Biochimica et Biophysica Acta, 1122 (1992) 107-112 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
107
BBAPRO 34~4
Characterisation of a tryptic peptide from human placental ribonuclease inhibitor which inhibits ribonuclease activity Isabelle Crevel-Thieffry a,1, Sue Cotterill b and E d m o n d Schuller a "Laboratoire de neuro-immunologie, H~pital de la Salp~td~re, INSERM 134, Paris (France) and h Department of BiochemiStry, Imperial College of Science, Technolog)" and Medicine, London (UK) (Received 19 February 1992)
Key words: Protein characterisation; Tryptic peptide; Ribonuclease inhibitor; HPLC
Affinity-purified human placental ribonuclease inhibitor (PRI) was digested by trypsin. Subsequent fractionation of the hydrolysate by HPLC yielded 44 fractions~ 3 of which retained the ability to inhibit ribonuclease. One of these, the most active, was a 15 amino acid peptide which had an amino acid composition corresponding to a tryptic fragment of PRI. This peptide was synthesised, and preliminary experiments were carried out on its interactions with ribonuclease. These experiments suggested that the behaviour of the peptide in terms of effect of pH, and effect of salt concentration were similar to the protein from which it was derived. These studies together with the strategic positioning of the peptide in the sequence of the ribonuclease inhibitor, suggest that this segment of PRI has an important role in the inhibitory activity of the intact protein.
Introduction
Ribonuclease inhihitor is a 50 kDa protein which inhibits ribonuclease by binding very tightly to it. In vivo as much as 95% of the ribonuclease can be complexed with inhibitor, however the inhihitor: ribonuclease ratio is clearly related to metabolism; elevated in ploliferating tissue [1,2], and diminished during catabolic metabolism [3]. This ratio has also been reported to be modified during Alzheimer's disease [4.5]. More recently ribonuclease inhibitor has also been shown to be involved in the regulation of another molecule: angiogenin, a blood vessel-inducing protein [6]. Due to the importance of the processes in which the inhibitor has been implicated a great deal of effort has been put into understanding the mechanism of the molecule. Ribonuclease inhibitor has been isolated from a number of systems, however, one of the richest sources of the protein is human placenta. In this tissue the protein (PRI), is mostly free from ribonuclease and therefore the yields of the protein obtained are large enough to allow comprehensive analysis of the molecule. Detailed studies have been performed on the interaction between purified inhibitor and pure ribonuclease, and the regions of ribonuclease involved
i Present address: Chester Beatty Laboratories, Fulham Road, London SW3, UK. Correspondence: !. CreveI-Thieffry, Chester Beatty Laboratories, Fulham Road, London SW3, UK.
in the interaction have been identified by analysis of the interactions of pure inhibitor with modified ribonuclease [7-9]. The regions of the inhibitor involved in the interaction are less well understood. However, recently a start has been made in this area; the gene for PRI and also that for the pig ribonuclease inhibitor (which is very closely related) have been cloned [10-12]. Analysis of deletion mutants of both of these proteins has identified regions which seem to be important for both the interaction of the inhibitor with the ribonuclease and also for inhibition activity [13-15]. In an effort to further analyse those regions important for ribonuclease inhibitor activity we were interested in identifying small regions of the inhibitor (-15-20 amino acids)which possessed inhibitory action in isolation. In this study we present the characterisation of one such molecule, a small peptide of only 15 amino acids made by tryptic digestion of the affinityp-~rified inhibitor. We show that this peptide alone possesses features similar to the intact protein, and discuss the relevance of this finding in relation to the data already available from other studies. Materials and Methods
Materials. Bovine pancreatic ribonuclease A and cytidyl-3',5'-guanosine (CpG) were from Sigma. Poly C was from Pharmacia. Trypsin, affinity-purified for protein sequencing was from Merck. All other chemicals were of the highest grade commercially available.
