BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1312-1317
Vol. 180, No. 3, 1991 November 14, 1991
I N T E R A C T I O N S OF A N T I S E N S E PEPTIDES W I T H O V I N E P R O L A C T I N Anil Bajpai, Kyle P. Hooper and Kurt E. Ebner Department of Biochemistry and Molecular Biology The University of Kansas Medical Center 39th and Rainbow Blvd. Kansas City, KS 66103 Received September 26, 1991
Two anfisense peptides were synthesized to a sense peptide corresponding to amino acid residues 23-35 of ovine prolactin. Both of the antisense peptides formed a saturable complex with the sense peptide and ovine prolactin. The sense peptide inhibited the interaction of ovine prolactin with the antisense peptides. Both of the antisense peptides ha re a common core sequence VMNV which can bind to ovine prolactin. The lactogenic hormones, rat prolactin and human growth hormone, compete with the binding of ovine prolactin to an antisense peptide whereas a nonlactogen, ovine growth hormone, does not compete indicating a degree of specificity in the interaction. ~ 1991 Academic Press, Inc.
Prolactin (PRL) is an anterior pituitary hormone which has diverse biological functions ranging from osmoregulation in fish to lactation in humans. Binding to a receptor is prerequiste for biological function (1). Based upon comparisons of the biological properties and chemical structures of a variety of prolactins, Kohmoto, e t al. (2) have suggested that Asp 20, His 27, Ser 62 and Thr 65 may be important for binding to receptor. Chemical modification of histidines 27 and 30 in ovine PRL (oPRL) completely abolished binding to receptor showing that these residues are critical for binding (3). Blalock and colleagues (4,5) have proposed a molecular recognition theory which suggests that appropriate peptides derived from complementary DNA sequence could assume conformations that allow for selective interactions between sense and antisense peptides. In particular, a reverse hydropathic relationship between complementary codons of complementary RNA strands was demonstrated and formed the basis of the interactions (6). The antisense peptide can be used in either the 5'-3' or 3'-5' direction since the central base of the codon principally specifies the hydropathy of the amino acid. Using this strategy, over a dozen antisense peptides, representing diverse systems, have been synthesized and shown to interact with their sense peptide (5,7-9 for references). The observation that the N-terminal region of oPRL, containing His 27 and 30, was a region essential for binding to receptor prompted the synthesis of antisense peptides to this region and examination of their interaction with the sense peptide and oPRL.
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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M A T E R I A L S AND M E T H O D S M a t e r i a l s - All general chemicals were obtained from Sigma Chemical Co. All t e r t butyloxycarbonyl amino acids for peptide synthesis were obtained from BioSearch. All hormones were obtained from the National Hormone and Pituitary Program, NIDDK. Synthesis of Antisense Peotides- Using the cDNA sequence of oPRL (10), two antisense peptides were synthesized corresponding to residues 23-35 of oPRL as described by Blalock (11). The antisense peptide, FRGEVMNVVGHHD, read from the complementary RNA in the 5'-3' direction, is, by convention (11), an anticomplementary peptide and is named oPRLAc23-35. Reading the complementary RNA in the 3'-5' direction gives the antisense peptide QYHRVMNVLERSV, which is a complementary peptide and is named oPRL¢23-35. These two peptides and the sense peptide, oPRL23_35 (VMVSHYIHNLSSE), were synthesized on a BioSearch SAM TWO solid phase peptide synthesizer as C-terminal amides using standard t e r t butyloxycarbonyl methodology as described by Merrifield (12). The peptide, VMNV, was synthesized manually as a C-terminal carboxylic acid using Merrifield (12) methodology as described by Stewart and Young (13). Peptides were purified by I-IPLC on a Vydac C-18 column and compositions confm'ned on an Applied Biosystems amino acid analyzer. Antisera Production- Antisera was produced to oPRLAc23_35and to