Biochimica et Biophysica Acta, 492 (1977) 95-101 © Elsevier/North-Holland Biomedical Press

BBA 37655

POLYALANYLATION OF BOVINE SOMATOTROPIN PEPTIDE 96-133

KENJI HARA and MARTIN SONENBERG Sloan-Kettering Institute for Cancer Research New York, N.Y. 10021 (U.S.A.)

(Received January 20th, 1977)

SUMMARY

Polyalanylation of bovine somatotropin peptide 96-133 has been investigated. Polyalanylated peptides contained an average of 3-50 additional alanines per mole of peptide. As indicated by circular dichroism, the parent peptide and the polyalanyl- ated peptides were helical at pH 4.0. The biological activity per mole of peptide with 50 additional alanines was approximately the same as that of the parent peptide.

INTRODUCTION

We have reported [1-3] that in experimental animals and human subjects limited tryptic digests of bovine somatotropin have significant biological activity. We have fractionated [4] these tryptic digests of bovine somatotropin by gel filtration in 50 ~ acetic acid and in 8 M urea, and have isolated two major peptides. The larger peptide (mol. wt. 16 000) originated from the amino-terminal position 1-95 and the carboxyl-terminal position 151-191 of the sequence of bovine somatotropin [5-7] which are linked by a disulfide bond. A smaller peptide (mol. wt. 5000), originating in residues 96-133 of the bovine somatotropin molecule (peptide (96-133)), was also isolated. Recently the amino acid sequence of peptide (96-133) has been deter- mined [8, 9]. It is homologous with region 96-133 of the sequence of bovine somato- tropin [5-7]. Because of the homology in the amino acid sequence between peptide (96-133) and a corresponding peptide occurring in human somatotropin, human chorionic somatomammotropin and ovine somatotropin greater than occurs in other parts of the molecules, we suggested [8, 9] that peptide (96-133) contains an amino acid sequence responsible in part for the biological activities of the intact native hormones.

These peptides are active in the weight gain and tibial width test in hypo- physectomized rats, but larger amounts of peptide are required for a response than with native bovine somatotropin [4, 10, 11]. The 16 000-dalton peptide is much less active in biological assays than the 5000-dalton peptide [4, 11]. Others [12, 13] have confirmed that sequence 96-133 has growth-promoting activity as does a synthetic sequence 96-136 of human somatotropin [14].

Peptide (96-133) may have reduced biological activity because of a secondary and tertiary structure significantly different from the native hormone from which it was derived. This could result in impaired affinity for its target cell receptor and/or

96

changes in its ability to stimulate a biological response. These considerations form the bases of separate studies and will be published subsequently.

The decrease growth response to peptide (96-133) may be related to its more rapid clearance and degradation. Clearance of proteins by the kidney is inversely related to their molecular weight [15]. We have found (Donner, D. and Sonenberg, M., unpublished) that peptide (96-133) is degraded more rapidly by isolated hepato- cytes than intact somatotropin.

Polyalanylation has been effectively employed to prepare protein and peptide derivatives of larger molecular weight and altered biological activity. Thus antigens have been found [17, 18] to have altered immunological reactivity. Human somato- tropin with 23 [19] or 265 [11] additional alanine residues per mol of 21 000 daltons retained full growth-promoting activity on a molar basis.

With the aforementioned considerations in mind we have prepared a series of polyalanylated derivatives of peptide (96-133) and report herein on their partial chemical and biological characterization.

EXPERIMENTAL

Materials Peptide (96-133) was prepared from a single component of tryptic digests of

bovine somatotropin as previously reported [4]. N-Carboxy-oL-alanine anhydride (Lot AL 86) was purchased from Miles-Yeda Ltd., Israel and was used immediately. All other reagents were of reagent grade.

Methods Samples of the peptide (96-133) were dissolved in 0.2 M phosphate buffer

(pH 7.6) and various amounts of a solution of N-carboxy-DL-alanine anhydride in dioxane (molar ratio of peptide (96-133):reagent was varied from 1:100 to 1:10) were added with stirring at 4 °C. The final dioxane concentrations were approximately 10-20~. After 24 h, the reaction was terminated by freezing the samples. In order to separate the poly(DL-Ala) peptide (96-133) from the unreacted N-carboxy-DL- alanine anhydride and the small polyalanine peptides, individual samples were gel- filtered on a Bio-Gel P-2 column equilibrated with 0.1 M ammonium bicarbonate buffer (pH 8.4). The samples collected from the column were directly lyophilized.