108
Tryptic digest of PRI. PRI was affinity-purified from human placentas according to Ref. 16. 16 nmol of the freeze dried protein was dissolved in 50 mM ammonium bicarbonate (pH 8.5) (freshly prepared). Trypsin was added in a ratio of 3.3% (w/w) to PRI (---0.75 mg/ml) and the reaction mixture was incubated at 37"C for 4 h. The mixture was fractionated by reverse-phase chromatography on a Beckman ultrapore RPMC 5 m column (4,6 mm diameter by 25 cm) - the elution being performed as described previously [10]. 180 tubes were collected and pooled to give 44 fractions. These were freeze dried and analysed as described below. Ribonuclease assay. For the determination of the active fractions following HPLC fractionation polyC was used as a substrate for ribonucleasc and the assay was performed as previously described [17]. For all other assays the substrate used was the dinucleotide CpG. This allows more meaningful kinetic analysis of the reaction, since it is a better defined template than polyC - the reaction of ribonuclease on this substrate involves the cleavage of only one bond. In addition it is possible to follow the reaction using a continuous assay. Degradation of the substrate is followed by moni-
o* t
toring increase of absorption at 245 nM in a PU 8720 Philips spectrophotometer. All reactions were performed in I ml of 0.1 M Tris (pH 7). The substrate (CpG) was used at concentrations between 27.5 and 82.5 mM, and bovine pancreatic ribonuclease was present at a concentration of 28 nM. The range of peptide concentration in the mixture was 0-12 fM. The K m of ribonuclease for this substrate is 0.36 x 10 - 3 M . l't should be noted that at pH 7 ribonuclease activity is not optimal (see results). However measurements at this pH remained linear over a period of 2 rain allowing accurate determination of the initial rates. At more optimal pH the reaction was too fast to allow accurate rate determinations. Amino acid analyses. Were performed on a Beckman 121 MB analyser on 18 h hydrolysates. Hydrolyses were carried out in evacuated sealed tubes at 110°C with 6 M HCI, in the presence of 1% phenol to avoid excessive degradation of tyrosine. Resul~:s In order to find a segment of the ribonuclease inhibitor which could inhibit ribonuclease, the protein
214aa
-
-
- | c H 3 e n ] t
0.1
--
--
--
15
/ f / /
/
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/ t
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wv'vvu~'~x~,,~ t J I
I
I
j
t
i
50
100
150
,
Ti.me 1Ln
[o
Fig. 1. HPLC profile. The elution was carried out as described in Materials and Methods. The position of active fractions are marked [1-3]. Fraction 3 was the one with the highest activity which was studied. Fraction 1 may correspond to two peptides not hydrolysed by trypsin.
109 was cleaved by each of the following treatments individually: 0.25 M acetic acid, endoproteinase Lys-C, ehymotrypsin (at pH 5) and trypsin. Purification of the resultant peptides was performed by HPLC. The best profile was consistently obtained with trypsin (Fig. 1). When the 44 HPLC fractions were tested for their ability to inhibit ribonuclease, three of them (labelled 1, 2, 3 - Fig. 1) showed inhibition. The', amino acid composition of fraction number 3 (the most active) corresponded well to the composition of a tryptie peptide predicted from the primary structure of the intact protein. This fragment is a peptide of 15 amino acids with N-terminal amino acid in position 287 in the sequence of PRI. The sequence of this peptide is: 287 E L S L A G N E L G D E G A R 3°l The amino acid composition of the other two fractions (1 and 2) suggested that they contained more than 1 tryptie peptide. We were not able to identify fraction 2 however, fraction 1 most likely corresponds to a 21 mer at position 173-194 in the sequence. The positions of both of these identified peptides are interesting as they clearly overlap the consensus sequence in the internal repeat structure of the PRI (see Discussion).
Characterisation of the peptide Although the fractions containing the peptide had clear inhibitory activity, it was formally possible that the observed inhibition was due to another minor como ponent of the fraction, or a combination of the observed peptide with another component of the fraction. We therefore synthesised the peptide chemically to show that this was not the case. The chemically synthesised peptide still inhibited the reaction with the same efficiency as the fraction, it therefore appears that it was this peptide alone that was responsible for the
30
.m
e~
10
0 5.0
6.0
7.0
8.0
9.0
pH Fig. 3. Influence of pH on RNase/peptide interactions (Unit = A absorbance/min×1000). Incubation was performed in Tris-HCl buffer with constant molarity: 0.1 M. Other incubation conditions were described in Materials and Methods. With peptide (e); without peptide ( [] ).
inhibitory activity of the fraction. The analysis was therefore continued with the chemically synthesised peptide.
Preincubation time It had been previously reported that the intact inhibitor n e e d s to be preincubated with ribonuclease prior to its addition to the reaction mixture [19]. We therefore looked at the effect of different times of preincubation for the peptide (Fig. 2). From the figure it can be seen that preincubation appears to be necessary, but after 10 min no further improvement in the inhibition could be achieved. This pattern is again similar to that observed for intact inhibitor.
Effect of pH 100
The assay was performed over a wide range of pH, both with and without the peptide. The results are shown in Fig. 3. These show that although the ribonuclease assay ~is affected by pH, with an optimum at pH 5.7, the interactions between the peptide and the ribonuclease are not affected by a change in pH.
so
60
~
40
Effect of diralent ions
zo
0 0
20
40
60
80
Time min Fig. 2. Effect of a preincubation time. The reaction was performed as described in Materials and Methods, but ribonuclease was preincubated for different times {as shown) with 3.7-10-15 M of peptide.
