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J. Biol. Chem., Vol. 278, Issue 34, 31796-31806, August 22, 2003
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From the
Departments of Biochemistry and
Ophthalmology, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226
Received for publication, March 7, 2003 , and in revised form, June 4, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Several biological activities of maspin have been characterized, which
suggest a role for maspin as a tumor suppressor and an inhibitor of
angiogenesis. Addition of recombinant maspin or transfection of the maspin
gene into carcinoma cells inhibits cell migration and invasion in
vitro and reduces tumor growth and metastasis in vivo
(3,
8). Maspin also inhibits the
in vitro migration and proliferation of endothelial cells and blocks
basic fibroblast growth factor-induced neovascularization in the rat corneal
pocket model (9). To date, the
underlying mechanisms of maspin action on inhibition of tumor invasion and
angiogenesis are not well established. Nonetheless, maspin can regulate
adhesion of cultured corneal stromal cells and carcinoma cells to
extracellular matrix
(ECM)1 molecules
(2,
10). Pretreatment with
recombinant maspin increases adhesion of cultured corneal stromal cells to
several ECM molecules, including type I and type IV collagen, laminin, and
fibronectin (2), whereas it
induces adhesion of carcinoma cells only to fibronectin and not to gelatin,
laminin, type I, and type IV collagens or fibrinogen
(10). Stimulation of cell-ECM
adhesion by maspin likely involves a mechanism by which maspin up-regulates
expression of integrins, because the level of 
5 integrin
(the component of the fibronectin receptor) on the cell surface is
induced in carcinoma cells pretreated with recombinant maspin
(10).
Maspin shares sequence homology with the serpins (serine protease inhibitors) of the ovalbumin-type subfamily, which includes ovalbumin, plasminogen activator inhibitor-2, squamous cell carcinoma antigen (SCCA), and bomapin (PI10) (11). Most serpins are inhibitors of specific proteases that react with an exposed reactive site loop (RSL) at the top of the molecule (see Fig. 1). Whether maspin is a protease inhibitor is still controversial. Early in vitro studies showed that maspin does not inhibit a number of representative proteases (12). One recent study demonstrated maspin can inhibit prostate carcinoma cell surface-associated plasminogen activation by urokinase type plasminogen activator (uPA) (13), however, another study, using uPA bound to its receptor on tumor cells and tPA bound to vascular smooth muscle cells, fibrin or the prion protein, reported maspin does not inhibit either of these two plasminogen activators (14).
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Although an intact RSL of maspin is required for inhibition of migration and invasion of mammary gland and prostate carcinoma cells (8), nothing is known about which region of the maspin molecule is required for induction of increased cell-ECM adhesion. Preincubation with blocking antibodies to the RSL of maspin (8), deletion from the P7' residue of the RSL to the C-terminal end (15), and cleavage of maspin at the putative P1 site Arg within the RSL (16) destroyed the ability of maspin to regulate the migration and invasion of carcinoma cells. We hypothesized that stimulation of cell-ECM adhesion by maspin also utilizes the RSL.
We investigated the structure-function relationship of maspin using maspin/ovalbumin chimeras rather than using maspin deletion mutants as previously studied, due to a concern about proper folding of the mutant molecules. A tertiary structure model of maspin based on the crystal structure of ovalbumin (Protein Data Bank: 1OVA [PDB] ) suggests maspin adopts a common serpin structure in which the C-terminal region (Strands B4 and B5) lies inside the molecule (Fig. 1). Deletion of this region could disrupt the overall serpin conformation resulting in an inactive molecule, whereas swapping equivalent regions between maspin and a homologous serpin would potentially preserve the overall structure. Expression of yeast recombinant maspin/ovalbumin chimeras as secreted proteins increases the probability the proteins are properly folded. Ovalbumin was chosen for swapping domains with maspin, because the physical properties of both molecules are similar. Maspin and ovalbumin are similar in size and share about 30% amino acid sequence identity. Neither molecule undergoes a urea-induced unfolding transition from a stressed (S) form to a heat-stable relaxed (R) form (12, 17). Furthermore, the hinge region (P8 to P12) of ovalbumin and maspin differs from that of inhibitory serpins in which a multiple alanine stretch is conserved (18). Most importantly, unlike maspin, ovalbumin does not induce cell-ECM adhesion (2).
