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Purification from Human Plasma of a Hexapeptide That Potentiates the Sulfation and Mitogenic Activities of Insulin-like Growth Factors
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
247, 587–591 (1998)
RC988834
Purification from Human Plasma of a Hexapeptide That Potentiates the Sulfation and Mitogenic Activities of Insulin-like Growth Factors Brigitte Dousset,*,1 Jean Straczek,* Fatima Maachi,* Dung Le Nguyen,† Christine Jacob,* Josette Capiaumont,* Pierre Nabet,* and Francine Belleville* *Laboratory of Biochemistry, Nancy Medical School, Henri Poincare´-Nancy I University, P.O. Box 184, 54505 Vandoeuvre Cedex, France; and †INSERM, Unit 376, University Hospital, Center Arnaud de Villeneuve, 34295 Montpellier Cedex, France
Received May 17, 1998
The human plasma contains small peptide molecules known as low molecular weight growth factors synergistically increasing certain biological actions of insulin-like growth factors. In the present work we isolated and characterized a hexapeptide with HWESAS as structure. This purified peptide was absolutely necessary for the sulfation activity of insulin-like growth factor-I on chick embryo pelvic cartilages and improved the mitogenic activity of both insulin-like growth factors. The effects of this hexapeptide were confirmed by using the homologous synthetic peptide, that exhibited similar biological effects. Other synthetic peptides with structure derived from hexapeptide were shown to be active: the pentapeptide HWESA appeared more potent than the tripeptide HWE, which is about 170 to 200 times less active than the hexapeptide. The sequence of hexapeptide HWESAS is identified in only one human protein that is C3f, a fragment of C3 complement. q 1998 Academic Press
The insulin-like growth factors (IGFs), IGF-I and IGF-II, regulate the proliferation and differentiation of several types of cell as well as a variety of other cell functions (1), such as the proteoglycan synthesis in cartilage (2). Early purification protocols for IGF-I and IGF-II were monitored by measuring the stimulation of 35SO4 uptake by rat (3), chick embryo (4) and pig (5) cartilage explants. However, it has been shown that the crude or partially purified IGF fractions stimulate the proteoglycan biosynthesis in cartilages contrary to the highly purified preparations or recombinant human IGFs (6, 7, 8). Therefore, Froesch et al. (6) suggested that an unidentified factor necessary for IGF 1
Corresponding author. Fax: 33.03.83.85.26 73.
activity was removed during purification. Also, Plet et al (9) observed that following dialysis, serum partially lost its capacity to stimulate cell growth while the addition of the serum filtrate leads to the restoration of the full serum activity. Heulin et al (8, 10) found that human serum contains one or several peptidic substances with molecular weights below 1 000 Da, called low molecular weight growth factors (LMWGF), which are absolutely necessary for the sulfation and mitogenic activity of IGFs (11). In addition, preliminary studies with crude LMW-GF prepared from serum ultrafiltrate (cut-off 1 000 Da) suggested that at least two active molecules were present (12). Recently, Straczek et al (13) have isolated and characterized a tetrapeptide (WGHE) synergistically activating the sulfation and mitogenic activities of IGF-1. However, such synergistic effects were also observed with other collected chromatography fractions indicating the presence of active substances, which are different from the aforementioned peptide and this study was undertaken to identify these factors. MATERIAL AND METHODS Purification. The pilot studies were done with small volumes of human plasma (0.8 - 11) (14), but the main purification was done with 15 liters of human plasma. The plasma was ultrafiltered through a PTAC membrane (1 000 Da cut-off; Millipore, Bedford, MA, USA) at 47 C, until the retentate volume was 1/3 of the original volume. The ultrafiltrate (10 liters) was extracted (v/v) with chloroform for 24 h at 47 C to remove organic solvent-soluble molecules such as steroids. The lyophilized aqueous phase containing 50 mg peptide/ml ultrafiltrate as determined by the method of Sargent (15) was placed on a 100 1 10 cm column of 200-400 mesh Biogel P2 (BioRad, Hercules, CA, USA). The column was eluted with distilled water at a flow rate of 150 ml/h. Fractions (20 min) were collected and their biological activities measured as described below. The fractions (P2b) containing activity eluted between Kav 0.45-0.65 (370-700 Da) were pooled and lyophilized for further purification by reverse-phase HPLC (13). Then, the sample was dissolved in 100 ml 0.1 % (v/v)
