pulse-chase
labeling experiments (data not shown). This indicates that GAG is added
to the
m-bikunin tandem protein before cleavage of the
protein (left panel, see bracket). The tandem protein
is proteolytically processed and
m and bikunin are
secreted 15-30 min after the onset of biosynthesis. Free
m was identified by radiosequencing and is seen in the
conditioned medium as a 25-kDa band (right panel). In the
control lanes a preimmune antiserum was used for the
immunoprecipitation step. The open arrows indicate bands
analyzed by radiosequencing.
m-bikunin tandem protein was
detected in the lysate as a 45-kDa band, similar to the band detected
using
m antiserum (Fig. 4). The diffuse band in
the left panel (see bracket) incorporated
radioactivity during metabolic sodium [
S]sulfate
pulse-chase labeling experiments (data not shown). The proteolytic
processing of the two proteins is apparent 15 min after the onset of
the biosynthesis. Several heterogeneous higher molecular weight bands
appear at the time in the cell lysate. These bands most likely
represent incomplete glycosylation and chain assembly. Free bikunin
appear in the conditioned medium early in the biosynthesis. The sizes
of the intracellular
m-bikunin (45 kDa) and the
secreted free bikunin (43 kDa) observed in the medium are similar. The
larger than expected site of the free bikunin is most likely due to the
addition of GAG. The diffuse bands (see bracket, right panel),
secreted into the medium 3 h after onset of the biosynthesis, was
identified as bikunin and HC2 after radiosequence analysis. The open arrows indicate bands analyzed by
radiosequencing.
m-bikunin band
incorporated radioactivity during metabolic sodium
[
S]sulfate pulse-chase labeling experiments
(data not shown), suggesting that the heterogeneity was due to the
addition of sulfated GAG (DeLuca et al., 1973; Delfert and
Conrad, 1985; Lohmander et al., 1986; von
Wrtemberg and Fries, 1989) to
m-bikunin before the proteolytic separation of the two
proteins. The assembly of the bikunin proteins was observed 15-30
min after the onset of the biosynthesis (Fig. 3A, left
panel). Following the assembly, free bikunin was observed in the
conditioned medium (Fig. 3B). The cleavage of
m-bikunin and assembly of heavy chain precursors and
bikunin appear to happen at the same time since free bikunin was never
detected in the cell lysate, as determined by radiosequence analysis.
Since the proteolytic processing of
m-bikunin occurs
in the trans-Golgi network or the secretory vesicles (Barr, 1991; Bratt et al., 1993) we suspect that the formation of the PGP
cross-link likewise occurs late in the secretary pathway.
I and I
I
are assembled intracellularly in the secretory vesicles and secreted.
In contrast to the transformed cell lines, the biosynthetic machinery
required for the correct synthesis, processing, assembly, and secretion
of the bikunin proteins are present in primary human hepatocytes.
Pulse-Chase Analysis of the PGP-mediated Chain
Assembly
The primary hepatocytes produce bikunin proteins
identical to the proteins identified in plasma. This includes the
formation of the PGP cross-link as evident by the dissociation of the
protein chains following gentle NaOH treatment (Fig. 3B,
panel B). The PGP cross-linking bikunin and heavy chains is
mediated by a chondroitin-4-sulfate chain originating from a typical O-glycosidic link to Ser of bikunin. The actual
cross-link is formed by esterification of the heavy chain C-terminal
Asp residue
-carbonyl and carbon-6 of an internal N-acetylgalactosamine of the GAG chain. The molecular
structure of the PGP cross-link is apparently shared by all bikunin
proteins (Enghild et al., 1991, 1993; Morelle et al.,
1994). Of note, the cDNA sequence of the bikunin protein heavy chains
encode a putative 30-kDa C-terminal extension (see Fig. 1). This
extension is not present in the mature assembled bikunin proteins
(Enghild et al., 1991, 1993).
-carbon of this Asp residue, the C-terminal extension
is displaced either before or at the same time as the formation of the
cross-link. The displacement of the C-terminal extension may involve
(i) proteolysis of the Asp-Pro peptide bond by a proteinase and
subsequent addition of GAG to the
-carbonyl by another enzyme or
(ii) cleavage of the Asp-Pro bond and simultaneous addition of GAG
catalyzed by the same novel enzyme. Alternatively, (iii) it is also
possible that the formation of the PGP cross-link is a nonenzymatic
process as previously suggested (Enghild et al., 1991).
