©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biosynthesis of Bikunin Proteins in the Human Carcinoma Cell Line HepG2 and in Primary Human Hepatocytes
POLYPEPTIDE ASSEMBLY BY GLYCOSAMINOGLYCAN (*)

(Received for publication, April 18, 1995; and in revised form, May 24, 1995)

Ida B. Th Jan J. Enghild (§)

From the Duke University Medical Center, Department of Pathology, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
SUMMARY
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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-alpha-inhibitor (IalphaI) (^1)(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 alpha3 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).


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 of bikunin and one or two heavy chains covalently bound to this carbohydrate chain. Bikunin is encoded by a tandem alpha(1)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.



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) IalphaI, composed of HC1, HC2, and bikunin, (ii) pre-alpha-inhibitor (PalphaI), 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-alpha-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, alpha(1)-microglobulin (alpha(1)m) and bikunin. A short dibasic connecting peptide separates the two proteins. Proteolysis of the connecting peptide release alpha(1)m and bikunin (Kaumeyer et al., 1986; Barr, 1991; Bratt et al., 1993, 1994). In humans, alpha(1)m is found in plasma complexed with IgA and albumin (Tejler and Grubb, 1976). In the rat, alpha(1)m is associated with alpha(1)-inhibitor 3 and fibronectin (Falkenberg et al., 1990, 1994).

Although the bikunin proteins are composed of more than one subunit, they resist dissociation in reduced SDS-PAGE. However, the subunits of IalphaI (Jessen et al., 1988; Enghild et al., 1989), PalphaI, 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 alpha-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).

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.


EXPERIMENTAL PROCEDURES

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), [^3H]leucine (179 Ci/mM), [^3H]isoleucine (111 Ci/mM), and [^3H]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. IalphaI, PalphaI, 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 IalphaI, PalphaI, 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(2) 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 [^3H]Ile (400 µCi/ml), [^3H]Leu (400 µCi/ml), or [^3H]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 ^3H 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 ^3H 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).


RESULTS AND DISCUSSION

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, alpha(1)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 IalphaI and PalphaI. HepG2 cells do not produce HC1, a component of IalphaI; however, they do secrete alpha(1)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.


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 IalphaI reacts with anti-HC1, anti-HC2, and bikunin antisera. Similarly, HC3 and bikunin antiserum reacted with PalphaI. The IalphaI antiserum recognized HC1, HC2, and bikunin. Since PalphaI contain bikunin this protein is also recognized by the IalphaI antiserum. The C-terminal extension antisera did not identify any plasma proteins. The expected dissociation of IalphaI and PalphaI 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 IalphaI 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 alpha(1)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.

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 alpha(1)m, suggesting that the band represents the alpha(1)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 PalphaI (125 kDa) and IalphaI (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 IalphaI and PalphaI (Fig. 10, panels A, B and C, D).


Figure 3: Biosynthesis of bikunin in primary human hepatocytes using a pulse-chase protocol. The cells were metabolically labeled with [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 alpha(1)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 IalphaI and PalphaI, 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.




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 [S]Met (closed bars), [^3H]Leu, and [^3H]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 alpha(1)m-bikunin band; Fig. 4, left panel, 45-kDa alpha(1)m-bikunin band; Fig. 5, left panel, 45-kDa alpha(1)m-bikunin band. Panel B,Fig. 4, right panel, 32-kDa free alpha(1)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.




Figure 10: Radiosequence analysis of proteins immunoprecipitated from primary human hepatocytes. Two double label experiments [S]Met/[^3H]Leu and [S]Met/[^3H]Val were performed to identify the three components of IalphaI. Similarly, as shown in the two lower panels, we performed two double label experiments to identify the two components of PalphaI. 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), [^3H]Leu, and [^3H]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 IalphaI band. Panels C and D, Fig. 3A, right panel, 125-kDa PalphaI band.




Figure 4: Pulse-chase kinetic analysis of alpha(1)m in HepG2 cells. The 45-kDa alpha(1)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 alpha(1)m-bikunin tandem protein before cleavage of the protein (left panel, see bracket). The tandem protein is proteolytically processed and alpha(1)m and bikunin are secreted 15-30 min after the onset of biosynthesis. Free alpha(1)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.




Figure 5: Pulse-chase analysis of bikunin synthesis in HepG2 cells. The alpha(1)m-bikunin tandem protein was detected in the lysate as a 45-kDa band, similar to the band detected using alpha(1)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 alpha(1)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.




