©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A 110-amino Acid Region within the A1-domain of Coagulation Factor VIII Inhibits Secretion from Mammalian Cells (*)

Kimberly A. Marquette (2), Debra D. Pittman (2), Randal J. Kaufman (1)(§)

From the (1) From The Howard Hughes Medical Institute and the Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48105 and (2) The Genetics Institute, Inc., Cambridge, Massachusetts 02140

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Factor VIII is the coagulation factor deficient in the X-chromosome-linked bleeding disorder hemophilia A. Factor VIII is homologous to blood coagulation factor V, both having a domain structure of A1-A2-B-A3-C1-C2. Previous transfection studies demonstrated that factor VIII is 10-fold less efficiently expressed than the homologous coagulation factor, factor V. The inefficient expression correlated with interaction of the factor VIII primary translation product with the protein chaperonin BiP in the lumen of the endoplasmic reticulum. In contrast, factor V was not detected in association with BiP and was secreted efficiently. To determine whether specific amino acid sequences within factor VIII inhibit secretion, we have studied the secretion of factor VIII deletion and factor VIII/factor V chimeric proteins upon transient transfection of COS-1 monkey cells. A chimeric factor VIII protein that contained the A1- and A2-domains of factor V was secreted with a similar efficiency as wild-type factor V, whereas the complementary chimera having the A1- and A2-domains of factor VIII was secreted with low efficiency, similar to wild-type factor VIII. These results suggested that sequences within the A1- and A2-domains were responsible for the low secretion efficiency of factor VIII. Secretion of A1-domain-deleted factor VIII was increased approximately 10-fold compared to wild-type factor VIII or A2-domain-deleted factor VIII. Expression of the factor VIII A1-domain alone did not yield secreted protein, whereas expression of the factor VIII A2-domain alone or the factor V A1-domain or A2-domain alone directed synthesis of secreted protein,. Secretion of a hybrid in which the carboxyl-terminal 110 amino acids of the A1-domain were replaced by homologous sequences from the factor V A1-domain was also increased 10-fold compared to wild-type factor VIII, however, the secreted protein was not functional and the heavy and light chains were not associated. These results localize a 110-amino acid region within the A1-domain that inhibits factor VIII secretion. This region is clustered with multiple short peptide sequences that have potential to bind BiP.


INTRODUCTION

Factor VIII functions in the intrinsic pathway of blood coagulation as a cofactor to accelerate the activation of factor X by factor IXa, a reaction that occurs on a negatively charged phospholipid surface in the presence of calcium ions. Factor VIII is synthesized as a single-chain polypeptide having the domain structure A1-A2-B-A3-C1-C2 (1, 2) . Upon secretion from the cell it is processed within the B-domain to yield a heterodimer consisting of a carboxyl-terminal-derived light chain of 80 kDa in a metal-ion dependent association with a 200-kDa amino-terminal-derived heavy chain fragment (3) . The domain structure of factor VIII is identical to that of the homologous coagulation factor, factor V (4, 5) . The factor VIII A-domains are 330 amino acids and have 40% amino acid identity with each other and to the A-domains of factor V and the plasma copper-binding protein ceruloplasmin (6) . Each C-domain is 150 amino acids and exhibits 40% identity to the C-domains of factor V, and to proteins that bind glycoconjugates and negatively charged phospholipids (7) . The B-domain is encoded by a single exon and exhibits little homology to the factor V B-domain (8, 9) . In plasma, the amino terminus of the factor VIII light chain is bound by noncovalent interactions to a primary binding site in the amino terminus of von Willebrand factor.

Although most evidence supports that the hepatocyte is the cell type that produces factor VIII in vivo (10, 11, 12, 13) , there are no known established or primary cell lines that express factor VIII. Thus, our knowledge of factor VIII expression is derived from interpretation of results from expression of the cDNA using expression vectors in transfected mammalian cells. Expression of factor VIII in these transfection systems is 2-3 orders of magnitude lower than that observed with other genes using similar vectors and approaches. Studies have identified three potential reasons that limit factor VIII expression (14) : 1) the factor VIII mRNA is inefficiently expressed (15, 16) , 2) the primary translation product is inefficiently transported from the endoplasmic reticulum (ER)() to the Golgi apparatus (17) , and 3) high levels of von Willebrand factor are required in the conditioned medium to promote stable accumulation of factor VIII (3) .