We tested the effect of Ca2+(CaCI2),Mn2+(MnCI2), Mg2+(MgCl2) and Zn2+(ZnCl2 ) on the peptide ribonuclease interactions. The effects of Cu2+(CuSO4 ) could not be tested since this solution interferes with the assay. In all cases the ions were used in the range of 0-30 mM, and peptide was added to a level where ribonudease activity was 70% inhibited. Fig. 4A shows the effect of these ions on the activity of the ribonuclease
110 and the interaction between peptide and ribonuclease. Calcium has no effect on the activity of ribonuclease. However, the presence of this ion seems to completely inhibit the effect of the peptide on the ribonuclease activity. With manganese there is a slight drop in ribonuclease activity at concentrations between 10 and 30 mM. As for calcium, addition of peptide does not inhibit ribonuclease activity, in fact, in this case there seems to be a small stimulation in activity, however the significance of a change of this magnitude is questionable. Magnesium, does not affect ribonuclease activity, and in the presence of this ion, even at high concentration, the peptide is still able to act as an efficient inhibitor. Zinc in the range of concentration used in the assay inhibits ribonuclease activity quite significantly. The addition of peptide further weakens this activity, suggesting that the inhibitor is still active, however, since the activity of the ribonuclease is already low, determinations of the exact magnitude of the inhibition cannot be made with high accuracy. Therefore, it seems that neither zinc or magnesium interfere with the interaction of the inhibitor with ribonuclease, however both manganese and calcium inhibit the inhibition completely. For the intact inhibitor only Ca 2+, Mg 2+ and Zn z+, have been tested, and at concentrations between
100
0.1 and 10 mM these ions have no effect on the ribonuclease: inhibitor interaction [19].
Effect of NaCi We also wanted to look at the effect of the ionic environment on the interaction between the peptide and the ribonuclease. For this purpose different concentrations of NaCI (0 to 100 mM) were added to the incubation mixture. The highest concentration of NaCI gave a higher ionic strength than the maximum (30 mM) used in the assay with divalent ions. The results of this experiment are shown in Fig. 4B. The presence of salt in this concentration range has a slight inhibitory effect on the activity of the ribonuclease, (this is consistent with earlier observations). The salt, however, does not seem to have any effect on the interaction between the peptide and ribonuclease. Similar results have been obtained for intact inhibitor [18,20]. Discussion
The data presented in this paper suggests that we have identified a small fragment of the ribonuclease inhibitor protein - just 15 amino acids long which appears to be able to bind to and inhibit ribonuclease. The interest of the peptide lies not only in its inhibitory activity, but also in its position in the amino acid sequence of the protein.
A IO0
B 80
•~
6o
40
20
o
~ 0
I0
20
Bivalent
30
ions (raM)
0
40
I
0
20
40
60
--
80
100
"
120
NaCI mM
Fig. 4. (A) Dependence of ribonuclease and peptide activity on bivalent ions concentration. The incubations were as described in Materials and Methods. The Ca 2. ( D ) Mn 2 ÷ ( • ), Mg 2 . ( o ) and Zn 2 + ( ~ ) given were the final concentrations in the incubation mixture. Ca" + + peptide ( • ), Mn 2+ + peptide ( ~ ) , Mg 2+ + peptide (e). "+ " Zn ~ + peptlde ( • ). (B) Dependence o f ribonuclease and peptide activity on NaCI concentration. The incubation conditions were as described in Materials and Method section. The NaCl concentrations were the final concentrations in the incubation mixture. ( • ) , with peptide; ( [] ), without peptide.
111 Although low concentrations of the peptide inhibit the reaction quite efficiently we were never able to achieve 100% inhibition of ribonuclease activity - the maximum we could reach was 70%. Addition of high concentrations of the peptide did, not improve the inhibition. On the contrary, with ve~'¢ high concentrations (100-1000 x more) the inhibitto ~ capacity of the peptide seemed to disappear. The r.ttost likely explanation for this is precipitation of the ~2ptide. An alternative would be absorption of the peptide on the reaction tube, however, this seems less likely since siliconisation of the tubes did not prevent the loss of inhibitory activity. The inhibition by the peptide was similar in many ways to that of the intact inhibitor. Comparable periods of preincubation were required, and the response of the the interaction to changes in pH and alterations in the concentrations of Na, Mg 2÷ and Zn -'+ were very similar. There is no data available on the effect of Mn 2+ on the interaction. The response to Ca 2+, however, was different. For the intact inhibitor it had been reported that there was no effect on the inhibition at C a 2+ concentrations between 0.1 and 10 mM, while for the peptide inhibition was seen at 10 raM. This effect must be specific since it does not occur with other divalent ions, or when the same ionic strength is provided with monovalent ions, however, the significance of this observation is not yet clear. A preliminary kinetic analysis of the mechanism of inhibition not presented here also suggests similarities between the peptide and the intact inhibitor. Our preliminary results implied that thc mechanism of inhibition was non-competitive and that the K i was very low. However, further kinetic analysis suggested that inhibition of ribonuclease by this peptide may be better described by a slow, tight-binding mechanism. In this case, as Morrison and Walsh have pointed out [21,22], analysis of results by Michaelis-Menten kinetics leads to misinterpretation of the results and a bias towards noncompetitive inhibition. Therefore, a much more detailed characterisation is needed to determine precise association/dissociation constants for the inhibitor and the nature of the inhibition. Thus, in this respect also the peptide may be the same as the intact inhibitor, in that early results suggested noncompetitive inhibition, whereas later interpretations favoured competitive inhibition [18,19]. The behaviour of the peptide suggests that it has an important role in the activity of the intact ribonuclease inhibitor. The position of the peptide in the amino acid sequence of the human placental ribonuclease inhibitor is also striking (sequence 287-301): PRI contains seven direct internal repeats [10], and our peptide has an amino acid sequence clearly overlapping this consensus (Fig. 5A). The position of the peptide corresponds well to regions defined as important in experi-
A I
EL-LS-N-LGDAG---LC-GL_
II
2~VELSLAGNEL~DEGAR!nl
IIII~3~TVSNNDINEAGVRVLCQGLK:O~
B 1
I
2
90
3
4
-~
144
6
2~7
7
J 'l
45~
/ 2~I
30I
Fig. 5. (A) Comparison of the amino acid sequence of the active peptides obtained after tryptic digest of the human placental ribonuclease inhibitor, with the consensus sequence of this protein proposed by Lee [10]. L Consensus sequence (173-194)of the human placental ribonuclease inhibitor [10]. I1. Sequence of the 15 amino acid peptide charaeterised in this study (HPLC fraction No. 3, in Fig. 1), IIl, Sequence of the peptide 173-194 which may correspond to two adjacent peptides not cleaved by trypsin (HPLC fraction No. 1, in Fig. 1). 1I and II! amino acids present in the consensus sequence are underlined. (B) Positioning of active peptide in the intact ribonuclease inhibitor. The sequence of the ribonuclease inhibitor is represented wi;b its seven internal repeats. ~, position of the peptide of the present study. Position of segments of the molecule deleted without affecting its inhibitory activity: ~ , residues !-90 or 93 deleted from pig ribonuclease inhibitor PRI [15]: D, residues 144-257 or 315-371 deleted from PR! [13,14].