In this study, we explored the function of the RSL domain of maspin on cell-ECM adhesion and tumor invasion using a region swapping approach between maspin and ovalbumin to preserve the serpin structure. The studies reported here document that 1) the maspin RSL domain, but not the C-terminal region, is required, 2) the RSL peptide is sufficient for induction of increased cell-ECM adhesion of corneal stromal cells and carcinoma cells and inhibition of carcinoma cell invasion, 3) replacement of the RSL of ovalbumin with that of maspin can convert ovalbumin to a maspin-like molecule, and 4) the RSL can compete for specific binding of maspin to carcinoma cells.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1.6 HIS3 lys2–208
trp1-101 ura3–52 gal2 can1) was obtained from Sigma
(St. Louis, MO). Escherichia coli strain JM 109 was purchased from
Promega (Madison, WI). ProSTARTM Ultra HF RT-PCR System and
PfuTurbo® Hotstart DNA polymerase were from Stratagene (La Jolla,
CA). All oligonucleotide primers were from Invitrogen Custom Primers
(Rockville, MD). The Ni-NTATM-agarose was obtained from Qiagen (Valencia,
CA). All restriction endonucleases were from New England BioLabs (Beverly,
MA). The CytoMatrixTM adhesion assay strips coated with human type I
collagen, fibronectin, or laminin were purchased from Chemicon International,
Inc. (Temecula, CA). Calcein acetoxymethyl ester (calcein AM), CyQuant®GR
dye, and BODIPY®-TR-X succinimidyl ester were obtained from Molecular
Probes (Eugene, OR). Clostripain (endoproteinase-Arg-C) was from Worthington
Biochemical Corp. (Lakewood, NJ). Cell-Essentials, Inc. (Boston, MA)
synthesized a 15-amino acid RSL polypeptide (P10–P5'). The purity
was greater than 95%, and the composition was confirmed by mass spectroscopy.
Human mammary gland carcinoma MDA-MB-231 cells and mouse T-lymphoma cells
transfected with pAc-neo-OVA, E.G7-OVA were obtained from American Type
Culture Collection (Manassas, VA). An immortalized normal human corneal
stromal cell line was a generous gift from Dr. M. A. Watsky (College of
Medicine, University of Tennessee, Memphis, TN). Leibovitz's L-15
medium, Dulbecco's modified Eagle's medium (with high glucose and
L-glutamine and without pyruvate), RPMI 1640 medium (with
L-glutamine), and neomycin (G418) were obtained from Invitrogen
(Carlsbad, CA). Defined fetal bovine serum (FBS) was from HyClone (Logan, UT).
MITO+TM serum extender and MatrigelTM were from BD
Biosciences (Bedford, MA). Polycarbonate membrane Transwell® chamber
inserts were purchased from Corning Costar Corp. (Cambridge, MA).
Ciprofloxacin was obtained from Bayer Corp. (Kankakee, IL). Unless specified,
all other reagents and medium components for the yeast and bacterial cultures
were purchased from Sigma. Cell Cultures—The MDA-MB-231 cells were maintained in Leibovitz's L-15 medium with 10% FBS and 10 µg/ml ciprofloxacin at 37 °C without CO2. The mouse T-lymphocyte E.G7-OVA cells were grown at 37 °C with 5% CO2 in RPMI 1640 medium with 2 mM L-glutamine, 18 mM sodium bicarbonate, 25 mM glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, 0.4 mg/ml G418, and 10% FBS. The immortalized normal human corneal stromal cells (19), which mimic activated migratory wound corneal stromal cells and have the same properties as passaged human corneal stromal cells, were routinely grown in Dulbecco's modified Eagle's medium containing 5% FBS, 1x MITO+, and 10 µg/ml ciprofloxacin in a humidified incubator with 5% CO2 at 34 °C.
The MDA-MB-231 human mammary carcinoma cells and immortalized corneal stromal cells do not synthesize maspin in contrast to normal mammary epithelial and corneal epithelial cells as determined by RT-PCR and Western blots using previously reported conditions for these assays (2).
