Processing of the Fibrillin-1 Carboxyl-terminal Domain*

Timothy M. RittyDagger , Thomas BroekelmannDagger , Clarina TisdaleDagger , Dianna M. Milewicz§, and Robert P. MechamDagger

From the Dagger  Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 and the § Department of Internal Medicine, University of Texas-Houston Medical School, Houston, Texas 77030

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To investigate the processing and general properties of the fibrillin-1 carboxyl-terminal domain, three protein expression constructs have been developed as follows: one without the domain, one with the domain, and one with a mutation near the putative proteolytic processing site. The constructs have been expressed in two eukaryotic model systems, baculoviral and CHO-K1. Post-translational modifications that normally occur in fibrillin-1, including glycosylation, signal peptide cleavage, and carboxyl-terminal processing, occur in the three constructs in both cell systems. Amino-terminal sequencing of secreted protein revealed leader sequence processing at two sites, a primary site between Gly-24/Ala-25 and a secondary site of Ala-27/Asn-28. Processing of the carboxyl-terminal domain could be observed by migration differences in SDS-polyacrylamide gel electrophoresis and was evident in both mammalian and insect cells. Immunological identification by Western blotting confirmed the loss of the expected region. The failure of both cell systems to process the mutant construct shows that the multi-basic sequence is the site of proteolytic processing. Cleavage of the fibrillin-1 carboxyl-terminal domain occurred intracellularly in CHO-K1 cells in an early secretory pathway compartment as demonstrated by studies with secretion blocking agents. This finding, taken with the multi-basic nature of the cleavage site and observed calcium sensitivity of cleavage, suggests that the processing enzyme is a secretory pathway resident furin-like protease.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fibrillin-1 and -2 are large, secreted glycoproteins known to be components of extracellular matrix microfibrils located in the vasculature, basement membrane, and various connective tissues and are often associated with a superstructure known as the elastic fiber (for reviews see Refs. 1 and 2). It has been clearly established that mutations in the FBN1 gene can result in the Marfan syndrome (for review see Ref. 3) and have been found in individuals with familial aortic aneurysm (4, 5), familial ectopia lentis (6), and Shprintzen-Goldberg syndrome (7). Studies using cell strains from individuals with the Marfan syndrome have suggested a dominant negative pathogenesis for FBN1 mutations, i.e. the product of the mutant allele disrupts the assembly of microfibrils (8, 9).

The two known fibrillin proteins are highly similar with modular structures consisting of repeating epidermal growth factor (EGF)1-like domains interspersed between 8 cysteine domains similar to those found in the latent transforming growth factor-beta -binding protein family. Of the 47 EGF-like domains, 43 contain the residues necessary for binding calcium. Near the amino terminus, fibrillin-1 contains a proline-rich domain that is unique to the molecule, whereas fibrillin-2 contains a glycine-rich domain in the analogous location. Both proteins have homologous carboxyl-terminal domains of about 185 residues. No functions have yet been ascribed to any fibrillin domains, and nothing is known about their role in the assembly of fibrillin-containing microfibrils.

The fibrillins are known to undergo several post-translational modifications that include beta -hydroxylation of Asn/Asp residues, N-linked glycosylation, and proteolytic cleavage of both the leader sequence and the carboxyl-terminal domain (10-12). Proteolytic cleavage of the fibrillin-1 carboxyl-terminal domain has been proposed to occur after the arginine at position 2731 and results in the removal of the last 140 residues. It has been suggested that a member of the Furin/PACE family, which cleaves multibasic sites, is responsible for the processing (12).

Due to the large size and great degree of similarity of fibrillin-1 and -2, it is difficult to distinguish between the two molecules much less experimentally evaluate the proteolytic processing of their carboxyl-terminal domains. To better understand the processing of the fibrillin-1 carboxyl-terminal domain, we have developed three truncated fibrillin expression constructs, one with the full-length FBN1 carboxyl-terminal domain, one with the domain deleted, and one with a mutation near the putative cleavage site. These protein constructs have been expressed in two eukaryotic model systems, baculoviral expression in SF9 insect cells and CHO-K1 mammalian cell lines. Both systems are able to produce and secrete each of the three constructs. Here we show that proteolytic processing of the fibrillin-1 carboxyl-terminal domain occurs in the constructs expressed in these systems. The loss of the expected region can be seen by SDS-PAGE migration differences and immunologically by Western blotting. Impaired cleavage observed in the construct with a mutated multi-basic site confirms the involvement of that region in normal processing. The use of secretion blocking agents shows that processing occurs intracellularly in the CHO-K1 cells and most likely by an early secretory pathway resident furin-like enzyme.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of LEE, LEEC, and LEEC-P1 FBN1 Sequences

The LEEC construct was amplified from human mRNA and was initially cloned in four parts (Figs. 1 and 2). The first part is "L," the leader sequence of FBN1 (exon 1). The second part is "E1," first half of the EGF-like and 8-cysteine domains (FBN1 exons 23-33). The third part is "E2," the second half of the EGF-like and 8-cysteine domains (FBN1 exons 30-44); and the fourth part is "C," the carboxyl-terminal domain (FBN1 exon 64-65). The FBN1 leader sequence and carboxyl-terminal domains were each amplified using the primers described below by reverse transcription of 2.0 µg of total RNA isolated from human foreskin fibroblasts with 20 units of Moloney murine leukemia virus reverse transcriptase (Boehringer Mannheim) followed by 30 cycles of polymerase chain reaction under standard conditions. Each cDNA was isolated from a low melting point agarose gel and ligated into the pCRII vector (Invitrogen, San Diego, CA). The E1 and E2 inserts were similarly amplified using 40 cycles of polymerase chain reaction and Vent polymerase and cloned into the pGEM-t vector (Promega, Madison, WI).

Description of Primers and cDNA Clones

cDNA Clone L-Exon 1 of FBN1-- These primers are designed to amplify the coding region of the first exon of the FBN1 gene. The sense primer begins with an EcoRI restriction site and Kozak consensus sequence followed by the initiating ATG codon. The antisense primer contains two other unique restriction sites, BspEI and BsiWI, that allow splicing to the next domain or the in frame replacement or removal of the leader sequence. Together they amplify a 195-bp product that encodes the first 56 amino acids of fibrilin-1 that contain the putative hydrophobic signal peptide leader sequence: leader sense primer, 5'-GGAATTCGCCACCATGCGTCGAGGGCGTCTG-3'; and leader antisense primer 5'-CCCCTCCGGATCCCGTACGGACATTG GGTCCTTTAAGC-3'.

cDNA Clone E1-Exon 23-33 of FBN1-- This primer pair is designed to amplify a 1,455-bp product that corresponds to the first codon of FBN1 exon 23 and continues into exon 33. The sense primer begins with a BsiWI restriction site to allow ligation to the 3' end of the leader sequence: E1 sense primer, 5'-GACTCGTACGGATATAGATGAATGTGAAGTGTTCC-3'; and E1 antisense primer, 5'-TCCTTCCTTGCACAGACAGCGG-3'.

