Expression and Purification of Recombinant Human SPARC Produced by Baculovirus

Molecular Cell Biology Research Communications 3, 345–351 (2000) doi:10.1006/mcbr.2000.0237, available online at http://www.idealibrary.com on

Expression and Purification of Recombinant Human SPARC Produced by Baculovirus Amy D. Bradshaw,* James A. Bassuk,† A. Francki,* and E. Helene Sage* ,1 *Department of Vascular Biology, Hope Heart Institute, Seattle, Washington 98104; and †Department of Urology, University of Washington, Seattle, Washington 98195

Received July 13, 2000

SPARC (secreted protein acidic and rich in cysteine/ osteonectin/BM-40), a matrix-associated protein, disrupts cell adhesion and inhibits the proliferation of many cultured cells. We report the expression of recombinant human protein (rhSPARC) in a baculovirus expression system. This procedure routinely yields ⬃1 mg of purified protein per 500 ml of culture supernate. rhSPARC produced by insect cells migrates at the appropriate molecular weight under reducing and nonreducing conditions. The rhSPARC purified from insect cell media appeared structurally similar to SPARC purified from mammalian tissue culture by the criterion of circular dichroism. In addition, a series of anti-SPARC and anti-SPARC peptide antibodies recognized insect cell rhSPARC. We also show that rhSPARC produced in this system is glycosylated and is biologically active, as assessed by inhibition of endothelial cell proliferation and induction of collagen I mRNA in mesangial cells. Significant amounts of rhSPARC can now be generated in the absence of contaminating mammalian proteins for structure/ function assays of SPARC activities. © 2000 Academic Press Key Words: osteonectin/BM-40; recombinant protein; baculovirus; matricellular.

SPARC (secreted protein acidic and rich in cysteine)/ BM-40/osteonectin is a member of the matricellular class of proteins. Matricellular proteins are modular, nonstructural components associated with the extracellular matrix that function to modulate cell behavior (1). SPARC, for example, is distributed in extracellular matrices through its binding to collagens (2, 3). Once in contact with cell surfaces, SPARC induces changes in cell shape, in part through the dissolution of focal adhesions (4). SPARC has also been shown to bind to a subset of mitogenic growth factors and to neutralize 1 To whom correspondence should be addressed at Department of Vascular Biology, Hope Heart Institute, 528 18th Avenue, Seattle, WA 98122. Fax: 206-903-2044. E-mail: [email protected].

their activity by prevention of receptor activation (5, 6). In addition, SPARC inhibits cell cycle progression in mid/late G1 (7), an activity that can be independent of mitogen inactivation mediated by SPARC-growth factor interaction (8). Recently, primary mesenchymal cells isolated from SPARC-null mice have been shown to exhibit an increased rate of proliferation that is consistent with a function for this protein in the regulation of cell cycle (9). Mammalian SPARC is comprised of 286 –302 amino acids and migrates on SDS–PAGE with an apparent M r of 43,000 under reducing conditions (10, 11). The modular nature of SPARC is defined by three separate domains: an acidic N-terminal, low-affinity Ca 2⫹binding domain, a central region with homology to follistatin, and a C-terminal domain with two highaffinity Ca 2⫹-binding EF hands (12–14). Separate domains of SPARC have been suggested to mediate different activities associated with the full-length protein (15). Peptides representing particular regions within SPARC have been employed to demonstrate that various activities attributed to this protein can be mimicked by fragments of the full-length polypeptide. For example, peptides representing the C-terminal domain of SPARC inhibit progression of the cell cycle and induce cell rounding in cultured cells (3). In contrast, peptides derived from amino acids 113 to 130 stimulate endothelial cell proliferation, an activity that is masked in the native protein (16). To confirm and extend these observations requires a recombinant source of SPARC suitable for mutational analysis. The baculovirus expression system allows for efficient production of active mammalian proteins with an ease of manipulation needed for the generation of different mutants. Production of rSPARC in E. coli, yeast and mammalian cells has been reported previously. However, the baculovirus expression system is potentially a superior method of production due to (1) the ability of insect cells to perform posttranslational modifications similar to those of mammalian cells, (2) the efficient secretion of recombinant protein under serum-free con-

