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J. Biol. Chem., Vol. 276, Issue 44, 40698-40703, November 2, 2001

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Novel Betacellulin Derivatives

SEPARATION OF THE DIFFERENTIATION ACTIVITY FROM THE MITOGENIC ACTIVITY^*

Takashi Itoh, Mitsuyo Kondo, Yoko Tanaka, Masayuki Kobayashi, Reiko Sasada, Kouichi Igarashi, Masato Suenaga, Nobuyuki Koyama, Osamu Nishimura, and Masahiko Fujino

From the Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Wadai-10, Tsukuba, Ibaraki 300-4293, Japan

Received for publication, July 13, 2001, and in revised form, August 20, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Betacellulin (BTC) is a member of the epidermal growth factor family. It has two biological activities: mitogenic activity in fibroblasts and vascular smooth muscle cells, and differentiation activity for the differentiation of pancreatic acinar AR42J cells into insulin-secreting cells. The previous finding that recombinant BTC promotes the neogenesis of -cells in a mouse model supports the possibility that BTC is a therapeutic protein. However, the mitogenic activity of BTC may not be needed for differentiation into -cells and may cause a side effect in clinical use. We prepared several derivatives of BTC to segregate the two activities, to decrease the mitogenic activity, and to maintain the differentiation activity. We succeeded in obtaining BTC derivatives segregated by the two biological activities by preparing truncated-type derivatives. A derivative of BTC, BTC24-76, with a truncated N-terminal 23 amino acids and C-terminal 4 amino acids, was 2.5-fold more active in differentiation and had one-tenth of the mitogenic activity. The derivatives described in the present study should be helpful in future applications as therapeutic proteins and in basic research for discovery of a BTC-specific receptor.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Betacellulin (BTC)¹ is a polypeptide growth factor that was initially described, purified, and cloned from a mouse insulinoma cell line, -TC-3 (1, 2). BTC belongs to the epidermal growth factor (EGF) family of peptide ligands that are characterized by a six-cysteine consensus motif that forms three intramolecular disulfide bonds crucial for binding the ErbB receptor family. The EGF family consists of at least 15 members, including EGF, transforming growth factor  (TGF-), amphiregulin (AR), heparin-binding EGF-like growth factor, epiregulin, heregulins, and other growth factors (3). The 80-amino acid mature BTC is proteolytically processed from a 177-amino acid membrane-anchored precursor. BTC is a potent mitogen for a wide variety of cell types, including vascular smooth muscle cells and pigment epithelial cells, with a potency nearly identical to that of EGF (1, 4). BTC also acts as a differentiation factor of the pancreatic tumor cell line AR42J (5), differentiating amylase-secreting cells into insulin-secreting cells in combination with activin A (6). This differentiation activity of BTC was not reproduced by applying a similar dose of EGF or TGF-. BTC is also required for the induction of insulin and glucokinase expression by PDX-1 in glucagonoma cells (7), and it mediates proliferation and differentiation in the rat insulinoma cell line INS-1 (8). The localization of BTC and the strong immunoreactivity to BTC detected in primitive duct cells of the fetal pancreas suggest that it may play physiologically important roles in the growth and differentiation of islet cells in the human pancreas (9, 10, 11). The administration of recombinant human BTC was shown to improve glucose tolerance in a selective alloxane perfusion model in the mouse by increasing the -cell volume in the islets (12). These findings suggest that BTC holds promise as a therapeutic tool for diabetics. The structure and biological functions of BTC were reviewed by Dunbar and Goddard (13).

Differentiation into -cells is not known in the EGF family, except for BTC. Since structural homology is highly conserved in the EGF family, including with BTC, these conserved amino acids may be unrelated to the differentiation activity of BTC. To determine whether it is possible to create a BTC derivative that exhibits differentiation activity but not mitogenic activity and whether such a derivative could become a useful therapeutic tool, we prepared in this study several derivatives of BTC and clarified the segregation of the two activities of BTC.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials

Recombinant human ¹²⁵I-EGF was purchased from Amersham Pharmacia Biotech. [³H]thymidine was purchased from DuPont-New England Nuclear (Boston, MA). G418 was purchased from Invitrogen Life Technologies (Groningen Netherlands). Recombinant human EGF was prepared by Bacillus brevis in the manner described by Yamagata et al. (14). A431 cells and Balb/c 3T3 (clone A31-714) cells were purchased from the American Type Culture Collection (Manassas, VA) and the Institute for Fermentation, Osaka (Osaka, Japan), respectively. AR42J cells were provided by Dr. I. Kojima of Gunma University (Maebashi, Japan).