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trifluoroacetic acid (TFA)/water and pumped (20 ml) at 2 ml/min onto a 250 1 10 mm Vydac C18 reverse-phase (RP) HPLC column (separations Group, Hesperia, CA, USA) equilibrated with the same buffer (0.1 % TFA in water : solvent A). The column was washed with solvent A for 10 min and the acetonitrile concentration increased to 10.5 % (v/v) over 35 min, and then to 17.5 % over the next (total time : 65 min) using linear gradients. Absorbance was monitored at 214 nm and 2 ml fractions were collected. The fractions eluted at 40–44 min exhibited biological activity (Fig. 1A). Such active fractions from five successive runs, were pooled, evaporated to dryness, dissolved in 500 ml 0.1 % TFA and concentrated by RP HPLC on an ˚ pore, 5 mm bead), analytical Vydac C18 column (250 1 4.6 mm, 300 A using 0.1 % TFA (solvent A) and 0.08 % TFA in acetonitrile (solvent B). Aliquots (50 ml) were loaded onto the column equilibrated with solvent A and the peptides eluted using the same gradient as above at 0.6 ml/min. The fractions corresponding to peaks 1, 2 and 3 were collected (Fig. 1B) and purified to apparent homogeneity by repeated passage through the same column. The acetonitrile concentration was raised to 15 % using a flatter linear gradient. Sample containing 250-400 pmol of peptides from each peak were then sequenced and analyzed by mass spectrometry. In peak 2 a tetrapeptide (WGHE) has been characterized (13) and peptide samples corresponding to peaks 1 and 3 were examined further. Peptide sequencing. Automated Edman degradation that was carried out on an Applied Biosystem (Foster City, CA, USA) 470 A protein sequencer. Phenylthiohydantoin (PTH) derivatives were identified by HPLC after automatic injection into an Applied Biosystem 120 A analyser coupled to the sequencer. The methods used have been described elsewhere (16). Electrospray mass spectrometry. Lyophilized samples of peaks 1 and 3 (Fig. 1B) were dissolved in 30 ml aqueous methanol (50 : 50 v/v) containing 1 % acetic acid. The analyses were carried out by injecting the sample at a flow rate of 2 ml/min into an HP 5989 mass spectrometer (Hewlett Packard, San Fernando, CA, USA) equiped with an electrospray ion source (Analytica of Brandford type) and scanning over a mass range of 200-1 100. The sum of several scans was used to obtain the final electrospray mass spectrum. The multiple-charged molecular ions obtained from a separately introduced sample of heart myoglobin (average molecular mass: 16 951.5 Da) were used for mass scale calibration. Synthetic peptide assembly. The synthesis of various peptides including the hexapeptide (HWESAS), the pentapeptide (HWESA), the tetrapeptide (HWES) and the tripeptide (HWE) was achieved by the solid phase method according to a Boc-HF strategy using BOP as the coupling reagent (17). The peptides were assembled manually using appropriate starting chloromethylated resins (CM-resin) : BocSer(Bzl)-CM-resin for HWESAS and HWES : Boc-Ala-CM-resin for HWESA : Boc-Glu(OcHx)-CM-resin for HWE. The resins contain 0.7 mmol of amino acid per gram. Amino acids are protected as follows: Boc-His(Dnp), Boc-Ser(Bzl) and Boc-Glu(OcHx); Boc-Trp was coupled unprotected. The peptidyl-resins obtained after peptide assembly were treated with thiophenol (50 % in DMF) for 30 min to remove the Dnp group prior to the final HF cleavage (1 h at 07 C in the presence of anisol). After trituration in ether, the crude compounds were recovered in a water/acetic acid mixture (10 : 90) and lyophilized. Purifications were achieved by HPLC : the crude products were injected onto a semi-preparative column (Merck Hibar, 25 1 250 nm, 10 mm beads) and eluted through a gradient using an acetonitrile/ water mixture containing 0.1 % TFA. The fractions with purity ú 95% as monitored by analytical HPLC were lyophilized as the purified peptides. Random peptides. Two peptides described as having sulfation growth factor activities were assayed: bursine (KHG) described by Kahn (18) and GHK described by Pickart (19) and Maquart (20). Bioassays. Purification steps were monitored by measuring the ability of samples to promote IGF-I induced [35 S] sulfate incorpora-
FIG. 1. Purification of low molecular weight growth factors by RP HPLC. A : Semi-preparative RP HPLC: bioactive Biogel P2 fractions were pooled, concentrated and chromatographed on semi-preparative Vydac C18 column using linear gradients of acetonitrile in 0.1 % TFA. Fractions collected at 39-49 min, were further purified. B: Analytical RP HPLC purification: the fractions collected during semi-preparative RP HPLC were applied to analytical Vydac C18 columns and eluted using a flatter acetonitrile gradient. Peaks 1 and 3 were sequenced and subjected to electrospray mass spectrometry analysis. The inset bar graphs show [35S] sulfate incorporation (arbitrary units per fraction).
tion into glycosaminoglycans (GAGs) in chick embryo pelvic cartilages (21). This sulfation bioassay was performed by using rhIGF (20 ng/ml) alone or combined with a reference human partially purified serum ultrafiltrate : P2b (1 000 Da cut-off), or with diluted purification fractions. The LMW-GF were active in the sulfation bioassay only in combination with rhIGF-I and did not stimulate rhIGF-II activity (22). For chromatogram fractions screening, the results were given in arbitrary units. Some purification steps were also monitored by evaluating the mitogenic activity, measured by [3H] thymidine incorporation into chick embryo fibroblasts. Pelvic cartilages from 11 day-old chick embryos were treated with 0.2 mg/ml collagenase and 0.25 % v/v trypsin and the fibroblasts collected. The fibroblasts were then grown in MEM/Hepes (Seromed), pH 7.2 supplemented with 5 % glutamine (200 mM); 5 % no-essential amino-acids (Flow Laboratories); 100 mg/ml streptomycin; 100 U/ml specillin and 10 % fetal calf serum (Flow Laboratories). The assays were conducted on 4 1 104 cells/ml, between the third and fifth passage. Cells were synchronized into
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FIG. 2. Electrospray mass spectrometry analysis. A: peak 3 (capillary exit voltage: 190 V), B: purified peak 1 (capillary exit voltage: 220 V).