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ditions, and (3) the generation of large amounts of protein. We now describe an expression system that yields active, recombinant human SPARC (rhSPARC) from serum-free insect cell culture. This system produces substantial amounts of human SPARC for biochemical and biological assays. MATERIALS AND METHODS Human rhSPARC cDNA (17), minus the signal sequence, was amplified by polymerase chain reaction (PCR) using sequence-specific primers designed to generate a single product with BamH1 sensitive 5⬘ and 3⬘ ends (Forward: 5⬘-GAATTCGGATCCTTAGATCACAAGATCCTTGTCGATA-3⬘ and Reverse: 5⬘-GAATTCGGATCCGCCCCTCAGCAAGAAGCCCTGCC-3⬘). The BamH1-digested PCR product was subcloned into the pAcGP67B baculovirus expression vector (Pharmingen, San Diego, CA) in frame with the viral gp67 signal sequence to allow for efficient protein secretion of rhSPARC by virally-transfected Spodoptera frugiperda 9 (Sf9) cells propagated in TMN-FH media supplemented with 10% fetal bovine serum (FBS, Pharmingen) at 27°C. The cloned product was dideoxysequenced in both directions to confirm proper insertion of the cDNA into the multiple cloning site of the plasmid. Recombinant baculovirus was generated by cotransfection of the pAcGP67/SPARC vector with linearized baculovirus (AcUW1.lacZ; Pharmingen) into the Sf9 cells. Cultured supernates were analyzed for rhSPARC expression by immunoblot analysis with four separate SPARC antibodies: a monoclonal antihuman platelet-osteonectin IgG (Haematological Tech., Essex Junction, VT), a rabbit anti-murine SPARC polyclonal IgG (2), a rabbit anti-murine peptide 4.2 polyclonal IgG (3), and a monoclonal antiSPARC IgG (18). Transfected cell supernates were subsequently used to generate high-titer stocks of recombinant virus for future infections of cells grown in protein-free media (Protein-free insect cell media, Pharmingen). For purification, 1/10 volume of 200 mM 3-(Nmorpholino)propane sulfonic acid (Mops, pH 6.5) was added to the starting material, and the pH was adjusted to 6.5. The supernate was applied to a Q-Sepharose Fast Flow (Pharmacia, Piscataway, NJ) anion-exchange resin (1.7 ⫻ 20 cm.) equilibrated in 200 mM LiCl, 20 mM Mops [pH 6.5]. rhSPARC was eluted from the column with a continuous salt gradient from 200 to 400 mM LiCl, 20 mM Mops [pH 6.5]. The peak fractions were determined by spectrophotometry and were confirmed by SDS–PAGE and staining with Coomassie brilliant blue R (19). Fractions containing rhSPARC were pooled, dialyzed against 0.1 N acetic acid, and lyophilized. rhSPARC samples were resuspended in gel filtration buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl) and were chromatographed over a