ments using mutant inhibitors. In addition all deletion constructs made from both PRI [13,14] and a ribonuclease inhibitor from pig. [15], which show binding and inhibition contain this sequence (Fig. 5B). The two other "active' fractions released from the original tryptic digest have not been as well characterised as fraction 3 since their amino acid composition suggested that they were mixed, and they show lower activity. However, one of these fractions (No. 1) most likely corresponds to two adjacent peptides, (173-187 and 188-194)which could be generated by incomplete cleavage after the arginine residue in position 187. Interestingly, this region of sequence shows several similarities to the peptide from fraction 3 and the consensus sequence for the repeats. The identification of this peptide is an important step towards elucidating the mechanism of the ribonuclease inhibitor, however, many details of the bchaviour of the peptide (e.g., the aggregation state of the protein required for binding, its position of contact
112 with ribonuclease compared to those of the intact inhibitor, its interaction with other molecules affected by PRI, e.g., angiogenin) are not yet understood. In addition its contribution to the activity of the intact protein, and its relationship to important regions of the molecule identified from experiments with mutants of the human placental ribonuclease inhibitor still needs to he defined. The roles of these other regions of the PRI are not yet known, however, even if they do not form the active site they could be involved in other important functions: e.g., maintaining the correct folding of the molecule (the occurrence of some deletion constructs which contain the 'active' peptide but do not show activity may be explained by incorrect folding of the molecule masking the effect of ~he peptide); making additional contacts with ribonuclease to strengthen the binding (e.g., it has been suggested that cysteines are involved in binding, as thiol blocking agents cause dissociation of the inhibitor, however our peptide contains no thiols); providing functions not yet tested for (specificity, interactions with other molecules, control of the levels of activity or degradation etc.). The identification of a region which appears to be central to the inhibition provides a good starting point from which to design other more detailed experiments. Further study of this peptide, its interaction with other molecules, and how these interactions compare to those observed with the intact inhibitor, should help to shed light on the functioning of this important molecule.
Acknowledgements We thank T. Day for helpful discussions, T. Etienne, D. Featherby and I. Blench for skillful technical assistance. This work was supported by a grant from ANRT (CIFRE No. 87275), with funds from the Centre de Recherche Delalande.