cDNA Clone E2-Exon 30-44 of FBN1-- These primers are designed to amplify a 1,771-bp product that begins in exon 30 and continues through exon 44 of FBN1. This sequence overlaps with clone E1 and can be joined to it by a common SmaI restriction site. The antisense primer contains a BspEI site for ligation to the carboxyl-terminal domain sequence: E2 sense primer, 5'-TTGTGTTATGATGGATTCATGG-3'; and E2 antisense primer, 5'-TCAGTCCGGAATTGCACTGTCCTGTGGA-3'.

cDNA Clone C-the Carboxyl-terminal Domain of FBN1-- These primers are designed to amplify 572-bp coding sequence that begins with the first amino acid after the last EGF-like domain of FBN1 and continues to the termination codon. The sense primer contains 5'-BspEI-NheI-3' sites to facilitate assembly and substitution or removal of the domain. The antisense primer contains 5'-XbaI-NheI-3' sites to facilitate vector transfer and carboxyl-terminal domain substitution or removal. The carboxyl-terminal sense primer is 5'-GTCATCCGGAGCTAGCTCTGGAATGGGCATGGG-3' and the carboxyl-terminal antisense primer is 5'-GCTCTAGAGCTAGCTTAATGAAGCAAAACCTGGAT-3'.

Assembly of the cDNA Clones

The FBN1 leader sequence clone insert was excised, gel-purified, and ligated to the 5' end of the EGF1 clone using XbaI and BsiWI restriction sites. As blue/white selection was already disrupted, positive clones were identified by colony lifts screened with polymerase chain reaction-generated radioactive probe. The LS/EGF1 cDNA sequence was excised, gel-purified, and ligated into position 5' of the EGF2 clone with 5'-ApaI and 3'-SmaI restriction sites thus producing the sequence referred to as "LEE." The LEE sequence was then inserted into the pcDNA3 expression vector (Invitrogen) using EcoRI and XbaI sites. The carboxyl-terminal domain was inserted into the pcDNA/LEE with 5'-BspEI and 3'-XbaI sites to yield the sequence referred to as "LEEC." The leader sequence and carboxyl-terminal domains were sequenced in both directions upon initial cloning. The E1 and E2 clones were sequenced at each end. After each assembly step, the junctions were sequenced to ensure the maintenance of the reading frame.

The -P1 mutant was produced by site-directed mutagenesis using the FBN1 carboxyl-terminal clone C. Primer 90 (5'-CGTTTGTGCTGCTCCGTTTC-3') and CT sense were used to amplify a 200-bp 5' fragment, and primer 89 (5'-GAAACGGAGCAGCACAAACG-3') and CT antisense were used to amplify a 400-bp 3' fragment. Equimolar amounts of the two fragments were then used as templates to amplify the full-length mutant carboxyl-terminal fragment using the following conditions: denaturation at 94 °C/2 min, annealing at 55 °C/3 min, and extension at 72 °C/1 min for six cycles. The carboxyl-terminal sense and antisense primers were then added to the reaction, and 30 additional cycles were carried out. Amplified fragments were cloned into pGEM-T and sequenced.

Bacterial Expression Construct

The FBN1 carboxyl-terminal domain was excised from the initial clone described above using the 5'-BspEI and 3'-HindIII sites and ligated into the pQE32 expression vector (Qiagen, Valencia, CA) using the 5'-XbaI and 3'-HindIII restriction sites. This vector contains an initiating ATG followed by a sequence that codes for six histidine residues, all of which are upstream of the inserted sequence. The resulting protein contains an amino-terminal His-tag that can bind a Ni2+ ion containing solid support resin to facilitate purification (Qiagen). The recombinant FBN1 carboxyl-terminal protein contains the following additional residues amino-terminal to the first residue of the domain: MRGSHHHHHHGIRMRARYPGAS + (FBN1-Ser-2688 to His-2871 terminus). The full-length recombinant peptide contains 206 amino acids with a predicted molecular mass of 23,266 Da and an isoelectric point of pH 8.4. The protein sequence was confirmed by amino-terminal sequencing of 15 residues.

Log phase cells growing in 500 ml of Luria broth at 37 °C were induced to produce protein with 1.5 mM isopropyl beta -D-thiogalactopyranoside. Five hours post-induction, the cultures were pelleted, lysed with M urea, pH 8.0, and centrifuged at 12,000 × g for 20 min. The recombinant protein with the 6× His tag was purified from the other bacterial proteins in the crude lysate by binding to Ni2+ resin column (Qiagen) and washing out non-bound contaminating proteins, followed by elution with a low pH, 8 M urea wash. The recombinant protein was then dialyzed in 12,000-14,000 molecular weight cut-off dialysis tubing (Spectrum Medical, Laguna Hills, CA) against a 0.5 M acetic acid solution and lyophilized.

Baculoviral/SF9 Cell Expression Constructs

The LEE, LEEC, and LEEC-P1 coding sequences were each cloned into the pFastbac shuttle vector using 5'-EcoRI and 3'-XbaI sites, and this was used to transform DH10' cells (Life Technologies, Inc.) containing bacmid. Plasmid/bacmid recombination occurred through bacterial transposon Tn7 regions flanking the coding insert, and positives were identified with blue/white selection. Bacmid DNA was prepared by alkaline lysis and transfected into serum-free SF9 cells using Lipofectin (Life Technologies, Inc.) for 4 h at 27 °C. Five days after lipofection, conditioned medium was removed, centrifuged to remove debris, and used to infect new cultures of SF9 cells. Cultures expressing protein were determined by Western blot of conditioned medium. Subsequent expression experiments were conducted in either serum-free SF-900IISFM (Life Technologies, Inc.) or IPL41 medium containing 10% fetal bovine serum.

Expression Constructs and Establishment of Three Stable Expression Lines in CHO-K1 Background

The LEE, LEEC, and LEEC-P1 coding sequences were each inserted into the pEE14 vector using the 5'-EcoRI and 3'-BclI sites. This vector employs the cytomegalovirus promoter/enhancer to drive expression and glutamine synthase minigene driven by the SV40 late promoter to provide selection with methionine sulfoximine (13). The cell lines were established as described previously (14). Briefly, three 60-mm dishes containing adherent CHO-K1 cells (ATCC, CCL-61) at approximately 50% confluency were transfected with 9 µg/dish of purified plasmid DNA that had been previously incubated with 30 µl of Lipofectin (Life Technologies, Inc.) in 200 µl of serum-free medium (Glasgow minimum essential medium from Washington University TC Support Center, St. Louis) for 30 min at room temperature. Total volume per dish was 2.0 ml, and the duration of transfection was 5 h after which 3.0 ml of normal serum-containing non-selective medium was added overnight. The following day, each 60-mm dish was subcultured into three 100-mm dishes in selective medium (Glasgow minimum essential medium without glutamine and containing 10% 10,000 molecular weight cut-off dialyzed FBS and 25 µM methionine sulfoximine). After 14 days under selection, clonal colonies were subcultured into 15-mm wells and screened for expression by Western blot of conditioned medium. Approximately 20 colonies were isolated for each clone and of those 10, 50, and 90% were positive by Western blot of conditioned medium for LEEC, LEE, and LEEC-P1, respectively. For each construct, one clone was chosen based on expression levels and was used in all subsequent experiments.