G0-G1 by placing them in serum-free medium for 48 hours, and then incubated with the fractions to be tested for 24 hours. [3H] thymidine was added for the last 6 hours. Cells were collected, washed twice with 0.15 M NaCl and counted. Mitogenic tests were carried out with rhIGFs or without rhIGFs. LMW-GF are more effective in mitogenic assays in combination with rhIGF-II that with rhIGF-I (data not shown). Statistical analysis. Each dose of tested product was assayed on 8 cartilages in the sulfation test and in six wells for mitogenic activity. The results of the bioassays were expressed as a stimulation ratio [SR : cpm mean of assay / cpm mean of blank (medium alone)], to allow comparisons. Significance was tested by variance analysis (ANOVA and Bonferroni t-test) using Sigmastat software (Jansen Scientific Software - Erkrath Germany).
RESULTS Purification. The biologically active fraction recovered from Biogel P2 gel permeation chromatography was purified by RP-HPLC. Although numerous peaks of peptides were resolved, the incorporation of [35 S] sulfate into chick embryo pelvic cartilages in presence of IGF-I was increased only by the addition of the frac-
tion corresponding to an inhomogeneous peak eluted with 11-12 % acetonitrile (Fig. 1A). This fraction was resolved into three well-defined peaks by analytical HPLC (Fig. 1B) and the stimulation of rhIGF-I sulfation activity was mainly noted with peaks 1 and 2 eluted fractions. Since the tetrapeptide (WGHE) has been isolated from the peak 1 eluate, the peak 3 fraction was considered and repeatedly subjected to the same analytical HPLC until the peptidic sample was apparently homogeneous. The third eluted fraction (peak 3) exhibited a less potent effect in combination with rhIGF-I and was therefore directly analyzed by mass spectrometry. Although the presence of high concentrations of peptides was observed in other area of the chromatograms, no biological activity was detected in the corresponding eluates. Mass spectrometry and sequence analysis. The peptide obtained from the peak 3 eluate was identified as a tripeptide with the following sequence HWE and an accurate molecular mass of 470.51 Da that agreed with the major peak mass at 471.6 Da identified on the elec-
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the hexapeptide (Fig. 3A) and between 70 and 76 % of the serum total activity was recovered. A synergistic effect on rhIGF-I was also observed with all other synthetic peptides (HWESA, HWES, HWE). The dose-response curves of these peptides are bell-shaped and the concentrations giving the maximal effect on sulfation activity are given in table I. In contrast, both random tripeptides, respectively KHG and GHK, had no biological action. Also, the mitogenic action of the synthetic hexapeptide was tested in combination with rhIGFs. Like in sulfation bioassay, a significant increase of [3H] thymidine incorporation into chick embryo fibroblasts was noted when the hexapeptide was associated to IGFs and specially to rhIGF-II (Fig. 3B) leading to the recovery from 60 to 65 % of total serum activity. DISCUSSION
FIG. 3. Stimulation ratio (SR) : cpm mean of assay/cpm mean of blank (medium alone). A : [35S] sulfate incorporation into cartilages of 11-day-old chick embryos following incubation in (a) medium alone, (b) medium with rh IGF-I (20 ng/ml), (c) 5 % human normal serum, (d) medium with rhIGF-I (20 ng/ml) / P2b (4 mg peptides/ ml), (e) medium with rhIGF-I (20 ng/ml)/ peak 1 (30 ng peptides/ ml), (f) medium with rhIGF-I (20 ng/ml)/ synthetic HWESAS (15 ng/ml). a, c, d, e and f were significantly different from b (t õ 0.05). B : [3H] thymidine incorporation into fibroblasts from chick embryo cartilages following incubation with (a) medium alone, (b) medium with rhIGF-II (25 ng/ml), (c) 5 % human normal serum, (d) medium with rhIGF-II (25 ng/ml) / P2b (4 mg peptides/ml), (e) medium with rhIGF-II (25 ng/ml)/ peak I (15 ng/ml), (f) medium with rhIGFII (25 ng/ml) / synthetic HWESAS (6,3 ng/m)l. c, d, e and f were significantly different from b (t õ 0.05).
trospray-mass spectrometry spectrum (Fig. 2A). The peptide sample of purified peak 1 contained a hexapeptide with HWESAS as sequence and predicted mass value of 715.78 Da corresponding to the highest peak (716.7 Da) on the acquired scan mass spectrum (Fig. 2B). Because the results of the sequence analysis only identified the hexapeptide, the unidentified component with the other major peak at 558.5 Da (Fig. 2B) might be the tetrapeptide HWES (accurate mass 557.6 Da). Biological activity. According to the results of mass spectrometry and sequence analysis, different peptides (HWESAS, HWESA, HWES and HWE) were synthesized and their biological effects were tested. While rhIGF-I alone had no sulfation activity, the incorporation of [35 S] in chick embryo pelvic cartilages was significantly stimulated in the concomitant presence of
The present study reports the purification and the identification from human serum of a hexapeptide with HWESAS as sequence. In combination with rhIGF-I, this peptide promoted the incorporation of [35 S] into pelvic cartilages of 11-day-old chick embryos by a synergistic effect, which was identical to that obtained with partially purified LMW-GFs (22). Also, the hexapeptide increased the [3H] thymidine incorporation into chick embryo fibroblasts when associated to rhIGFs although, in this case, the degree of stimulation was less with rhIGF-I than with rhIGF-I. The actions of the natural hexpaptide were confirmed by using the synthetic analogue, which was found to exert similar biological activities. In contrast, both random peptides (KHG and HGK) had no action. Moreover, in the last step of purification two peptides with a structure related to that of the hexapeptide were also found including a large quantity of the identified tripeptide HWE and small amounts of a peptide (molecular mass Å 558.6 Da) that might correspond to the tetrapeptide HWES. These peptides could be generated during purification, or could result from proteolysis in vivo. All of them stimulated the rhIGF-I sulfation activity although the maximal action was observed at HWE concentration approximatively 200 times lower than HWESAS concentration.