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 alpha(1)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 alpha(1)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 alpha(1)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 alpha(1)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.

The data described above suggest that the liver, in vivo, secrete the assembled bikunin proteins. The bikunin proteins PalphaI and IalphaI 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 alpha-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).

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 alpha-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 alpha-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).

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.

HepG2 Cells Secrete alpha(1)m

The pulse-chase protocol and alpha(1)m-specific antiserum were employed to investigate the biosynthesis of the alpha(1)m-bikunin tandem protein produced by HepG2 cells (Fig. 4). Radiosequencing of immunoprecipitated alpha(1)m demonstrated the presence of the alpha(1)m-bikunin tandem protein in the cell lysates of HepG2 cultures (Fig. 9, panel A). Approximately 15 min after the onset of protein synthesis, alpha(1)m-bikunin was cleaved as demonstrated by the appearance of free alpha(1)m in cell lysate immunoprecipitates (Fig. 4, left panel, 15-min chase, 32-kDa band). Prior to secretion of the proteins, diffuse alpha(1)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 alpha(1)m-bikunin before the proteolytic cleavage event as seen with the primary human hepatocytes (Fig. 3). The cleaved alpha(1)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 alpha(1)m-bikunin cleavage products was confirmed by radiosequence analysis of both intracellular and secreted alpha(1)m species (Fig. 9, panels A and B).

These observations suggest that processing of alpha(1)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 alpha(1)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 alpha(1)m-bikunin tandem protein (Fig. 9, panel A). The intracellular proteolytic processing of the alpha(1)m-bikunin tandem protein was described in the previous section and similar data was obtained using the bikunin antiserum. Briefly, the processing of the alpha(1)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 alpha(1)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 alpha(1)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.

Secreted bikunin migrated in SDS-PAGE as a protein of approximately 43-kDa (Fig. 5, right panel) similar to the 45-kDa intracellular alpha(1)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 alpha(1)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.

Biosynthesis of wild type bikunin proteins should result in the appearance of three polypeptide species of 225, 125, and 43 kDa representing IalphaI, PalphaI, 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 alpha(1)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.

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 alpha-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 PalphaI, but Secrete HC3 Precursor

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.

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.

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 PalphaI ( 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.


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 PalphaI, 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.




SUMMARY

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 IalphaI since HC1 is a component of this protein, (ii) HepG2 cells do not secrete PalphaI, but they do secrete the HC3 precursor alone. Additionally (iii) the HepG2 cells secrete alpha(1)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 alpha-carbonyl of the accessible C-terminal Asp residue by a novel enzyme.

Primary human hepatocytes produce bikunin proteins identical to the proteins detected in plasma (Fig. 3, 8, and 10). GAG is added to Ser of bikunin before the proteolytic mediated dissociation of alpha(1)m-bikunin. Subsequent to the addition of GAG, the bikunin proteins were assembled intracellularly and immediately secreted. This suggests that cleavage of the alpha(1)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 alpha(1)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).


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL49542. 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: Box 3712, Duke University Medical Center, Durham NC., 27710. Tel.: 919-684-2872; Fax: 919-684-2920.

^1
The abbreviations used are: IalphaI, inter-alpha-inhibitor; alpha(1)m, alpha(1)-microglobulin; GAG, glycosaminoglycan; HC, heavy chain; IHRP, inter-alpha-trypsin inhibitor family heavy chain related protein; PalphaI, pre-alpha-inhibitor; PGP cross-link, protein glycosaminoglycan protein cross-link; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

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.


