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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 4 1570-1576
Copyright © 2003 by The Endocrine Society

A Method for Identification of the Peptides That Bind to a Clone of Thyroid-Stimulating Antibodies in the Serum of Graves’ Disease Patients

Chan Hyun Na, Mi Hwa Lee, Bo Youn Cho and Chi-Bom Chae

Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea; and Department of Internal Medicine, Seoul National University College of Medicine (C.B.Y.), Seoul 110-744, Korea

Address all correspondence and requests for reprints to: Dr. Chi-Bom Chae, Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea. E-mail: cbchae{at}postech.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A method was developed for identification of the peptide sequences that bind to thyroid-stimulating antibody (TSAb) clones from phage-displayed peptide library. Immunoglobulin G (IgG) was purified from the serum of a Graves’ disease patient that stimulates the synthesis of cAMP in the cells that express TSH receptor (TSHR). The IgG that binds to TSHR was purified by an affinity column packed with the resin cross-linked with the extracellular domain of human TSHR. The receptor-binding IgG was then mixed with phages that display linear or cyclic peptides at the end of tail protein pIII. The bound phages were eluted with acidic glycine after extensive washing. From sequencing of the pIII gene of the bound phages, one can deduce the sequences of the peptides that bind to the receptor-binding IgG. Each peptide sequence was then tested for inhibition of the synthesis of cAMP from thyroid cells induced by the serum of a Graves’ patient. In this way, one can obtain the peptides that bind to a clone of TSAb. We obtained a peptide sequence that inhibits the action of TSAb at an extremely low concentration (<10-14 M). Such a peptide will be useful for various studies on TSAb.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GRAVES’ DISEASE (GD) is an autoimmune disease characterized by overproduction of thyroid hormones due to the continuous stimulation of TSH receptor (TSHR) on the surface of thyroid cells by autoimmune antibodies with consequent hyperthyroidism. TSHR is composed of 764 amino acids, containing a large extracellular domain of 396 residues and a signal peptide, together with the remaining residues forming the 7 transmembrane regions and short intracellular tail (1, 2, 3, 4). There are many types of autoantibodies for TSHR, such as thyroid-stimulating antibody (TSAb), TSH binding inhibitory antibody, and thyroid stimulating blocking antibody (5). TSAb acts as a TSHR agonist and induces excessive thyroid hormone secretion, leading to hyperthyroidism (2, 6, 7, 8). TSAbs are thought to be polyclonal and recognize several different epitopes on the TSHR (9, 10, 11, 12). Therefore, determination of the epitopes of TSAbs is of great importance not only in understanding the pathogenesis of GD, but also in developing new therapeutic methods for GD.

Many laboratories have adopted different strategies to map the epitopes of autoantibody binding to TSHR. Some important regions have been identified by substitution, deletion mapping, and chimeric receptor approach. In these studies the TSHR extracellular domain (TSHRE) with the mutated region was used (13, 14, 15, 16, 17). Another method in mapping the epitopes is the use of synthetic peptides composed of a section of human TSHR to probe the interaction of the receptor and its ligand (17, 18, 19, 20). According to these studies, the epitopes of TSAbs appear to exist in the large extracellular domain of TSHR and to be critically dependent on the three-dimensional conformational structure of TSHR (1, 2, 21, 22, 23). Therefore, it is difficult to obtain epitopes for different clones of TSAb. There have been several attempts to solve this problem using a combinatorial peptide library (10, 24).

In a previous study we employed a peptide library to identify the peptide sequences that inhibit the synthesis of cAMP induced by immunoglobulin G (IgG) from a GD patient (GD IgG) (24). The peptide identified from this approach does not necessarily bind to TSAb. Another approach we used was identification of the peptide sequences that bind to monoclonal TSAb (10). However, the availability of different types of TSAb clone is limited. In this report we present a new method that allows identification of the peptide sequences that bind to a clone of TSAb in the serum of GD patients. The method basically involves the following: enrichment of IgG that binds to TSH receptor, binding of phage-displayed peptide libraries to the TSHR-binding IgG, and selection of the peptide sequences that inhibit the synthesis of cAMP induced by GD IgG.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Expression and purification of TSHRE

Human TSHRE spanning from 1–414 amino acids and the six-histidine tag at the C terminus was expressed in HeLa S3 cells by infecting them with recombinant vaccinia virus as described previously (25).