oPRL23_35. Peptides, one mg each, were conjugated to keyhole limpet hemocyanin (KLH) by the method of Goldsmith (14). The resulting KLH conjugates were emulsified with an equal volume of Freund's complete adjuvant. For the antisera to oPRLAc23-35, 3.0 ml of emulsified oPRLAc23_35-KLH were injected in multiple intradermal sites in the upper back region of two male New Zealand white rabbits (3-4 kg each). Two weeks later,, each rabbit received a booster immunization with material prepared identically except that half as much was injected in Freund's incomplete adjuvant. Two weeks later rabbits were bled for immune serum. Thereafter, animals recieved a booster every fourth week and bled two weeks after the booster. For antisera to oPRL23_35,3.0 ml of emulsified oPRL23.35KLH was injected in multiple subdermal sites in the inguinal area of two male New Zealand white rabbits (3-4 kg each). A booster injection of half the volume (1.5 ml) of oPRL23_35-KLH emulsified in incomplete adjuvant was injected into multiple intradermal sites along the back of the rabbit two weeks after initial injection. Two weeks later, the rabbit was bled for immune serum. Thereafter, animals recieved a booster every second week and bled before each booster. ELISA Solid-Phase Binding Assay- All assays were performed with Dynatech Immulon II microtiter plates. Total and nonspecific binding were determined in triplicate whereas nonimmune binding was done in duplicate. Specified quantities of peptide were immobilized to each well at 37°C for 2 h in 100 ~tl of 50 mM sodium carbonate, pH 9.1. To determine nonspecific adsorption of peptide or antisera, 100 ~tl of a 2% porcine gelatin solution were added to wells with no peptide. The gelatin solution was prepared in 50 mM sodium carbonate, pH 9.1, and was used initially at a temperature of approximately 45°C. Following incubation, the wells were washed three times with TBST (20 mM Tris-HCl,pH 7.5, 137 mM NaC1, 3 mM KC1, 0.05% Tween-20) followed by the addition of 200 ~tl of the gelatin solution for 2 h at 37 °C. Following incubation, the wells were washed as before. Secondary peptide or protein was then allowed to react with the bound peptide for 2 h at ambient room temperature in 100 gl of 10 mM Tris-HC1, pH 7.5. The wells were washed as before. Total and nonspecific binding was assessed with 100 Ixl of immune serum. For anti-oPRL(7) and anti-oPRLAc~3_35, a dilution of 1:10000 was used and for antioPRL23-35, a 1:1000 dilution was used. Nonimmune binding was assayed with the same dilution and volume of preimmune sera. The plates were incubated overnight at 4"C and, then, washed six times with TBST. Next, 100 ~l of a 1:1000 dilution of goat anti-rabbit IgG alkaline phosphataseconjugate were added per well. All antibody dilutions were made in TBST. After 1 h at 37°C, the plate was washed six times with TBST followed by the addition of 100 ~tl of a 1 mg/ml solution of Sigma 104 alkaline phosphatase substrate per well in 100 mM glycine, pH 10.4, 2 mM MgC12, 2 mM ZnCI/. Following 30 min of color reaction at 37°C, the absorbance at 405 nm was read on a Dynatech MR-580 Microelisa Autoreader. Where indicated, the error bars are the standard deviation of the mean. RESULTS The two antisense peptides to oPRL23_35 were tested for their ability to interact with the sense peptide, oPRL23_35 (Fig. 1). The antisense peptide oPRLA¢23-35 demonstrated saturation binding to plate-bound oPRL23_35 and, conversly, the sense peptide oPRL23_35 exhibited saturation binding to plate-bound oPRLc23.35. Other experiments showed that the sense peptide
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0.6 0.5 0.4 o= ,=~ 0.3 0.20.1. 0,0!
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Figol, Interaction of antisense peptides to oPRL with the sense peptide, oPRL23.35. Five txg of either oPRL23.35 or oPRLc23-35 are immobilized to microtiter plates and reacted with varying concentrations of oPRLAc23_35(closedcircles) or oPRLc23_35 (closedsquares), respectively. The binding of oPRLAc23.35 to oPRL23_35 is recognized with anti-oPRLAc23_35 and binding of oPRL23.35 to oPRLc23.35 is recognized with anti-oPRL23.35. Nonimmune binding is represented by the open circles.