Construction of Maspin/Ovalbumin Swap Mutants—The human maspin gene was previously obtained from human corneal epithelium by RT-PCR as described by Ngamkitidechakul et al. (2). Total RNA from the T lymphocyte cell line containing the ovalbumin gene was extracted using TRI reagent. The full-length ovalbumin gene was specifically amplified by RT-PCR using the ProSTARTM Ultra HF RT-PCR system according to the manufacturer's procedure (Stratagene) in the Mastercycler® personal thermocycler (Eppendorf, Westbury, NY). In the RT step, a specific ovalbumin antisense primer (Table I) was used to generate ovalbumin cDNA. PCR amplification of the cDNA was performed using two primers in which the EcoRI site was incorporated into the 5'-end of the sense primer and BglII into the 5'-end of the antisense primer. The annealing temperature for the PCR was 65 °C. The gel-purified 1.2-kb PCR product was digested with EcoRI and BglII, and ligated into the YEpHF expression vector (20). DNA sequencing at the MCW Protein and Nucleic Acid Facility confirmed the nucleotide sequences of both human maspin and chicken ovalbumin cDNA. Three ovalbumin (OV)/maspin-RSL swap mutants and one chimeric maspin/OV-RSL swap mutant were constructed by the overlap extension PCR method of Ho et al. (21) using the primers listed in Table I. For each mutant construction, two sets of primers were utilized to amplify two separate overlapping PCR products that contain the mutation (or insertion) together with either upstream or downstream sequences. These PCR products were amplified using PfuTurbo® Hotstart DNA polymerase according to the manufacturer's instructions using the optimal annealing temperature at 60 °C. Two PCR fragments were joined together through overlapping sequences and PCR using primers from the 5'- and 3'-ends of the appropriate gene. An annealing temperature of 55 °C was used. The full-length PCR products were cut with EcoRI and BglII and cloned directly into the yeast expression vector YEpHF. The MCW Protein and Nucleic Acid Facility confirmed the reading frame and sequences of all mutants.
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To construct a site-specific P1 R to Q and R to A maspin mutants, the YEpHF-Maspin template vector was directly amplified by the method of Weiner et al. (22) using two primers containing the mutation sequences (Table I) and PfuTurbo® Hotstart DNA polymerase. After 18 cycles of PCR amplification (55 °C annealing), the PCR reaction was treated with DpnI (1 unit) at 37 °Cfor1hto remove the parental template. The purified PCR products were transformed into JM 109 E. coli cells. A colony containing the plasmid with the desired mutation was chosen, and the mutated sequences were confirmed by sequencing.
Expression and Purification of Yeast Recombinant HIS/FLAG-tagged
Proteins—All recombinant proteins were produced as N-terminal
HIS/FLAG tag fusion proteins in a yeast system. The yeast expression vector
YEpHF was modified from the original vector YEp-FLAG-1 by adding a
six-histidine tag upstream of the FLAG peptide tag
(20). Due to the presence of
an -factor leader peptide sequence, the recombinant proteins were
expressed and secreted outside the S. cerevisiae yeast BJ3505 into
the buffered expression medium (2% peptone, 1% yeast extract, 3% glycerol, 1%
glucose, and 100 mM K2HPO4, pH 6.4). After
growing at 30 °C for 72 h, 1 liter of the culture medium was collected and
concentrated to 200 ml by ultrafiltration (Amicon) under nitrogen gas at 60
p.s.i. The concentrate was pH-adjusted to 8.0.
The yeast rHIS/FLAG-tagged protein was purified by mixing the concentrated yeast conditioned medium with 10 ml of Ni-NTATM-agarose resin overnight at 4 °C. The resin was washed with 50 mM phosphate buffer, pH 8.0, containing 300 mM NaCl and 10 mM imidazole. Subsequently, the recombinant protein was eluted with 50 mM phosphate buffer, pH 7.0, containing 300 mM NaCl and 250 mM imidazole. The eluted protein was concentrated using an Ultrafree spin concentrator (Millipore), and dialyzed in phosphate buffered saline (PBS), pH 7.4, to remove the imidazole. Identical results were obtained in the presence and absence of the N-terminal His tag and the FLAG sequence for the maspin functions measured here.