Purification of IgG that binds to TSHRE

Total IgG was purified from the serum of GD patients by binding to a protein A column. Purified GD IgG was loaded onto a column in which TSHRE was coupled to nickel-chelate-nitrilotriacetic acid (Ni-NTA) agarose (25). The bound IgG was eluted with 4 M MgCl2 (pH 4.8) (26). The activity of purified anti-TSHR Ab was confirmed by its ability to induce cAMP synthesis as previously described (24, 25).

Assay for the effect on binding of [125I]TSH to thyroid membrane by antibody

The TSH binding inhibitory Ig (TBII) assay kits were purchased from RSR Ltd. (Pentwyn, UK). The principle of this assay is based on the ability of autoantibodies to compete for [125I]TSH binding to the TSHR solubilized from porcine thyroid membrane (27). The TBII assay mixture contains 50 µl solubilized porcine thyroid membrane fractions, 100 µl [125I]TSH (0.36 KBq), and 50 µl sera or IgG of GD patients. The tube contents were mixed vigorously by vortexing for about 5 sec. After 2-h incubation at room temperature, 1 ml 16.5% polyethylene glycol/1.6 M NaCl was added to each tube and incubated for 30 min at room temperature. Each tube was centrifuged at 1500 x g for 30 min at 4 C, and the pellet was counted for radioactivity in a {gamma}-scintillation counter.

Screening of peptides that bind to anti-TSHR Abs with a phage display peptide library

In this study we used a Ph.D.-12mer phage display peptide library kit (New England Biolabs, Inc., Beverly, MA). The Ph.D.-12mer phage display peptide library is a combinatorial library of random dodecapeptides fused to the N terminus of a minor coat protein (pIII) of M13 phage. The library consists of 2.7 x 109 diverse sequences that were amplified once to yield about 50 copies of each peptide sequence in 10 µl of the supplied phages. The biopanning process consisted of 3 rounds of affinity selection. A microtiter well was coated with the enriched IgG (250 ng), followed by blocking with 0.5% BSA/NaHCO3 (pH 8.6). In the first round of biopanning, 50 µl of the phage library (2.5 x 1011 plaque-forming units) were incubated with 50 µl normal human IgG (0.1 mg/ml) in 0.5% BSA/NaHCO3 for preclearing before being added to the well coated with the enriched IgG. After 2-h incubation at room temperature, the well was washed 10 times with Tris-buffered saline containing 0.1% Tween 20, and the bound phages were eluted with 0.1 M glycine/0.1% BSA (pH 2.2) and subjected to the next round of screening after being amplified. In the second round, washing stringency was elevated by increasing the Tween 20 concentration from 0.1% to 0.5% and increasing the washing steps (20 times). In the third round, the wells were washed 50 times with Tris-buffered saline containing 0.1% Tween 20 (0.5% Tween 20) with gentle shaking. The phage fractions in all biopanning procedures were titrated to determine the degree of enrichment.

Assay for specificity of the affinity-selected phages

Each well of an ELISA plate was coated with 10 µg (in 10 µl) of either normal or GD IgG by incubation at 4 C overnight and then blocked with 5% skim milk/PBS (pH 7.4). Affinity-selected phages (1010 plaque-forming units in 50 µl 5% skim milk/PBS) were added to each well. After washing with PBS with 0.1% Tween 20, the bound phages were detected by incubation with sheep anti-M13 antibodies conjugated with horseradish peroxidase (Amersham Pharmacia Biotech, Arlington Heights, IL). The activity of the bound horseradish peroxidase was determined by incubation with 2,2'-azidobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (Kirkegaard \|[amp ]\| Perry Laboratories, Gaithersburg, MD) as a substrate. The reaction was stopped by the addition of an equal volume of 1% sodium dodecyl sulfate, and absorbance at 405 nm was determined in an automated ELISA reader (model EL 312e, Bio-Tek Instruments, Inc., Winooski, VT).