Protein Expression and Purification

Baculoviral-- SF9 cells were infected in either serum-free medium (SF900II from Life Technologies, Inc.) or in the presence of 10% FBS in IPL41 medium (Life Technologies, Inc.). Protein could be detected as early as 24 h postinfection and continued to be produced up to 90 h postinfection. To protect against degradation under serum-free conditions, medium was collected after 48 h postinfection and (4-amidinophenyl)methanesulfonyl fluoride (Life Technologies, Inc.) to 2 mM was added followed by centrifugation at 16,000 × g for 15 min at 4 °C prior to purification. Harvest of protein from serum-containing medium was performed at 90 h postinfection.

Various methods were used to purify and concentrate protein from serum-free conditioned medium; these include dialysis in 12,000-14,000 molecular weight cut-off tubing (Spectrum) against PBS and then lyophilization, 40% ammonium sulfate precipitation, followed by dialysis and lyophilization, trichloroacetic acid precipitation followed by EtOH:ether wash and resuspension, and concentration by 50,000 molecular weight cut-off centrifuge filters (Spectrum). Highest purity and yield were achieved with pressurized ultrafiltration through Diaflow YM100 molecular weight cut-off filters (Amicon, Beverly, MA) followed by fast protein liquid chromatography size fractionation using a Superose 6 column (Amersham Pharmacia Biotech) at 0.5 ml/min 100 mM ammonium bicarbonate, pH 7.5.

Stable CHO-K1 Lines-- To produce conditioned medium containing recombinant protein, LEE-, LEEC-, or LEEC-P1-expressing cells were grown in selective medium until confluency and switched to Hy-Q CCM5 serum-free medium (Life Technologies, Inc.) where they were grown for up to 4 days. After conditioning, the medium was withdrawn and centrifuged at 15,000 × g for 30 min to pellet debris. Next the medium underwent stirred ultrafiltration under 2.5 pounds/square inch at 4 °C through a YM100 molecular weight cut-off membrane (Amicon) as a purification and concentration step.

Fetal Bovine Cells-- Cell cultures established from fetal bovine nuchal ligament, fetal bovine vascular smooth muscle (BSMC), and fetal bovine chondrocytes (FBC) were used as a source of fibrillin for positive controls. Protein was harvested in a manner similar to that described for the CHO-K1 lines with the exception that when grown in serum-free medium, DMEM with ITS+3 (Sigma) was used instead. The conditioned medium was used directly or concentrated approximately 25× with ultrafiltration at 2.5 pounds/square inch through XM300K molecular weight cut-off filters (Amicon).

Antibody Production

The peptides Fib15D (YLDIRPRGDNGDTAC) and FBN1-386 (GNEDGFFKINOKEGC) were made using an Applied Biosystems 431A protein synthesizer by standard FMOC (N-(9-fluorenyl)methoxycarbonyl) chemistry (Fig. 1 and 2). The peptides were purified and cross-linked to rabbit serum albumin (Sigma) using the heterobifunctional cross-linker m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce) at a molar ratio of approximately 15:1 peptide to rabbit serum albumin. One milligram of peptide-rabbit serum albumin conjugate was emulsified in Freund's complete adjuvant for the initial injection into rabbits and Freund's incomplete adjuvant for subsequent injections at 2-week intervals. After 6 weeks, the resulting polyclonal antisera were collected and subjected to a caprylic acid precipitation step, followed by dialysis against PBS, lyophilization, and affinity purification against the immunogen peptide linked to Sulfa-link support (Pierce). After elution with 100 mM glycine, pH 2.3, the purified antibody was dialyzed, lyophilized, and resuspended in PBS at approximately 1.0 mg/ml.

The recombinant FBN1 carboxyl-terminal domain was expressed in bacteria, column purified as described above, and used to generate polyclonal antibodies. The immune serum was subjected to a caprylic acid precipitation followed by affinity purification against the protein bound to the Ni2+ resin support. After elution with 8 M urea, pH 8.0, the purified antibody was dialyzed, lyophilized, and resuspended in PBS at approximately 1.0 mg/ml.

Protein Radiolabeling

Baculoviral-- Medium was removed 24 h postinfection from a 35-mm well containing 1 × 106 SF9 cells, and the infected cells were washed with and then incubated for a 2-h starvation in Cys-free Grace's medium with 5% dialyzed FBS. This was then replaced with Cys-free Grace's medium supplemented with 100 µCi/1.0 ml/well [35S]Cys and 5% dialyzed FBS. Labeling continued for 24 h after which the conditioned medium was collected.

CHO Stables-- Confluent 15-mm wells were incubated in Cys-free DMEM with 5% dialyzed FBS for a 2-h starvation period and then incubated for various times in the presence of 50 µCi of [35S]Cys/200 µl medium/well. Chase medium was non-selective Glasgow minimum essential medium 175 µl/well with 5% FBS. The chase medium was collected after various times points. To collect cell lysate, cells were washed twice with serum-free medium and lysed with 175 µl/15-mm well of 1% Nonidet P-40, 50 mM Tris, pH 8.0, with 1.5 mM (4-amidinophenyl)methanesulfonyl fluoride, 1.0 µM aprotinin, 100 µM leupeptin, and 4 mM Pefabloc (Life Technologies. Inc.). Cellular debris was removed by centrifugation at 16,000 × g for 5 min at 4 °C. For SDS-PAGE analysis, 15 ml per lane of labeled medium or supernatant was typically loaded.

PNGse F Digestions

Baculoviral Protein-- 5.0 ml of conditioned medium was collected for 24 h from SF9 cells infected with LEEC baculovirus and immunoprecipitated with FBN1 carboxyl-terminal antibody at 1:250 overnight at 4 °C. The IgG-protein complex was bound to solid support with 100 µl of protein A-Trisacryl (Pierce) by a 1-h incubation at room temperature followed by centrifugation and two washes with 0.05% Tween 20 in PBS. For the PNGase F digestion, 5 µl of the Trisacryl beads were added to each of two parallel tubes containing 9 µl of water and 1.5 µl of 10× denaturing buffer and boiled for 10 min. The tubes were centrifuged, and the supernatant was removed to other tubes with 1.0 µl of 10× buffer, 1.0 µl of 10× Nonidet P-40 solution, and 1.0 µl of PNGase (500 New England Biolabs units) and digested at 37 °C for 1.5 h. The reaction was stopped by the addition of reducing loading buffer, and the contents were electrophoresed through a 7.5% SDS-polyacrylamide gel and detected by Western blot.