HepG2 Cells Secrete
The pulse-chase protocol and
m
m-specific antiserum were employed to investigate the
biosynthesis of the
m-bikunin tandem protein produced
by HepG2 cells (Fig. 4). Radiosequencing of immunoprecipitated
m demonstrated the presence of the
m-bikunin tandem protein in the cell lysates of HepG2
cultures (Fig. 9, panel A). Approximately 15 min after
the onset of protein synthesis,
m-bikunin was cleaved
as demonstrated by the appearance of free
m in cell
lysate immunoprecipitates (Fig. 4, left panel, 15-min
chase, 32-kDa band). Prior to secretion of the proteins, diffuse
m-bikunin bands became apparent (Fig. 4, left panel, see bracket). This band incorporated
sulfate, a component of most GAG, during sodium
[
S]sulfate metabolic labeling experiments (data
not shown). Apparently GAG is added to
m-bikunin
before the proteolytic cleavage event as seen with the primary human
hepatocytes (Fig. 3). The cleaved
m is only
transiently observed just prior to its extracellular translocation. It
appears in the conditioned medium immediately following the proteolytic
cleavage reaction (Fig. 4, right panel). The identity
of these bands as
m-bikunin cleavage products was
confirmed by radiosequence analysis of both intracellular and secreted
m species (Fig. 9, panels A and B).
m-bikunin in HepG2 cells is similar to the processing
seen in the primary hepatocytes. We conclude that the components
required for the proteolytic cleavage of the
m-bikunin
tandem protein is retained by HepG2 cells.
The Intracellular Assembly of Bikunin and Heavy Chains Is
Incomplete in HepG2 Cells
The pulse-chase analysis using a
bikunin-specific antiserum allowed a more direct comparison to the
biosynthesis of the bikunin proteins in primary human hepatocytes.
Similar to the data obtained by immunoblotting (see Fig. 2B), the analysis of anti-bikunin
immunoprecipitates demonstrates that wild type bikunin proteins are not
produced by HepG2 cells (Fig. 5, right panel). As seen
in the left panel of Fig. 5, SDS-PAGE of
immunoprecipitates from cell lysates and conditioned medium results in
a variety of discrete and heterogeneous bands. N-terminal radiosequence
analysis (see open arrow) was used to confirm the identity of
the 45-kDa band in the left panel as
m-bikunin tandem protein (Fig. 9, panel
A). The intracellular proteolytic processing of the
m-bikunin tandem protein was described in the previous
section and similar data was obtained using the bikunin antiserum.
Briefly, the processing of the
m-bikunin tandem
protein was observed 15 min after the onset of protein synthesis (Fig. 5, left panel, 15 min chase). The processing
appeared to be somewhat incomplete as
m-bikunin is
observed in the cell lysate several hours after the onset of
biosynthesis (Fig. 5, left panel). Several
heterogeneous higher molecular weight protein bands become visible
before the cleavage of the
m-bikunin tandem protein (Fig. 5, left panel). These diffuse bands (Fig. 5, left panel, see brackets)
incorporated radioactivity during metabolic sodium
[
S]sulfate pulse-chase labeling experiments
(data not shown) suggesting that the heterogeneity is due to the
addition of sulfated GAG. We suspect that the addition of GAG to
bikunin alters the migration in SDS-PAGE due to the large hydrodynamic
volume of the GAG.
m-bikunin tandem
protein prior to proteolytic processing (Fig. 5, left
panel). The 43-kDa bikunin immunoprecipitated from HepG2
conditioned medium does not contain the
m polypeptide
as confirmed by radiosequence analysis (Fig. 9, panel
C). The migration of secreted bikunin (43 kDa) is higher than
expected and is most likely due to the addition of GAG as described
above.
I, P
I, and bikunin, respectively (Fig. 2A and Fig. 3A, right panel).
Whereas a 43-kDa band representing bikunin is observed, discrete bands
at 225 and 125 kDa are not observed in HepG2 conditioned media (Fig. 5, right panel), confirming that HepG2 cells are
not capable of assembling mature bikunin proteins.. However, the
processing of
m-bikunin, proteolytic processing, and
addition of GAG appear to be maintained in HepG2 cells.
The HC2 Translation Product in HepG2 Cells Contains the
C-terminal 30-kDa Extension
To examine the processing of the
heavy chains and the addition of GAG to the C-terminal Asp residue we
prepared specific antisera to each heavy chain as well as specific
antisera directed against synthetic peptide components of the putative
C-terminal extensions of the heavy chain precursor.-carbonyl of the C-terminal Asp by
another novel enzyme. However, in HepG2 cells some of the
proteolytically cleaved mature HC2 escape the second cross-linking
reaction. Apparently, in this defective cell line some of the
proteolytically processed HC2 evades cross-linking.