Arginine-specific Proteolysis of Recombinant Maspin and the R340Q and R340A Mutants—Endoproteinase-Arg-C (clostripain) was incubated with the yeast recombinant wild-type maspin the R340Q and R340A mutants to confirm the mutagenesis. The enzyme was preactivated at room temperature for 2 h in 1 mM calcium acetate containing 2.5 mM dithiothreitol. The recombinant proteins (50 µg) in 50 mM ammonium bicarbonate, 0.2 mM calcium acetate, and 2.5 mM dithiothreitol were incubated with the enzyme (50:1 molar ratio) at 37 °C. At various time points, aliquots were removed and E-64 (10 µM) was added to stop the enzymatic reaction. For zero time points, 5 µg of the proteins was mixed with E-64 at 10 µM final concentration and then the enzyme was added. SDS sample buffer was added to the reaction samples and then boiled. The samples were subjected to SDS-PAGE, and the proteins were visualized by Coomassie Brilliant Blue staining. After 2 min of incubation with the protease, the wild-type maspin was cleaved with the appearance of a band corresponding to the size expected for a cleavage at Arg-340, however, the maspin R340Q and R340A mutants were not cleaved over the first 60 min (data not shown). However, with time additional identical smaller products were noted in all samples.
Cell-ECM Adhesion Assay—MDA-MB-231 cells or corneal stromal cells were incubated with serum-free medium containing either 0.5 µM (MDA-MB-231 cells) or 1.0 µM (corneal stromal cells) of maspin, the maspin/ovalbumin chimeras or the RSL peptide for 18 h prior to cell adhesion assay performed according to the protocol previously used by us and others (2, 10). The maspin RSL peptide was used at 1 µM final concentration in both cell types. The adhesion assay was performed using CytoMatrix adhesion strips as previously described (2). Briefly, after preincubation with maspin, the cells were harvested using 2 mM EDTA in PBS. Both cell types were resuspended in RPMI 1640 containing 1x MITO+. The MDA-MB-231 cells and the corneal stromal cells were plated at a density of 2 x 104 cells/well and allowed to attach for 1 h at 37 °C on fibronectin-coated wells for the carcinoma cells and on type I collagen-, fibronectin-, or laminin-coated wells for the corneal stromal cells. Fibronectin was used for the MDA-MB-231 cells, because maspin stimulates adhesion only to this ECM molecule (10). Collagen type I was used for the corneal stromal cell studies, because it is the major extracellular molecule present in the corneal stroma (2). Fibronectin and laminin, ECM molecules found at lower amounts, also were used for selected experiments. Following washing with PBS, the adherent cells were stained with crystal violet and quantified using a microplate reader (Bio-Tek Instruments, Winooski, VT). The experiments were repeated four or five times with three to five replicates per experiment.
In Vitro Tumor Cell Invasion Assay—The invasion assay was performed using the standard Matrigel method of Sternlicht et al. (23) with several modifications. Briefly, polycarbonate (8-µm) Transwell inserts were precoated with 2 mg/ml Matrigel. MDA-MB-231 cells (5 x 104) were incubated in the absence or presence of 0.5 µM rHIS/FLAG maspin, ovalbumin, the maspin/ovalbumin chimeras, the R340Q and R340A maspin mutants or the RSL peptide in Leibovitz's L-15 medium containing 1% FBS, 1x MITO+, and 10 µg/ml ciprofloxacin. After 48 h of incubation at 37 °C in a humidified incubator without CO2, the cells in the bottom well were labeled with the fluorogenic vital dye calcein AM (5 µM final concentration). Fluorescence was measured using a CytoFluorTM fluorescence microplate reader (Millipore) with excitation of 480-nm and emission of 530-nm filters. Cells in duplicate wells without Transwell inserts served as controls for cell proliferation and/or death during the incubation period. Invasion was calculated by dividing the relative fluorescence value of invading cells by that of total cells plated in duplicate wells without Transwell inserts. Invasion of the control was set at 100%. The experiments were repeated four or five times with three to five replicates per experiment.