References 1 Shortman, K. (1962) Biochim. Biophys. Acla. 61, 50-55. 2 Kraft, N. and Shortman, K. (1970) Biochim. Biophys. Acta 217, 164-175. 3 Quirin-Stricker, C., Gross, M. and Mandel, P (1968) Biochim. Biophys. Acta 159, 75-80 4 Sajdel-Sulkowska, E.M. and Marotta C.A. (1984) Science 225, 947-949. 5 Majocha, R.E., SajdeI-Sulkowska, E.M., Rodenrys A., VentosaMichelman, M. and Marotta, C.A. (1987) Soc. Neurosei. Abstr. 13, 819. 6 Shapiro, R. and VaUee, B.L. (1987) Proc Natl Acad Sci USA 84, 2238-2241. 7 Blackburn, P. and Jailkhani, B.J. (1979) J. Biol. Chem. 254, 12488-12493. 8 Blackburn, P. and Gavilanes, J.G. (1982) J. Biol. Chem. 257, 316-321. 9 Blackburn, P. and Gavilanes, J.G. (1982) J. Biol. Chem. 255, 10959-10965. 10 Lee, F.S., Fox, E.A., Zhou, H.M., Strydom, D.J. and Vatlee, B.L. (1988) Biochemistry 27, 8545-8553. 11 Schneider, R., Schneider-Scherzer, E., Thurner, M., Auer, B. and Schweiger, M. (1988) Embo. J 7, 4151-4156. 12 Hofsteenge, J., K/effer, B., Matthies, R., Hemmings, B.A. and Stone, S.R. (1988) Biochemistry 27, 8537-8544. 13 Lee, F.S. and Vallee, B.L. (1990) Proc. Natl. Acad. Sci. USA 87, 1879-1883. 14 Lee, F.S. and Vallee, B.L. (1990) Biochemistry 29, 6633-6638. 15 Hofsteenge, J., Vincentini, A. and Stone, S.R. (1991) Biochem. J. 275, 541-543. 16 Blackburn, P. (1979) J. Biol. Chem. 254, 24, 12484-12487. 17 Allinquant, B., Mussenger, C. and Schuller, E. (1984) Acta Neurol. Scand. 70, 12-19. 18 Lee, F.S., Shapiro, S. and Vallee, B.L.(I989) Biochemistry 28, 225-230. 19 Blackburn, P., Wilson, G. and Moore, S. (1977) J. Biol. Chem. 252, 5904-5910 20 Gribnau, A.A.M., Shoenmakers, J.G.G., Van kraaikamp, M., Hilak, M. and BIoemendal, H. (1970) Biochim. Biophys. Acta 224, 55-62. 21 Morrison, J'.F. (1982) Trends Biochem. 7, 102-105. 22 Morrison, J.F. and Walsh, C.T. (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61,201-301,
107
BBAPRO 34~4
Characterisation of a tryptic peptide from human placental ribonuclease inhibitor which inhibits ribonuclease activity Isabelle Crevel-Thieffry a,1, Sue Cotterill b and E d m o n d Schuller a "Laboratoire de neuro-immunologie, H~pital de la Salp~td~re, INSERM 134, Paris (France) and h Department of BiochemiStry, Imperial College of Science, Technolog)" and Medicine, London (UK) (Received 19 February 1992)
Key words: Protein characterisation; Tryptic peptide; Ribonuclease inhibitor; HPLC
Affinity-purified human placental ribonuclease inhibitor (PRI) was digested by trypsin. Subsequent fractionation of the hydrolysate by HPLC yielded 44 fractions~ 3 of which retained the ability to inhibit ribonuclease. One of these, the most active, was a 15 amino acid peptide which had an amino acid composition corresponding to a tryptic fragment of PRI. This peptide was synthesised, and preliminary experiments were carried out on its interactions with ribonuclease. These experiments suggested that the behaviour of the peptide in terms of effect of pH, and effect of salt concentration were similar to the protein from which it was derived. These studies together with the strategic positioning of the peptide in the sequence of the ribonuclease inhibitor, suggest that this segment of PRI has an important role in the inhibitory activity of the intact protein.
Introduction
Ribonuclease inhihitor is a 50 kDa protein which inhibits ribonuclease by binding very tightly to it. In vivo as much as 95% of the ribonuclease can be complexed with inhibitor, however the inhihitor: ribonuclease ratio is clearly related to metabolism; elevated in ploliferating tissue [1,2], and diminished during catabolic metabolism [3]. This ratio has also been reported to be modified during Alzheimer's disease [4.5]. More recently ribonuclease inhibitor has also been shown to be involved in the regulation of another molecule: angiogenin, a blood vessel-inducing protein [6]. Due to the importance of the processes in which the inhibitor has been implicated a great deal of effort has been put into understanding the mechanism of the molecule. Ribonuclease inhibitor has been isolated from a number of systems, however, one of the richest sources of the protein is human placenta. In this tissue the protein (PRI), is mostly free from ribonuclease and therefore the yields of the protein obtained are large enough to allow comprehensive analysis of the molecule. Detailed studies have been performed on the interaction between purified inhibitor and pure ribonuclease, and the regions of ribonuclease involved
i Present address: Chester Beatty Laboratories, Fulham Road, London SW3, UK. Correspondence: !. CreveI-Thieffry, Chester Beatty Laboratories, Fulham Road, London SW3, UK.
in the interaction have been identified by analysis of the interactions of pure inhibitor with modified ribonuclease [7-9]. The regions of the inhibitor involved in the interaction are less well understood. However, recently a start has been made in this area; the gene for PRI and also that for the pig ribonuclease inhibitor (which is very closely related) have been cloned [10-12]. Analysis of deletion mutants of both of these proteins has identified regions which seem to be important for both the interaction of the inhibitor with the ribonuclease and also for inhibition activity [13-15]. In an effort to further analyse those regions important for ribonuclease inhibitor activity we were interested in identifying small regions of the inhibitor (-15-20 amino acids)which possessed inhibitory action in isolation. In this study we present the characterisation of one such molecule, a small peptide of only 15 amino acids made by tryptic digestion of the affinityp-~rified inhibitor. We show that this peptide alone possesses features similar to the intact protein, and discuss the relevance of this finding in relation to the data already available from other studies. Materials and Methods
Materials. Bovine pancreatic ribonuclease A and cytidyl-3',5'-guanosine (CpG) were from Sigma. Poly C was from Pharmacia. Trypsin, affinity-purified for protein sequencing was from Merck. All other chemicals were of the highest grade commercially available.