DNA sequencing and peptide synthesis

The phage clones were amplified and precipitated with polyethylene glycol, and the precipitate was suspended in iodide buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 4 M NaI], followed by the addition of 2.5 vol ethanol. Precipitated phage DNA was washed with 70% ethanol, followed by resuspension in TE buffer [10 mM Tris-Cl (pH 8.0) and 1 mM EDTA; New England Biolabs, Inc.]. The phage DNA was sequenced with the -96 gIII sequencing primer supplied with the Ph.D.-12mer phage display peptide library kit, and the amino acid sequences of the N-terminal region of pIII were deduced. The deduced peptides were synthesized at Research Institute, Hyundai Pharmaceuticals (Bucheon, Korea), and PeptidoGenic Research & Co., Inc. (Livermore, CA).

Measurement of cAMP

The activity of TSAb in GD IgG was assessed by determining the amount of cAMP produced from Chinese hamster ovary cells transfected with TSHR (CHO-TSHR cells) (12). The CHO-TSHR cells were seeded at 105 cells/well in 48-well plates for 24 h before the cAMP assay. The CHO-TSHR cells were maintained under 5% CO2 at 37 C and cultured in Ham’s F-12 mixture medium supplemented with 10% bovine calf serum and 0.5 mg/ml geneticin (G418, Life Technologies, Inc., Gaithersburg, MD). After the culture medium was removed, cells were preincubated with NaCl-free isotonic Hanks’ balanced salt solution (HBSS) for 30 min at 37 C. The GD IgG (2 mg/ml) was added to the CHO-TSHR cells in 100 µl NaCl-free isotonic HBSS containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) for 3 h at 37 C. Then the intracellular cAMP was extracted with ice-cold absolute ethanol, and the amount of cAMP was determined with a commercial RIA kit (Amersham Pharmacia Biotech, Aylesbury, UK) (8).

Inhibitory activity of TSHRE and synthetic peptides for synthesis of cAMP

The activity of purified TSHRE was confirmed by its inhibitory activity against the generation of cAMP induced by GD IgG in the cAMP assay system as described previously (25).

For the investigation of the inhibitory activity of peptides on the synthesis of cAMP induced by GD IgG, IgG (2 mg/ml) was preincubated with various amounts of peptides in 100 µl NaCl-free isotonic HBSS containing 0.5 mM IBMX for 1 h at room temperature before being added to the culture dish containing CHO-TSHR cells. The preincubated mixture was then added to the cells in culture, the cells were incubated for 3 h, and the amount of generated cAMP was determined as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Purification of TSHRE and anti-TSHR Ab

For selection of the peptides that bind to GD IgG, we first enriched IgG that binds to TSHRE. TSHRE was obtained by use of a recombinant vaccinia virus system as described by Lee et al. (25). The activity of purified TSHRE was confirmed by its inhibitory activity against cAMP generation by GD IgG as described by Lee et al. (25) (data not shown). The IgG purified by protein A-agarose was applied to a column of TSHRE coupled to Ni-NTA. After extensive washing, the bound IgG was eluted with 4 M MgCl2. The activity of purified anti-TSHR Abs was confirmed by cAMP generation assay (Fig. 1Go) and TBII assay (Fig. 2Go). The results presented in Fig. 1Go suggest that the purified IgG induces cAMP synthesis in dose-dependent manner, and more than 1000-fold enrichment of TSAb was achieved by TSHRE affinity chromatography. The results presented in Fig. 2Go suggest that the purified IgG has inhibitory activity on TSH binding to TSHR at about a 1000-fold lower concentration of total GD IgG.



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  		Figure 1. TSAb activity of purified anti-TSHR Ab. Total GD IgG or unbound IgG (A), or purified anti-TSHR Ab (B) in 100 µl NaCl-free isotonic HBSS and 0.5 mM IBMX was added to CHO-TSHR cells (105 cells) in culture in 48-well plates. After 3-h incubation at 37 C, the amount of cAMP produced was determined as described in Materials and Methods. Each point represents the mean of duplicate determinations along with the SD indicated.