REFERENCES

  1. Balduyck, M., Mizon, C., Loutifi, H., Richet, C., Roussel, P., and Mizon, J.(1986) Eur. J. Biochem.158,417-422 [Abstract]
  2. Barr, P.(1991) Cell66,1-3 [Medline] [Order article via Infotrieve]
  3. Bourguignon, J., Sesboue, R., Diarra-Mehrpour, M., Daveau, M., and Martin, J. P.(1989) Biochem. J.261,305-308 [Medline] [Order article via Infotrieve]
  4. Bourguignon, J., Diarra-Mehrpour, M., Thiberville, L., Bost, F., Sesboue, R., and Martin, J. P.(1993)Eur. J. Biochem.212,771-776 [Abstract]
  5. Bratt, T., Olsson, H., Sjberg E. M., Jergil, B., and , B.(1993)Biochem. Biophys. Acta1157,147-154 [Medline] [Order article via Infotrieve]
  6. Bratt, T., Cedervall, T., and B.(1994)FEBS Lett.354,57-61 [CrossRef][Medline] [Order article via Infotrieve]
  7. Broze, G. J., Jr., Girard, T. J., and Novotny, W. F.(1990)Biochemistry 29,7539-7546 [Medline] [Order article via Infotrieve]
  8. Bury, A.(1981) J. Chromatogr.213,491-500 [CrossRef]
  9. Castillo, G. M., and Templeton, D. M.(1993)FEBS Lett.318,292-296 [CrossRef][Medline] [Order article via Infotrieve]
  10. Camaioni, A., Hascall, V. C., Yanagishita, M., and Salustri, A.(1993)J. Biol. Chem.268,20473-20481 [Abstract/Free Full Text]
  11. Chawla, R. K., Lawson, D. H., Ahmad, M., and Travis, J.(1992)J. Cell. Biochem.50,277-236
  12. Chamberlain, J. P.(1979)Anal. Biochem.98,132-135 [Medline] [Order article via Infotrieve]
  13. Chen, L., Mao, S. J., and Larsen, W. J.(1992)J. Biol. Chem.267,12380-12386 [Abstract/Free Full Text]
  14. Chen, L., Mao, S. J., McLean, L. R., Powers, R. W., and Larsen, W. J.(1994) J. Biol. Chem.269,28282-28287 [Abstract/Free Full Text]
  15. Choi-Miura, N., Sano, Y., Oda, E., Nakono, Y., Tobe, T., Yanagishita, T., Taniyama, M., Katagiri, T., and Tomita, M.(1995)J. Biochem. (Tokyo)117,400-407 [Abstract]
  16. Chu, M. L., Zhang, R. Z., Pan, T. C., Stokes, D., Conway, D., Kuo, H. J., Glanville, R., Mayer, U., Mann, K., Deutzmann, R., and Timpl, R.(1990) EMBO J.9,385-393 [Abstract]
  17. Delfert, D. M., and Conrad, H. E.(1985)J. Biol. Chem.260,14446-14451 [Abstract/Free Full Text]
  18. DeLuca, S., Richomond, M. E., and Silbert, J. E.(1973)Biochemistry 12,3911-3915 [Medline] [Order article via Infotrieve]
  19. Diarra-Mehrpour, M., Bourguignon, J., Bost, F., Sesboue, R., Muschio, F. Sarafan, N., and Martin, J. P.(1992)Biochem. Biophys. Acta 1132,114-118 [Medline] [Order article via Infotrieve]
  20. Enghild, J. J., Th, I. B., Pizzo, S. V., and Salvesen, G.(1989) J. Biol. Chem.264,15975-15981 [Abstract/Free Full Text]
  21. Enghild, J. J., Th, I. B., Pizzo, S. V., and Salvesen, G. (1990) in Serine Proteases and Serpins in the Nervous System (Festoff, B., eds) pp. 79-91, Plenum Press, New York
  22. Enghild, J. J., Salvesen, G., Hefta, S. A., Th, I. B., Rutherfurd, S., and Pizzo, S. V.(1991)J. Biol. Chem. 266,747-751 [Abstract/Free Full Text]
  23. Enghild, J. J., Salvesen, G., Th, I. B., Valnickova, Z., Pizzo, S. V., and Hefta, S. A.(1993)J. Biol. Chem.268,8711-8716 [Abstract/Free Full Text]
  24. Falkenberg, C., Grubb, A., and , B.(1990) J. Biol. Chem.265,16150-16157 [Abstract/Free Full Text]
  25. Falkenberg, C., Enghild, J. J., Th, I. B., Salvesen, G., and , B.(1994)Biochem. J.301,745-751 [Medline] [Order article via Infotrieve]
  26. Gebhard, W., Schreitmller, T., Hochstrasser, K., and Wachter, E.(1988)FEBS Lett.229,63-67 [CrossRef][Medline] [Order article via Infotrieve]