TABLE I
Concentrations of Synthetic Peptide Giving the Maximum Response in Combination with 20 ng/ml IGF-I in Sulfation Test on Chick Embryo Pelvic Cartilages Peptides
Concentration (ng/ml) giving the maximal response
t value
HWESAS HWESA HWES HWE
15 ng/ml 30 ng/ml 500 ng/ml 2.5 mg/ml
5.04 2.73 4.65 3.44
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The HWESAS motif appears to be present in only one human protein called C3f that is a fragment of the third component of human complement (C3) (data bank consultation: data base Swiss PROT and PIR, Fastal Software). This heptadecapeptide (SSKITHRIHWESASLLR) is released when the fragment C3b is converted to C3bi (23). The HWESA structure is also found in bovine cytochrome P450 (P450-C21) whereas that of the tripeptide (HWE) is present in the precursors of various human proteins such as the a 2 macroglobulin, the a galactosidase A, the apolipoprotein B100, the class II major histocompatibility antigen (DQ4, DQ5, DQ6) a chains. The physiological role of these natural peptides is yet unknown. However in the course of our work on the physiological activities and tissular production of LMW-GF, observations in humans suggested that the kidney was involved in their production with a particular role for the tubule. By using the same sulfation bioassay, it was shown that LMW-GF bioactivity is present in normal human serum ultrafiltrates while it is absent in serum ultrafiltrates of patients with end stage chronic renal failure and restored to normal by a successful kidney transplantation (24). These findings were confirmed by an experimental study in the pig (25). IGF-I is produced in the kidney and IGF-I receptors have been identified throughout the organ, particularly in the proximal tubule (26). After ischemic acute renal failure in rats the recovery of renal function as well as the tubular integrity are markedly increased in animals treated by IGF-I (27). HWESAS might reinforce the effect of IGF-I, especially in the tubule. The IGF activity is also modulated by several IGF binding proteins (28, 29, 30) and it is therefore possible that the hexapeptide (HWESAS) and the tetrapeptide (WGHE) could regulate the IGF activity on cells. Since HWESAS could be generated in vivo by the action of endopeptidase(s) on complement fragment C3f and it can be speculated that this heptadecapeptide is also a growth promoting factor. ACKNOWLEDGMENTS This work was supported by The Conseil Regional de Lorraine.
REFERENCES 1. Jones, J. I., and Clemmons, R. R. (1995) Endocrine Rev. 16, 3– 34. 2. Froesch, E. R., Schmid, C., Schwander, J., and Zapf, J. (1985) Annu. Rev. Physiol. 47, 443–467. 3. Svoboda, M. E., Van Wyck, J. J., Klapper, D. G., Bellow, R. E., Grissom, F. E., and Schlueter, R. J. (1980) Biochemistry 19, 790– 797. 4. Fryklund, L., Uthne, K., Sievertsson, H., and Westermark, B. (1974) Biochem. Biophys. Res. Commun. 61, 950–956.
5. Van Den Brande, J. L., and Du Caju, M. V. L. (1974) Acta Endocrinol. (Copenh). 75, 233–242. 6. Froesch, E. R., Zapf, J., Andhya, T. K., Ben Porath, E., Segen, B. J., and Gibson, K. D. (1976) Proc. Natl. Acad. Sci. USA 73, 2904–2908. 7. Silbergeld, A., Mamet, R., Laron, Z., and Nevo, Z. (1981) Acta Endocrinol. (Copenh). 97, 503–507. 8. Heulin, M. H., Artur, M., Malaprade, D., Straczek, J., Pierson, M., Stoltz, J. F., Belleville, F., Paysant, P., and Nabet, P. (1981) Biochem. Biophys. Res. Commun. 99, 644–653. 9. Plet, A., and Berwald-Netter, Y. (1980) Biochem. Biophys. Res. Commun. 94, 744–754. 10. Heulin, M. H., Rajoelina, J., Artur, M., Geschier, C., Straczek, J., Lasbennes, A., Belleville, F., and Nabet, P. (1987) Life Sci. 41, 297–304. 11. Heulin, M. H., Artur, M., Straczek, J., Belleville, F., Nabet, P., Hermann, A., Lebeurre, M. O., and Schimpff, R. M. (1982) J. Cell. Sci. 57, 129–137. 12. Heulin, M. H., Rajoelina, J., Lasbennes, A., Geschier, C., Lamotte, F., Straczek, J., Gelot, M. A., Artur, M., Belleville, F., and Nabet, P. (1989) in Biotechnology of Plasma Proteins: Colloque INSERM (Stoltz, J. F., and Rivat, C., Ed.), Vol. 175, pp. 129– 134, Libbey, London. 13. Straczek, J., Maachi, F., Le Nguyen, D., Becchi, M., Heulin, M. H., Nabet, P., and Belleville, F. (1995) FEBS Lett. 373, 207– 211. 