 


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  		Figure 2. Inhibition of TSH binding to the TSHR by GD IgG and enriched IgGs. Total GD IgG, unbound IgG, or purified anti-TSHR Ab was incubated with both solubilized porcine thyroid membrane and [125I]TSH for 2 h at room temperature. The complex of [125I]TSH and thyroid membrane was precipitated with polyethylene glycol, and the radioactivity in the precipitate was determined as described in Materials and Methods. Data are expressed as the percent inhibition of [125I]TSH binding to solubilized porcine thyroid membrane compared with an equal amount of normal serum. Each point represents the mean of duplicate determinations along with the SD indicated.

 
Selection of phages binding to anti-TSHR Ab

To select the phage clones that bind to anti-TSHR Ab, a phage-displayed peptide library (2.5 x 1011 plaque-forming units) was added to the anti-TSHR Ab (250 ng) coated on a microtiter well. After 2-h incubation at room temperature, the well was washed, and the bound phages were eluted with acidic glycine. The eluted phages were amplified and reapplied to the anti-TSHR Ab coated on a well. The yield of eluted phages for each round did not show appreciable increase. However, as will be shown later, phages with specific sequences were enriched.

Binding activities of selected phages to anti-TSHR Ab and their deduced peptide sequences

On the assumption that the final outputs from the three rounds of biopanning would contain a significant amount of phage clones that bind to anti-TSHR Ab with high affinity, the phage clones selected after the third round of biopanning were picked, amplified, and individually tested by ELISA for their preference for GD IgG over normal IgG. Forty-eight phage clones were assayed by the ELISA method, and many clones showed preference for GD IgG over normal IgG (Fig. 3Go). DNA from the 14 candidate phage clones were sequenced, and their deduced amino acid sequences are shown in Table 1Go. Among 14 phage clones examined, 4 clones expressed HKMHSHPRLTSP (peptide A), and 3 clones expressed HWKHNRHDPSPP (peptide B) at the N terminus of pIII (Table 1Go). Among 14 clones, 9 showed histidine at the first and fourth positions. Of these, 4 showed HKMH, 4 showed HWKH, and 1 showed the HLKH motif. All 9 different peptide sequences were synthesized.



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  		Figure 3. Binding activities of selected phages to GD IgG. Phage clones, rescued from the third round of biopanning, were individually tested by ELISA for their ability to bind to normal IgG ({square}) and GD IgG ({blacksquare}). Normal IgG and GD IgG were immobilized in plastic wells, the selected phages were added to both wells, and binding of the phage to IgG was determined by treatment with polyclonal sheep anti-M13 antibodies conjugated with horseradish peroxidase. {downarrow}, Sequenced clones (14 clones).

 

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  		Table 1. Amino acid sequences deduced from the phage clones selected after three rounds of biopanning

 
Effects of candidate peptides on the cAMP synthesis induced by GD IgG

To investigate whether the selected peptide sequences inhibit the cAMP synthesis induced by anti-TSHR Ab, we determined the amount of cAMP produced in response to GD IgG in the presence and the absence of peptides. The peptide was preincubated for 1 h at room temperature with 2 mg/ml GD IgG before being added to CHO-TSHR cells. After treatment with GD IgG alone or GD IgG plus selected peptides, the level of cAMP synthesized in CHO-TSHR cells was determined as described in Materials and Methods. Only peptides A and B among nine selected peptides were tested first. Both of these peptides inhibited cAMP synthesis in CHO-TSHR cells induced by anti-TSHR Ab at 10 µM. Due to the lack of a sufficient amount of GD IgG, only peptide B was further characterized. Peptide B showed half-maximal inhibitory activities at about 10-14 M (Fig. 4Go). Peptide B (10 µM) did not inhibit the cAMP synthesis induced by TSH (data not shown). To investigate the sequence specificity of peptide B, the reverse sequence of peptide B (reverse B) was synthesized. Reverse B did not show any inhibitory activity (Figs. 4Go and 5Go).