also bound to oPRLAc23_35. Maximum binding at room temperature occured by 2 h and between pH 5 to 8. Of the proteins tested (BSA, ovalbumin, powdered skim milk, lysozyme) 2% gelatin was most effective in reducing nonspecific binding, which was determined for each data point, and was generally less than 20% of the total binding. The binding of oPRLAc23-35 to two unrelated peptides (IEIEEENKRLL and QSPPEIHKCR) was linear and nonsaturating and was not significantly different from nonspecific absorption to gelatin. Since a high titer antisera to oPRL was available (10), the binding to oPRL to the antisense peptides and the tetrapeptide VMNV, common to both antisense peptides, was determined (Fig. 2). Maximum binding occured by 2 h at room temperature, oPRL demonstrated saturation binding to plate-bound oPRLAc23-35 (Fig. 2, top), oPRLc23-35 (Fig. 2, middle) and VMNV (Fig. 2,
bottom). The data in Fig. 3 shows that 20 pg of the sense peptide oPRL23_35 inhibited the binding of 5 pg oPRL to 1 ~tg of plate bound antisense peptide oPRLAc23-35 by 50% in a linear, dosedependent manner. It was also possible to test if other lactogenic or nonlactogenic hormones could compete with the binding of oPRL to plate bound oPRLAc23-35. The data in Fig. 4 shows that the lactogenic hormones rat PRL (rPRL) and human growth hormone (hGH) could compete with oPRL binding but ovine GH (oGH), a structurally related nonlactogen, did not compete. DISCUSSION One of the predictions of the molecular recognition theory is that antisense peptides derived from the corresponding complementary mRNA will bind to the sense peptide or the protein containing the sense peptide. Such interactions have been described in a number of diverse protein-protein interactions (5,7-9) though some investigators have not been successful (15). Clearly, it is important to choose a region of a protein which has the potential for such an interaction to occur. The observation that chemical modification of His 27 and His 30 of oPRL (3) 1314
Vol. 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1,2 •
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Fig. 2. Binding of oPRL to immobilized antisense peptides. Varying concentrations of oPRL are reacted with 50 ng of oPRLAc23_35 (top), 50 ng of oPRLc23_35 (middle) or 100 ng of VMNV (bottom). The binding of oPRL to the antisense peptides is recognized by the anti-oPRL sera. Specific binding is represented by the closed circles and nonimmune binding is represented by the open circles.
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Fig. 3. Inhibition of oPRL binding to the antisense peptide (oPRLAc23_35) by the sense peptide (oPRL23_35). One rtg o f oPRLAc2).35 were immobilized per well and reacted with 5 txg of oPRL
in the presence of increasing amounts of oPRL23-35. The oPRL bound was recognized by antioPRL serum preabsorbed to oPRL23_35. The closed circles represent specific binding and the open circles represent nonimmune binding. Fig. 4. Competition of rPRL, hGH and oGH for oPRL binding to oPRLAc23-35. Two I~g of oPRLAc23_35 were immobilized per well and reacted with 5 pg of oPRL in the presence of increasing amounts or rPRL (closed circles), hGH (open circles) or oGH (closed squares). Binding of oPRL in recognized by anti-oPRL serum. Nonimmune binding has been subtracted for each point and the data represents specific binding. 1315
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resulted in complete loss of the ability to bind to receptor and, thus, this region had a high probability of being important in the binding interaction. Hence, the sense peptide oPRL23_35 was chosen to span the region containing His 27 and His 30. The two antisense peptides to this region (oPRL23_35) were determined from the complementary mRNA strand in both the 5'-3' (designated as oPRLA¢23-35) and 3'-5' (designated as oPRLc23_35) direction. Antibodies to the peptides oPRLAc23-35 and oPRL23_35 were generated for assay purposes. As shown in Fig. 1, both of the antisense peptides interacted with the sense peptide oPRL23-35. The studies were further extended to examine the binding of the antisense peptides to native oPRL and, again, both antisense peptide bound to native oPRL presumably in the region containing amino acids 23-35. The sequence oPRL27_30 is H Y I H and the corresponding sequence in both the complementary and anticomplementary antisense peptides was VMNV. Examination of the hydropathy profiles of the sense and antisense peptides showed that the region 27-30 gave the maximum difference in hydropathy suggestive of an important interaction site (11). Indeed, the antibody generated to
oPRLAc23_35 clearly recognizes the peptide VMNV and this peptide also bound to oPRL (Fig. 2) suggesting the importance of this region in the binding interactions. As predicted, the sense peptide (oPRL23-35) competed with oPRL for binding to the antisense peptide oPRLAc23-35 (Fig. 3). All prolactins contain His at positions 27 and 30 and hGH, a lactogen, has His at 18 and 21 which is analogous to His 27 and 30 of the prolactins.