Polyacrylamide gel disc electrophoresis was carried out by the methods of Ornstein [20], Davis [21], and Reisfeld et al. [22]. Electrophoresis was carried out at pH 4.3 in 7.5 ~o polyacrylamide gel.

Peptide samples were hydrolyzed in vacuo with 6 M HC1 at 110 °C for 24 h. The hydrolysates were analyzed on an automatic amino acid analyzer Beckman Model 121 according to the method of Spackman et al. [23].

Circular dichroism determinations were made in the far-ultraviolet range (250-195 nm) with a Cary Model 60 recording spectropolarimeter with circular dichroism attachment 6002. Aliquots of the peptide solutions were taken for Lowry- Folin [24] determination of protein concentration for each experiment.

The growth responses of the poly(DL-Ala) peptide (96-133) samples, peptide (96-133) and the international somatotropin standard were determined in hypophy- sectomized rats [25] by the tibial width response test [26], which was performed by Biomatics Laboratories, Chicago, Ill., 60642, U.S.A.

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RESULTS

Samples of peptide (96-133) were dissolved in 0.2 M phosphate buffer (pH 7.6) and various amounts of a solution of N-carboxy-DL-alanine anhydride in dioxane were added. The experimental details pertaining to each preparation of poly(DL-Ala) peptide (96-133) are given in Table I. The formation of poly(DL-Ala) peptide (96-133) was allowed to proceed at 4 °C for 24 h with stirring. After gel-filtration on Bio-Gel P-2 to remove salts, N-carboxy-DL-alanine anhydride, small oligopeptides of alanine, and dioxane, the resulting fraction was lyophilized.

TABLE I

PREPARATION AND CHEMICAL CHARACTERIZATION OF poly(DL-Ala) PEPTIDE (96- 133)

Sample Native Buffer A m o u n t Dioxane Number peptide volume of volume of (96-133) (ml ) monomer (ml) added (mg) (mg) alanines

poly(DL-Ala) peptide (96-133)-1 80 8 184 2 50.5

poly(DL-Ala) peptide (96-133)-2 80 8 92 2 19.5

poly(DL-Ala) peptide (96-133)-3 80 8 46 1 6.5

poly(DL-Ala) peptide (96--133)-4 80 8 18.4 1 3.0

Aliquots of native peptide (96-133) and the poly(DL-Ala) peptide (96-133) samples were subjected to acid hydrolysis followed by amino acid analysis. The amino acid composition of the control was in good agreement with that of peptide (96-133). Analysis of poly(DL-Ala) peptide (96-133) revealed that poly(DL-Ala) peptide (96-133) contained an average of 3-50.5 extra alanine residues per mol of protein (Table I).

On polyacrylamide gel disc electrophoresis unreacted peptide (96-133), and poly(DL-Ala) peptide (96-133) samples had similar electrophoretic mobilities although the latter demonstrated broader protein-staining bands.

The circular dichroism spectra (Fig. 1) of poly(oL-Ala) peptide (96-133) and peptide (96-133) in 0.025 M sodium formate buffer (pH 4.0) showed troughs at 208 nm and 222 nm. The trough at 222 nm was shallower for poly(DL-Ala) peptide (96-133)-1 with 50 additional alanine groups than derivatives with fewer additional groups or the non-derivatized peptide (96-133). The trough of 208 nm was similar in amplitude, 0 ~ 13 000, for the unreacted and polyalanylated peptides.

Bioassay results of the polyalanylated derivatives compared to the parent peptide and the international standard are summarized in Table II. The parent pep- tide (96-133) retained significant capacity to increase tibia width of hypophysectomized rats as a function of the administered dose. A tibia width of 296 ~ 12/~m was noted with a total dose per rat of 4.5 mg. The slopes of the dose response curves, i.e. tibia

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TABLE II

BIOLOGICAL RESPONSE OF poly(oL-Ala) PEPTIDE (96-133)

5 rats per group; PI P value of slope compared to international standard; P2 P value of slope compared to peptide (96-133); S.E. standard error; S.D. standard deviation; N.S. not significant.