14. Heulin, M. H., Rajoelina, A., Lasbennes, A., Lamotte, F., Straczek, J., Gelot, M. A., Artur, M., Belleville, F., and Nabet, P. (1988) in Blood Growth Factors of Very Low Molecular Weight: Evidence Purification and Characterization of Several Molecules Active in Vitro on Cartilage Metabolism and Fibroblast Multiplication: Colloques INSERM (Aubry, A., Marrand, M., and Vittoux, B., Eds.), Vol. 174, pp. 69–72, Libbey, London. 15. Sargent, M. G. (1987) Anal. Biochem. 163, 476–481. 16. Straczek, J., Heulin, M. H., Chenut, A. M., Denoroy, L., Barenton, B., Blanchard, M., Charrier, J., Martal, J., Belleville, F., and Nabet, P. (1990) J. Chromatogr. 533, 35–46. 17. Le Nguyen, D., Heitz, A., and Castro, B. (1987) J. Chem. Soc. Perkin Trans I, 1915–1919. 18. Kahn, A. (1986) Me´decine/Science 2, 335–337. 19. Pickart, L., and Thaler, M. M. (1973) Nature New Biol. 243, 85– 87. 20. Maquart, F. X., Pickart, L., Laurent, M., Gillery, P., Monboisse, J. C., and Borel, J. P. (1988) FEBS lett. 238, 343–346. 21. Heulin, M. H., Artur, M., Geschier, C., Straczek, J., Vescovi, G., Belleville, F., Paysant, P., and Nabet, P. (1984) Acta Endocrinol. (Copenh). 106, 43–51. 22. Maachi, F., Straczek, J., Jacob, C., Belleville, F., and Nabet, P. (1995) Life Sci. 56, 2273–2284. 23. Lambris, J. D. (1993) in Complement Today (Cruse, J. M., and Lewis, R. D., Ed.), pp. 16–45, Karger, Basel. 24. Jacob, C., Maachi, F., El Farricha, O., Dousset, B., Kessler, M., Belleville, F., and Nabet, P. (1993) Clin. Nephrol. 39, 327–334. 25. Jacob, C., Hubert, J., Maachi, F., Punga-Maole, A., Dousset, B., Junke, E., and Belleville, F. (1995) Renal Failure 17, 339–347. 26. Feld, R. A., and Hirchberg, R. (1996) TEM. 7, 85–93. 27. Hirschberg, R., and Ding, H. (1996) J. Am. Soc. Nephrol. 7, 1826– 1835. 28. Binoux, M., and Hossenlopp, P. (1988) J. Clin. Endocrinol. Metab. 67, 509–514. 29. Tsao, T., Wang, J., Frevenza, F. C., Vu, T. H., Jin, I. H., Hofman, A. R., and Rabkin, R. (1995) Kidney Int. 47, 1658–1668. 30. Rechler, M. (1997) Endocrinology 138, 2645–2647.
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247, 587–591 (1998)
RC988834
Purification from Human Plasma of a Hexapeptide That Potentiates the Sulfation and Mitogenic Activities of Insulin-like Growth Factors Brigitte Dousset,*,1 Jean Straczek,* Fatima Maachi,* Dung Le Nguyen,† Christine Jacob,* Josette Capiaumont,* Pierre Nabet,* and Francine Belleville* *Laboratory of Biochemistry, Nancy Medical School, Henri Poincare´-Nancy I University, P.O. Box 184, 54505 Vandoeuvre Cedex, France; and †INSERM, Unit 376, University Hospital, Center Arnaud de Villeneuve, 34295 Montpellier Cedex, France
Received May 17, 1998
The human plasma contains small peptide molecules known as low molecular weight growth factors synergistically increasing certain biological actions of insulin-like growth factors. In the present work we isolated and characterized a hexapeptide with HWESAS as structure. This purified peptide was absolutely necessary for the sulfation activity of insulin-like growth factor-I on chick embryo pelvic cartilages and improved the mitogenic activity of both insulin-like growth factors. The effects of this hexapeptide were confirmed by using the homologous synthetic peptide, that exhibited similar biological effects. Other synthetic peptides with structure derived from hexapeptide were shown to be active: the pentapeptide HWESA appeared more potent than the tripeptide HWE, which is about 170 to 200 times less active than the hexapeptide. The sequence of hexapeptide HWESAS is identified in only one human protein that is C3f, a fragment of C3 complement. q 1998 Academic Press
The insulin-like growth factors (IGFs), IGF-I and IGF-II, regulate the proliferation and differentiation of several types of cell as well as a variety of other cell functions (1), such as the proteoglycan synthesis in cartilage (2). Early purification protocols for IGF-I and IGF-II were monitored by measuring the stimulation of 35SO4 uptake by rat (3), chick embryo (4) and pig (5) cartilage explants. However, it has been shown that the crude or partially purified IGF fractions stimulate the proteoglycan biosynthesis in cartilages contrary to the highly purified preparations or recombinant human IGFs (6, 7, 8). Therefore, Froesch et al. (6) suggested that an unidentified factor necessary for IGF 1
Corresponding author. Fax: 33.03.83.85.26 73.