Tumor Cell-Maspin Binding and Competition Assay—Yeast recombinant maspin was conjugated with the fluorescent dye BODIPY®-TR-X, succinimidyl ester and purified, and the degree of labeling, five BODIPY molecules per maspin molecule, was determined according to the manufacturer's procedure (Molecular Probes). To perform the binding assay, increasing amounts of BODIPY-TR-X-maspin (0.2–8.0 µM) were incubated in the dark with MDA-MB-231 cells at 4 °C for 90 min in serum-free medium containing 0.1% bovine serum albumin (BSA). Next, the cells were washed three times with ice-cold PBS with 0.1% BSA. The binding was determined by measuring fluorescence using the CytoFluor microplate reader (excitation: 580 nm; emission: 640 nm). The fluorescence values were converted to maspin concentration using a linear BODIPY maspin reference standard curve. To determine nonspecific binding, the assay was performed in the presence of a 50-fold excess of non-labeled maspin. Specific binding was calculated by subtracting the nonspecific binding from the total binding. Determination of the number of cells was carried out using CyQuant® GR as described in the manufacturer's protocol (Molecular Probes) with an excitation of 485 nm and an emission of 530 nm and a linear standard cell curve. A non-linear regression curve for specific binding was calculated using five sets of data and plotted using SigmaPlot with the Enzyme Kinetics/Pharmacology Module (SPSS, Chicago, IL). Kd and Bmax values and curve statistics were determined from non-linear regression analysis using the same program.
Competition assays of cell-maspin binding were conducted using 4 µM fluorescent-labeled maspin. The RSL peptide at 2-, 10-, 20-, or 100-fold excess was added to cells along with BODIPY-TR-X-maspin. Nonspecific binding was determined by adding a 50x excess of unlabeled maspin. Bound labeled maspin was quantified in the presence and absence of each competitor as described above. The experiments were repeated three times with three to five replicates per experiment.
Statistical Analysis—Overall differences among the treatment groups were determined using one-way analysis of variance, and differences between individual treatments were determined using the Student-Newman-Keuls test by SigmaStat software (SPSS Inc.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Effect of OV/Maspin-RSL Chimeras on Cell-ECM Adhesion—In contrast to maspin, the Maspin/OV-RSL/OV-C swap mutant containing the RSL to the C-terminal end of ovalbumin did not stimulate adhesion of either the corneal stromal cells to type I collagen (Fig. 2A) or the carcinoma cells to fibronectin (Fig. 2B). However, when only the C-terminal domain of ovalbumin (Maspin/Masp-RSL/OV-C-Term) was swapped into the maspin molecule, adhesion was stimulated in both cell systems with all ECMs, suggesting the C-terminal portion of maspin is not directly associated with this activity. In contrast, replacement of the RSL of maspin alone with that of ovalbumin (Maspin/OV-RSL/Maspin-C-Term) abolished the effect on adhesion in both the corneal stromal cells to type I collagen (Fig. 2A) and the carcinoma cells to fibronectin. Therefore, the RSL of maspin is required for enhancement of cell-ECM adhesion.
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Effect of Mutation of the Putative P1 Residue, Arg-340, on Cell Adhesion—To determine whether arginine at the putative P1 site within the RSL of maspin is required for maspin activity on cell-ECM adhesion, we mutated the Arg-340 at the P1 site to a Gln in one mutant and to an Ala in a second mutant. The R340Q mutant and wild type maspin, increased adhesion of corneal stromal cells to type I collagen (Fig. 3A) and of mammary carcinoma cells to fibronectin (Fig. 3B). The R340Q mutant was significantly more active than maspin for the carcinoma cells. In contrast, the R340A mutant lost activity. This mutant failed to induce adhesion of the corneal stromal cells (Fig. 3A) but showed intermediate activity between maspin and the control for the carcinoma cells (Fig. 3B). This loss of activity is not due to a major change in conformation, because limited proteolysis using endoproteinase-Arg-C over 6 h showed the same degradation pattern for this mutant as that for the active R340Q mutant. Thus, Gln can replace Arg at the putative P1 site but Ala cannot fully substitute for Arg at this site.