All other growth hormones are not
lactogenic and contain a Gin at position 18. Perhaps oPRLA¢23-35 could differentiate between lactogens and nonlactogens based upon this structural difference. The results presented in Fig. 4 show a degree of specificity in that the lactogenic hormone, rPRL and hGH, did compete for the binding of oPRL to oPRLAc23-35 whereas oGH did not compete under these conditions. The antisera to oPRL recognizes oGH almost as well as oPRL. Thus, if oGH had bound to the immobilized antisense peptide, oPRLAc23-35, there would be an increase in absorbance and this was not observed indicating that oGH did not bind to the antisense peptide. The antisera to oGH had virtually no cross-reactivity with either rPRL or hGH. The two antisense peptides did interact with the corresponding sense peptide and oPRL and this interaction had some degree of specificity. These studies are consistent with the predictions of the molecular recognition theory and provide the basis for a detailed study on the nature of these interactions at both the peptide and protein level.
ACKNOWLEDGMENTS Supported in part by NIH grant HD 18584. The authors wish to thank Mr. Richard Grabbe for synthesizing and purifying the peptides.
REFERENCES 1. 2. 3. 4.
Cooke, N.E. (1989) In Endocrinology (L.J. DeGroot, Ed.) Vol. I, pp. 384-407. WB Saunders, Philadelphia, PA. Kohmoto, K., Tsummasaur, S., and Sakiya, F. (1984) Eur. J. Biochem. 138, 227-239. Anderson, T.T. and Ebner, K.E. (1979) J. Biol. Chem. 254, 10995-10999. Blalock, J.E. (1990) Trends Biotechnol. 8, 140-144. 1316
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5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Clark, B.L. and Blalock, J.E. (1991) In Antisense Nucleic Acids and Proteins: Fundamentals and Applications (J.N.M. Mol and A.R. van der Krol, Eds.) pp. 169-185. Marcel Dekker, New York. Markus, G., Tritsch, G.L. and Parthasarathy, R. (1989) Arch. Biochem. Biophys. 272, 433-439. Lu, F.X., Aiyar, N. and Chaiken, I. (1991) Proc. Natl Acad. Sci. USA 88, 3642-3646. Ghiso, J., Saball, E., Leoni, J., Rostagno, A. and Frangione, B. (1990) Proc. Natl Acad Sci. USA 87, 1288-1291. Slootstra, J.W. and Roubos, E.W. (1991) In Antisense Nucleic Acids and Proteins: Fundamentals and Applications (J.N.M. Mol and A.R. van der Krol, Eds.) pp. 205-228. Marcel Dekker, New York. Varrna, S., Kwok, S. and Ebner, K.E. (1989) Gene 77, 349-359. Bost, K.L. and Blalock, J.E. (1989) Methods Enzymol. 168, 16-28. Merrifield, R.B. (1963) J. Amer. Chem. Soc. 85, 2149-2154. Stewart, J.M. and Young, J.D. (1984) Solid Phase Peptide Synthesis, pp. 71-95. Pierce Chemical Co., Rockford, IL. Goldsmith, P., Glerschik, P., Milligan, G., Unson, C.G., Vinitsky, R., Malech, H.L. and Spiegel, A.M. (1987) J. Biol. Chem. 262, 14683-14688. Eberle, A.N. and Huber, M. (1991) In Antisense Nucleic Acids and Proteins: Fundamentals and Applications (J.N.M. Mol and A.R. van der Krol, Eds.) pp. 187-203. Marcel Dekker, New York.