activity was removed during purification. Also, Plet et al (9) observed that following dialysis, serum partially lost its capacity to stimulate cell growth while the addition of the serum filtrate leads to the restoration of the full serum activity. Heulin et al (8, 10) found that human serum contains one or several peptidic substances with molecular weights below 1 000 Da, called low molecular weight growth factors (LMWGF), which are absolutely necessary for the sulfation and mitogenic activity of IGFs (11). In addition, preliminary studies with crude LMW-GF prepared from serum ultrafiltrate (cut-off 1 000 Da) suggested that at least two active molecules were present (12). Recently, Straczek et al (13) have isolated and characterized a tetrapeptide (WGHE) synergistically activating the sulfation and mitogenic activities of IGF-1. However, such synergistic effects were also observed with other collected chromatography fractions indicating the presence of active substances, which are different from the aforementioned peptide and this study was undertaken to identify these factors. MATERIAL AND METHODS Purification. The pilot studies were done with small volumes of human plasma (0.8 - 11) (14), but the main purification was done with 15 liters of human plasma. The plasma was ultrafiltered through a PTAC membrane (1 000 Da cut-off; Millipore, Bedford, MA, USA) at 47 C, until the retentate volume was 1/3 of the original volume. The ultrafiltrate (10 liters) was extracted (v/v) with chloroform for 24 h at 47 C to remove organic solvent-soluble molecules such as steroids. The lyophilized aqueous phase containing 50 mg peptide/ml ultrafiltrate as determined by the method of Sargent (15) was placed on a 100 1 10 cm column of 200-400 mesh Biogel P2 (BioRad, Hercules, CA, USA). The column was eluted with distilled water at a flow rate of 150 ml/h. Fractions (20 min) were collected and their biological activities measured as described below. The fractions (P2b) containing activity eluted between Kav 0.45-0.65 (370-700 Da) were pooled and lyophilized for further purification by reverse-phase HPLC (13). Then, the sample was dissolved in 100 ml 0.1 % (v/v)
587
0006-291X/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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trifluoroacetic acid (TFA)/water and pumped (20 ml) at 2 ml/min onto a 250 1 10 mm Vydac C18 reverse-phase (RP) HPLC column (separations Group, Hesperia, CA, USA) equilibrated with the same buffer (0.1 % TFA in water : solvent A). The column was washed with solvent A for 10 min and the acetonitrile concentration increased to 10.5 % (v/v) over 35 min, and then to 17.5 % over the next (total time : 65 min) using linear gradients. Absorbance was monitored at 214 nm and 2 ml fractions were collected. The fractions eluted at 40–44 min exhibited biological activity (Fig. 1A). Such active fractions from five successive runs, were pooled, evaporated to dryness, dissolved in 500 ml 0.1 % TFA and concentrated by RP HPLC on an ˚ pore, 5 mm bead), analytical Vydac C18 column (250 1 4.6 mm, 300 A using 0.1 % TFA (solvent A) and 0.08 % TFA in acetonitrile (solvent B). Aliquots (50 ml) were loaded onto the column equilibrated with solvent A and the peptides eluted using the same gradient as above at 0.6 ml/min. The fractions corresponding to peaks 1, 2 and 3 were collected (Fig. 1B) and purified to apparent homogeneity by repeated passage through the same column. The acetonitrile concentration was raised to 15 % using a flatter linear gradient. Sample containing 250-400 pmol of peptides from each peak were then sequenced and analyzed by mass spectrometry. In peak 2 a tetrapeptide (WGHE) has been characterized (13) and peptide samples corresponding to peaks 1 and 3 were examined further. Peptide sequencing. Automated Edman degradation that was carried out on an Applied Biosystem (Foster City, CA, USA) 470 A protein sequencer. Phenylthiohydantoin (PTH) derivatives were identified by HPLC after automatic injection into an Applied Biosystem 120 A analyser coupled to the sequencer. The methods used have been described elsewhere (16). Electrospray mass spectrometry. Lyophilized samples of peaks 1 and 3 (Fig. 1B) were dissolved in 30 ml aqueous methanol (50 : 50 v/v) containing 1 % acetic acid. The analyses were carried out by injecting the sample at a flow rate of 2 ml/min into an HP 5989 mass spectrometer (Hewlett Packard, San Fernando, CA, USA) equiped with an electrospray ion source (Analytica of Brandford type) and scanning over a mass range of 200-1 100. The sum of several scans was used to obtain the final electrospray mass spectrum. The multiple-charged molecular ions obtained from a separately introduced sample of heart myoglobin (average molecular mass: 16 951.5 Da) were used for mass scale calibration. Synthetic peptide assembly. The synthesis of various peptides including the hexapeptide (HWESAS), the pentapeptide (HWESA), the tetrapeptide (HWES) and the tripeptide (HWE) was achieved by the solid phase method according to a Boc-HF strategy using BOP as the coupling reagent (17). The peptides were assembled manually using appropriate starting chloromethylated resins (CM-resin) : BocSer(Bzl)-CM-resin for HWESAS and HWES : Boc-Ala-CM-resin for HWESA : Boc-Glu(OcHx)-CM-resin for HWE. The resins contain 0.7 mmol of amino acid per gram. Amino acids are protected as follows: Boc-His(Dnp), Boc-Ser(Bzl) and Boc-Glu(OcHx); Boc-Trp was coupled unprotected. The peptidyl-resins obtained after peptide assembly were treated with thiophenol (50 % in DMF) for 30 min to remove the Dnp group prior to the final HF cleavage (1 h at 07 C in the presence of anisol). After trituration in ether, the crude compounds were recovered in a water/acetic acid mixture (10 : 90) and lyophilized. Purifications were achieved by HPLC : the crude products were injected onto a semi-preparative column (Merck Hibar, 25 1 250 nm, 10 mm beads) and eluted through a gradient using an acetonitrile/ water mixture containing 0.1 % TFA. The fractions with purity ú 95% as monitored by analytical HPLC were lyophilized as the purified peptides. Random peptides. Two peptides described as having sulfation growth factor activities were assayed: bursine (KHG) described by Kahn (18) and GHK described by Pickart (19) and Maquart (20). Bioassays. Purification steps were monitored by measuring the ability of samples to promote IGF-I induced [35 S] sulfate incorpora-
FIG. 1. Purification of low molecular weight growth factors by RP HPLC. A : Semi-preparative RP HPLC: bioactive Biogel P2 fractions were pooled, concentrated and chromatographed on semi-preparative Vydac C18 column using linear gradients of acetonitrile in 0.1 % TFA. Fractions collected at 39-49 min, were further purified. B: Analytical RP HPLC purification: the fractions collected during semi-preparative RP HPLC were applied to analytical Vydac C18 columns and eluted using a flatter acetonitrile gradient. Peaks 1 and 3 were sequenced and subjected to electrospray mass spectrometry analysis. The inset bar graphs show [35S] sulfate incorporation (arbitrary units per fraction).