Superdex-70 gel filtration column (Pharmacia). Peak fractions were determined by spectrophotometry (0.8 A 280 ⫽ 1 mg/ml) and were confirmed by SDS–PAGE. Typically, approximately 1 mg of rhSPARC per 500 ml of cultured Sf9 supernate was obtained by this method. Purified rhSPARC was subjected to N-terminal amino acid sequence analysis to confirm proper initiation and translation of the rhSPARC. As expected, a sequence of five additional amino acids (ADLGS) was present prior to the start of the N-terminal sequence (APQ. . .) of human SPARC, due to nucleotides present at the 5⬘end of the cDNA to allow in-frame cloning of rhSPARC with the GP67 signal sequence. To determine the extent of N-glycosylation, we incubated 2 ␮g of purified rhSPARC with 0.5 U of recombinant N-glycanase (Peptide-N-Glycosidase F, Oxford Glycoscience, Bedford MA) in 1⫻ N-glycanase buffer (20 mM sodium phosphate [pH 7.5], 50 mM EDTA) for 20 h at 37°C. For comparison, a parallel digestion was performed with 1 ␮g of SPARC isolated from parietal yolk sac (PYS) cells as described in Sage et al. (2). The digests were analyzed by SDS–PAGE followed by staining with Coomassie blue. Circular dichroism spectroscopy was carried out as described by Bassuk et al. (17). 500 ␮g of rhSPARC from a Q-Sepharose fractionation was resuspended in Chelex-treated gel filtration buffer and subjected to gel filtration chromatography. rhSPARC (95% free of contaminating proteins and 100% free of divalent cations) was scanned repetitively (5–12 times) to generate normalized spectra. Typical conditions included a scan range of 290 to 195 nm in a Shiatsu J700 circular dichroism spectrometer at ambient temperature in starting buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl). Additional scans were performed in starting buffer containing 50 mM CaCl 2, or in starting buffer containing 100 ␮M EDTA. The spectra of buffer alone were subtracted from the spectra containing protein. Proliferation assays were carried out as described in Kupprion et al. (6). Briefly, low passage (5–10) human dermal microvascular endothelial cells (HMEC, Clonetics, San Diego, CA) were plated at equal density in a 24-well tissue culture plate precoated with 1.5% gelatin (Sigma). The cells were incubated in the absence of serum (MCDB 131 (Gibco-BRL), 2 mM L-glutamine, 20 ␮g/ml heparin, 50 ␮g/ml endothelial cell growth supplement (ECGS, Collaborative Res., Bedford MA), and 500 U/ml penicillin G, 500 U/ml streptomycin-SO 4) for 24 h to establish quiescence and were subsequently stimulated with 2% FBS. rhSPARC was added at the specified concentrations concomitantly with FBS. [ 3H]Thymidine (Amersham, Piscataway, NJ, 3.7 ⫻ 10 4 Bq/ml) was added at 2 ␮Ci/ml for the last 4 h of the total 18-h incubation time. Incorporation was quantified by scintillation counting of cell extracts precipitated with trichloroacetic acid. All conditions were performed in triplicate.

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FIG. 1. rhSPARC produced in Sf 9 insect cells migrates at the appropriate molecular weight. Supernatant proteins from baculovirus-infected cultures were separated by SDS–PAGE under reducing (lanes 1– 4) and nonreducing conditions (lanes 5– 8), transferred to nitrocellulose, and probed with an anti-SPARC antibody. Lanes 1 and 5, supernate from cells infected with wild-type virus; lanes 2– 4 and 6 – 8, increasing amounts of recombinant virusinfected cell supernates (lanes 2 and 5, 5 ␮l; lanes 3 and 7, 10 ␮l; lanes 4 and 8, 25 ␮l). Molecular mass markers are indicated in kDa.

Reverse transcriptase polymerase chain reaction (RT-PCR) experiments were carried out as described in (20). Briefly, 1 ␮g of total RNA was subjected to RTPCR with the Access RT-PCR system (Promega, Madison, WI). Oligonucleotide primers complementary to murine ␤-tubulin, murine SPARC, and murine ␣2 (I) collagen designed according to the Entrez nucleotide query program (URL: http://www.ncbi.nlm.nih.gov) and the TM 3 oligonucleotide search program (URL: http://www-genome.wi.mit.edu), were used to amplify specific sequences of the respective cDNAs. The optimal number of cycles for detection of specific cDNAs prior to saturation was established at 24 for the ␤-tubulin standard, and all subsequent reactions were performed at this cycle number. Amplification of newly synthesized first strand cDNA was performed in a Thermolyne Temptronic Thermal Cycler. Equivalent aliquots of each amplification reaction were separated on a 1.2% agarose gel containing 0.5 ␮g/ml ethidium bromide (EtBr).

lecular weight for human SPARC (⬃40,000). The band shifted under nonreducing conditions as predicted to M r 38,000 (Fig. 1, lanes 5– 8). The higher molecularweight components (especially apparent in Fig. 1, lane 8) are likely multimeric forms of SPARC, as only the monomer is present after reduction (lane 4). Four separate anti-SPARC antibodies, specific for either the full-length protein or peptides derived from separate domains of the protein, produced similar results (data not shown). Thus, rhSPARC was expressed and secreted by the recombinant baculovirus-infected cells in a form that was recognized by antibodies previously shown to be specific for SPARC synthesized by mammalian cells. We developed a two-step process to isolate rhSPARC from the supernate of infected Sf9 cells grown in protein-free media, an approach similar to that used to purify mammalian SPARC from bovine aortic endothelial cells (21). First, supernates from infected cells were passed over an anion-exchange column in the presence of 200 mM LiCl; rhSPARC was eluted with a continuous salt gradient (200 – 400 mM LiCl). A representative preparation is shown in Fig. 2A. The majority of proteins in the supernate did not bind to the column in 200