Fraction Total dose Tibia width Linear Slope PI P~ (per rat per 4 days) (#m ± S.E.) correlation ( ± S.D.) ~g) (mol X 10 -9) coefficient

176 f 9 Solvent International standard

somatotropin 25 1.2 233 ± 11 0.999 44.1 i l . 8 75 3.6 278± 3

225 10.7 330± 11 Peptide (96-133) 500 100 204± 7 0.998 41 .9±2 .6 >0.1

1500 300 255± 14 4500 900 296~z12

poly(DL-Ala) peptide (96-133)-1 500 58 209± 5 0.959 29.3 ±8 .6 <0.05

1500 175 227~ 6 4500 525 277~ 10

poly(DL-Ala) peptide (96-133)-2 500 78 251 ± 16 0.999 18.6±0.35 <0.001

1500 234 274± 6 4500 702 294± 4

poly(DL-Ala) peptide (96-133)-3 500 92 255 ± 14 0.911 16.3 ± 7.3 <0.001

1500 276 260 ± 12 4500 828 293 i 9

poly(DL-Ala) peptide (96-133)-4 500 91 238 ± 7 0.999 25.2 ~ 1.0 <0.001

1500 273 266± 7 4500 819 2 9 6 ± 1 0

N.S.

<0.001

<0.001

<0.001

width vs. log dose, were s imilar for the paren t pep t ide and the in te rna t iona l somato- t r op in s tandard . Thus pept ide (96-133) on a weight basis was app rox ima te ly 4 ~ as active and on a m o l a r basis app rox ima te ly 1 Yoo as active as the in te rna t iona l s tandard .

A l l poly(DL-Ala) pept ide (96-133) derivatives p roduced significant increases in t ib ia width and there was a dose response re la t ionship. Al l the derivatives had significantly shal lower s lopes o f the dose response curves than the bovine somato - t r op in s tandard . W h e n c o m p a r e d to the pa ren t pep t ide (96-133), the slopes of the response curve were diss imil i lar except for poly(DL-Ala) pept ide (96-133)-1. The increase in t ib ia l width for the pa ren t pept ide (96-133) and poly(DL-Ala) pep t ide (96-133)-1 were s imi lar when c o m p a r e d on a mo la r basis. Because o f the diss imilar s lopes o f pa ren t pept ide (96-133) and the remain ing po lya lany la t ed derivatives o f pep t ide (96-133) po tency ra t ios could no t be es t imated.

DISCUSSION

Po lya lany la t ed h u m a n s o m a t o t r o p i n which conta ined an average o f 23 extra alanines per mol o f p ro te in was as active on a weight or mo la r basis as the or iginal

99

+5

I I I I I I

/ / . / ~ / , /

x

-10 /]

-15 - I I I I I I

190 200 210 220 230 240 250 Wavelength (nm)

Fig. 1. Circular dichroism spectra of peptide (96-133) ( . . . . . . ) poly(DL-Aia) peptide (96-133)-1 ( ) and peptides of poly(DL-Ala) peptide (96-133)-(2, 3, 4) ( . . . . -) in 0.025 M sodium formate buffer (pH 4.0).

starting preparation of human somatotropin [19]. Human somatotropin which had introduced 265 additional alanine residues retained approximately 50~ of the growth-promoting activity on a weight basis or full activity on a molar basis [11]. Polyalanylated bovine somatotropin which contained 139 additional alanine groups per mol of bovine somatotropin was completely without growth-promoting ac- tivity [11]. Furthermore when peptide (96-133) was polyanalylated at pH 7.8 for 72 h with an average of 51 additional alanine groups and not further fractionated there was complete loss of growth-promoting activity when assayed at doses approximately 10 9/0 of those employed in the present study [11]. In the present experiments, under the reaction conditions mentioned in Methods and Table I, peptide (96-133) was polyalanylated with a smaller number, 3.0, 6.5, 19.5, and 50.5 additional alanine groups per mol of peptide (96-133). These retained growth-promoting activity with a linear log dose response. On the basis of weight poly(DL-Ala) peptide (96--133)-1 retained about 40 ~ the growth-promoting activity as peptide (96-133); on a molar basis the growth-promoting activity was approximately the same as peptide (96--133) (Table II). In the present experiments, polyalanylation at pH 7.6 for 24 h with sub- sequent fractionation gave a derivative, poly(DL-Ala) peptide (96-133)-1 with 50.5 additional alanines and retention of biological activity. It would appear that the different conditions of polyalanylation without fractionation and perhaps more likely the much smaller doses employed in the earlier study [11] may have resulted in no detectable biological activity as opposed to the retention of biological activity noted in the present study.

The fact that at pH 4.0 the circular dichroism spectra of the series of poly- (DL-Ala) peptide (96-133) derivatives were helical in nature as was that of peptide (96-133) suggests that the presence of additional alanine side-chains with essentially no change in charge compared to peptide (96-133) does not impair the ability of this

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peptide to form a helix at pH 4.0. Without addit ional structural information e.g. circular dichroism at other pH values ionic strengths, tertiary structures as indicated by spectrofluorescence o f tyrosine and the relationship o f such solution properties to structure in vivo, it is difficult to relate these data to biological activity.