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Effect of the Maspin RSL Polypeptide on Cell-ECM Adhesion—Because the RSL of maspin is required for the increase in adhesion of cells to ECM, we next examined whether the RSL is sufficient to induce this activity. A synthetic polypeptide corresponding to the 15 amino acid residues (P10–P5', aa 331–345) within the RSL of maspin was assayed to determine its effects on adhesion of cells to ECMs. The RSL peptide induced adhesion similar to maspin for both the corneal stromal cells and the carcinoma cells (Fig. 4, A and B).
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Conversion of Ovalbumin to a Maspin-like Molecule—To further characterize the role of the RSL of maspin, we generated and tested a chimeric ovalbumin mutant in which the RSL of maspin was exchanged for the RSL of ovalbumin. As shown in Fig. 5, the Ovalbumin/Masp-RSL/OV-C-Term was also able to stimulate adhesion of stromal cells to type I collagen and carcinoma cells to fibronectin. Thus, ovalbumin can be converted by the replacement of only the RSL into a maspin-like molecule that induces cell-ECM adhesion.
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Requirement of the Presence of the RSL of Maspin for Induction of Adhesion of Corneal Stromal Cells to Laminin and Fibronectin—Maspin not only stimulates adhesion of human corneal stromal cells to type I collagen but also to laminin and fibronectin (2). To determine whether the RSL of maspin is critical for the stimulation of maspin adhesion to multiple extracellular matrix molecules, two mutants, the Maspin/OV-RSL/Masp-C and the Ovalbumin/Masp-RSL/OV-C were chosen for use in the adhesion assays. The ability of maspin to stimulate adhesion of the corneal stromal cells to fibronectin and laminin in addition to type I collagen was lost upon removal of the maspin RSL and its replacement with the RSL of ovalbumin (Fig. 6). In addition, the RSL of maspin was sufficient to convert ovalbumin into a molecule that can stimulate adhesion of the corneal stromal cells not only to type I collagen but also to fibronectin and laminin. Thus, the RSL of maspin is critical for the stimulation of adhesion of human corneal cells to multiple extracellular matrix molecules.
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Effect of the Chimeric Maspin/OV Mutants, the Putative P1 Mutants, and the Maspin RSL Polypeptide on Carcinoma Cell Invasion—The RSL of maspin was previously shown to be important for inhibition of carcinoma cell invasion (8, 15, 16). Deletion of the P7' residue through the C-terminal end of maspin (aa 347–375) and trypsin cleavage of maspin at the P1 site Arg within the RSL resulted in loss of the activity. However, these modifications can potentially alter the overall structure of maspin leading to an inactive molecule. To better demonstrate the requirement of the RSL of maspin for inhibition of carcinoma cell invasion, we tested the maspin/ovalbumin chimeras using an in vitro tumor cell invasion assay. As predicted, the RSL of maspin is required, because substitution of the RSL of maspin with that of ovalbumin (Masp/OV-RSL/Masp C) abolished the activity (Fig. 7). Substitution of Arg at the putative P1 site with Gln (RQ) retained inhibitory activity, whereas this activity was lost in the Ala mutant (RA). The ovalbumin/maspin containing the RSL of maspin (OV/Masp-RSL/OV C) inhibited invasion of the MDA-MB-231 mammary gland carcinoma cells further establishing the role of the RSL. These results suggest inhibition of carcinoma cell invasion by maspin utilizes the P10–P5' residues within the RSL. The RSL peptide was tested, and the results confirmed a functional role of this region. Similar to the effect on increased cell-ECM adhesion, this peptide was sufficient to inhibit invasion of the carcinoma cells through Matrigel.