tion into glycosaminoglycans (GAGs) in chick embryo pelvic cartilages (21). This sulfation bioassay was performed by using rhIGF (20 ng/ml) alone or combined with a reference human partially purified serum ultrafiltrate : P2b (1 000 Da cut-off), or with diluted purification fractions. The LMW-GF were active in the sulfation bioassay only in combination with rhIGF-I and did not stimulate rhIGF-II activity (22). For chromatogram fractions screening, the results were given in arbitrary units. Some purification steps were also monitored by evaluating the mitogenic activity, measured by [3H] thymidine incorporation into chick embryo fibroblasts. Pelvic cartilages from 11 day-old chick embryos were treated with 0.2 mg/ml collagenase and 0.25 % v/v trypsin and the fibroblasts collected. The fibroblasts were then grown in MEM/Hepes (Seromed), pH 7.2 supplemented with 5 % glutamine (200 mM); 5 % no-essential amino-acids (Flow Laboratories); 100 mg/ml streptomycin; 100 U/ml specillin and 10 % fetal calf serum (Flow Laboratories). The assays were conducted on 4 1 104 cells/ml, between the third and fifth passage. Cells were synchronized into
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FIG. 2. Electrospray mass spectrometry analysis. A: peak 3 (capillary exit voltage: 190 V), B: purified peak 1 (capillary exit voltage: 220 V).
G0-G1 by placing them in serum-free medium for 48 hours, and then incubated with the fractions to be tested for 24 hours. [3H] thymidine was added for the last 6 hours. Cells were collected, washed twice with 0.15 M NaCl and counted. Mitogenic tests were carried out with rhIGFs or without rhIGFs. LMW-GF are more effective in mitogenic assays in combination with rhIGF-II that with rhIGF-I (data not shown). Statistical analysis. Each dose of tested product was assayed on 8 cartilages in the sulfation test and in six wells for mitogenic activity. The results of the bioassays were expressed as a stimulation ratio [SR : cpm mean of assay / cpm mean of blank (medium alone)], to allow comparisons. Significance was tested by variance analysis (ANOVA and Bonferroni t-test) using Sigmastat software (Jansen Scientific Software - Erkrath Germany).
RESULTS Purification. The biologically active fraction recovered from Biogel P2 gel permeation chromatography was purified by RP-HPLC. Although numerous peaks of peptides were resolved, the incorporation of [35 S] sulfate into chick embryo pelvic cartilages in presence of IGF-I was increased only by the addition of the frac-
tion corresponding to an inhomogeneous peak eluted with 11-12 % acetonitrile (Fig. 1A). This fraction was resolved into three well-defined peaks by analytical HPLC (Fig. 1B) and the stimulation of rhIGF-I sulfation activity was mainly noted with peaks 1 and 2 eluted fractions. Since the tetrapeptide (WGHE) has been isolated from the peak 1 eluate, the peak 3 fraction was considered and repeatedly subjected to the same analytical HPLC until the peptidic sample was apparently homogeneous. The third eluted fraction (peak 3) exhibited a less potent effect in combination with rhIGF-I and was therefore directly analyzed by mass spectrometry. Although the presence of high concentrations of peptides was observed in other area of the chromatograms, no biological activity was detected in the corresponding eluates. Mass spectrometry and sequence analysis. The peptide obtained from the peak 3 eluate was identified as a tripeptide with the following sequence HWE and an accurate molecular mass of 470.51 Da that agreed with the major peak mass at 471.6 Da identified on the elec-
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the hexapeptide (Fig. 3A) and between 70 and 76 % of the serum total activity was recovered. A synergistic effect on rhIGF-I was also observed with all other synthetic peptides (HWESA, HWES, HWE). The dose-response curves of these peptides are bell-shaped and the concentrations giving the maximal effect on sulfation activity are given in table I. In contrast, both random tripeptides, respectively KHG and GHK, had no biological action. Also, the mitogenic action of the synthetic hexapeptide was tested in combination with rhIGFs. Like in sulfation bioassay, a significant increase of [3H] thymidine incorporation into chick embryo fibroblasts was noted when the hexapeptide was associated to IGFs and specially to rhIGF-II (Fig. 3B) leading to the recovery from 60 to 65 % of total serum activity. DISCUSSION
FIG. 3. Stimulation ratio (SR) : cpm mean of assay/cpm mean of blank (medium alone). A : [35S] sulfate incorporation into cartilages of 11-day-old chick embryos following incubation in (a) medium alone, (b) medium with rh IGF-I (20 ng/ml), (c) 5 % human normal serum, (d) medium with rhIGF-I (20 ng/ml) / P2b (4 mg peptides/ ml), (e) medium with rhIGF-I (20 ng/ml)/ peak 1 (30 ng peptides/ ml), (f) medium with rhIGF-I (20 ng/ml)/ synthetic HWESAS (15 ng/ml). a, c, d, e and f were significantly different from b (t õ 0.05). B : [3H] thymidine incorporation into fibroblasts from chick embryo cartilages following incubation with (a) medium alone, (b) medium with rhIGF-II (25 ng/ml), (c) 5 % human normal serum, (d) medium with rhIGF-II (25 ng/ml) / P2b (4 mg peptides/ml), (e) medium with rhIGF-II (25 ng/ml)/ peak I (15 ng/ml), (f) medium with rhIGFII (25 ng/ml) / synthetic HWESAS (6,3 ng/m)l. c, d, e and f were significantly different from b (t õ 0.05).