In the growth response curve the slopes o f the poly(DL-Ala) peptide (96-133) preparat ions except poly(DL-Ala) peptide (96-133)-1 were not parallel to that of peptide (96-133). The lack o f parallelism may reflect a decrease in affinity between poly(DL-Ala) peptide (96-133) and its receptor. It would also suggest that the smaller size o f peptide (96-133) alone with possibly increased clearance compared to bovine somatot ropin is but a small factor in the decreased biological activity.

Thus modification o f peptide (96-133) by polyalanylat ion under the conditions described herein, decreases the slope o f the dose response curves i.e. tibia width vs. log dose. Where the slopes were comparable e.g. peptide (96-133) and poly(DL-Ala) peptide (96-133)-1 with 50 addit ional alanine groups there would appear to be no change in growth-promot ing activity on a molar basis. On a weight basis the growth response o f the polyalanylated derivative was reduced. It would appear that all or addit ional port ions of the bovine somato t rop in molecule are necessary for full biological activity.

ACKNOWLEDGEMENTS

This study was supported in part by grants CA-08748 of the Nat ional Cancer Institute and BC-147 of the American Cancer Society.

REFERENCES

1 Sonenberg, M., Free, C., Dellacha, J. M., Bonadonna, G., Haymowitz, A. and Nadler, A. C. (1965) Metab. (Clin. Exp.) 14, 1189-1213

2 Nadler, A. C., Sonenberg, M., New, M. I. and Free, C. A. (1967) Metab. (Clin. Exp.) 16,830-845 3 Sonenberg, M., Kikutani, M., Free, C. A., Nadler, A. C. and Dellacha, J. M. (1968) Ann. N.Y.

Acad. Sci. 148, 532-558 4 Yamasaki, N., Kikutani, M. and Sonenberg, M. (1970) Biochemistry 9, 1107-1114 5 Santom6, J. A., Dellacha, J. M., Paladini, A. C., Pefia, C., Bicsoglio, M. J., Daurat, S. T., Poskeno,

E. and Wolfenstein, C. E. M. (1973) Eur. J. Biochem. 37, 164-170 6 Wallis, M. (1973) FEBS Lett. 35, 11-14 7 Grail, L. and Li, C. H. (1974) Biochem. Biophys. Res. Commun. 56, 168-176 8 Yamasaki, N., Kangawa, K., Kobayashi, S., Kikutani, M. and Sonenberg, M. (1972) J. Biol.

Chem. 247, 3874-3880 9 Yamasaki, N., Simanaka, J. and Sonenberg, M. (1975) J. Biol. Chem. 250, 2510-2514

10 Swislocki, N. I., Sonenberg, M. and Yamasaki, N. (1970) Endocrinology 87, 900-904 11 Sonenberg, M., Yamasaki, N., Kikutani, M., Swislocki, N. I., Levine, L. and New, M. I. (1972)

Second International Symposium on Growth Hormone, Milan, Italy, Excerpta Medica Inter- national Congress Series 244, 75-91

12 Graf, L. and Li, C. H. (1974) Biochemistry 13, 5408-5415 13 Graf, L., Barat, E., Borvendeg, J., Hermann, I. and Patthy, A. (1976) Eur. J. Biochem. 64, 333-340 14 Blake, J. and Li, C. H. (1973) Int. J. Peptide Protein Res. 5, 123-125 15 Renkin, E. M. and Gilmore, J. P. (1973) in Handbook of Physiology, Section 8, p. 185. American

Physiological Society, Washington, D.C. 16 Reference deleted 17 Sela, M. (1966) Adv. Immunology 5, 29-129 I8 Bauminger, S., Schechter, I. and Sela, M. (1967) lmmunochemistry 4, 169-181

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19 Canfield, R. E. (1968) Ann. N.Y. Acad. Sci. 148, 293-307 20 Ornstein, L. (1964) Ann. N.Y. Acad. Sci. 121,321-349 21 Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404-427 22 Reisfeld, R. A., Lewis, U. J. and Williams, D. E. (1962) Nature 196, 281-283 23 Spackman, D. H., Stein, W. H. and Moore, S. (1958) Anal. Chem. 30, 1190-1206 24 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193,265-

275 25 Marx, W., Simpson, M. E. and Evans, H. M. (1942) Endocrinology 30, 1-10 26 Geschwind, I. I. and Li, C. H. (1955) in Hypophyseal Growth Hormone, Nature and Actions

(Smith, R. W., Gaebler, O. H. and Long, C. N. H., eds.), pp. 25-58 McGraw-Hill, New York