RESULTS The cDNA encoding human endothelial umbilical vein SPARC (17) was subcloned into the baculovirus expression vector pAcGP67 and was cotransfected with wild-type virus into Sf9 cells maintained in media containing 10% FBS. Supernate was harvested from the transfected cells and was assayed for recombinant virus encoding rhSPARC by immunoblot analysis. Figure 1 shows increasing amounts of supernatant proteins, resolved by SDS–PAGE under reducing and nonreducing conditions, after transfer to nitrocellulose and reaction with a monoclonal antibody generated against human platelet osteonectin. Cells cotransfected with the SPARC-encoding vector and wild-type virus exhibited an immunoreactive band at the appropriate mo-

FIG. 2. Purification of rhSPARC. Supernates from recombinant virus-infected cells were collected and subjected to anion-exchange chromatography, as described under Materials and Methods. Eluted fractions were separated by SDS–PAGE (10% gel) under reducing conditions and were visualized by Coomassie blue staining, as shown in A. SPARC-containing fractions were pooled and subjected to gel filtration chromatography. Eluted fractions were separated by SDS– PAGE (10% gel) under reducing conditions and were visualized by staining with Coomassie blue, as shown in B. Molecular mass markers are indicated in kDa on the right. Lines represent the top and bottom of the gel.

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Purification of rSPARC from Insect Cell Supernate

Culture supernate (⬃200 ml) Anion exchange chromatography Gel filtration chromatography

Total protein (mg)

% SPARC

Yield

5000 1.3 0.370

0.084 85 100

26% a 8.8%

a Minimal levels of SPARC are detected in the flowthrough or wash fractions. The majority of SPARC immunoreactivity, not associated with the eluted fractions, is present in the 2 M LiCl strip performed following the elution gradient.

mM LiCl. rhSPARC was eluted near the middle of the gradient, with several contaminating proteins. A gel filtration column was employed as the second step of the isolation process to remove remaining contaminants (Fig. 2B). The predominant band at M r 38,000 – 40,000 appears variably as a doublet, a result of heterogeneous glycosylation (see below). The two-step purification typically yields ⬃1 mg of purified protein per 500 ml of infected supernate (Table 1). 500 ␮g of rhSPARC (⬎95% free of contaminating proteins) was subjected to circular dichroism (CD) spectrophotometry to ascertain whether secondary structure was dependent upon Ca 2⫹. The CD spectra are shown in Fig. 3. Similar to mammalian SPARC and to rhSPARC produced in E. coli (17, 22), Sf9 rhSPARC showed a characteristic signal at ⬃220 nm, indicative of ␣-helix within the protein. The addition of EDTA resulted in a shift of the spectrum at this wavelength, whereas the addition of supplemental Ca 2⫹ restored

FIG. 3. Induction of ␣-helix in rhSPARC by Ca 2⫹. 50 ␮g of rhSPARC in a Chelex-treated buffer of 10 mM Tris–HCl (pH 8.0) that contained 0.15 M NaCl was scanned from 270 to 195 nm in a circular dichroism spectrometer. The average of 12 repetitive scans in the presence of excess Ca 2⫹ or excess EDTA, after subtraction of the spectrum in buffer solution, is shown.

FIG. 4. rhSPARC expressed in insect cells is glycosylated. SPARC purified from murine parietal yolk sac cells (lanes 1 and 2) and purified rhSPARC (lanes 3 and 4) were subjected to digestion by N-glycanase. The proteins were separated by SDS–PAGE (10% gel) under reducing conditions and were visualized by staining with Coomassie blue. Molecular mass markers are indicated in kDa.