The inefficient secretion of factor VIII correlates with binding to the protein chaperonin identified as the immunoglobulin binding protein (BiP), also known as the glucose-regulated protein of 78 kDa (GRP78) (18) within the lumen of the ER (19) . BiP is a member of the heat-shock protein family that exhibits a peptide-dependent ATPase activity (20) . BiP expression is induced by the presence of unfolded protein or unassembled protein subunits within the ER (21, 22) . High level factor VIII expression-induced BiP transcription (23) . In addition, factor VIII release from BiP and transport out of the ER required high levels of intracellular ATP (24) . In contrast, the homologous coagulation protein, factor V, did not associate with BiP and did not require high levels of ATP for secretion (25) . Deletion of the factor VIII B-domain yielded a protein that bound BiP to a lesser degree and was more efficiently secreted (17) . To evaluate whether the factor VIII B-domain was responsible for BiP interaction, factor V and factor VIII chimeric cDNA molecules were constructed in which the B-domain sequences were exchanged (26) . A factor VIII hybrid harboring the B-domain of factor V was expressed and secreted as a functional molecule, although the secretion efficiency of the hybrid was poor and similar to wild-type factor VIII (26) . This indicated that the difference in secretion efficiency between factors V and VIII was not directly attributable to specific sequences within the factor VIII B-domain, the most divergent region between these homologous coagulation factors. To further identify regions within factor VIII that inhibit secretion, we have analyzed the expression of factor VIII deletion and factor VIII/factor V chimeric molecules. The results identify a 110-amino acid region within the factor VIII A1-domain that inhibits secretion.


EXPERIMENTAL PROCEDURES

Materials

Rabbit anti-factor V polyclonal antibody was purchased from Dako Corp. (Carpinteria, CA). Factor V monoclonal antibody E9 directed against the factor V light chain (27) was kindly provided by Dr. K. G. Mann (University of Vermont, Burlington, VT). Anti-factor VIII heavy chain monoclonal antibody F-8 was described previously (28) . Anti-factor VIII light chain monoclonal antibody was obtained from Hybritech Corp. (La Jolla, CA). Anti-factor VIII polyclonal antibody (R-305) was provided by Genetics Institute, Cambridge, MA. Soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, and aprotinin were purchased from Sigma. Human -thrombin and factor Xa were obtained from Hematological Technology (Burlington, VT). [S]Methionine (>1000 Ci/mmol) was obtained from Amersham Corp. (Arlington Heights, IL).

Plasmid Mutagenesis and Hybrid Construction

The mammalian cell expression vector pMT2 (29) utilizes the adenovirus major late promoter with SV40 enhancer and origin of replication. The factor VIII expression vector pMT-VIII has a unique XhoI site upstream of the initiation codon and a unique SalI site at nucleotide 8379, where nucleotide 1 is at the 5` end of the SV40 enhancer (26) . The factor V expression vector pMT-V has two SalI sites flanking the factor V cDNA at nucleotides 1088 and 7932 (26) . To facilitate domain exchanges and deletions, site-directed oligonucleotide-mediated mutagenesis was performed by the heteroduplex procedure (30) using different sized (approximately 50 bases) mutagenic oligonucleotides to introduce unique MluI restriction sites at arginine residues 226, 336, 372, and 740 within factor VIII to yield expression plasmids designated p226 MluI, p336 MluI, p372 MluI, and p740 MluI. Plasmids p372 MluI and p740 MluI were previously described (28) . Unique MluI restriction sites were introduced into the factor V cDNA at arginine residues 198, 313, and 709 within factor V to yield p198 MluI, p313 MluI, and p709 MluI. Double MluI mutations were sequentially introduced into pMT2-V at amino acids 313 and 709 to yield p313/709 MluI. Chimeric and deletion molecules were constructed by restriction endonuclease digestion, gel electrophoresis in low-gelling temperature agarose, and ligation of the isolated fragments. The resulting plasmids were purified over cesium chloride and characterized by extensive restriction mapping and partial DNA sequencing.

The factor VIII deletion pVIIIA1 was constructed by digesting p372 MluI with XhoI and MluI and ligation to complementary phosphorylated oligonucleotides encoding the factor VIII signal peptide and flanked by unique XhoI (5`) and MluI (3`) restriction sites having the sequence: 5`-TCGAGAGCTTCGACCACCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTA-3`. The factor VIII deletion molecule pA1-ac deletes amino acids 1 through 372 and does not contain the factor VIII acidic region (residues 336-372). A linker comprised of two complementary synthetic oligonucleotides encoding the acidic region from 336 to 372 flanked by the 5` and 3` MluI restriction sites was ligated into the unique MluI site of pA1-ac to yield pVIIIA1. The hybrid pVIIIA1V containing the A1-domain of factor V (amino acids 1-312) was constructed by ligation of the 11,026-bp XhoI/ MluI fragment from pVIIIA1 to the 1,067-bp SalI/ MluI fragment from p313 MluI and designated pVIIIA1V-ac. pVIIIA1V was generated by ligation of the 108-bp MluI fragment (336-372 acidic region) from pVIIIA1 to MluI linearized pVIIIA1V-ac to yield pVIIIA1V.