Construction of BTC Derivatives Expression Vectors

Structural genes of the 80-amino acid mature BTC (BTC80) and its truncated derivatives were prepared by polymerase chain reaction from pBO41 (10) and inserted into pTCII expression vector containing the replication origin and tetracycline-resistance gene of pBR322, T7 promoter, and T7 terminator of pET3c (Novagen, Madison, WI). To construct the C-terminal truncated mutants, we used an upper primer, 5'-CATATGGATGGGAATTCCACCAGAAGTCCTG-3', containing an NdeI site and ATG as the start codon. The lower primers containing the stop codon and BamHI site were 5'-GGATCCCTAGTCAACTCTCTCACACCTTGCTCC-3' for BTC77, 5'-GGATCCCTAGTCAACTCTCTCACACCTTGCTCC-3' for BTC76, 5'-GGATCCCTAAACTCTCTCACACCTTGCTCCAAT-3' for BTC75, 5'-GGATCCCTATCTCTCACACCTTGCTCCAATGTA-3' for BTC74, and 5'-GGATCCCTACTCACACCTTGCTCCAATGTAGCC-3' for BTC73. To construct BTC2-76 and BTC24-76, we used 5'-CAGCATATGGGGAATTCCACCAGAAGTCCT-3' and 5'-CAGCATATGGCTACCACCACACAATCAAAG-3' as the upper primers, respectively. The lower primer to construct BTC2-76 and BTC24-76 was the same as that used in BTC76. Structural genes of BTCR72K, substituting Lys for Arg⁷², and of BTCL78A, substituting Ala for Leu⁷⁸, were prepared by site-directed mutagenesis with using a QuikChangeT^M site-directed mutagenesis kit (Stratagene, La Jolla, CA). In BTCR72K, the codon AGG coding for Arg⁷² was mutated to AAG using 5'-CTACATTGGAGCAAAGTGTGAGAGAGTTGAC-3' and 5'-GTCAACTCTCTCACACTTTGCTCCAATGTAG-3' as primers. In BTCL78A, the codon TTG coding for Leu⁷⁸ was mutated to GCG using 5'-GGTGTGAGAGAGTTGACGCGTTTTACTAGTG-3' and 5'-CACTAGTAAAACGCGTCAACTCTCTCACACC-3' as primers. Structural genes of BTCR72K and BTCL78A were also subcloned into pTCII expression vector. Each plasmid was introduced into Escherichia coli MM294 (DE3) to obtain recombinant E. coli for protein expression.

Cultivation of Recombinant E. coli

Recombinant E. coli cells were cultured in modified M9 medium at 37 °C. The expression of recombinant protein was induced by adding isopropyl -thiogalactoside to a final concentration of 0.1 mM. Cultivation was continued for an additional 4 h. After cultivation, E. coli cells were harvested by centrifugation.

Purification of BTC and BTC Derivatives

Recombinant BTC80 and BTC derivatives were expressed as inclusion bodies. Recombinant protein was extracted from E. coli cells with extraction buffer (100 mM Tris-HCl, 7 M guanidine hydrochloride, 1 mM EDTA, and 1 mM (p-amidinophenyl)methane sulfonyl fluoride, at pH 8.0). After incubation with vigorous stirring at 4 °C for 1 h, the extracted solution was cleared by centrifugation, followed by 25-fold dilution with the refolding buffer (50 mM Tris-HCl, 2 M urea, 1 mM EDTA, 0.5 mM glutathione in oxidized form, 1 mM glutathione in reduced form, and 0.1 M arginine hydrochloride at pH 8.0). After renaturation at 4 °C for 15 h, the refolded solution was clarified by centrifugation, and the supernatant was concentrated by ultra filtration (molecular weight cut off: 3,000). After urea was added at a concentration of 2 M and the pH was adjusted to 5.0, the solution was applied to an SP-TOYOPEARL 650 M column (2.2 × 12 cm) (Tosoh, Tokyo, Japan) equilibrated with 50 mM acetate buffer (pH 5.0). After absorption, the column was washed with the same buffer, and the protein was eluted with a linear gradient of NaCl (0-1 M). The desired fractions were collected, and the eluate was concentrated by ultrafiltration. The concentrated solution was applied to an Asahipak C4P-50 column (1.0 × 25 cm) (Showa Denko, Tokyo, Japan) equilibrated with 0.1% trifluoroacetic acid. After absorption, the column was washed with 0.1% trifluoroacetic acid, and the protein was eluted with a linear gradient of acetonitrile (0-28% for BTC80, BTCR72K, and BTCL78A; and 0-13% for the deletion-type derivatives BTC77, BTC76, BTC75, BTC74, and BTC73). The eluted fractions were collected and re-chromatographed on an Asahipak C4P-50 column. The purified protein was dialyzed against distilled water and lyophilized.