108
Tryptic digest of PRI. PRI was affinity-purified from human placentas according to Ref. 16. 16 nmol of the freeze dried protein was dissolved in 50 mM ammonium bicarbonate (pH 8.5) (freshly prepared). Trypsin was added in a ratio of 3.3% (w/w) to PRI (---0.75 mg/ml) and the reaction mixture was incubated at 37"C for 4 h. The mixture was fractionated by reverse-phase chromatography on a Beckman ultrapore RPMC 5 m column (4,6 mm diameter by 25 cm) - the elution being performed as described previously [10]. 180 tubes were collected and pooled to give 44 fractions. These were freeze dried and analysed as described below. Ribonuclease assay. For the determination of the active fractions following HPLC fractionation polyC was used as a substrate for ribonucleasc and the assay was performed as previously described [17]. For all other assays the substrate used was the dinucleotide CpG. This allows more meaningful kinetic analysis of the reaction, since it is a better defined template than polyC - the reaction of ribonuclease on this substrate involves the cleavage of only one bond. In addition it is possible to follow the reaction using a continuous assay. Degradation of the substrate is followed by moni-
o* t
toring increase of absorption at 245 nM in a PU 8720 Philips spectrophotometer. All reactions were performed in I ml of 0.1 M Tris (pH 7). The substrate (CpG) was used at concentrations between 27.5 and 82.5 mM, and bovine pancreatic ribonuclease was present at a concentration of 28 nM. The range of peptide concentration in the mixture was 0-12 fM. The K m of ribonuclease for this substrate is 0.36 x 10 - 3 M . l't should be noted that at pH 7 ribonuclease activity is not optimal (see results). However measurements at this pH remained linear over a period of 2 rain allowing accurate determination of the initial rates. At more optimal pH the reaction was too fast to allow accurate rate determinations. Amino acid analyses. Were performed on a Beckman 121 MB analyser on 18 h hydrolysates. Hydrolyses were carried out in evacuated sealed tubes at 110°C with 6 M HCI, in the presence of 1% phenol to avoid excessive degradation of tyrosine. Resul~:s In order to find a segment of the ribonuclease inhibitor which could inhibit ribonuclease, the protein
214aa
-
-
- | c H 3 e n ] t
0.1
--
--
--
15
/ f / /
/
J
/ t
.:'1 O,OS 3
wv'vvu~'~x~,,~ t J I
I
I
j
t
i
50
100
150
,
Ti.me 1Ln
[o
Fig. 1. HPLC profile. The elution was carried out as described in Materials and Methods. The position of active fractions are marked [1-3]. Fraction 3 was the one with the highest activity which was studied. Fraction 1 may correspond to two peptides not hydrolysed by trypsin.
109 was cleaved by each of the following treatments individually: 0.25 M acetic acid, endoproteinase Lys-C, ehymotrypsin (at pH 5) and trypsin. Purification of the resultant peptides was performed by HPLC. The best profile was consistently obtained with trypsin (Fig. 1). When the 44 HPLC fractions were tested for their ability to inhibit ribonuclease, three of them (labelled 1, 2, 3 - Fig. 1) showed inhibition. The', amino acid composition of fraction number 3 (the most active) corresponded well to the composition of a tryptie peptide predicted from the primary structure of the intact protein. This fragment is a peptide of 15 amino acids with N-terminal amino acid in position 287 in the sequence of PRI. The sequence of this peptide is: 287 E L S L A G N E L G D E G A R 3°l The amino acid composition of the other two fractions (1 and 2) suggested that they contained more than 1 tryptie peptide. We were not able to identify fraction 2 however, fraction 1 most likely corresponds to a 21 mer at position 173-194 in the sequence. The positions of both of these identified peptides are interesting as they clearly overlap the consensus sequence in the internal repeat structure of the PRI (see Discussion).
Characterisation of the peptide Although the fractions containing the peptide had clear inhibitory activity, it was formally possible that the observed inhibition was due to another minor como ponent of the fraction, or a combination of the observed peptide with another component of the fraction. We therefore synthesised the peptide chemically to show that this was not the case. The chemically synthesised peptide still inhibited the reaction with the same efficiency as the fraction, it therefore appears that it was this peptide alone that was responsible for the
30
.m
e~
10
0 5.0
6.0
7.0
8.0
9.0
pH Fig. 3. Influence of pH on RNase/peptide interactions (Unit = A absorbance/min×1000). Incubation was performed in Tris-HCl buffer with constant molarity: 0.1 M. Other incubation conditions were described in Materials and Methods. With peptide (e); without peptide ( [] ).
inhibitory activity of the fraction. The analysis was therefore continued with the chemically synthesised peptide.
Preincubation time It had been previously reported that the intact inhibitor n e e d s to be preincubated with ribonuclease prior to its addition to the reaction mixture [19]. We therefore looked at the effect of different times of preincubation for the peptide (Fig. 2). From the figure it can be seen that preincubation appears to be necessary, but after 10 min no further improvement in the inhibition could be achieved. This pattern is again similar to that observed for intact inhibitor.