The factor VIII deletion molecule pVIIIA2 (336-739) has a unique MluI site at the junction of the deletion and was described previously (28) . The factor VIII chimera containing the factor V A2-domain (factor V amino acid residues 313-708) was constructed by linearizing pVIIIA2 with MluI and ligating it to the 1188-bp MluI fragment from p313/709 MluI.

The factor VIII deletion plasmid p1-225 was made by ligation of the 3859-bp ClaI/ MluI fragment from pVIIIA1 to the 7685-bp ClaI/ MluI fragment from p226 MluI. The factor VIII chimera pV, replacing factor VIII amino acids 1-225 with residues 1-197 from factor V, was assembled by ligation of the 11,468-bp XhoI/ MluI fragment from p1-225 to the 722-bp SalI/ MluI fragment from p198 MluI. The factor VIII deletion p226-371 removes amino acids 226-371 and was constructed by ligation of the 4,531-bp ClaI/ MluI fragment from p226 MluI to the 7,243-bp ClaI/ MluI fragment from p372 MluI. The factor VIII chimera pV, replacing factor VIII amino acids 226-335 with factor V amino acids 198-312, was constructed by a 3-way ligation of MluI linearized p226-371, the 106-bp MluI/ SacI fragment from p198 MluI and the 238-bp SacI/ MluI fragment from p313 MluI. Then the factor VIII acidic region (336-372) was isolated from pVIIIA1 on a 108-bp MluI fragment and ligated into the unique MluI site.

The factor VIII chimera (pVIIIhcV) containing residues 1-708 from factor V was constructed by ligation of the 9,922-bp XhoI/ MluI fragment from p740 MluI to the 2,260-bp SalI/ MluI fragment from p709 MluI. The factor V hybrid molecule (pVhcVIII), harboring the heavy chain of factor VIII (amino acid residues 1-739) was constructed by ligating the 7,206-bp SalI/ MluI fragment from p740 MluI to the 4,553-bp SalI/ MluI fragment from p709 MluI. The hybrid pVIIIA1V containing the A1-domain of factor V (amino acids 1-312) was constructed by ligation of the 11,026-bp XhoI/ MluI fragment from pVIIIA1 to the 1,067 bp SalI/ MluI fragment from p313 MluI. To construct the factor VIII chimera containing the A2-domain (amino acids 313-709) of factor V(pVIIIA2V), pVIIIA2 was linearized with MluI and ligated to the 1,188-bp fragment from p313/709 MluI.

The expression vector encoding the factor VIII A1-domain alone (pVIIIA1) was constructed by ligation of the 6102-bp MluI/ SalI fragment from p372 MluI to the following annealed phosphorylated oligonucleotide MluI- SalI linker: 5`-CGCGTTGATGAG-3` and 5`-TCGACTCATCAA-3`, which contains two termination codons. The expression vector encoding the factor VIII A2-domain (pA2) was described previously (28) . The factor V A1-domain alone was assembled by ligating the 4924-bp XhoI/ MluI fragment from pVIIIA1 to the 1067-bp SalI/ MluI fragment from p313 MluI. The factor V A2-domain alone was constructed by a 3-way ligation of the 3859-bp ClaI/ MluI fragment from pVIIIA1, the 1188-bp MluI fragment from p313/709 MluI, and the 1141-bp ClaI/ MluI fragment from pVIIIA1. The factor V heavy chain alone (94-kDa) was constructed by ligation of the 4924-bp XhoI/ MluI fragment from pVIIIA1 to the 2281-bp SalI/ MluI fragment from p709 MluI.