CHO-K1 Protein-- CHO-K1 cell line stably expressing the LEE construct was washed with and incubated in Cys-free DMEM for 2 h (1 × 106 cells/35-mm well) and then incubated with 125 µCi of [35S]Cys/1.0 ml of medium/well for 24 h in Cys-free DMEM with 5% dialyzed FBS. Medium was collected and spun through a 50,000 molecular weight cut-off column to remove unincorporated label and resuspended in fresh medium at the original volume. Two 20-µl aliquots of conditioned medium were processed in parallel. Each tube received 2.0 µl of denaturing buffer, was boiled for 10 min, centrifuged, and then received 2.0 µl each of Buffer G7 and Nonidet P-40 10× (New England Biolabs, Beverly, MA), and one tube also received 2.5 µl of PNGase F (1,250 NEB New England Biolabs units), and both were incubated at 37 °C for 2 h. The reaction was stopped by the addition of reducing loading buffer, and the contents were electrophoresed through a 5.0% SDS-polyacrylamide gel, fixed, dried, and exposed to x-ray film for 6 h.

Amino-terminal Sequencing

Baculoviral- and CHO-K1-expressed protein was collected from conditioned medium and concentrated by ultrafiltration as described above. Twenty µl of concentrated sample was loaded with reducing buffer onto SDS-PAGE and run at 20 mA for 1.5 h. Proteins were transferred onto ProBlott polyvinylidene difluoride support (Applied Biosystems, Foster City, CA) and detected with Coomassie stain. Appropriate bands were excised and sequenced on an Applied Biosystems model 473A automated sequencer using Edman degradation.

Secretion Blocks

CHO-K1 cells expressing LEEC were grown to confluency with normal medium in 15-mm wells. Before the introduction of [35S]Cys pulse, all wells underwent a 2-h Cys-free starvation as described above. Thirty min prior to the pulse, A23187 was added to a final concentration of 2 µM to the appropriate wells. Fifteen min before the pulse, brefeldin A, ammonium chloride, and monensin were added to the appropriate wells in final concentrations of 10 µg/ml, 50 mM, and 10 µM, respectively. All cells were then pulsed for 30 min as described above. After the pulse, the wells were washed once with normal medium containing blocking agents and then incubated with 175 µl of normal cold chase medium containing blocking agents for 2 h. Chase medium was retained and analyzed directly by SDS-PAGE and autoradiography. Cells were washed 3× with PBS then lysed as described above. Cell lysate was microcentrifuged 15,000 × g at 4 °C for 10 min, and the supernatant was analyzed by SDS-PAGE and autoradiography.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning and Assembly of Expression Constructs-- Using the primers described under "Experimental Procedures," each of the target sequences was amplified and assembled as described in Figs. 1 and 2. All three constructs, LEE, LEEC and LEEC-P1, begin with the first 56 amino acids (exon 1) of fibrillin-1. This is spliced to exons 23-44 of fibrillin-1 which contains 17 calcium binding EGF-like domains, three LTBP-like repeats, one RGD sequence, and eight potential sites of N-linked glycosylation. In the LEEC and LEEC-P1 proteins, exon 44 is spliced to the entire carboxyl-terminal domain of FBN1 which contains the putative cleavage site and three potential sites of N-linked glycosylation. The carboxyl-terminal domain of LEEC-P1 contains the Arg to Ser mutation at the tetrabasic cleavage site. The entire LEEC construct contains 1189 amino acids, has a pI of pH 4.6, and a predicted molecular mass of 129,907 daltons. As seen in other proteins containing several EGF-like domains, all constructs run at a higher than expected molecular weight on SDS-PAGE.


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Fig. 1.   Domain structure of fibrillin-1 and expression constructs. Diagram illustrating the domain organization of fibrillin-1 and the three expression constructs LEE, LEEC, and LEEC-P1. The regions recognized by three polyclonal antibodies are indicated. The -P1 mutant is depicted in the box at bottom left and domain types are illustrated at bottom right.

Baculoviral Expression System Produces, Secretes, and Correctly Processes LEE, LEEC, and LEEC-P1 Constructs-- Each of the three fibrillin-1 constructs was expressed in SF9 insect cell lines. All were produced at high levels with the expected post-translational modifications (see below). Protein was produced in SF9 cells under both serum-containing and serum-free conditions. Time course experiments revealed that peak protein production occurred between 24 and 48 h postinfection, although a significant amount of production continued out to 90 h (data not shown). Harvest of protein from serum-containing medium was performed at 90 h postinfection and was often used for Western blots without further purification. In general, production in the absence of serum resulted in higher levels of protein degradation but had greater ease of purification due to lower amounts of background serum proteins. The extent of degradation was related to the progression of infection and subsequent lysis of the host SF9 cells. Protein produced under both serum-free and serum-containing conditions was used for glycosylation analysis, amino-terminal sequencing, Western blots, and the solubility experiments. The FIB15D, FBN1-CT, and FBN1-386 antibodies recognized the appropriate constructs on Western blot (Fig. 3).

CHO-K1 Cells Stably Transfected with pEE14 Expression Vector Produce, Secrete, and Correctly Process LEE, LEEC, and LEEC-P1 Constructs-- Transfection of each of the three pEE14 expression vector-based constructs into CHO-K1 cells yielded several positive clonal cell lines, with the exception of LEEC for which only two positive clones were identified. Positive clones were selected for further experiments based upon protein expression levels as identified by Western blots of conditioned medium. The LEE and LEEC-P1 cell lines produced sufficient protein to identify by Western blot of medium conditioned for 24 h. The LEEC line often required a concentration step before immunological recognition by Western blot.

The use of serum-free conditions greatly facilitated purification by ultrafiltration as described. Protein stability problems analogous to the baculoviral system were not experienced under these conditions. These preparations were used for amino-terminal sequencing and Western blotting.

Characterization of Antibodies to Regions of the Carboxyl-terminal Domain of FBN1-- Bacterial expression of the FBN1 carboxyl-terminal cDNA followed by 6His-tag/Ni2+ column purification yielded mg/ml quantities of purified protein (data not shown). These preparations were used to produce the rabbit polyclonal antibody (FBN1-CT antibody) to the fibrillin-1 carboxyl-terminal domain.

The FBN1-CT rabbit polyclonal antibody recognizes the LEEC and LEEC-P1 proteins by Western blot and immunoprecipitation in both baculoviral and CHO-K1 systems (Fig. 3). Importantly, it recognizes both the uncleaved and cleaved carboxyl-terminal domain as 44 residues of the domain remain after processing. As expected, it does not recognize LEE. The antibody also immunoprecipitates native, full-length fibrillin from FBC (Fig. 3A), fetal calf ligament, or BSMC (not shown)-conditioned medium and detects fibrillin by Western blot.

Antibody Fib15D, generated to a peptide sequence in the fourth 8-cysteine domain of fibrillin-1, recognizes all three constructs as well as both processed and unprocessed forms of LEEC (Fig. 3B). Antibody FBN1-386, in contrast, recognizes an epitope in the region of the carboxyl-terminal domain that is lost after cleavage (see map in Fig. 2). This antibody only recognizes LEEC-P1 and the larger, unprocessed form of the LEEC constructs as expressed in both baculoviral and CHO-K1 cell systems (Fig. 3C). The FBN1-386 antibody does not detect fibrillin in conditioned medium from FBC or BSMC cultures (data not shown).