Physicochemical Analysis

SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (15). Amino acid composition analysis was carried out using a HITACHI L-8500 amino acid analyzer. Approximately 10 µg of protein was hydrolyzed for 24 or 48 h at 110 °C in 5.7 N HCl containing 4% thioglycolic acid. Protein sequencing was performed on an Applied Biosystems Model 477A protein sequencer. C-terminal amino acid analysis was performed using a Hitachi L-8500 amino acid analyzer after treated vapor-phase hydrazinolysis for 3.5 h at 100 °C (16).

3T3 Cells Mitogenic Assay

The 3T3 cells mitogenic assay was performed as described previously (17). Briefly, Balb/c 3T3 cells were plated on 96-well plates in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS) (5 × 10² cells/well). The medium was replaced with DMEM containing 0.5% FBS at day 1, and serial 2-fold dilutions of BTC80, BTC derivatives, EGF, or medium alone were added at day 4. After 18 h of cultivation, DNA synthesis was monitored by pulse labeling with [³H]thymidine for 6 h. The cells were washed three times with phosphate-buffered saline and then dissolved with 0.1 ml of 5% SDS for 15 min at 37 °C. The amounts of radioactivity incorporated into the cells were determined by liquid scintillation counting.

Differentiation of AR42J Cells into Insulin-Secreting Cells

Immunofluorescence Microscopy Assay-- We performed a differentiation assay of the AR42J cells becoming insulin-secreting cells by BTC80 and BTC derivatives, as described by Mashima (6), except the assay was performed in the absence of Activin A. AR42J cells were plated on an 8-well Lab-Tek^TM II Chamber Slide (Nalge Nunc, Naperville, IL) in DMEM containing 10% FBS (5 × 10⁴ cells/well) with BTCs and were then cultured for 5 days. The cells were fixed with 10% paraformaldehyde in phosphate-buffered saline at 4 °C for 16 h and then treated with 0.1% Triton X-100 in phosphate-buffered saline for 5 min. The fixed cells were incubated sequentially with Block Ace (Snow Brand, Tokyo, Japan), with anti-insulin antibody (Advanced Immunochemical, Long Beach, CA), and fluorescein isothiocyanate-labeled anti-mouse IgG antibody (Kappel, West Chester, PA). The cells were examined using a fluorescent microscope.

Alkaline Phosphatase-reporter (AP-reporter) Assay-- To measure the differentiation of AR42J cells to insulin-secreting cells quantitatively, we established an AR42J clone, AR1898-0192, which contained a secreted alkaline phosphatase (SEAP) (18) gene downstream of rat insulin II gene promoter (19). Rat insulin II gene promoter was cloned by polymerase chain reaction from rat tail genomic DNA and inserted upstream of the SEAP gene to construct plasmid pTB1898. Plasmid pTB1898 contains rat insulin II gene promoter, SEAP, SV40 splicing junction, SV40 poly(A), and ampicillin resistance. Plasmid pTB1898 was co-transfected into AR42J cells with pMCI neomycin poly(A) (Stratagene, La Jolla, CA) containing neomycin resistance gene using Trans IT^TM-LT1 (Mirus, Madison, WI), and the cells were cultured in the presence of G418 (800 µg/ml). The stable transfectant clones were screened for SEAP activity in the culture media in response to adding BTC. A clone AR1898-0192 was obtained as one of the higher responding transfectants. AR1898-0192 cells were plated on 96-well plates in DMEM containing 10% FBS (1 × 10⁴ cells/well) and cultured for 5 days in the presence of serial dilutions of BTCs. The culture supernatant was then collected and treated at 65 °C for 30 min to inactivate endogenous alkaline phosphatase activity derived from serum and was incubated with 1 M diethanolamine, 0.5 mM MgCl₂, 10 mM L-homoarginine, and 2 mg/ml p-nitrophenyl phosphate (pNPP) at 37 °C for 24 h. Optical density at 405 nm was measured by a micro-plate reader (Molecular Device, Sunnyvale, CA).