Effect of pH 100
The assay was performed over a wide range of pH, both with and without the peptide. The results are shown in Fig. 3. These show that although the ribonuclease assay ~is affected by pH, with an optimum at pH 5.7, the interactions between the peptide and the ribonuclease are not affected by a change in pH.
so
60
~
40
Effect of diralent ions
zo
0 0
20
40
60
80
Time min Fig. 2. Effect of a preincubation time. The reaction was performed as described in Materials and Methods, but ribonuclease was preincubated for different times {as shown) with 3.7-10-15 M of peptide.
We tested the effect of Ca2+(CaCI2),Mn2+(MnCI2), Mg2+(MgCl2) and Zn2+(ZnCl2 ) on the peptide ribonuclease interactions. The effects of Cu2+(CuSO4 ) could not be tested since this solution interferes with the assay. In all cases the ions were used in the range of 0-30 mM, and peptide was added to a level where ribonudease activity was 70% inhibited. Fig. 4A shows the effect of these ions on the activity of the ribonuclease
110 and the interaction between peptide and ribonuclease. Calcium has no effect on the activity of ribonuclease. However, the presence of this ion seems to completely inhibit the effect of the peptide on the ribonuclease activity. With manganese there is a slight drop in ribonuclease activity at concentrations between 10 and 30 mM. As for calcium, addition of peptide does not inhibit ribonuclease activity, in fact, in this case there seems to be a small stimulation in activity, however the significance of a change of this magnitude is questionable. Magnesium, does not affect ribonuclease activity, and in the presence of this ion, even at high concentration, the peptide is still able to act as an efficient inhibitor. Zinc in the range of concentration used in the assay inhibits ribonuclease activity quite significantly. The addition of peptide further weakens this activity, suggesting that the inhibitor is still active, however, since the activity of the ribonuclease is already low, determinations of the exact magnitude of the inhibition cannot be made with high accuracy. Therefore, it seems that neither zinc or magnesium interfere with the interaction of the inhibitor with ribonuclease, however both manganese and calcium inhibit the inhibition completely. For the intact inhibitor only Ca 2+, Mg 2+ and Zn z+, have been tested, and at concentrations between
100
0.1 and 10 mM these ions have no effect on the ribonuclease: inhibitor interaction [19].
Effect of NaCi We also wanted to look at the effect of the ionic environment on the interaction between the peptide and the ribonuclease. For this purpose different concentrations of NaCI (0 to 100 mM) were added to the incubation mixture. The highest concentration of NaCI gave a higher ionic strength than the maximum (30 mM) used in the assay with divalent ions. The results of this experiment are shown in Fig. 4B. The presence of salt in this concentration range has a slight inhibitory effect on the activity of the ribonuclease, (this is consistent with earlier observations). The salt, however, does not seem to have any effect on the interaction between the peptide and ribonuclease. Similar results have been obtained for intact inhibitor [18,20]. Discussion
The data presented in this paper suggests that we have identified a small fragment of the ribonuclease inhibitor protein - just 15 amino acids long which appears to be able to bind to and inhibit ribonuclease. The interest of the peptide lies not only in its inhibitory activity, but also in its position in the amino acid sequence of the protein.
A IO0
B 80
•~
6o
40
20
o
~ 0
I0
20
Bivalent
30
ions (raM)
0
40
I
0
20
40
60
--
80
100
"
120
NaCI mM
Fig. 4. (A) Dependence of ribonuclease and peptide activity on bivalent ions concentration. The incubations were as described in Materials and Methods. The Ca 2. ( D ) Mn 2 ÷ ( • ), Mg 2 . ( o ) and Zn 2 + ( ~ ) given were the final concentrations in the incubation mixture. Ca" + + peptide ( • ), Mn 2+ + peptide ( ~ ) , Mg 2+ + peptide (e). "+ " Zn ~ + peptlde ( • ). (B) Dependence o f ribonuclease and peptide activity on NaCI concentration. The incubation conditions were as described in Materials and Method section. The NaCl concentrations were the final concentrations in the incubation mixture. ( • ) , with peptide; ( [] ), without peptide.