trospray-mass spectrometry spectrum (Fig. 2A). The peptide sample of purified peak 1 contained a hexapeptide with HWESAS as sequence and predicted mass value of 715.78 Da corresponding to the highest peak (716.7 Da) on the acquired scan mass spectrum (Fig. 2B). Because the results of the sequence analysis only identified the hexapeptide, the unidentified component with the other major peak at 558.5 Da (Fig. 2B) might be the tetrapeptide HWES (accurate mass 557.6 Da). Biological activity. According to the results of mass spectrometry and sequence analysis, different peptides (HWESAS, HWESA, HWES and HWE) were synthesized and their biological effects were tested. While rhIGF-I alone had no sulfation activity, the incorporation of [35 S] in chick embryo pelvic cartilages was significantly stimulated in the concomitant presence of
The present study reports the purification and the identification from human serum of a hexapeptide with HWESAS as sequence. In combination with rhIGF-I, this peptide promoted the incorporation of [35 S] into pelvic cartilages of 11-day-old chick embryos by a synergistic effect, which was identical to that obtained with partially purified LMW-GFs (22). Also, the hexapeptide increased the [3H] thymidine incorporation into chick embryo fibroblasts when associated to rhIGFs although, in this case, the degree of stimulation was less with rhIGF-I than with rhIGF-I. The actions of the natural hexpaptide were confirmed by using the synthetic analogue, which was found to exert similar biological activities. In contrast, both random peptides (KHG and HGK) had no action. Moreover, in the last step of purification two peptides with a structure related to that of the hexapeptide were also found including a large quantity of the identified tripeptide HWE and small amounts of a peptide (molecular mass Å 558.6 Da) that might correspond to the tetrapeptide HWES. These peptides could be generated during purification, or could result from proteolysis in vivo. All of them stimulated the rhIGF-I sulfation activity although the maximal action was observed at HWE concentration approximatively 200 times lower than HWESAS concentration.
TABLE I
Concentrations of Synthetic Peptide Giving the Maximum Response in Combination with 20 ng/ml IGF-I in Sulfation Test on Chick Embryo Pelvic Cartilages Peptides
Concentration (ng/ml) giving the maximal response
t value
HWESAS HWESA HWES HWE
15 ng/ml 30 ng/ml 500 ng/ml 2.5 mg/ml
5.04 2.73 4.65 3.44
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The HWESAS motif appears to be present in only one human protein called C3f that is a fragment of the third component of human complement (C3) (data bank consultation: data base Swiss PROT and PIR, Fastal Software). This heptadecapeptide (SSKITHRIHWESASLLR) is released when the fragment C3b is converted to C3bi (23). The HWESA structure is also found in bovine cytochrome P450 (P450-C21) whereas that of the tripeptide (HWE) is present in the precursors of various human proteins such as the a 2 macroglobulin, the a galactosidase A, the apolipoprotein B100, the class II major histocompatibility antigen (DQ4, DQ5, DQ6) a chains. The physiological role of these natural peptides is yet unknown. However in the course of our work on the physiological activities and tissular production of LMW-GF, observations in humans suggested that the kidney was involved in their production with a particular role for the tubule. By using the same sulfation bioassay, it was shown that LMW-GF bioactivity is present in normal human serum ultrafiltrates while it is absent in serum ultrafiltrates of patients with end stage chronic renal failure and restored to normal by a successful kidney transplantation (24). These findings were confirmed by an experimental study in the pig (25). IGF-I is produced in the kidney and IGF-I receptors have been identified throughout the organ, particularly in the proximal tubule (26). After ischemic acute renal failure in rats the recovery of renal function as well as the tubular integrity are markedly increased in animals treated by IGF-I (27). HWESAS might reinforce the effect of IGF-I, especially in the tubule. The IGF activity is also modulated by several IGF binding proteins (28, 29, 30) and it is therefore possible that the hexapeptide (HWESAS) and the tetrapeptide (WGHE) could regulate the IGF activity on cells. Since HWESAS could be generated in vivo by the action of endopeptidase(s) on complement fragment C3f and it can be speculated that this heptadecapeptide is also a growth promoting factor. ACKNOWLEDGMENTS This work was supported by The Conseil Regional de Lorraine.
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