EGF Receptor Binding Assay

EGF receptor binding assay was performed using human epidermal carcinoma A431 cells (20). The A431 cells were plated on 48-well plates in DMEM containing 10% FBS (3 × 10⁴ cells/well). At day 2, each well was washed three times with binding media (DMEM containing 20 mM HEPES and 0.1% bovine serum albumin). The cells were then incubated with ¹²⁵I-EGF (0.02 pM) for 90 min at 4 °C in the presence of serial 2-fold dilutions of BTCs and washed three times with 0.5 ml of binding media followed by lysis with 0.1 N NaOH containing 1% SDS. The amounts of radioactivity bound to the cells were determined by a gamma counter.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The mature form of BTC consists of 80 amino acid residues, and the EGF motif in the C-terminal domain consists of 50 amino acid residues. Both forms of BTC have been reported to have the same mitogenic activity on Balb/c 3T3 cells (4). Fig. 1 shows the amino acid sequence alignment of the conserved EGF motif of the EGF family: human, bovine, mouse, and rat BTC (2, 21, 22); human and rat TGF (23, 24); human and mouse EGF (25, 26); human heparin-biding EGF (27); human epiregurin (ER) (28); human amphiregulin (AR) (29); and human heregulin (HRG) 1 (30). This family shares high sequence similarity. In particular, six cysteine residues that form three disulfide bonds and a characteristic three-loop structure are completely conserved in the EGF family. Two glycine and arginine residues are also completely conserved. Highly conserved amino acid residues are inferred to be important in biological activities. The essential amino acid residues for the mitogenic activity were examined in detail for EGF and TGF- using recombinant DNA technology. In EGF, the importance of Tyr²⁹, Arg⁴¹, Asp⁴⁶, and Leu⁴⁷ for mitogenic and binding activities was shown using site-directed mutagenesis (31-35). The truncated form of the mouse EGF, mEGF (1-46), in which the C-terminal polypeptide segment containing Leu⁴⁷ is deleted, exhibited diminished binding activity (36). In TGF-, the Gly¹⁹, Val³³, Tyr³⁸, Gly⁴⁰, Arg⁴², Asp⁴⁷, and Leu⁴⁸ were shown to be important for both mitogenic and binding activity (21, 22, 37, 38). In these residues, which are crucial for biological activities, Arg (41 in EGF, 42 in TGF-) and Leu (47 in EGF, 48 in TGF-) are highly conserved in the EGF family. Although the conserved Leu adjacent to the C terminus is absent in AR, the importance of Leu at that position in mitogenic activity was shown by Thompson et al. (39). They prepared AR with an extended C terminus containing the conserved Leu adjacent to the C terminus, AR-DLLA. AR-DLLA showed higher mitogenic activity than mature AR. The Leu residue is also absent in HRG1, which has no affinity to epidermal growth factor receptor (EGFR), ErbB1.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Amino acid sequence alignment of the conserved EGF motif of the EGF family. Cysteine residues conserved in the EGF family are indicated by boxes. Other conserved amino acids are indicated by asterisks. HB, heparin-biding; ER, epiregurin, HRG, human heregulin.