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Vol. 180, No. 3, 1991 November 14, 1991
I N T E R A C T I O N S OF A N T I S E N S E PEPTIDES W I T H O V I N E P R O L A C T I N Anil Bajpai, Kyle P. Hooper and Kurt E. Ebner Department of Biochemistry and Molecular Biology The University of Kansas Medical Center 39th and Rainbow Blvd. Kansas City, KS 66103 Received September 26, 1991
Two anfisense peptides were synthesized to a sense peptide corresponding to amino acid residues 23-35 of ovine prolactin. Both of the antisense peptides formed a saturable complex with the sense peptide and ovine prolactin. The sense peptide inhibited the interaction of ovine prolactin with the antisense peptides. Both of the antisense peptides ha re a common core sequence VMNV which can bind to ovine prolactin. The lactogenic hormones, rat prolactin and human growth hormone, compete with the binding of ovine prolactin to an antisense peptide whereas a nonlactogen, ovine growth hormone, does not compete indicating a degree of specificity in the interaction. ~ 1991 Academic Press, Inc.
Prolactin (PRL) is an anterior pituitary hormone which has diverse biological functions ranging from osmoregulation in fish to lactation in humans. Binding to a receptor is prerequiste for biological function (1). Based upon comparisons of the biological properties and chemical structures of a variety of prolactins, Kohmoto, e t al. (2) have suggested that Asp 20, His 27, Ser 62 and Thr 65 may be important for binding to receptor. Chemical modification of histidines 27 and 30 in ovine PRL (oPRL) completely abolished binding to receptor showing that these residues are critical for binding (3). Blalock and colleagues (4,5) have proposed a molecular recognition theory which suggests that appropriate peptides derived from complementary DNA sequence could assume conformations that allow for selective interactions between sense and antisense peptides. In particular, a reverse hydropathic relationship between complementary codons of complementary RNA strands was demonstrated and formed the basis of the interactions (6). The antisense peptide can be used in either the 5'-3' or 3'-5' direction since the central base of the codon principally specifies the hydropathy of the amino acid. Using this strategy, over a dozen antisense peptides, representing diverse systems, have been synthesized and shown to interact with their sense peptide (5,7-9 for references). The observation that the N-terminal region of oPRL, containing His 27 and 30, was a region essential for binding to receptor prompted the synthesis of antisense peptides to this region and examination of their interaction with the sense peptide and oPRL.
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
M A T E R I A L S AND M E T H O D S M a t e r i a l s - All general chemicals were obtained from Sigma Chemical Co. All t e r t butyloxycarbonyl amino acids for peptide synthesis were obtained from BioSearch. All hormones were obtained from the National Hormone and Pituitary Program, NIDDK. Synthesis of Antisense Peotides- Using the cDNA sequence of oPRL (10), two antisense peptides were synthesized corresponding to residues 23-35 of oPRL as described by Blalock (11). The antisense peptide, FRGEVMNVVGHHD, read from the complementary RNA in the 5'-3' direction, is, by convention (11), an anticomplementary peptide and is named oPRLAc23-35. Reading the complementary RNA in the 3'-5' direction gives the antisense peptide QYHRVMNVLERSV, which is a complementary peptide and is named oPRL¢23-35. These two peptides and the sense peptide, oPRL23_35 (VMVSHYIHNLSSE), were synthesized on a BioSearch SAM TWO solid phase peptide synthesizer as C-terminal amides using standard t e r t butyloxycarbonyl methodology as described by Merrifield (12). The peptide, VMNV, was synthesized manually as a C-terminal carboxylic acid using Merrifield (12) methodology as described by Stewart and Young (13). Peptides were purified by I-IPLC on a Vydac C-18 column and compositions confm'ned on an Applied Biosystems amino acid analyzer. Antisera Production- Antisera was produced to oPRLAc23_35and to oPRL23_35. Peptides, one