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Determination of the Kinetics of Maspin Binding to Mammary Gland Carcinoma Cells and the Ability of the RSL Peptide to Inhibit Maspin Binding—The ability of the RSL peptide to mimic maspin activity led us to hypothesize that the effects induced by maspin are initiated by maspin binding to the cells through the RSL. Maspin binding to mammary gland carcinoma cells is specific and saturable (Fig. 8). The data fit best a one binding site model with an r2 of 0.98. The kd determined by non-linear regression was 367 ± 67 nM and the Bmax was 5.44 ± 0.24 pmol/105 cells. The calculated number of maspin binding sites per cell was 32.0 ± 2.2 x 106. These values indicate that maspin binds to the carcinoma cells through a high number of low affinity sites. Using near saturating amounts of labeled maspin (4 µM), the RSL peptide even at 2x (8 µM) competed for specific binding of maspin to the carcinoma cells (Fig. 9). The degree of inhibition of specific maspin binding did not significantly change from 2 and 100x of the RSL peptide. These results suggest that maspin can bind to the cell surface through the RSL.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In contrast to the effect of substitution of Arg-340 with Gln on cell-ECM adhesion and invasion, the putative P1 Arg of maspin cannot be substituted with this residue for inhibition of carcinoma cell migration (9). Although both this study and ours used multiple cell types and ECMs, the results within each set of experiments are consistent. The migration of the mammary carcinoma cells through Matrigel-coated membranes was inhibited by maspin but not by an R340Q mutant (9). The same results were shown for endothelial cell migration through membranes coated with gelatin and skin fibroblast and keratinocyte migration though non-coated membranes. The R340Q mutant retained the ability to stimulate adhesion of the carcinoma cells to fibronectin and the corneal stromal cells with type I collagen, fibronectin, and laminin (data not shown). This mutant also inhibited invasion of mammary carcinoma cells through Matrigel, the same matrix used for the migration assay. Thus, the differences probably are due to the mode of interaction of maspin with the cells rather than the extracellular matrix molecule present. In the adhesion assay, the cells were preincubated with maspin for 18 h, the medium was removed, and the cells were released with EDTA and replated in serum- and maspin-free medium on ECM-coated wells for 1 h (10). In the invasion assay, the cells were mixed with maspin, placed into the Transwell inserts and allowed to migrate for 48 h. In the migration assay, maspin was placed in the chamber on the opposite side of the membrane from the cells (9), which results in the establishment of a concentration gradient across the membrane. Thus, the observed differences for the R340Q mutant may be due to the specific experimental conditions of the different assays or to distinct mechanisms of action of maspin.
The C-terminal region of maspin is not required for adhesion-promoting
activity, because the maspin/ovalbumin chimera containing the C-terminal end
(aa 346–375) of ovalbumin remains fully active. Previous studies showed
that deletion of the P7' residue through the C-terminal results in the
loss of maspin activity (9,
15). In this deletion mutant,
the internal 
-strands B4 and B5 and the exposed -strand C1
(Fig. 1) are missing. These
results suggest the C-terminal region, which is a highly conserved region of
serpins (27,
28), is required for proper
conformation of the molecule, including the RSL.
Not only is the RSL of maspin required, it is sufficient to induce cell-ECM
adhesion in both cell systems and to inhibit carcinoma cell invasion. Both the
RSL peptide and the ovalbumin mutant containing the RSL of maspin can
efficiently inhibit carcinoma cell invasion and induce increased adhesion of
carcinoma cells to fibronectin and corneal stromal cells to several ECMs. The
sufficiency of the RSL peptide is unusual, because the serpin inhibition
mechanism requires additional regions of the protein. This mechanism involves
proteolytic cleavage at the P1 site, insertion of the RSL into -sheet A,
and translocation of the attached protease to the opposite side of the
molecule (29). Thus, the
stimulation of cell-matrix adhesion and the inhibition of invasion probably do
not involve the serpin mechanism of protease inhibition. However, a role of
the RSL as a canonical protease inhibitor cannot be ruled out.
Conversion of inactive ovalbumin to a maspin-like molecule by swapping the
RSL is atypical for the serpin system. A number of studies have explored the
possibility of exchanging activities and/or specificities among serpins by
swapping the RSL regions between inhibitory serpins such as
1-antitrypsin and 1-antichymotrypsin
(30). Except for the highly
identical (90%) serpins SCCA1 and SCCA2
(31), replacement of the RSL
is not sufficient to transfer the specificity of one serpin to another
(30). However, the specificity
of the inhibitory activity of serpins has been changed by site-specific
mutagenesis within the RSL
(32,
33).