According to the homology of the EGF motif in BTC to EGF and TGF-, we attempted to identify the amino acids crucial for the mitogenic activity of BTC, as shown in Fig. 2. Arg⁷² and Leu⁷⁸ seem to contribute to the mitogenic activity of BTC, and the mutation of these residues will decrease the activity. We proposed that if the amino acids are unrelated to the differentiation activity of BTC, it would be possible to create a BTC derivative exhibiting the differentiation activity and not the mitogenic activity. Fig. 3 shows the BTC derivatives used in the present study. BTCR72K and BTCL78A are mutated derivatives with Lys replacing Arg⁷² and Ala replacing Leu⁷⁸. BTC77, BTC76, BTC75, BTC74, and BTC73 are truncated derivatives lacking the C-terminal peptides of 3, 4, 5, 6, and 7 amino acid residues, respectively. BTC2-76 and BTC24-76 are truncated derivatives of BTC76 lacking the N-terminal peptides of 1 and 23 amino acid residues, respectively. The BTC80 and BTC derivatives having a proper N terminus had an additional N-terminal Met when expressed in E. coli. Although we already developed a technology to remove an additional Met at the N-terminal of the recombinant protein (40), in this case we did not remove the N-terminal additional Met. The presence of a Met residue does not seem to be vital to the biological activity of BTC, as reported by Seno et al. (10). All preparations showed high purities in SDS-polyacrylamide gel electrophoresis and reversed phase-high pressure liquid chromatography and good agreement with the theoretical values determined by amino acid analysis, C-terminal amino acid analysis, and N-terminal amino acid sequencing (data not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Schematic representation of the structure of BTC-80 and conserved amino acids in EGF and TGF-. The EGF motif of BTC is indicated by a box. The conserved amino acid residues in BTC, EGF, and TGF- are indicated by shaded circles. The target amino acid residues Arg72 and Leu78 for mutation or deletion are indicated with asterisks.

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Amino acid sequences of human Betacellulin and its derivatives. BTC and BTC derivatives containing the proper amino terminus have an additional methionine residue at the amino terminus when expressed by E. coli. BTCR72K and BTCL78A are mutated-type derivatives substituting Lys for Arg72 and Ala for Leu78, respectively. BTC77, BTC76, BTC75, BTC74, and BTC73 are truncated-type derivatives lacking a C-terminal peptide consisting of 3, 4, 5, 6, and 7 residues, respectively. BTC2-76 and BTC24-76 are truncated-type derivatives of BTC76 with deleted N-terminal 1 and 23 amino acids, respectively.

We first prepared the BTC80 and BTC derivatives BTCR72K and BTC77 and assayed for biological activities. Fig. 4 shows the results of the mitogenic assay with Balb/c 3T3 cells. BTC80 was equally active with EGF, while BTCR72K and BTC77 were less active than BTC80. BTCR72K was almost inactive and the mitogenic activity of BTC77 was ~1/100 that of BTC80. Fig. 5 shows the results of the differentiation assay with AR42J cells according to the immunofluorescence method using anti-insulin antibody. In the case of the culture without BTC80 or with EGF, no AR42J cell was strongly stained. BTCR72K was also inactive in the differentiation assay. BTC77 was equally as active as BTC80 on the differentiation of AR42J. These results showed that Arg⁷² is essential for both activities and the C-terminal peptide containing Leu⁷⁸ is essential for mitogenic activity and contribute less to differentiation activity. We also prepared BTCL78A, replacing Leu⁷⁸ with Ala and assayed the activities. BTCL78A showed the same behavior as BTC77: the mitogenic activity was decreased and the differentiation activity was maintained (data not shown). The manipulations of Leu⁷⁸ lead BTC80 to interesting molecules with decreased mitogenic activity and a maintained differentiation activity. Although the manipulated mutation and deletion are both effective in decreasing the mitogenic activity, we concluded that deletion-type derivatives are the most promising for further investigation. Because of the potential of BTC derivatives as therapeutic tools, deletion is more favorable than mutation since it is essentially a wild type BTC and causes no antigenicity or immunogenicity. We also prepared the deletion-type derivatives BTC76, BTC75, BTC74, and BTC73. To measure the activity of BTC derivatives on the differentiation of AR42J cells into insulin-secreting cells, we established stable transfectant AR1898-0192 cells. Since the SEAP gene was inserted downstream of insulin promoter II, the differentiated cells by BTC secreted not only insulin but also SEAP, and the SEAP activity in the culture medium was detected as a reporter. Figs. 6 and 7 show the mitogenic and differentiation activities of the BTC80 and BTC derivatives. As shown in Fig. 6, deletion of the C-terminal peptides decreased the mitogenic activity drastically. The derivatives BTC74 and BTC73 retained almost no mitogenic activity. BTC75 retained less than 1% of that of BTC, and BTC77 and BTC76 retained only a low percentage. In the differentiation activity, the derivatives BTC75, BTC74, and BTC73 retained almost no activity, but BTC77 and BTC76 retained approximately one third of the differentiation activity of BTC. The deletion of the C-terminal 3 or 4 amino acid residues decreased the differentiation activity, but the extent of the decrease was relatively low compared with that of the mitogenic activity.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Mitogenic assay of the EGF, BTC, and the BTC derivatives BTCR72K and BTC77 using Balb/c 3T3 cells. EGF (solid circle), BTC80 (open circle), BTCR72K (open triangle), and BTC77 (open square) were assayed for their ability to stimulate DNA synthesis in Balb/c 3T3 cells.