DNA Transfection and Analysis

Plasmid DNA was transfected into COS-1 cells by the DEAE-dextran procedure (29) . Conditioned medium was harvested 60 h post-transfection in the presence of 10% heat-inactivated fetal bovine serum for factor VIII assay. Primary translation products were analyzed by pulse labeling cells for 15 min with [S]methionine (250 µCi/ml in methionine-free medium) and preparing cell extracts in a Nonidet P-40 lysis buffer (31) . Protein secretion was monitored by metabolically pulse labeling cells with [S]methionine (250 µCi/ml for 15 min) and chasing for increasing periods of time in medium containing excess unlabeled methionine as described (30) . Cell extracts and conditioned medium were harvested as described previously (30, 31) . The factor VIII was quantitatively immunoprecipitated with an anti-factor VIII monoclonal antibody F-8 (28) coupled to CL-4B Sepharose or with anti-factor VIII light chain monoclonal antibody coupled to Affi-Gel. Factor V was immunoprecipitated with either monoclonal antibody E-9 specific to the factor V light chain (27) or a rabbit polyclonal antibody coupled to Affi-Gel (Bio-Rad). The antibodies were tested prior to the experiments to determine the amount of antibody required for quantitative immunoprecipitation. In all cases excess antibody was used. The immunoprecipitates were washed as described (31) . Immunoprecipitated proteins from the conditioned medium were resuspended 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2.5 mM CaCl, and 5% glycerol for electrophoresis on an SDS-low bis-8% polyacrylamide gel (32) . In addition to analysis by immunoprecipitation, equal quantities of [S]methionine-labeled conditioned medium were analyzed by SDS-PAGE. Proteins were visualized by autoradiography after fluorography by treatment with EnHance (Dupont, Boston, MA). The band intensities were quantitated by scanning the lanes using an LKB UltroScan XL laser densitometer (Pharmacia LKB Biotechnology Inc., Uppsala, Sweden). The total amount of secreted protein was estimated by adding the amount of single-chain to the amount of processed fragments.

Chain association was measured by co-immunoprecipitation experiments. The conditioned medium was immunoprecipitated with the factor VIII heavy chain monoclonal antibody (F8). The antibody-factor VIII complexes were collected by centrifugation and the supernatant fluid was subsequently immunoprecipitated with the light chain-specific monoclonal antibody. The immunoprecipitated proteins were analyzed by SDS-polyacrylamide gel electrophoresis as described above.

Factor VIII activity was measured by a chromogenic assay (33) or in a clotting assay using factor VIII-deficient plasma (34) . Factor V activity was measured in a clotting assay using factor V-deficient plasma.

Total RNA was isolated from transfected COS-1 cells at 60 h post-transfection using the guanidine thiocyanate method (35) . Northern blot hybridization was performed using a dihydrofolate reductase cDNA probe as described previously (26) . The Northern blots were also probed with a -actin cDNA probe to insure that the lanes were equally loaded.


RESULTS

The A1-domain of Factor VIII Inhibits Secretion

Previous studies in transfected mammalian cells demonstrated that factor V was more efficiently secreted than factor VIII (25, 26) . In addition, secretion of the factor VIII 90-kDa heavy chain fragment compared to the 80-kDa light chain fragment, each fused to the factor VIII signal peptide, was significantly reduced, although both proteins were translated at similar levels in the cell (36) .() These results suggested that the factor VIII light chain can be efficiently secreted in the absence of the heavy chain and that the heavy chain expressed alone is inefficiently secreted, similar to wild-type factor VIII. To further localize a region(s) within the factor VIII heavy chain that may inhibit secretion, the expression of A1-domain (residues 1-336) deleted factor VIII (A1) and of A2-domain (residues 373-740) deleted factor VIII (A2) was studied. In addition, a series of chimeric proteins were constructed that replaced different regions of factor VIII heavy chain with factor V residues. The factor V and factor VIII cDNAs were engineered to contain unique MluI restriction sites (encoding Thr-Arg) at the Arg residues 226, 336, 372, and 740 in human factor VIII and at Arg residues 198, 313, and 709 in human factor V. In this way, homologous sequences were exchanged by DNA fragment exchanges and chimeric molecules were produced by transient DNA transfection of COS-1 monkey kidney cells. These particular residues were chosen because they are proteolytic cleavage sites that are likely on the outer surfaces of these proteins and therefore chimeric proteins that harbor novel junctions at these sites are more likely to fold properly. Analysis of factor VIII expression upon transient transfection of COS-1 monkey cells demonstrated that introduction of the MluI restriction sites did not affect the synthesis, secretion, functional activity, or the ability of the molecules to be cleaved by thrombin (data not shown).