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  		Figure 4. Effect of peptide B on the cAMP synthesis induced by GD IgG. Serially diluted peptide B (•) or reverse B ({circ}) was preincubated with GD IgG (100 µg) in 100 µl NaCl-free isotonic HBSS containing 0.5 mM IBMX for 1 h at room temperature. The mixtures were added to CHO-TSHR cells and incubated for 3 h, and the level of cAMP was determined as described in Materials and Methods. The amount of cAMP generated in the presence of assay buffer only was 1.29 ± 0.22 pmol, and the amount produced by GD IgG was 2.52 ± 0.12 pmol after correction for background. Each point represents the mean of duplicate determinations along with the SD indicated.

 


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  		Figure 5. Effect of peptide B on the cAMP synthesis induced by the IgG of various GD patients. IgGs from five different GD patients were added to CHO-TSHR cells after preincubation with or without 10 µM peptide B at room temperature for 1 h as described in Fig. 4Go, and the level of cAMP was determined as described in Materials and Methods.

 
Effect of peptide B on the cAMP synthesis induced by IgG of various GD patients and effects of other selected peptides on GD IgG

The effect of peptide B on other GD patients’ IgGs was examined. We assayed the level of cAMP produced by IgGs of five GD patients in the presence or absence of peptide B. cAMP production by two of five GD IgGs was inhibited by peptide B (Fig. 5Go). The effects of other selected peptides on cAMP synthesis were also investigated. Among the nine peptide sequences listed in Table 1Go, all of the sequences containing histidine at the first and fourth positions showed complete inhibition of the synthesis of cAMP induced by GD IgG. An additional sequence, HATGTHGLSLSH, that has histidine at the first and sixth positions also showed complete inhibition (Fig. 6Go).



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  		Figure 6. Inhibitory effect of selected peptide sequences on the cAMP synthesis induced by a GD IgG. The IgG of patient E in Fig. 4Go was preincubated with or without 10 µM of each of 9 peptide sequences (see Table 1Go) including peptide B at room temperature for 1 h, and the level of cAMP was determined as described in Materials and Methods. Only 6 amino acids from the N terminus of each peptide (total of 12 amino acids; see Table 1Go) are indicated. The amount of cAMP generated in the presence of assay buffer only was 2.27 ± 0.13 pmol, and the amount produced by GD IgG was 1.88 ± 0.11 pmol after correction for background. Each point represents the mean of duplicate determinations along with the SD indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our goal in this study was development of a method for identification of the peptides that bind and inhibit the function of TSAb clones with use of a phage-displayed peptide library. Because of the presence of various classes of thyroid autoantibodies in the serum of patients with GD, the availability of pure TSAb clones would be desirable for screening peptides that inhibit the function of TSAb. This was achieved by molecular cloning of some human monoclonal TSAb genes. However, to date two clones were obtained (9), and the clones appeared not to be prevalent among patients with GD (10). Therefore, we have devised a method for identifying the peptides that bind to a clone of TSAb in the serum of patient with GD. In this method, the IgG that binds to human TSH receptor was enriched by recombinant human TSHRE cross-linked to Ni-NTA resin, and the peptides that bind IgG for TSHR were identified from a phage-displayed peptide library. Individual peptides were then investigated for inhibition of cAMP synthesis induced by GD IgG. In theory, the peptides that either bind to the abundant clones of GD IgG or have higher affinity for certain clones of GD IgG would be identified. The GD IgG clones may not necessarily be those of TSAb. However, for GD IgG to show stimulatory activity for synthesis of cAMP in thyroid cells, the concentration of a TSAb clone should be substantial, and the clone may be dominant among various GD IgGs in the serum of GD patients. As we used the ectodomain of the TSHR, we cannot rule out the possibility that we might have missed some TSAb that may not bind to the isolated ectodomain of TSH receptor due to the possible changes in conformation in the absence of the rest of the receptor molecule.