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Fig. 2.   Amino acid sequence of expression constructs. The sequences of LEE, LEEC, and LEEC-P1 are identical from residue 1 to 1002. The LEEC and LEEC-P1 proteins also contain the FBN1 carboxyl-terminal domain. The Arg to Ser substitution present in the LEEC-P1 mutant is shown in bold above the sequence. Residues introduced by restriction sites are shown in bold letters. The putative FBN1 carboxyl-terminal domain cleavage site is boxed. The peptide sequences used for the Fib15D and 386 antibodies are underlined. The first 54 residues of FBN1 (exon 1) were used as the leader sequence in all of our constructs. Protein secreted by CHO and SF9 cells contains two populations of amino-terminally processed LEEC as indicated by arrows.


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Fig. 3.   Antibody recognition of secreted proteins from baculoviral and CHO-K1 expression systems and FBC cells. A, left, Western blot of LEE, LEEC, and LEEC-P1 protein from baculoviral conditioned medium using FBN1-CT antibody (Ab). A, right, immunoprecipitation of radiolabeled fibrillin (arrow) from FBC conditioned medium. CM, total conditioned medium. IP, immunoprecipitation using the carboxyl-terminal antibody. B, Western blot of LEE, LEEC, and LEEC-P1 protein from baculoviral and CHO-K1 conditioned medium using the Fib15D antibody. C, Western blot of LEE, LEEC, and LEEC-P1 protein from baculoviral and CHO-K1 conditioned medium using the 386 antibody. For all Western blots, each gel was loaded with identical samples. Upper and lower arrows indicate cleaved and uncleaved proteins, respectively.

Carboxyl-terminal Processing and Other Post-translational Modifications-- The coding sequence of each of the constructs, LEE, LEEC, and LEEC-P1, begins with exon 1 of fibrillin-1. To determine the signal peptide cleavage site, amino-terminal sequencing of protein expressed in both baculoviral and CHO-K1 systems was performed. Interestingly, both systems process exon 1 of fibrillin 1 in the same way using a preferred cleavage site between Gly-24/Ala-25 and a secondary site of Ala-27/Asn-28 (Fig. 2). The primary site is used 4-5 times more frequently than the secondary site.

Cleavage of the carboxyl-terminal domain also occurred in both cell systems, as demonstrated by an electrophoretic mobility shift and the loss of immuno-recognition by the FBN1-386 antibody (Fig. 3). [35S]Cys pulse/chase experiments with CHO-K1 cells displayed no significant differences in the secretion rates of LEE, LEEC, and LEEC-P1 proteins at the time points studied (Fig. 4). Approximately 50% of the labeled protein was secreted by 1 h, and most was secreted by 5 h. In both expression systems, neither failure to cleave (LEEC-P1) nor the absence of the carboxyl-terminal domain (LEE) disrupted secretion. Although there was no difference in the ability of the cell to secrete the P1 mutant compared with LEEC, processing of the carboxyl-terminal domain of LEEC-P1 was less efficient (Fig. 5) in both SF9 and CHO cells. It is important to note that PACE/furin family members are known to be present and active in SF9 cells and correctly process multi-basic sites (15-17).


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Fig. 4.   Pulse/chase of untransfected CHO-K1 and transfected cell lines stably expressing LEE, LEEC, and LEC-P1 constructs. Each cell line was grown and labeled, and samples were prepared as described under "Experimental Procedures." Time = 0 corresponds to the end of the 30-min pulse and the beginning of the cold chase. The length of the chase is indicated in hours below each lane. Molecular weight markers are indicated at the sides. Arrows indicate unprocessed and processed protein. Each figure contains cell lysate and medium from untransfected CHO-K1 cells (A). B, CHO-K1 cells expressing LEE. C, CHO-K1 cells expressing LEEC. The lane designated "trypsin" indicates lysate from cells treated with trypsin at T = 0 h. D, CHO-K1 cells expressing LEEC-P1. LEEC is included for size reference.


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Fig. 5.   Inefficient processing of LEEC-P1 mutant. Western blot with the FBN1 carboxyl-terminal antibody of conditioned medium from the baculoviral system (A) and the CHO-K1 system (B). Both systems demonstrated less efficient processing of the LEEC-P1 mutant. Upper and lower arrows indicate unprocessed and processed forms, respectively.

It is interesting to note that we were sometimes able to detect unprocessed LEEC in conditioned medium from the baculoviral system. The amount detected varied according to the amount of virus used for infection, i.e. high viral titers resulted in large amounts of unprocessed protein, whereas lower viral levels resulted in more efficient processing. This is likely to be an artifact of the expression system, given that a baculoviral infection causes the shutdown of host cell gene transcription-translation by 24 h. Thus, the normal complement of host SF9 cell furins may be overwhelmed by 72-96 h of high levels of transgene expression and not able to efficiently process all of the protein produced (15). Unprocessed LEEC was never detected in CHO-K1 cell conditioned medium.

PNGase F cleaves the linkage between an asparagine side chain and the first N-acetylglucosamine unit of an attached polysaccharide chain. Protein produced by both systems was digested with PNGase F, and this resulted in faster migration on SDS-PAGE (Fig. 6). These data indicate that both expression systems are glycosylating asparagine residues. After PNGase F digestion, the CHO-K1 LEEC-P1 protein, which contains 11 of the 14 potential N-glycosylation sites of full-length fibrillin 1, changes SDS-PAGE migration so that it is approximately equal to the untreated LEE protein which has a calculated molecular mass that is 19 kDa less.


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Fig. 6.   PNGase digestion of glycosylated protein from baculoviral and CHO-K1 systems. A, Western blot of LEEC protein immunoprecipitated from the baculoviral system with the FBN1-CT antibody and digested with PNGase. Both the processed and unprocessed forms are sometimes visible in conditioned medium from the baculoviral system (see text). B, autoradiography of [35S]Cys-labeled LEE and LEEC-P1 protein isolated from the CHO-K1 system and digested with PNGase. The PNGase-treated proteins from both expression systems migrate more rapidly than untreated protein due to the removal of Asn-linked sugars.

Mapping Intracellular Cleavage Compartment Using Secretion Blocking Agents-- The presence of processed LEEC in cell lysates suggested that cleavage of the carboxyl-terminal domain occurs intracellularly. To rule out the possibility that processing was occurring extracellularly on the cell surface, CHO cells were treated with trypsin prior to lysis to remove surface-bound proteins. Fig. 4C shows that both processed and unprocessed LEEC were still evident in the cell lysate after trypsin treatment, confirming that both forms of the protein were inaccessible to trypsin digestion.