  27. Gebhard, W., Schreitmller, T., Hochstrasser, K., and Wachter, E.(1989)Eur. J. Biochem.181,571-576 [Abstract]
  28. Gebhard, W., Hochstrasser, K., Fritz, H., Enghild, J. J., Pizzo, S. V., and Salvesen, G.(1990)Biol. Chem. Hoppe-Seyler 371,113-122
  29. Greenspan, D. S.(1993)Hum. Mol. Genet.2,273-278 [Abstract]
  30. Heron, A., Bourguignon, J., Calle, A., Borghi, H., Sesboue, R., Diarra-Mehrpour, M., and Martin, J. P.(1994)Biochem. J.302,573-580 [Medline] [Order article via Infotrieve]
  31. Huang, L., Yoneda, M., and Kimata, K. A.(1993)J. Biol. Chem.268,26725-26730 [Abstract/Free Full Text]
  32. Hunkapiller, M. W., Lujan, E., Ostrander, F., and Hood, L. E.(1983) Methods Enzymol.91,227-236 [Medline] [Order article via Infotrieve]
  33. Itoh, H., Ide, H., Ishikawa, N., and Nawa, Y.(1994)J. Biol. Chem. 269,3818-3822 [Abstract/Free Full Text]
  34. Jessen, T. E., Faarvang, K. L., and Ploug, M.(1988)FEBS Lett.230,195-200 [CrossRef][Medline] [Order article via Infotrieve]
  35. Kagen, A., and Glick, M.(1979) in ``Oxytocin'', Methods of Hormone Radioimmunoassay (Jaffa, B. B., and Behrman, H. R., eds) pp. 328-329, Academic Press, New York
  36. Kaumeyer, J. F., Polazzi, J. O., and Kotick, M. P.(1986)Nucleic Acids Res.14,7839-7850 [Abstract]
  37. Kido, H., Yokogoshi, Y., and Katunuma, N.(1988)J. Biol. Chem.263,18104-18107 [Abstract/Free Full Text]
  38. Kitagawa, T., and Aikawa, T.(1976)J. Biochem. (Tokyo) 79,233-239 [Abstract]
  39. Lohmander, L. S., Hascall, V. C., Yanagishita, M., Kuettner, K. E., and Kimura, J. H.(1986)Arch. Biochem. Biophys.250,211-227 [Medline] [Order article via Infotrieve]
  40. Matsudaira, P.(1987)J. Biol. Chem.262,10035-10038 [Abstract/Free Full Text]
  41. Morelle, W., Capon, C., Balduyck, M., Sautiere, P., Kouach, M., Michalski, C., Fournet, B., and Mizon, J.(1994)Eur. J. Biochem.221,881-888 [Abstract]
  42. , L.(1992)Int. J. Biochem.24,215-222 [Medline] [Order article via Infotrieve]
  43. Perlmutter, D. H., Dinarello, C. A., Punsal, P. I., and Colten, H. R.(1986) J. Clin. Invest.78,1349-1354 [Medline] [Order article via Infotrieve]
  44. Rubenstein, D. S., Th, I. B., Pizzo, S. V., and Enghild, J. J.(1993) Biochem. J.290,85-95 [Medline] [Order article via Infotrieve]
  45. Saguchi, K., Tobe, T., Hashimoto, K., Sano, Y., Nakano, T., Miura, N., Tomita, M.(1995) J. Biochem. (Tokyo)117,14-18 [Abstract]
  46. Salier, J. P.(1990)Trends Biochem. Sci.15,435-439 [CrossRef][Medline] [Order article via Infotrieve]
  47. Salvesen, G., and Enghild, J. J.(1990)Biochemistry29,5304-5308 [Medline] [Order article via Infotrieve]
  48. Sjberg, E. M., and Fries, E.(1992)Arch. Biochem. Biophys. 295,217-222 [Medline] [Order article via Infotrieve]
  49. Tanzi, R. E., McClatchey, A. I., Lamperti, E. D., Villa-Komaroff, L., and Gusella, J. F.(1988)Nature33,528-530
  50. Tejler, L., and Grubb, A. O.(1976)Biochim. Biophys. Acta.439,82-94 [Medline] [Order article via Infotrieve]
  51. von Wrtemberg, M. M., and Fires, E.(1989) Biochemistry28,4088-4093 [Medline] [Order article via Infotrieve]
  52. Wisniewski, H. G., Burgess, W. H., Oppenheim, J. D., and Vilcek, J.(1994) Biochemistry33,7423-7429 [Medline] [Order article via Infotrieve]
  53. Wun, T. C., Kretzmer, K. K., Girard, T. J., Miletich, J. P., and Broze, G. J., Jr.(1988) J. Biol. Chem.263,6001-6004 [Abstract/Free Full Text]

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