mg each, were conjugated to keyhole limpet hemocyanin (KLH) by the method of Goldsmith (14). The resulting KLH conjugates were emulsified with an equal volume of Freund's complete adjuvant. For the antisera to oPRLAc23-35, 3.0 ml of emulsified oPRLAc23_35-KLH were injected in multiple intradermal sites in the upper back region of two male New Zealand white rabbits (3-4 kg each). Two weeks later,, each rabbit received a booster immunization with material prepared identically except that half as much was injected in Freund's incomplete adjuvant. Two weeks later rabbits were bled for immune serum. Thereafter, animals recieved a booster every fourth week and bled two weeks after the booster. For antisera to oPRL23_35,3.0 ml of emulsified oPRL23.35KLH was injected in multiple subdermal sites in the inguinal area of two male New Zealand white rabbits (3-4 kg each). A booster injection of half the volume (1.5 ml) of oPRL23_35-KLH emulsified in incomplete adjuvant was injected into multiple intradermal sites along the back of the rabbit two weeks after initial injection. Two weeks later, the rabbit was bled for immune serum. Thereafter, animals recieved a booster every second week and bled before each booster. ELISA Solid-Phase Binding Assay- All assays were performed with Dynatech Immulon II microtiter plates. Total and nonspecific binding were determined in triplicate whereas nonimmune binding was done in duplicate. Specified quantities of peptide were immobilized to each well at 37°C for 2 h in 100 ~tl of 50 mM sodium carbonate, pH 9.1. To determine nonspecific adsorption of peptide or antisera, 100 ~tl of a 2% porcine gelatin solution were added to wells with no peptide. The gelatin solution was prepared in 50 mM sodium carbonate, pH 9.1, and was used initially at a temperature of approximately 45°C. Following incubation, the wells were washed three times with TBST (20 mM Tris-HCl,pH 7.5, 137 mM NaC1, 3 mM KC1, 0.05% Tween-20) followed by the addition of 200 ~tl of the gelatin solution for 2 h at 37 °C. Following incubation, the wells were washed as before. Secondary peptide or protein was then allowed to react with the bound peptide for 2 h at ambient room temperature in 100 gl of 10 mM Tris-HC1, pH 7.5. The wells were washed as before. Total and nonspecific binding was assessed with 100 Ixl of immune serum. For anti-oPRL(7) and anti-oPRLAc~3_35, a dilution of 1:10000 was used and for antioPRL23-35, a 1:1000 dilution was used. Nonimmune binding was assayed with the same dilution and volume of preimmune sera. The plates were incubated overnight at 4"C and, then, washed six times with TBST. Next, 100 ~l of a 1:1000 dilution of goat anti-rabbit IgG alkaline phosphataseconjugate were added per well. All antibody dilutions were made in TBST. After 1 h at 37°C, the plate was washed six times with TBST followed by the addition of 100 ~tl of a 1 mg/ml solution of Sigma 104 alkaline phosphatase substrate per well in 100 mM glycine, pH 10.4, 2 mM MgC12, 2 mM ZnCI/. Following 30 min of color reaction at 37°C, the absorbance at 405 nm was read on a Dynatech MR-580 Microelisa Autoreader. Where indicated, the error bars are the standard deviation of the mean. RESULTS The two antisense peptides to oPRL23_35 were tested for their ability to interact with the sense peptide, oPRL23_35 (Fig. 1). The antisense peptide oPRLA¢23-35 demonstrated saturation binding to plate-bound oPRL23_35 and, conversly, the sense peptide oPRL23_35 exhibited saturation binding to plate-bound oPRLc23.35. Other experiments showed that the sense peptide
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0.6 0.5 0.4 o= ,=~ 0.3 0.20.1. 0,0!
0
10
20
30 ~Lg Peptide
40
0
50
60
Figol, Interaction of antisense peptides to oPRL with the sense peptide, oPRL23.35. Five txg of either oPRL23.35 or oPRLc23-35 are immobilized to microtiter plates and reacted with varying concentrations of oPRLAc23_35(closedcircles) or oPRLc23_35 (closedsquares), respectively. The binding of oPRLAc23.35 to oPRL23_35 is recognized with anti-oPRLAc23_35 and binding of oPRL23.35 to oPRLc23.35 is recognized with anti-oPRL23.35. Nonimmune binding is represented by the open circles.