The phage-displayed peptide library is a powerful tool for the development of pharmacologically active peptide agonists or antagonists (28). It has been known that epitopes for TSAbs are not linear, but 3-dimensional, consisting of amino acids of distant sites (1, 2, 9, 21, 22, 23). A random peptide library may have peptide sequences that recognize TSAb. In this study we used a commercially available phage-displayed peptide library. Our approach was the selection of phages that bind to IgG for TSHR in the serum of GD patients. For this purpose we enriched TSHR IgG by use of purified human TSHRE. Phages were then bound to the enriched IgG. We attempted to elute the bound phages with TSHRE during the biopanning procedure. However, we found that phage clones that have high affinity for anti-TSHR Abs were not eluted by TSHRE (data not shown). Therefore, the bound phages were eluted at low pH. Most of the selected phage clones showed preference for the IgG of GD patients over the IgG of normal subjects for binding. After three rounds of biopanning, most of the phages that were eluted from anti-TSHR Ab had the consensus motives in their sequences. Among 14 phage clones examined, 9 had histidine residues at the first and fourth positions. Among the 9 sequences, 4 had lysine at the second position, and 5 clones had lysine at the third position. There are no sequences in the extracellular domain of TSHR that match the peptide sequences. Recently, Kajava et al. (29) proposed a 3-dimensional model structure for the extracellular domain of TSHR. However, the report lacks sufficient details that will allow determination of 3dimensional epitopes for GD IgG.

Due to the availability of limited amount of GD IgG, only peptide B was further characterized. The concentration of peptide B for half-maximal inhibition of the synthesis of cAMP induced by GD IgG was about 10-14 M (Fig. 4Go). When similar experiment was carried out with IgG of another patient (patient E of Fig. 5Go), peptide B also exhibited inhibitory activity at similar concentration (data not shown). The extremely low inhibitory concentration of peptide B is very unusual. It may reflect unusually high affinity of the peptide for a clone of TSAb. Work is in progress to clarify this point.

When the effect of peptide B on the IgG of other patients was investigated, peptide B inhibited the stimulatory effect of two of five GD IgG, suggesting that not all GD patients contain the TSAb clone that is inhibited by peptide B. This is in agreement with the reports that TSAb is polyclonal, and each GD patient may have a different clone(s) of TSAb (9, 10, 11, 12, 24). Peptide B does not inhibit cAMP synthesis induced by TSH. To further confirm the specificity of peptide B, the reverse sequence of peptide B (reverse B) was synthesized. Reverse B does not inhibit the cAMP generation induced by GD IgG at the effective concentration of peptide B. When all peptide sequences were investigated for their inhibitory activity for cAMP generation induced by GD IgG, five of nine peptide sequences, including peptide B, inhibited the synthesis of cAMP. Four of the five sequences showed a similar sequence pattern at the N terminus: histidine at the first and fourth positions, and one other sequence that had histidine at the first and sixth positions. Four sequences had lysine at the second or third position. Consequently, it appears that HKXH or HXKH is an important motif.

In summary, we have devised a new method that identifies the peptide sequences that bind to GD IgG and inhibit the function of TSAb clones in the serum of GD patient. This method will enable us to identify the peptide sequences that interact with different clones of TSAb, and such a peptide may be useful for various purposes: classification of different clones of TSAb, purification of TSAb, diagnosis of GD, prognosis of different GD patients, and development of drugs for treatment of GD.


   Footnotes
 
Abbreviations: CHO-TSHR, Chinese hamster ovary cells transfected with TSH receptor; GD, Graves’ disease; HBSS, Hanks’ balanced salt solution; IBMX, 3-isobutyl-1-methylxanthine; IgG, immunoglobulin G; Ni-NTA, nickel-chelate-nitrilotriacetic acid; TBII, TSH binding inhibitory immunoglobulin; TSAb, thyroid-stimulating antibody; TSHR, TSH receptor; TSHRE, extracellular domain of TSH receptor.

Received April 8, 2002.

Accepted January 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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F. Latrofa, G. D. Chazenbalk, P. Pichurin, C.-R. Chen, S. M. McLachlan, and B. Rapoport
Affinity-Enrichment of Thyrotropin Receptor Autoantibodies from Graves' Patients and Normal Individuals Provides Insight into Their Properties and Possible Origin from Natural Antibodies
J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4734 - 4745.
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