To better characterize the location and timing of intracellular processing of the human fibrillin-1 carboxyl-terminal domain present in the LEEC protein, four well known secretion blocking agents were employed. Ammonium chloride is a weak base that blocks the secretory pathway by neutralizing the pH of acidifying compartments of the trans-Golgi and secretory vesicles. Monensin mediates a one-for-one exchange of a proton for a monovalent cation such as sodium or potassium (18). The use of ammonium chloride and monensin in pulse-chase experiments resulted in the build-up of processed LEEC (Fig. 7). The pH neutralization that results from the use of agents such as ammonium chloride and monensin have been shown to disrupt processing of secreted proteins normally carried out by the enzymes that reside in late compartments (19, 20). The presence of processed LEEC under these conditions is consistent with the idea that proteolytic processing occurs before and not in these late secretory compartments.


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Fig. 7.   Secretion blocking of LEEC in CHO-K1 cells. Cells were preincubated with the various blocking agents, pulsed for 30 min with [35S]Cys, and chased in the presence of blocking agents for 2 h as described under "Experimental Procedures." Gel A contains cell lysate. Gel B contains conditioned medium. For both gels: lane 1, control with no blocking agents; lane 2, brefeldin (10 µg/ml); lane 3, A23187 (2 µM); lane 4, brefeldin (10 µg/ml) + A23187 (2 µM); lane 5, monensin (10 µM); lane 6, ammonium chloride (50 mM). Upper and lower arrows indicate unprocessed and processed forms, respectively.

Brefeldin A (BFA) disrupts anterograde transport from the ER to the cis-Golgi by blocking the ability of ADP-ribosylation factors to exchange GTP for GDP and become activated (21). Under circumstances when forward transport is blocked, retrograde transport becomes visibly apparent, and this leads to the redistribution of many Golgi resident proteins back to the ER (for review see Ref. 22). The use of BFA in pulse-chase experiments resulted in the build up of processed LEEC (Fig. 7) indicating that in CHO-K1 cells, the cleavage occurs in the ER or results from a Golgi resident enzyme that acts after the fusion of the two compartments.

To refine further the cleavage compartment of LEEC in CHO-K1 cells, the calcium ionophore calcimycin (A23187), which causes the equilibration of calcium levels between the ER, Golgi, and cytoplasm, was employed. There are strict calcium requirements for trafficking from the ER to the Golgi (see Ref. 23 and for review see Ref. 24). By disrupting this balance, calcimycin blocks protein exit from the ER (25, 26) without altering protein synthesis or inhibiting glycosyltransferases (27, 28). When calcimycin is used in combination with BFA, retrograde flow of Golgi contents to the ER is efficiently blocked (28).

The use of calcimycin alone or calcimycin with BFA in the pulse/chase protein labeling studies results in the intracellular retention of both unprocessed and processed LEEC (Fig. 7). Importantly, treatment with calcimycin + BFA results in a form of LEEC with a SDS-PAGE migration suggestive of incomplete glycosylation confirming that the inhibition of retrograde flow from the Golgi has been successfully inhibited. Because LEEC is already proteolytically processed at the point of this block, we can conclude that processing is carried out by an enzyme that resides early in the secretory pathway.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To study the processing of the carboxyl-terminal domain of fibrillin-1, protein from three truncated fibrillin-1 constructs was produced by baculoviral expression in SF9 cells and stable expression from the pEE14 vector in CHO-K1 cells. In these expression systems, each of the three constructs (LEE, LEEC, and LEEC-P1) was produced at high levels with the expected post-translational modifications of leader sequence cleavage, glycosylation, and carboxyl-terminal domain processing.

Amino-terminal sequencing of protein from both expression systems revealed leader sequence processing at two sites: a primary site between Gly-24/Ala-25 and a secondary site of Ala-27/Asn-28 (Fig. 2). The secondary site was predicted by Pereira et al. (29) to be the signal peptide cleavage site from primary sequence analysis of the cloned cDNA. Cleavage at Gly-24/Ala-25, however, is an acceptable site based upon signal sequence structural requirements and signal peptidase specificity. It has been reported that a multibasic site in the FBN1 leader sequence was intracellularly processed when expressed in HT1080 cells (11). These constructs, however, employed the BM40 leader sequence inserted before the FBN1 leader sequence. This may explain the differences between our results and the previously reported observations.

Proteolytic processing of the FBN1 carboxyl-terminal domain in the LEEC construct occurred in both the baculoviral and CHO-K1 cell systems. The inability of the FBN1-386 antibody to recognize processed LEEC confirms that the region is lost after processing (Fig. 3). Although the exact cleavage site was not determined, it is likely to occur at the multibasic sequence indicated in Figs. 1 and 2. Tetrabasic sequences of this type have been shown to be sites of cleavage by members of the Furin/PACE family of proteases, and it has been suggested that these enzymes are responsible for the processing event in native fibrillin (12).

There are seven known mammalian members of the furin/PACE family that are related to the bacterial subtilisins (30). Takahashi et al. (31) have assigned a site preference order for one furin family member (PACE) using in vivo expression data coupled with information from in vitro incubations with purified PACE. They found the following: RXRXKR > XXRXKR >/= RXRXXR > RXXXKR > XXRXXR with an arginine in the -P1 position being required. Sites with XXXXKR and RXXXXR sequences were not processed. These rules are specific only for PACE. The other family members that have been examined, e.g. PACE4, have slightly different preferences (32, 33).

In agreement with these established site preferences, the LEEC-P1 mutant, which replaces the required arginine with a serine in the -P1 position, shows less efficient processing than LEEC. It should be noted that an acceptable site still exists in the -P1 sequence if the recognition frame is shifted one residue upstream. In this frame, however, the -P4 basic residue is then lost which would be expected to lower the suitability of the site.

It is interesting to compare the putative processing site of fibrillin-1 (PKRGRKRRS) to that of fibrillin-2 (PKKDSRQKRS). Data from Milewicz et al. (12) indicates that the enzyme that normally processes fibrillin-1 has a -P6 basic residue requirement. However, there is not a basic residue in the -P6 position of fibrillin-2. This suggests that fibrillin-1 and fibrillin-2 may be processed by different enzymes, or with different efficiencies, or in different compartments within the same cell. Given the wide tissue- and cell-specific differences of expression in the furin family, it may be that the cleavage of the carboxyl-terminal domain of fibrillins is not confined to only one subcellular compartment or carried out by only one protease and may vary by cell and tissue type. Fibrillin maturation within different compartments of the secretory pathway may affect ECM assembly by preventing or allowing interactions with chaperones or other matrix components. In this way, tissue-specific or temporal expression of the furin responsible for fibrillin processing may afford a level of post-translational control of the maturation and matrix deposition of the molecule.

To date, all known members of the PACE family are calcium-dependent (34, 35), and modeling studies have predicted a calcium-binding site, conserved from subtilisin, within their catalytic domains. Raghunuth et al. (36) reduced the efficiency of fibrillin cleavage in human fibroblasts by the addition of 5 mM EGTA to culture medium. This would be expected to hinder the activity of intracellular PACE which has half-maximal activity at a calcium concentration of 0.2 mM (37). Also, calcimycin-induced calcium depletion has been shown to inhibit a Golgi-localized furin (38). Indeed, the calcium sensitivity of the processing enzyme in the CHO-K1 protein expression system described here is evident as the disruption of normal calcium levels by calcimycin reduced the efficiency of the processing enzyme and leads to the retention of the unprocessed form of LEEC that is still visible in the samples incubated with calcimycin. This is consistent with the idea that a furin family member is responsible for processing in this cell type.