the curve to that typical of the native conformation of SPARC (17, 22). These spectra confirm that Sf9produced rhSPARC can bind Ca 2⫹ which stabilizes the ␣-helical structure of the protein. To determine whether Sf9 cells produced a glycosylated rhSPARC, we digested 2 ␮g of rhSPARC with N-glycanase. Sf9 insect cells are known to N-glycosylate protein; however, processing of core oligosaccharides to an appreciable extent does not take place (23, 24). As shown in Fig. 4, rhSPARC produced by Sf9 cells was sensitive to N-glycanase. In comparison to SPARC purified from murine parietal yolk sac (PYS) cells, Sf9 rhSPARC was modified somewhat differently, although both proteins contained oligosaccharide that was sensitive to N-glycanase, as assessed by SDS–PAGE (compare lanes 1 and 2 to lanes 3 and 4). PYS-SPARC also appears to have a more extensive glycosylation, as the N-glycanase-digested PYSSPARC (lane 2, arrow) was approximately the same size as the Sf9-SPARC prior to treatment with N-glycanase (lane 3). Mammalian SPARC has been shown previously to inhibit the synthesis of DNA and proliferation of cells in culture (8). The ability of Sf9 cell rhSPARC to inhibit DNA synthesis was assayed by [ 3 H]thymidine incorporation into human microvascular endothelial cell DNA. As shown in Fig. 5A, increasing concentrations of rhSPARC inhibited the proliferation of these cells in a concentration-dependent manner. Approximately 50% inhibition was achieved at 40 ␮g/ml (1.2 ␮M), a higher concentration than that previously reported for SPARC synthesized by PYS cells (8) but less than that reported for rhSPARC produced in E. coli (17). SPARC has also been shown to induce cell rounding in a variety of cultured cells. We have reported that Sf 9 rhSPARC affected cell shape in a similar manner (9). In addition, rhSPARC has been

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DISCUSSION

FIG. 5. rhSPARC produced by baculovirus has biological activity. (A) rhSPARC expressed in insect cells inhibits the proliferation of endothelial cells. rhSPARC, purified as described under Materials and Methods, was resuspended in PBS. The control was composed of non-rhSPARC-containing fractions eluted from the same column, treated identically and added to cells at the same volume as the highest concentration of rhSPARC. Cells exposed to increasing concentrations of rhSPARC exhibited progressive levels of inhibition. 50% inhibition (ED 50) was seen at ⬃40 ␮g/ml. Error bars represent the standard error of the mean. CPM, counts per minute. The results shown are representative of three separate experiments. (B) Equal numbers of wild-type and SPARC-null mesangial cells were stimulated for 6 h with 30 ␮g/ml rhSPARC. Total RNA was extracted and subjected to RT-PCR. PCR products were separated by agarose gel electrophoresis and stained with EtBr. Primers against ␤-tubulin mRNA were used as an internal control. Lanes 1 and 3, unstimulated cells; lanes 2 and 4, cells stimulated with rhSPARC. rhSPARC stimulation resulted in a 136% increase in wild-type cells and a 245% increase in SPARC-null cells over that of respective unstimulated cells. These results were reproducible with four separate preparations of mesangial cells.

previously shown to increase levels of ␣1 (I) collagen mRNA and type I collagen protein in SPARC-null mesangial cells (20). Collagen I is a trimer composed of two ␣1 subunits and one ␣2 subunit. As shown in Fig. 5B, the addition of rhSPARC to wild-type and SPARC-null mesangial cells resulted in increased levels of collagen ␣2 (I) mRNA as well. The increase in collagen ␣1 (I) mediated by rhSPARC has been shown to act through a TGF-␤-dependent mechanism (20). Since the colIa2 gene also contains a TGF-␤ activation element in the promoter (25), the elevated levels of ␣2 (I) collagen, as seen in Fig. 5B, are consistent with the increase in TGF-␤ activity that has been observed upon the addition of rhSPARC (20).