Chimeric molecules were constructed replacing factor VIII residues 1-740 with factor V residues 1-709 (designated VIIIhcV), factor VIII residues 1-336 with factor V residues 1-313 (designated VIIIA1V), and factor VIII residues 373-740 with factor V residues 314-709 (designated VIIIA2V). Transfected cells were metabolically labeled with [S]methionine and cell extracts were analyzed for primary translation products by immunoprecipitation with an anti-factor VIII polyclonal antibody. Total radiolabeled conditioned medium was analyzed directly to rule out the possibility that the mutant proteins were not efficiently recognized by the antibody. In addition, total cellular RNA was prepared for analysis by Northern blot hybridization to quantitate factor VIII mRNA levels. Analysis of total conditioned medium from wild-type factor V-transfected cells detected migration of factor V at 330 kDa, just below a background polypeptide (Fig. 1 A, lane 4). In contrast, wild-type factor VIII was barely detectable above background bands (Fig. 1 A, lane 1). Deletion of the A1-domain (Fig. 1 A, lane 2) or replacement of the factor VIII heavy chain (Fig. 1 A, lane 6) or the A1-domain (Fig. 1 A, lane 7) with corresponding sequences of factor V increased the level of factor VIII polypeptides in the conditioned medium significantly above the wild-type factor VIII. In addition, the increased expression permitted direct detection of radiolabeled light chain in the total conditioned medium from cells transfected with the A1, VIIIhcV, or VIIIA1V (Fig. 1 A, lanes 2, 6, and 7). In contrast, deletion of the A2-domain (Fig. 1 A, lane 3) or replacement of the factor VIII A2-domain with factor V residues (Fig. 1 A, lane 8) did not increase the level of factor VIII polypeptides detected in the conditioned medium. Secretion of a factor V chimera that had factor V residues 1-709 replaced for factor VIII residues was reduced compared to wild-type factor V (Fig. 1 A, lane 5), suggesting the factor VIII residues 1-740 inhibit accumulation of the VhcVIII chimera in the conditioned medium. In addition, a factor V chimeric molecule that had the factor V light chain (residues 1546-2196) replaced by the factor VIII light chain (residues 1649-2332) accumulated to levels similar to wild-type factor V, indicating that the light chain of factor VIII did not inhibit factor V accumulation in the conditioned medium (data not shown). Analysis of mRNA (Fig. 1 B) and primary translation products (Fig. 1 C) indicated that the mRNAs encoding the deletion and chimeric factor VIII proteins were expressed at similar levels and were translated with similar efficiencies. Although there were minor variations in the mRNA levels detected, they did not account for the major difference in the amount of protein secreted into the conditioned medium. In addition, differences in methionine content could not account for the increased secretion associated with the absence of the A1-domain of factor VIII. These results indicate that specific sequences within the factor VIII heavy chain inhibit accumulation of factor VIII in the conditioned medium.


Figure 1: The A1-domain limits factor VIII expression at a post-translational level. COS-1 cells were transfected with the indicated plasmid DNAs encoding wild-type factor V (V), wild-type factor VIII (VIII), or the indicated deletion or chimeric molecules. At 60 h post-transfection cells were labeled for 6 h with [S]methionine and equivalent amounts of total conditioned medium were directly loaded onto a reducing 6.5% polyacrylamide-SDS gel and analyzed by autoradiography ( panel A). In parallel, RNA was isolated for Northern blot hybridization analysis using a dihydrofolate reductase probe that reacts with all vector-derived mRNA species ( panel B). Cells were also pulse-labeled with [S]methionine for 15 min and lysates prepared for immunoprecipitation with an anti-factor VIII polyclonal antibody ( panel C). Migration of factor V, factor VIII related polypeptides ( VIII), and the factor VIII light chain ( LC) is shown. Molecular weight markers are shown on the left.



To determine whether the increased factor VIII accumulation was due to increased secretion from the cell or increased stability in the conditioned medium, more detailed [S]methionine pulse labeling and chase analysis was performed by immunoprecipitation with an anti-factor VIII light chain-specific monoclonal antibody. The factor VIII wild-type (Fig. 2 A, lane 1), the A1- (A1; Fig. 2 B, lane 1) and A2- (A2; Fig. 2 B, lane 10) domain deletion molecules, and a chimeric factor VIII replacing residues 1-740 for factor V amino acids (1-709) (VIIIhcV; Fig. 2A, lane 10) were all translated at similar levels. The majority of the translation products for the wild-type, A1, and VIIIhcV disappeared from the cell after 2 h, whereas the A2 was detectable in the cell after 4 h. Analysis of the conditioned medium detected factor VIII polypeptides secreted after 2 h as a 250-kDa single chain and a 80-kDa light chain doublet for wild-type, A1, and VIIIhcV factor VIII molecules (Fig. 2). Secretion of A2-domain-deleted factor VIII was slightly delayed, appearing at 4 h. However, the secretion of both the A1 and VIIIhcV were increased approximately 5-10-fold, compared to wild-type or A2 factor VIII and there were also significant amounts of a 160-kDa carboxyl-terminal light chain that was previously characterized to extend to residue 1313 (25) . All proteins were relatively stable in the conditioned medium over a 6-h period with some degradation occurring after 24 h. Thrombin digestion of the A1 and A2 deletion molecules yielded the expected products migrating at 73, 50, and 43 kDa, whereas the 50- and 43-kDa fragments were absent in their respective deletion molecules, indicating that neither the A1-domain nor the A2-domain were not required for thrombin cleavage (data not shown). The analysis of the A1- and A2-domain deletion molecules show that neither the A1-domain or the A2-domain are required for factor VIII secretion. Replacement of factor VIII heavy chain residues 1-740 with homologous factor V residues increased the secretion efficiency approximately 10-fold, to a level similar to the A1 factor VIII. These results suggest that sequences within the factor VIII A1-domain inhibit factor VIII secretion.