also bound to oPRLAc23_35. Maximum binding at room temperature occured by 2 h and between pH 5 to 8. Of the proteins tested (BSA, ovalbumin, powdered skim milk, lysozyme) 2% gelatin was most effective in reducing nonspecific binding, which was determined for each data point, and was generally less than 20% of the total binding. The binding of oPRLAc23-35 to two unrelated peptides (IEIEEENKRLL and QSPPEIHKCR) was linear and nonsaturating and was not significantly different from nonspecific absorption to gelatin. Since a high titer antisera to oPRL was available (10), the binding to oPRL to the antisense peptides and the tetrapeptide VMNV, common to both antisense peptides, was determined (Fig. 2). Maximum binding occured by 2 h at room temperature, oPRL demonstrated saturation binding to plate-bound oPRLAc23-35 (Fig. 2, top), oPRLc23-35 (Fig. 2, middle) and VMNV (Fig. 2,
bottom). The data in Fig. 3 shows that 20 pg of the sense peptide oPRL23_35 inhibited the binding of 5 pg oPRL to 1 ~tg of plate bound antisense peptide oPRLAc23-35 by 50% in a linear, dosedependent manner. It was also possible to test if other lactogenic or nonlactogenic hormones could compete with the binding of oPRL to plate bound oPRLAc23-35. The data in Fig. 4 shows that the lactogenic hormones rat PRL (rPRL) and human growth hormone (hGH) could compete with oPRL binding but ovine GH (oGH), a structurally related nonlactogen, did not compete. DISCUSSION One of the predictions of the molecular recognition theory is that antisense peptides derived from the corresponding complementary mRNA will bind to the sense peptide or the protein containing the sense peptide. Such interactions have been described in a number of diverse protein-protein interactions (5,7-9) though some investigators have not been successful (15). Clearly, it is important to choose a region of a protein which has the potential for such an interaction to occur. The observation that chemical modification of His 27 and His 30 of oPRL (3) 1314
Vol. 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1,2 •
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Fig. 2. Binding of oPRL to immobilized antisense peptides. Varying concentrations of oPRL are reacted with 50 ng of oPRLAc23_35 (top), 50 ng of oPRLc23_35 (middle) or 100 ng of VMNV (bottom). The binding of oPRL to the antisense peptides is recognized by the anti-oPRL sera. Specific binding is represented by the closed circles and nonimmune binding is represented by the open circles.
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~tg Hormone
Fig. 3. Inhibition of oPRL binding to the antisense peptide (oPRLAc23_35) by the sense peptide (oPRL23_35). One rtg o f oPRLAc2).35 were immobilized per well and reacted with 5 txg of oPRL
in the presence of increasing amounts of oPRL23-35. The oPRL bound was recognized by antioPRL serum preabsorbed to oPRL23_35. The closed circles represent specific binding and the open circles represent nonimmune binding. Fig. 4. Competition of rPRL, hGH and oGH for oPRL binding to oPRLAc23-35. Two I~g of oPRLAc23_35 were immobilized per well and reacted with 5 pg of oPRL in the presence of increasing amounts or rPRL (closed circles), hGH (open circles) or oGH (closed squares). Binding of oPRL in recognized by anti-oPRL serum. Nonimmune binding has been subtracted for each point and the data represents specific binding. 1315
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resulted in complete loss of the ability to bind to receptor and, thus, this region had a high probability of being important in the binding interaction. Hence, the sense peptide oPRL23_35 was chosen to span the region containing His 27 and His 30. The two antisense peptides to this region (oPRL23_35) were determined from the complementary mRNA strand in both the 5'-3' (designated as oPRLA¢23-35) and 3'-5' (designated as oPRLc23_35) direction. Antibodies to the peptides oPRLAc23-35 and oPRL23_35 were generated for assay purposes. As shown in Fig. 1, both of the antisense peptides interacted with the sense peptide oPRL23-35. The studies were further extended to examine the binding of the antisense peptides to native oPRL and, again, both antisense peptide bound to native oPRL presumably in the region containing amino acids 23-35. The sequence oPRL27_30 is H Y I H and the corresponding sequence in both the complementary and anticomplementary antisense peptides was VMNV. Examination of the hydropathy profiles of the sense and antisense peptides showed that the region 27-30 gave the maximum difference in hydropathy suggestive of an important interaction site (11). Indeed, the antibody generated to
oPRLAc23_35 clearly recognizes the peptide VMNV and this peptide also bound to oPRL (Fig. 2) suggesting the importance of this region in the binding interactions. As predicted, the sense peptide (oPRL23-35) competed with oPRL for binding to the antisense peptide oPRLAc23-35 (Fig. 3). All prolactins contain His at positions 27 and 30 and hGH, a lactogen, has His at 18 and 21 which is analogous to His 27 and 30 of the prolactins.