HepG2 Cells Do Not Produce P
Anti-HC3 immunoprecipitates from cell lysates and
conditioned medium revealed a single polypeptide of 100 kDa (Fig. 7). The identity of this polypeptide as HC3 was confirmed
by radiosequence analysis (Fig. 9, panels F and G). Analogous to the synthesis of HC2, the predicted
N-terminal propeptide was not identified in the cell lysates (0 min
chase time). The propeptides are probably removed very early in
biosynthesis, either prior to, or concomitant with the removal of the
signal peptides. HC3 immunoprecipitated from both HepG2 cell lysate and
conditioned medium, migrated in SDS-PAGE as a band of 100 kDa
throughout the chase (Fig. 7, A and B).
Moreover, antiserum to the HC3 C-terminal extension precipitates a
polypeptide from both cell lysates and conditioned medium that migrates
in SDS-PAGE as a band of 100 kDa. This suggests that unlike HC2, no
C-terminal processing occurs with HC3 in these cells. As before, the
identity of these polypeptides was confirmed by radiosequence analysis (Fig. 9, panels F and G). No other HC3 related
products were detected, indicating that only HC3 precursor is produced
by HepG2 cells.I, but Secrete HC3
Precursor
Are Primary Hepatocytes Secreting Heavy Chain
Precursors?
As described above, transformed liver cells secrete
large amounts of heavy chain precursors. Precursors of HC3 are not seen
in normal plasma; which may reflect the possibility that (i) they are
not secreted by normal hepatocytes, (ii) they are sequestered or
removed from the plasma rapidly, or (iii) the heavy chain precursors
are assembled into mature bikunin protein with high efficiency outside
the hepatocyte.I ( Fig. 3and Fig. 8). We were unable to specifically
immunoprecipitate proteins in the conditioned medium using this HC3
precursor specific antiserum. The data suggests that the primary human
hepatocytes are not secreting HC3 precursor and that all components of
the biosynthetic machinery required for authentic assembly of the
bikunin proteins are present intracellularly in primary human
hepatocytes. Consequently, we conclude that the bikunin proteins
detected in human plasma are assembled intracellularly in hepatocytes.
I, approximately 30 min after the onset of biosynthesis. The
displaced 30-kDa C-terminal extension was not detected in the lysate or
in the conditioned medium. Immunoprecipitation from the conditioned
medium did not show any significant bands suggesting that primary
hepatocytes are not secreting free heavy chains. This is in agreement
with the lack of free heavy chains in normal human plasma (Fig. 2). The open arrows indicate bands analyzed by
radiosequencing.
I since HC1 is a component of this
protein, (ii) HepG2 cells do not secrete P
I, but they do secrete
the HC3 precursor alone. Additionally (iii) the HepG2 cells secrete
m, free bikunin, HC2, HC2 precursors, and a
heterogeneous protein composed of HC2 and bikunin (Fig. 2,
4-6, and 9). The cross-linking of bikunin and HC2 is most likely
mediated by the PGP cross-link as it can be dissociated by gentle NaOH
treatment. The formation of the PGP cross-link by HepG2 cells was
significantly impaired since the cell secreted large amounts of heavy
chain precursor. The secretion of mature unassembled HC2 suggests that
the formation of the PGP cross-link involves at least two steps: (i) a
proteolytic cleavage of the Asp-Pro peptide bond and (ii) followed by
the addition of GAG to the
-carbonyl of the accessible C-terminal
Asp residue by a novel enzyme.
of bikunin
before the proteolytic mediated dissociation of
m-bikunin. Subsequent to the addition of GAG, the
bikunin proteins were assembled intracellularly and immediately
secreted. This suggests that cleavage of the
m-bikunin
protein and formation of the PGP cross-link occur in the latter part of
the biosynthetic pathway, most likely in the secretory vesicles. In
addition to the secretion of assembled bikunin protein, the primary
cells secreted large amounts of free
m and
glycosylated bikunin. Free bikunin is not found in significant levels
in plasma. This may be due to rapid renal filtration of this protein.
If this is true, free bikunin could be the source of urinary trypsin
inhibitor (Balduyck et al., 1986). Alternatively, free bikunin
may enter other compartments of the body (Chawla et al., 1992;
Chen et al., 1992; Castillo and Templeton 1993; Wisniewski et al., 1994; Chen et al., 1994).
I, inter-
-inhibitor;
m,
-microglobulin; GAG,
glycosaminoglycan; HC, heavy chain; IHRP, inter-
-trypsin inhibitor
family heavy chain related protein; P
I, pre-
-inhibitor; PGP
cross-link, protein glycosaminoglycan protein cross-link; PAGE,
polyacrylamide gel electrophoresis.
We thank David Rubenstein, Tim Oury, Charleen Chu,
SChristensen, and Eva Olsen for helpful discussions and
insightful comments on the manuscript. We also thank Salvatore Pizzo
for support and encouragement throughout the course of this study.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.