The use of the RSL in a role that does not involve the serpin mechanism of
inhibition is also unusual. Although the RSL of non-inhibitory serpins such as
pigment epithelial-derived factor (PEDF) and thyroxine-binding globulin (TBG)
acts as a substrate rather than an inhibitor for proteases, the RSL is not a
functional domain responsible for their activities. Cleavage of the exposed
RSL does not affect the neurite-promoting function of PEDF
(34). Because the
hormone-binding site in TBG resides in -sheets B and C, cleaved TBG
still can bind to thyroxine
(35). The RSL of cortisol
binding globulin is also not a hormone-binding domain, yet cleavage of the RSL
by elastase reduces the binding affinity for cortisol
(36). In contrast, maspin uses
the RSL for induction of increased cell-matrix adhesion of corneal stromal
cells and carcinoma cells and inhibition of invasion of carcinoma cells.
Although the RSL of maspin is sufficient for these activities of maspin, other
domains are essential for other functions of maspin. The anti-angiogenic
effect involves the N-terminal domain (aa 1–139) of maspin
(9), and the collagen-binding
domain of maspin maps to amino acid residues at positions 84–112
(37). Therefore, several
different parts of the maspin molecule may play distinctive and/or cooperative
roles supporting many functions of maspin.
Mammary carcinoma cells bound maspin specifically and saturably through low affinity binding sites. The kd for maspin binding to the cells is similar to the concentration required for maximal biological activity. The maspin concentration for maximal inhibition of invasion of mammary carcinoma cells is 170–770 nM (16), which is in the same range of the kd determined here for the binding of maspin to the carcinoma cells. The relationship of the kd value to the concentration required to exert maximal biological activity has also been observed for another serpin, PEDF. The kd for binding of PEDF to Tyr-79 retinoblastoma cells and to cerebellar granule neurons is similar to the concentration required for neurotrophic activity (38).
Not only is the 15-amino acid RSL peptide required and sufficient to mimic activity of maspin, it also competes for maspin binding to cells. The RSL does not completely inhibit 100% of maspin binding to the mammary carcinoma cells, yet the maspin binding experiments indicate only one binding site on the cells. If the binding site on maspin is larger than just the RSL, there is the possibility that, when the RSL is bound to the cell surface, complete steric hindrance of maspin binding is not achieved; therefore, residual maspin binding activity may still be observed. Nevertheless, the inhibition of binding of maspin to the carcinoma cells by the RSL suggests the RSL of maspin is involved in binding of the protein to the carcinoma cell surface.
In summary, the RSL of maspin, but not the C-terminal domain (aa 346–375), is required for stimulation of corneal stromal and carcinoma cell adhesion to ECM and inhibition of carcinoma cell invasion in vitro. The putative P1 Arg can be substituted with Gln but not Ala and retain its full ability to increase adhesion suggesting this residue is involved in a hydrogen bond. Ovalbumin can be converted to a fully active maspin-like molecule by substitution of the RSL of ovalbumin with that of maspin. Maspin binds to the carcinoma cell surface with a kd value in the range needed for biological activity. The RSL of maspin can compete with the full maspin molecule for this binding. The sufficiency of the RSL for stimulation of adhesion and inhibition of invasion suggests the serpin mechanism of inhibition is not involved.
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FOOTNOTES |
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¶ Recipient of a Senior Scientific Award from Research to Prevent Blindness,
Inc.
|| To whom correspondence should be addressed: Dept. of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-8431; Fax: 414-456-6510; E-mail: stwining{at}mcw.edu.
1 The abbreviations used are: ECM, extracellular matrix; aa, amino acid(s);
BSA, bovine serum albumin; FBS, fetal bovine serum; Masp/Masp-RSL/OV-C, maspin
mutant containing the C-terminal domain of ovalbumin; Masp/OV-RSL/OV-C, maspin
mutant containing the RSL and the C-terminal domain of ovalbumin;
Masp/OV-RSL/Masp-C, maspin mutant containing only the RSL of ovalbumin;
OV/Masp-RSL/OV-C, ovalbumin mutant containing the RSL of maspin; PAI,
plasminogen activator inhibitor; PBS, phosphate-buffered saline; PEDF, pigment
epithelial-derived factor; rHIS/FLAG, recombinant histidine/FLAG tag; R340Q,
maspin Arg to Glu P1 mutant; RSL, reactive site loop; RT, reverse
transcriptase; serpin, serine protease inhibitor; SCCA, squamous cell
carcinoma antigen; TBG, thyroxine binding globulin; uPA, urokinase-type
plasminogen activator.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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