All other growth hormones are not
lactogenic and contain a Gin at position 18. Perhaps oPRLA¢23-35 could differentiate between lactogens and nonlactogens based upon this structural difference. The results presented in Fig. 4 show a degree of specificity in that the lactogenic hormone, rPRL and hGH, did compete for the binding of oPRL to oPRLAc23-35 whereas oGH did not compete under these conditions. The antisera to oPRL recognizes oGH almost as well as oPRL. Thus, if oGH had bound to the immobilized antisense peptide, oPRLAc23-35, there would be an increase in absorbance and this was not observed indicating that oGH did not bind to the antisense peptide. The antisera to oGH had virtually no cross-reactivity with either rPRL or hGH. The two antisense peptides did interact with the corresponding sense peptide and oPRL and this interaction had some degree of specificity. These studies are consistent with the predictions of the molecular recognition theory and provide the basis for a detailed study on the nature of these interactions at both the peptide and protein level.
ACKNOWLEDGMENTS Supported in part by NIH grant HD 18584. The authors wish to thank Mr. Richard Grabbe for synthesizing and purifying the peptides.
REFERENCES 1. 2. 3. 4.
Cooke, N.E. (1989) In Endocrinology (L.J. DeGroot, Ed.) Vol. I, pp. 384-407. WB Saunders, Philadelphia, PA. Kohmoto, K., Tsummasaur, S., and Sakiya, F. (1984) Eur. J. Biochem. 138, 227-239. Anderson, T.T. and Ebner, K.E. (1979) J. Biol. Chem. 254, 10995-10999. Blalock, J.E. (1990) Trends Biotechnol. 8, 140-144. 1316
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5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
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Clark, B.L. and Blalock, J.E. (1991) In Antisense Nucleic Acids and Proteins: Fundamentals and Applications (J.N.M. Mol and A.R. van der Krol, Eds.) pp. 169-185. Marcel Dekker, New York. Markus, G., Tritsch, G.L. and Parthasarathy, R. (1989) Arch. Biochem. Biophys. 272, 433-439. Lu, F.X., Aiyar, N. and Chaiken, I. (1991) Proc. Natl Acad. Sci. USA 88, 3642-3646. Ghiso, J., Saball, E., Leoni, J., Rostagno, A. and Frangione, B. (1990) Proc. Natl Acad Sci. USA 87, 1288-1291. Slootstra, J.W. and Roubos, E.W. (1991) In Antisense Nucleic Acids and Proteins: Fundamentals and Applications (J.N.M. Mol and A.R. van der Krol, Eds.) pp. 205-228. Marcel Dekker, New York. Varrna, S., Kwok, S. and Ebner, K.E. (1989) Gene 77, 349-359. Bost, K.L. and Blalock, J.E. (1989) Methods Enzymol. 168, 16-28. Merrifield, R.B. (1963) J. Amer. Chem. Soc. 85, 2149-2154. Stewart, J.M. and Young, J.D. (1984) Solid Phase Peptide Synthesis, pp. 71-95. Pierce Chemical Co., Rockford, IL. Goldsmith, P., Glerschik, P., Milligan, G., Unson, C.G., Vinitsky, R., Malech, H.L. and Spiegel, A.M. (1987) J. Biol. Chem. 262, 14683-14688. Eberle, A.N. and Huber, M. (1991) In Antisense Nucleic Acids and Proteins: Fundamentals and Applications (J.N.M. Mol and A.R. van der Krol, Eds.) pp. 187-203. Marcel Dekker, New York.
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