111 Although low concentrations of the peptide inhibit the reaction quite efficiently we were never able to achieve 100% inhibition of ribonuclease activity - the maximum we could reach was 70%. Addition of high concentrations of the peptide did, not improve the inhibition. On the contrary, with ve~'¢ high concentrations (100-1000 x more) the inhibitto ~ capacity of the peptide seemed to disappear. The r.ttost likely explanation for this is precipitation of the ~2ptide. An alternative would be absorption of the peptide on the reaction tube, however, this seems less likely since siliconisation of the tubes did not prevent the loss of inhibitory activity. The inhibition by the peptide was similar in many ways to that of the intact inhibitor. Comparable periods of preincubation were required, and the response of the the interaction to changes in pH and alterations in the concentrations of Na, Mg 2÷ and Zn -'+ were very similar. There is no data available on the effect of Mn 2+ on the interaction. The response to Ca 2+, however, was different. For the intact inhibitor it had been reported that there was no effect on the inhibition at C a 2+ concentrations between 0.1 and 10 mM, while for the peptide inhibition was seen at 10 raM. This effect must be specific since it does not occur with other divalent ions, or when the same ionic strength is provided with monovalent ions, however, the significance of this observation is not yet clear. A preliminary kinetic analysis of the mechanism of inhibition not presented here also suggests similarities between the peptide and the intact inhibitor. Our preliminary results implied that thc mechanism of inhibition was non-competitive and that the K i was very low. However, further kinetic analysis suggested that inhibition of ribonuclease by this peptide may be better described by a slow, tight-binding mechanism. In this case, as Morrison and Walsh have pointed out [21,22], analysis of results by Michaelis-Menten kinetics leads to misinterpretation of the results and a bias towards noncompetitive inhibition. Therefore, a much more detailed characterisation is needed to determine precise association/dissociation constants for the inhibitor and the nature of the inhibition. Thus, in this respect also the peptide may be the same as the intact inhibitor, in that early results suggested noncompetitive inhibition, whereas later interpretations favoured competitive inhibition [18,19]. The behaviour of the peptide suggests that it has an important role in the activity of the intact ribonuclease inhibitor. The position of the peptide in the amino acid sequence of the human placental ribonuclease inhibitor is also striking (sequence 287-301): PRI contains seven direct internal repeats [10], and our peptide has an amino acid sequence clearly overlapping this consensus (Fig. 5A). The position of the peptide corresponds well to regions defined as important in experi-
A I
EL-LS-N-LGDAG---LC-GL_
II
2~VELSLAGNEL~DEGAR!nl
IIII~3~TVSNNDINEAGVRVLCQGLK:O~
B 1
I
2
90
3
4
-~
144
6
2~7
7
J 'l
45~
/ 2~I
30I
Fig. 5. (A) Comparison of the amino acid sequence of the active peptides obtained after tryptic digest of the human placental ribonuclease inhibitor, with the consensus sequence of this protein proposed by Lee [10]. L Consensus sequence (173-194)of the human placental ribonuclease inhibitor [10]. I1. Sequence of the 15 amino acid peptide charaeterised in this study (HPLC fraction No. 3, in Fig. 1), IIl, Sequence of the peptide 173-194 which may correspond to two adjacent peptides not cleaved by trypsin (HPLC fraction No. 1, in Fig. 1). 1I and II! amino acids present in the consensus sequence are underlined. (B) Positioning of active peptide in the intact ribonuclease inhibitor. The sequence of the ribonuclease inhibitor is represented wi;b its seven internal repeats. ~, position of the peptide of the present study. Position of segments of the molecule deleted without affecting its inhibitory activity: ~ , residues !-90 or 93 deleted from pig ribonuclease inhibitor PRI [15]: D, residues 144-257 or 315-371 deleted from PR! [13,14].
ments using mutant inhibitors. In addition all deletion constructs made from both PRI [13,14] and a ribonuclease inhibitor from pig. [15], which show binding and inhibition contain this sequence (Fig. 5B). The two other "active' fractions released from the original tryptic digest have not been as well characterised as fraction 3 since their amino acid composition suggested that they were mixed, and they show lower activity. However, one of these fractions (No. 1) most likely corresponds to two adjacent peptides, (173-187 and 188-194)which could be generated by incomplete cleavage after the arginine residue in position 187. Interestingly, this region of sequence shows several similarities to the peptide from fraction 3 and the consensus sequence for the repeats. The identification of this peptide is an important step towards elucidating the mechanism of the ribonuclease inhibitor, however, many details of the bchaviour of the peptide (e.g., the aggregation state of the protein required for binding, its position of contact
112 with ribonuclease compared to those of the intact inhibitor, its interaction with other molecules affected by PRI, e.g., angiogenin) are not yet understood. In addition its contribution to the activity of the intact protein, and its relationship to important regions of the molecule identified from experiments with mutants of the human placental ribonuclease inhibitor still needs to he defined. The roles of these other regions of the PRI are not yet known, however, even if they do not form the active site they could be involved in other important functions: e.g., maintaining the correct folding of the molecule (the occurrence of some deletion constructs which contain the 'active' peptide but do not show activity may be explained by incorrect folding of the molecule masking the effect of ~he peptide); making additional contacts with ribonuclease to strengthen the binding (e.g., it has been suggested that cysteines are involved in binding, as thiol blocking agents cause dissociation of the inhibitor, however our peptide contains no thiols); providing functions not yet tested for (specificity, interactions with other molecules, control of the levels of activity or degradation etc.). The identification of a region which appears to be central to the inhibition provides a good starting point from which to design other more detailed experiments. Further study of this peptide, its interaction with other molecules, and how these interactions compare to those observed with the intact inhibitor, should help to shed light on the functioning of this important molecule.
Acknowledgements We thank T. Day for helpful discussions, T. Etienne, D. Featherby and I. Blench for skillful technical assistance. This work was supported by a grant from ANRT (CIFRE No. 87275), with funds from the Centre de Recherche Delalande.
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