View larger version (77K): [in this window] [in a new window]   Fig. 5.   AR42J cells differentiation assay of BTC and the BTC derivatives BTCR72K and BTC77. AR42J cells were incubated for 5 days with 2 ng/ml of EGF, BTC80, or the BTC derivatives BTCR72K and BTC77 and were stained with anti-insulin antibody. A, without BTC. B, BTC80. C, BTCR72K. D, BTC77. E, EGF.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Mitogenic assay of BTC and the BTC derivatives BTC77, BTC76, BTC75, BTC74, and BTC73 using Balb/c 3T3 cells. BTC80 (open circle), BTC77 (solid circle), BTC76 (open square), BTC75 (solid square), BTC74 (open triangle), BTC73 (solid triangle), and EGF (closed diamond) were assayed for their ability to stimulate DNA synthesis in Balb/c 3T3 cells.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   AR42J Cells Differentiation Assay of BTC and the BTC derivatives BTC77, BTC76, BTC75, BTC74, and BTC73 by AP-reporter assay. AR1898-0192 cells were differentiated to insulin-secreting cells by BTC80 (open circle), BTC77 (solid circle), BTC76 (open square), BTC75 (solid square), BTC74 (open triangle), and BTC73 (solid triangle).

We previously reported that an N terminus-truncated mutant of BTC, lacking residues 1-30, had a mitogenic activity that was equipotent with BTC80 (4). We also investigated the N terminus truncation of BTC in combination with C terminus truncation. In BTC2-76 and BTC24-76, the N-terminal 1 and 23 amino acid residues were truncated from BTC76 and were constructed to avoid the addition of Met at the N terminus when expressed by E. coli. BTC80 and BTC derivatives with a proper N-terminal amino acid, Asp, had an additional Met at the N terminus. BTC2-76 and BTC24-76 with the N-terminal amino acid residues Gly and Ala, respectively, had no additional Met at the N terminus, as confirmed by N-terminal amino acid sequencing. Figs. 8 and 9 show the results of the receptor binding assay and AP-reporter assay, respectively. The specific binding of the BTC derivatives to EGFR was based on the ability of various concentrations of the derivatives to compete with ¹²⁵I-labeled hEGF in the radioreceptor binding assay. Indeed, the reduction of mitogenic activity was observed using Balb/c 3T3 cells. However, it is important to clarify at which step the reduction occurred, either by lowering the affinity to EGFR or by affecting the subsequent signal transduction steps. The derivative having no mitogenic activity or affinity to EGFR is the most promising because it is less likely to be affected by EGFR and can be deliver into the pancreas where the differentiation activity of BTC is most important. The concentration of the competitor causing a 50% binding inhibition (IC₅₀) was estimated for each derivative from the curve represented in Fig. 8. The relative binding affinity was calculated from IC₅₀ values compared with that of BTC80. BTC80 bound to EGFR on A431 cells with an IC₅₀ value of 1.2 nM. The IC₅₀ values and relative activity for BTC76, BTC2-76, and BTC24-76 were 95 nM (1.3%), 38 nM (3.2%), and 11 nM (10.9%), respectively. As these results indicate, BTC76 has decreased mitogenic activity and binding affinity to EGFR, and it is in the preferred form for being a promising therapeutic candidate. Similarly, the effective concentration of each derivative resulting in 50% maximal differentiation of AR42J cells (EC₅₀) was estimated for each derivative from the curve represented in Fig. 9. The relative differentiation activity for each derivative was calculated by comparison of this value with that of BTC80. BTC80 differentiated AR42J cells into insulin-secreting cells with an EC₅₀ value of 0.2 nM. The EC₅₀ values and relative activity for BTC76, BTC2-76, and BTC24-76 were 0.6 nM (33.3%), 0.2 nM (100%), and 0.08 nM (250%), respectively. The binding affinity of BTC76 was reduced to 1/100 of that of BTC80, whereas the differentiation activity was reduced only to one third that of BTC80. The truncation of the N terminus of BTC76 showed higher affinities to EGFR than BTC76. The affinities are 1/30 (BTC2-76) and 1/10 (BTC24-76) that of BTC80. The differentiation activity also increased: BTC2-76 has the same activity as BTC80 and BTC24-76 has 2.5 times the activity of BTC80. These values and the calculated ratio of the differentiation activity to the binding affinity to EGFR are listed in Table I.