We have described an efficient, inexpensive method for the production and purification of rhSPARC by the use of a baculovirus expression system. The secretion of rhSPARC by insect cells in a serum-free culture generates sufficient amounts of protein for biochemical and biological analyses. In addition, this system is ideally suited for the expression and purification of mutant forms of SPARC. Many of the activities assigned to SPARC have been demonstrated with mammalian protein purified from the murine PYS-2 cell line. A disadvantage of this source is the levels of contaminating proteins such as laminin and serum albumin which copurify with SPARC. Additional steps must be employed to remove these proteins during the purification of SPARC from mammalian cells. In addition, because SPARC binds to a variety of different growth factors, SPARC purified from mammalian tissue culture must be assayed for levels of growth factor(s) that can affect biological assays (5, 6). Thus a recombinant source of SPARC is beneficial for provision of significant amounts of purified protein in the absence of contaminating mammalian proteins. rhSPARC produced in insect cells migrated at a size consistent with the predicted molecular weight, and more rapidly under nonreducing conditions, as observed previously for mammalian SPARC. In addition, rhSPARC exhibited a similar elution profile from an anion-exchange column, but without the contaminating secreted mammalian proteins which copurify with PYS-SPARC. Antibodies generated against peptides derived from both N-terminal and C-terminal domains of SPARC were immunoreactive with Sf9 cell rhSPARC. In addition, two antibodies generated against full-length protein also recognized rhSPARC produced by insect cells. We have shown that rhSPARC was sensitive to N-glycanase; however, the extent of glycosylation was less than that of PYSSPARC. This difference could account for the lower specific activity associated with Sf9 SPARC, in comparison to nonrecombinant mammalian SPARC, with respect to the inhibition of endothelial cell proliferation (Fig. 4), although a mutant form of rSPARC produced in yeast without glycosylation at Asn-98 was previously shown to inhibit the proliferation of endothelial cells (26). Another possibility is that murine SPARC might be more effective than human SPARC on bovine endothelial cells. The degree of glycosylation can affect the collagen binding activity of SPARC. The function of the oligosaccharide moiety in SPARC is not completely understood, although its heterogeneity is characteristic (23). Platelet SPARC is modified with more complex sugar moieties compared to that of bone, which is primarily high-mannose type oligosaccharide with little branching (24). Presumably the difference in glycosylation of platelet versus bone SPARC accounts for their

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reported disparate binding constants for association with collagen V, for which bone SPARC exhibits a higher affinity (27). We have not examined the capacity of rhSPARC from insect cells to bind collagen V, although preliminary experiments indicate that rhSPARC does bind collagen I (Bradshaw, Carbon, and Sage, in preparation). We have also shown that rhSPARC produced in insect cells evinced similar dependency of secondary structure on Ca 2⫹ binding as SPARC produced in E. coli, PYS cells, and transfected mammalian cells (16, 20, 29). Two previous reports have described the generation of recombinant SPARC in E. coli and yeast. Although these systems yielded SPARC with biological activity, both were subsequently found to have limitations. In addition to the lack of glycosylation by E. coli, a substantial amount of bacterially expressed rhSPARC was not soluble; a refolding step was therefore necessary to generate functional protein but was associated with substantial decreases in recovery (30). rhSPARC from E. coli has the potential for contamination by endotoxin, a highly undesirable feature for biological assays, especially those involving endothelial cells (31). The yeast system, on the other hand, did not generate sufficient quantities of secreted protein to provide a worthwhile source of rSPARC. Furthermore, a preponderance of contaminating proteins was found in the yeast growth medium which hampered purification of the rSPARC (26). Recombinant SPARC has also been produced by stable transfection of human cell lines (20, 29). While rSPARC produced by human cells exhibited similar properties to PYS-SPARC in terms of electrophoretic mobility and circular dichroism, no detectable biological activity by assay of cell shape changes or inhibition of proliferation was associated with rSPARC produced by human cells (29). The baculovirus system is ideally suited for the production of rhSPARC, as it provided substantial yields of active protein without mammalian or bacterial contamination that might confound interpretation of biological assays. Other advantages of the baculovirus expression system include the suitability of insect cells for spinner cultures, as these cells are able to grow and produce protein in the absence of cell attachment, and the ease with which the system can be manipulated to generate mutant forms of the protein. Thus, the production of rhSPARC by baculovirus can be scaled up to yield amounts of protein greater than described here. We are pursuing the generation of mutant forms of this protein to define more precisely the active regions within SPARC in the context of the native protein. ACKNOWLEDGMENTS This work was supported in part by National Institutes of Health Grants GM 40711, HL 18645, DK 47459, and GM 18705 (to A.D.B.), by the University of Washington Royalty Research Fund (to J.A.B.),

by the Seattle Diabetes Research Council, and by National Institutes of Health Training Grant DK 07467 (to A.D.B.).

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