Figure 2: The A-1 domain inhibits secretion of factor VIII. Expression vectors encoding wild-type, A1-domain deleted (A1), and A2-domain deleted (A2), and the heavy chain chimeric (VIIIhcV) factor VIII were transfected into COS-1 cells and analyzed [S]methionine pulse labeling and chase as described under ``Experimental Procedures.'' Cell extracts and conditioned medium were immunoprecipitated with a monoclonal antibody reactive with the factor VIII light chain. Molecular weight markers are shown on the left.



An Independently Expressed Factor VIII A1-domain Is Not Secreted

The above results indicate that A1-domain within factor VIII either actively retains factor VIII in the cell or otherwise prevents efficient protein folding to inhibit factor VIII secretion. Insight into whether the A1-domain contains a retention signal was obtained by comparing expression of the factor VIII A1-domain alone to the factor VIII A2-domain alone, each fused to the signal peptide of factor VIII to direct transport into the ER. Whereas the A1-domain was not secreted, most of the translated A2-domain was efficiently secreted (Fig. 3 A). The A1-domain in the cell extract migrated as a triplet that was converted to a single species by digestion with N-glycanase, indicating the different species result from heterogeneity in N-linked glycosylation (data not shown). The secretion of the intact factor V 94-kDa heavy chain, the factor V A1-domain, or the factor V A2-domain were also evaluated after a 15-min pulse and a 6-h chase in complete medium (Fig. 3 B, lanes 1-8). Analysis of the conditioned medium after 6 h (Fig. 3 B, lanes 9-11), demonstrated that the factor V heavy chain or individual domains were all detectably secreted compared to the factor VIII A1-domain. These results show that the factor VIII A1-domain is uniquely not secreted from the cell and suggest that the factor VIII A1-domain polypeptide is directly responsible for inhibiting factor VIII secretion. Residues 227-336 in the A1-domain Inhibit Factor VIII Secretion-To further localize the region that inhibits factor VIII secretion, additional internal deletions within the A1-domain (residues 1-226 or 227-336) were analyzed and found not to be secreted, suggesting that smaller deletions affected folding of the protein to inhibit secretion. Therefore, to preserve proper polypeptide folding, we studied the secretion of factor VIII chimeric proteins containing portions of the factor V A1-domain. Two factor VIII chimeric molecules were constructed that had residues 1-226 or 227-336 replaced by homologous sequences within factor V (residues 1-197 or 198-313, respectively). Analysis of pulse-labeled cell extracts indicated that the wild-type and both A1-domain chimeric proteins were translated with equal efficiencies (Fig. 4, lanes 1-3). In contrast, analysis of the conditioned medium demonstrated that the secretion of the chimeric factor VIII protein that had factor VIII residues 227-336 replaced by factor V residues was increased approximately 5-fold (Fig. 4, lane 7) compared to wild-type factor VIII (Fig. 4, lane 5) or a chimera that had factor VIII residues 1-226 exchanged for the homologous factor V sequences (Fig. 4, lane 9). These results demonstrate that residues 227-336 within the factor VIII A1-domain inhibit secretion. Loss of Procoagulant Activity in the V Hybrid Correlates with Chain Dissociation-Analysis of factor VIII activity in the conditioned medium indicated that all the factor VIII and factor V chimeric proteins described in this report were inactive, although they were all susceptible to thrombin cleavage (data not shown). Further analysis by co-immunoprecipitation with factor VIII light chain- and heavy chain-specific monoclonal antibodies demonstrated reduced association of the heavy and light chains for the A1-domain chimeric proteins. Immunoprecipitation with the heavy chain-specific antibody detected single chain, heavy chain, and associated light chain at 80 kDa for wild-type factor VIII (Fig. 4, lane 5). Immunoprecipitation of the supernatant with a light chain-specific monoclonal antibody also detected the the single chain and some 80-kDa light chain, indicating that approximately 50% of the light chain was not associated (Fig. 4, lane 6), similar to previous observations (3) . In contrast, similar comparison of the amount of light chain co-immunoprecipitation for either the V(Fig. 4, lanes 9 and 10) or the Vhybrid (Fig. 4, lanes 7 and 8) demonstrated the majority of the light chain did not co-immunoprecipitate with the heavy chain monoclonal antibody. The 160-kDa species reactive with the light chain antibody for the 227-336 hybrid represents a carboxyl-terminal light chain polypeptide extending to residue 1313. These results show that both the Vand Vchimeras display reduced chain association that could contribute to the loss of procoagulant activity. Factor VIII Residues 227-336 Contain Multiple Peptides with Potential to Bind BiP-Our results show that factor VIII contains unique sequences between residues 227 and 336 that inhibit secretion that are not observed in factor V. We hypothesize that this region contains a high affinity BiP-binding site. Based on the ability of phage displaying random peptides to bind BiP, Blond-Elguindi et al. (38) provided a statistical method to predict the potential for any 7-mer peptide to bind BiP. We applied this algorithm to the sequences within the 227-336 region within the A1-domain of factors VIII and V (Fig. 5). The major difference between factor VIII and factor V is in a region in the factor VIII A1-domain that contains a hydrophobic cluster having several peptides between residues Ileand Phe( arrows in Fig. 5 ) with scores greater than 10, a score with high probability to bind BiP. Analysis of the factor VIII A2-domain or the A3-domain did not identify a region with multiple BiP-binding peptides (data not shown). These results suggest this hydrophobic pocket may represent a high affinity BiP-binding site.