View larger version (29K): [in this window] [in a new window]   Fig. 8.   EGF receptor binding assay of BTC and the BTC derivatives BTC76, BTC2-76, and BTC24-76 using A431 cells. BTC80 (open circle), BTC76 (open triangle), BTC2-76 (solid circle), and BTC24-76 (solid triangle) were assayed for their ability to compete with 125I-labeled EGF for binding to EGFRs exposed on the surface of A431 cells.

View larger version (26K): [in this window] [in a new window]   Fig. 9.   AR42J cells differentiation assay of BTC and the BTC derivatives BTC76, BTC2-76, and BTC24-76 by AP-reporter assay. AR1898-0192 cells were differentiated to insulin-secreting cells by BTC80 (open circle), BTC76 (open triangle), BTC2-76 (solid circle), and BTC24-76 (solid triangle).

The ratio of the differentiation activity to the binding affinity to EGFR of the derivatives BTC76, BTC2-76, and BTC24-76 were all in excess of 20. Although the differentiation activity and the binding affinity to EGFR of each derivative differ with the status of the N terminus, the extent of the separation of the differentiation activity from the binding affinity does not differ. Regarding the site specific mutation of Leu⁴⁷ of EGF, the affinity to EGFR decreased drastically but the reduction in mitogenic activity was lower (35). While the truncation of the C-terminal peptide of BTC drastically decreased both activities, the reduction of the mitogenic activity was more drastic than that of the binding affinity to EGFR. Further characterization of these differences will yield a better understanding of their binding and signaling mechanisms.

We segregated the two activities of BTC while maintaining the differentiation activity and decreasing the mitogenic activity. The receptors of BTC reported are ErbB-1 homodimer, ErbB-4 homodimer, and heterodimers including ErbB-1 or ErbB-4 as well as ErbB2/ErbB3 (41-44). Moreover, previous studies have suggested the possible existence of a specific receptor for BTC, besides the known ErbB family, in AR42J cells (45). Truncation of the C-terminal peptides greatly decreased the affinity to ErbB-1 with little effect on the affinity to ErbB-4 or the specific receptor of BTC on AR42J cells. Alterations in the conformation of BTC and BTC derivatives may also be useful in future research on receptor binding. Further, crystallographic investigations of BTC80 and BTC76 may prove useful. In the present study, we produced both mutations and deletions of amino acid residues. The truncated-type derivatives are more promising as a therapeutic tool than the derivatives having one or more mutated amino acid residues because the former are free from risk of immunogenicity and antigenicity. BTC80 was previously shown to improve glucose tolerance in a mouse model (12), and the BTC derivatives are expected to improve tolerance even more effectively than BTC80 because of their decreased affinity to the EGF receptor. Since the truncated-type derivatives with high differentiation activity are composed of the amino acid sequence that exists in the mature BTC, such derivative might be generated naturally by proteolytic digestion of BTC80 in vivo. It is our hope that the present findings will lead to the discovery of a novel biological function of BTC and, perhaps, to the development of BTC as a therapeutic tool for diabetes.

	ACKNOWLEDGEMENTS

We thank Dr. Y. Sumino and Dr. H. Sawada for their continuing encouragement.

	FOOTNOTES

^* 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: Discovery Research Laboratories I, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Wadai-10, Tsukuba, Ibaraki 300-4293, Japan. Tel.: 81-298-64-5040; Fax: 81-298-64-5000; E-mail: Ito1_Takashi@takeda.co.jp.

Published, JBC Papers in Press, August 24, 2001, DOI 10.1074/jbc.M106603200

	ABBREVIATIONS

The abbreviations used are: BTC, betacellulin; EGF, epidermal growth factor; TGF, transforming growth factor; AR, amphiregulin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; AP, alkaline phosphatase; SEAP, secreted alkaline phosphatase; pNPP, p-nitrophenyl phosphate; EGFR, epidermal growth factor receptor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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