Raghunath et al.2 added in vitro transcribed and translated LEEC (referred to as "mini-fibrillin") to medium conditioned by cells transfected with PACE, PACE4, PC1/3, and PC2 mutated by the removal of their normal transmembrane domains to result in secretion. Each of the enzymes tested was able to process LEEC. This work demonstrates that the tetra-basic site in the fibrillin-1 carboxyl-terminal domain can be recognized by these proteases. Wild type PACE is thought to localize to the trans-Golgi network and therefore would be expected to process proteins of the constitutive secretory pathway (for review see Refs. 35, 39, and 40). The intracellular localization of other widely expressed PACE family members such as PACE4, PC5/6, and PC8 has not been determined but is also thought to be somewhere in the constitutive secretory pathway (30, 33, 41).

The compartment in which the processing of full-length fibrillin occurs is still unknown. Immunoprecipitation of radiolabeled fibrillin from conditioned FBC medium yields only one band by autoradiography (Fig. 3A). Additionally, immunoprecipitated fibrillin is not detected by Western blot with the FBN1-386 antibody made against an epitope in the carboxyl-terminal fragment that is cleaved from the full-length protein, yet the FBNI-386 antibody readily detects the unprocessed domain of the LEEC-P1 mutant. The lack of significant amounts of unprocessed fibrillin in conditioned medium indicates that processing either occurs just after secretion in a quick and efficient manner or occurs intracellularly in a way similar to the processing undergone by our constructs in the CHO and SF9 systems.

Milewicz et al. (12) have proposed that carboxyl-terminal domain processing must occur before fibrillin can be incorporated into a linear microfibril. Studying a cell line that secretes fibrillin with the entire carboxyl-terminal domain intact because of an arginine to tryptophan mutation at position 2726, the authors found that the unprocessed fibrillin was not incorporated into the matrix. Yet three Marfan causing mutations have been described that result in the omission of 69, 83, and 90% of the region normally lost after the processing step. Two mutations, R2756X and R2776X, result in a premature translation stop and the omission of the last 116 and 96 residues of the domain, respectively (42, 43). A third mutation is a two nucleotide deletion (8237 delGA) that results in a frameshift and a premature stop. This allele codes correctly through residue 2746 and then ends with an out of frame stop at 2757 (44). Importantly, premature intracellular polymerization of fibrillin has not been reported for cells expressing these mutations. These mutations are different from all other fibrillin-1 mutations in that they affect a part of the molecule that is not present in the mature protein. They show that this region somehow plays an important role in the assembly or matrix incorporation of the molecule.

Handford et al. (45) has proposed a model of fibrillin assembly in which monomers assemble into an aligned bundle. It may be that the carboxyl-terminal domain of fibrillin serves to aid the intracellular association of monomers into multimeric fibrillin building blocks (e.g. dimers or larger multimers), and once that function has been accomplished, the domain can be processed, and other assembly steps can proceed. Other matrix molecules are known to assemble intracellularly into large multimeric structures and then undergo secretion, most notably laminin (46), tenascin (47), and several of the collagens (48).

With the constructs described here, we have been able to more closely observe the proteolytic processing of the fibrillin-1 carboxyl-terminal domain. The loss of the expected region is shown by SDS-PAGE migration differences and immunologically by Western blotting. Impaired processing observed in the mutant LEEC-P1 construct confirms the involvement of the multi-basic site in normal processing. Use of secretion blocking agents has shown that cleavage occurs intracellularly in the CHO-K1 cells and is likely carried out by an early secretory pathway resident furin-like enzyme.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL53325, HL29594, HL41926 (to R. P. M.), and RO1 AR43626 (to D. M. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, Box 8228, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2254; Fax: 314-362-2252; E-mail: bmecham{at}cellbio.wustl.edu.