Figure 3: Independently expressed factor VIII A1-domain is not secreted. COS-1 cells were transfected with expression vectors encoding the factor VIII A1-domain (residues 1-336) or the A2-domain (373-740) each fused to the factor VIII signal peptide ( panel A). The secretion of the factor V heavy chain (94 kDa, residues 1-709), the factor V A1-domain (residues 1-313), or the factor V A2-domain (residues 314-709) each fused to the factor VIII signal peptide was also evaluated ( panel B). Cells were pulse-labeled with [S]methionine for 15 min and chase performed for the indicated periods of time ( panel A) or for 6 h ( panel B) as described under ``Experimental Procedures.'' Cell extracts and conditioned medium were analyzed using a factor VIII polyclonal antibody for immunoprecipitation ( panel A) or a factor V polyclonal antibody ( panel B). The mock sample represents COS-1 cells that did not receive DNA. The molecular weight markers are indicated on the left.




Figure 4: Residues 226-336 inhibit factor VIII secretion. Expression vectors encoding wild-type factor VIII ( wt) and the hybrids containing factor V exchanges of factor VIII residues 1-226 (V) or 227-336 (V) were transfected into COS-1 cells. Mock represents cells that did not receive DNA. After 60 h the cells were pulse-labeled with [S]methionine for 15 min and chased in complete medium for 6 h, and then analyzed as described under ``Experimental Procedures.'' Cell extracts and conditioned medium were immunoprecipitated with the anti-factor VIII heavy chain monoclonal antibody (F8) ( h). The supernatants from the anti-factor VIII heavy chain antibody immunoprecipitation of the conditioned medium were subsequently immunoprecipitated with anti-factor VIII light chain monoclonal antibody ( l). The migration of single chain ( s.c.), heavy chain ( h.c.), light chain, and the 160-kDa extended light chain ( x-l.c.) are shown. Molecular weight markers are shown on the left.




Figure 5: BiP scores for the 227-336 region of factor VIII and the homologous region in factor V. The amino acid sequence from residues 227-336 is shown for factor VIII (above) and the homologous residues in factor V are shown below. The BiP scores for all 7-mer peptides are shown as bars that occur at the initial amino acid of the peptide. Black bars, factor VIII. Gray bars, factor V. The solid bar indicates the position of +10, a highly significant score for BiP binding. The asterisk indicates the residues involved in type 1 copper binding. The arrows indicate Ileand Phe.