2 M. Raghunath, E. A. Putnam, T. M. Ritty, D. Hamstra, E-S. Park, M. Tschodrich-Rotter, R. Peters, A. Rehemtulla, and D. M. Milewicz, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: EGF, epidermal growth factor; BFA, brefeldin A; BSMC, bovine smooth muscle cell; ER, endoplasmic reticulum; FBC, fetal bovine chondrocyte; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PNGase, peptide N-glycosidase F; FBS, fetal bovine serum; bp, base pair; DMEM, Dulbecco's modified Eagle's medium; CHO, Chinese hamster ovary.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Rosenbloom, J., Abrams, W. R., and Mecham, R. (1993) FASEB J. 7, 1208-1218[Abstract/Free Full Text]
  2. Kielty, C. M., and Shuttleworth, C. A. (1995) Int. J. Biochem. & Cell Biol. 27, 747-760[CrossRef][Medline] [Order article via Infotrieve]
  3. Dietz, H. C., and Pyeritz, R. E. (1995) Hum. Mol. Genet. 4, 1799-1809[Abstract]
  4. Francke, U., Berg, M. A., Tynan, K., Brenn, T., Liu, W., Aoyama, T., Gasner, C., Miller, D. C., and Furthmayr, H. (1995) Am. J. Hum. Genet. 56, 1287-1296[Medline] [Order article via Infotrieve]
  5. Milewicz, D. M., Michael, K., Fisher, N., Coselli, J. S., Markello, T., and Biddinger, A. (1996) Circulation 94, 2708-2711[Abstract/Free Full Text]
  6. Kainulainen, K., Karttunen, L., Puhakka, L., Sakai, L., and Peltonen, L. (1994) Nat. Genet. 6, 64-69[Medline] [Order article via Infotrieve]
  7. Sood, S., Eldadah, Z. A., Krause, W. L., McIntosh, I., and Dietz, H. C. (1996) Nat. Genet. 12, 209-211[Medline] [Order article via Infotrieve]
  8. Aoyama, T., Francke, U., Dietz, H. C., and Furthmayr, H. (1994) J. Clin. Invest. 94, 130-137[Medline] [Order article via Infotrieve]
  9. Dietz, H. C., McIntosh, I., Sakai, L. Y., Corson, G. M., Chalberg, S. C., Pyeritz, R. E., and Francomano, C. A. (1993) Genomics 17, 468-475[CrossRef][Medline] [Order article via Infotrieve]
  10. Glanville, R. W., Qian, R. Q., McClure, D. W., and Maslen, C. L. (1994) J. Biol. Chem. 269, 26630-26634[Abstract/Free Full Text]
  11. Reinhardt, D. P., Keene, D. R., Corson, G. M., Poschl, E., Bachinger, H. P., Gambee, J. E., and Sakai, L. Y. (1996) J. Mol. Biol. 258, 104-116[CrossRef][Medline] [Order article via Infotrieve]
  12. Milewicz, D. M., Grossfield, J., Cao, S. N., Kielty, C., Covitz, W., and Jewett, T. (1995) J. Clin. Invest. 95, 2373-2378[Medline] [Order article via Infotrieve]
  13. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1992) in Current Protocols in Molecular Biology (Janssen, K., ed), pp. 16.14.6-16.14.13, John Wiley & Sons, Inc., New York
  14. Crouch, E., Chang, D., Rust, K., Persson, A., and Heuser, J. (1994) J. Biol. Chem. 269, 15808-15813[Abstract/Free Full Text]
  15. O'Reilly, D. O., Miller, L. K., and Lucklow, V. A. (eds) (1992) Baculovirus Expression Vectors, pp. 218-219, W. H. Freeman and Co., New York
  16. Hu, S. I., Kosowski, S. G., and Schaaf, K. F. (1987) J. Virol. 61, 3617-3620[Medline] [Order article via Infotrieve]
  17. Ramabhadran, T. V., Gandy, S. E., Ghiso, J., Czernik, A. J., Ferris, D., Bhasin, R., Goldgaber, D., Frangione, B., and Greengard, P. (1993) J. Biol. Chem. 268, 2009-2012[Abstract/Free Full Text]
  18. Pressman, B. C., and Fahim, M. (1982) Annu. Rev. Pharmacol. Toxicol. 22, 465-490[CrossRef][Medline] [Order article via Infotrieve]
  19. Beers, M. F. (1996) J. Biol. Chem. 271, 14361-14370[Abstract/Free Full Text]
  20. Martin, B. L., Schrader-Fischer, G., Busciglio, J., Duke, M., Paganetti, P., and Yankner, B. A. (1995) J. Biol. Chem. 270, 26727-26730[Abstract/Free Full Text]
  21. Lippincott-Schwartz, J., Donaldson, J. G., Schweizer, A., Berger, E. G., Hauri, H. P., Yuan, L. C., and Klausner, R. D. (1990) Cell 60, 821-836[Medline] [Order article via Infotrieve]
  22. Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz, J. (1992) J. Cell Biol. 116, 1071-1080[Medline] [Order article via Infotrieve]
  23. Beckers, C. J., and Balch, W. E. (1989) J. Cell Biol. 108, 1245-1256[Abstract]
  24. Balch, W. E. (1990) Trends Biochem. Sci. 15, 473-477[CrossRef][Medline] [Order article via Infotrieve]
  25. Beccari, T., Datti, A., Orlacchio, A., Farinelli, S., and Blasi, E. (1992) Biochem. Int. 27, 783-791[Medline] [Order article via Infotrieve]
  26. Zhang, C. Y., Fujinaka, Y., Yokogoshi, Y., and Saito, S. (1995) Biochem. Biophys. Res. Commun. 207, 238-243[CrossRef][Medline] [Order article via Infotrieve]
  27. Coukell, M. B., Cameron, A. M., and Adames, N. R. (1992) J. Cell Sci. 103, 371-380[Abstract/Free Full Text]
  28. Ivessa, N. E., De Lemos-Chiarandini, C., Gravotta, D., Sabatini, D. D., and Kreibich, G. (1995) J. Biol. Chem. 270, 25960-25967[Abstract/Free Full Text]
  29. Pereira, L., D'Alessio, M., Ramirez, F., Lynch, J. R., Sykes, B., Pangilinan, R., and Bonadio, J. (1993) Hum. Mol. Genet. 2, 961-968[Abstract]
  30. Bruzzaniti, A., Goodge, K., Jay, P., Taviaux, S. A., Lam, M. H., Berta, P., Martin, T. J., Moseley, J. M., and Gillespie, M. T. (1996) Biochem. J. 314, 727-731[Medline] [Order article via Infotrieve]
  31. Takahashi, S., Hatsuzawa, K., Watanabe, T., Murakami, K., and Nakayama, K. (1994) J. Biochem. (Tokyo) 116, 47-52[Abstract]
  32. Rehemtulla, A., Barr, P. J., Rhodes, C. J., and Kaufman, R. J. (1993) Biochemistry 32, 11586-11590[Medline] [Order article via Infotrieve]
  33. Creemers, J. W., Kormelink, P. J., Roebroek, A. J., Nakayama, K., and Van de Ven, W. J. (1993) FEBS Lett. 336, 65-69[CrossRef][Medline] [Order article via Infotrieve]
  34. Davidson, H. W., Rhodes, C. J., and Hutton, J. C. (1988) Nature 333, 93-96[CrossRef][Medline] [Order article via Infotrieve]
  35. Halban, P. A., and Irminger, J. C. (1994) Biochem. J. 299, 1-18[Medline] [Order article via Infotrieve]
  36. Raghunath, M., Kielty, C. M., and Steinmann, B. (1995) J. Mol. Biol. 248, 901-909[CrossRef][Medline] [Order article via Infotrieve]
  37. Molloy, S. S., Bresnahan, P. A., Leppla, S. H., Klimpel, K. R., and Thomas, G. (1992) J. Biol. Chem. 267, 16396-16402[Abstract/Free Full Text]
  38. Oda, K. (1992) J. Biol. Chem. 267, 17465-17471[Abstract/Free Full Text]
  39. Smeekens, S. P. (1993) Bio/Technology 11, 182-186[Medline] [Order article via Infotrieve]
  40. Denault, J. B., and Leduc, R. (1996) FEBS Lett. 379, 113-116[CrossRef][Medline] [Order article via Infotrieve]
  41. Rehemtulla, A., Dorner, A. J., and Kaufman, R. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8235-8239[Abstract]
  42. Kainulainen, K., Sakai, L. Y., Child, A., Pope, F. M., Puhakka, L., Ryhanen, L., Palotie, A., Kaitila, I., and Peltonen, L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5917-5921[Abstract]
  43. Hayward, C., Porteous, M. E., and Brock, D. J. (1994) Hum. Mutat. 3, 159-162[Medline] [Order article via Infotrieve]
  44. Nijbroek, G., Sood, S., McIntosh, I., Francomano, C. A., Bull, E., Pereira, L., Ramirez, F., Pyeritz, R. E., and Dietz, H. C. (1995) Am. J. Hum. Genet. 57, 8-21[Medline] [Order article via Infotrieve]
  45. Handford, P., Downing, A. K., Rao, Z., Hewett, D. R., Sykes, B. C., and Kielty, C. M. (1995) J. Biol. Chem. 270, 6751-6756[Abstract/Free Full Text]
  46. Lissitzky, J. C., Charpin, C., Bignon, C., Bouzon, M., Kopp, F., Delori, P., and Martin, P. M. (1988) Biochem. J. 250, 843-852[Medline] [Order article via Infotrieve]
  47. Redick, S. D., and Schwarzbauer, J. E. (1995) J. Cell Sci. 108, 1761-1769[Abstract/Free Full Text]
  48. Engvall, E., Hessle, H., and Klier, G. (1986) J. Cell Biol. 102, 703-710[Abstract]


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