DISCUSSION

The results of these studies show that a 110-amino acid region within the factor VIII A1-domain inhibits secretion of factor VIII. Expression of independent heavy and light chains of factor VIII previously demonstrated that the heavy chain was inefficiently secreted compared to the light chain (36) . To determine whether a specific region within the heavy chain was responsible for the inefficient secretion, analysis of in-frame deletions within the heavy chain was performed. Secretion of A1-domain-deleted factor VIII was increased approximately 10-fold compared to wild-type or A2-domain-deleted factor VIII. In addition, deletion of the 36-amino acid acidic region between the A1- and A2-domains of factor VIII did not affect secretion efficiency (data not shown). Additional internal deletions within the A1-domain were not secreted, likely due to polypeptide misfolding. Thus, to further localize the region, chimeric molecules were made between factor VIII and the homologous coagulation protein, factor V, that is efficiently secreted. Whereas exchange of factor VIII residues 1-226 for the homologous residues of factor V did not increase the secretion efficiency, similar exchange of factor VIII residues 227-336 improved secretion 5-10-fold in different experiments. To determine if the A1-domain directly inhibits secretion, we studied the secretion of individual domains. Compared to the factor VIII A1-domain that was not detectably secreted, the factor VIII A2-domain and the factor V A1-domain were effectively secreted. Analysis of the data obtained from the different approaches by characterizing the secretion of factor VIII deletion mutants, factor VIII and factor V hybrid molecules, and expression of single A-domains independent of the rest of the molecule implicated sequences between residues 227 and 336 within the A1-domain as inhibiting secretion.

Previous studies indicated that the inefficient secretion of factor VIII correlated with a stable interaction with BiP and a requirement for high levels of intracellular ATP for BiP release and secretion (17, 24) . In contrast, factor V was efficiently secreted, was not detected in association with BiP, and did not require high levels of ATP for secretion (25) . We have now localized a region that inhibits factor VIII secretion between residues 227 and 336 within the factor VIII A1-domain and propose that this represents a high affinity BiP-binding site. Although it is likely that BiP can interact with multiple sites within unfolded proteins, the Escherichia coli chaperonin GroEL specifically interacts with a distinct binding site on human granulocyte ribonuclease (37) . To date, a unique high affinity BiP-binding site has not been identified within any protein transiting the secretory pathway. Two studies (38, 39) have reported on the amino acid requirements for peptide binding to BiP. Both approaches show that aliphatic amino acids are enriched in BiP-binding peptides. However, one study using filamentous phage display (38) also detected a preponderance of aromatic amino acids (Trp and Phe), that were not observed by direct sequencing of random-bound peptides (39) . Gething and co-workers (38) , provided a statistical method to predict the BiP binding potential of a particular 7-mer peptide. We applied this statistical program to the 227-336 region of the A1-domains of factor VIII and factor V. Residues Ileto Phehave multiple BiP binding scores greater than 10, indicating an extremely high probability of binding BiP (38) that were not present in the factor V A1-domain or in the A2- or A3-domains of factor VIII. Present studies are directed to analyze the effect on secretion of specific mutations in this region.

At present it is not known what structural features are required for the metal-ion dependent factor VIII heavy and light chain association but it likely occurs through interactions between the A1-domain and A3-domain. It is interesting that exchange of either factor VIII amino acid residues 1-226 or 227-336 for homologous factor V residues destroyed heavy and light chain association. This suggests that chain association requires specific factor VIII residues between 1 and 226 as well as between 227 and 336. Analogous to the postulated role of BiP in binding and retaining unassociated subunits of heavy and light chain immunoglobulins (40, 41) , and chains of the major histocompatibility complex (42) , and nicotinic receptor subunits (43) , we propose that BiP binds the factor VIII A1-domain in the amino terminus until association occurs with the A3-domain in the carboxyl terminus. It is particularly intriguing that the A-domains of ceruloplasmin and factor VIII (44) are structurally related to ancient copper-binding proteins, such as azurin (45, 46) , nitrite reductase (47) , and ascorbate oxidase (48) . These ancient copper-binding proteins have a similar three-dimensional folding pattern similar to the immunoglobulin variable domains (46, 49) consisting of a double -barrel, each of which is composed of 7 anti-parallel -strands. The structural homologies between immunoglobulin variable regions and these copper-binding proteins suggest similarities in folding intermediates and domain association.


FOOTNOTES

*
Portions of this work were supported by National Institutes of Health Grant HL53777 (to R. J. K.). 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: Tel.: 313-763-9037; Fax: 313-763-9323.

The abbreviations used are: ER, endoplasmic reticulum; BiP, immunoglobulin binding protein; bp, base pair(s).

K. A. Marquette, D. D. Pittman, and R. J. Kaufman, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Mary-Jane Gething for applying the BiP-score algorithm to the A-domains and Dr. Alnawaz Rehemtulla for advice and discussion during the course of these studies.


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