- Email: [email protected]
Interaction of a synthetic peptide of the interferon α-2 C-terminal part with human blood leukocytes
December 199 I
FEBS 10509 Volume 295. number 12.3. 70-71 0 1991 Federa1ion of European Biochemical Socic1ics 00145793i9l/S3.50
Interaction
of a synthetic peptide of the interferon a-2 C-terminal part with human blood leukocytes Binding to peripheral blood mononuclear
A.V. .i)anilkovitch’,
A.I. Kharitonenkov’, K.V. Freze’, A.F. Shevalier’, A.F. Kirkin’ and M.V. Gusev’
Received 30 September
cells
0-V. Kolosova’, T.V. Bulargina’,
199 I
the 124-138 amino acid scquencc ol11lc human intcrfcron a-_7 (IFN a-2) n~olcculc, which is known IO possessIFN-like antiproliferative activity. specifically binds 10 human blood leukocytes. Scalchard plots rcvcal 1~0 ditTcrcn1 K,, V~IUCS, for the ‘low’ and ‘high’ arfiniiy binding. The inlcrac1ioo of the “51-labcllcd peplid, n 2438 with ~hecclls is no1 impnircd by human IFN a-2 or chokrd
A biologically aclivc syn1hctic pcptidc. 2438. representing
tosin.
IFS a-2: Synlhctic pcp1ide; Specific binding:
In this study, we report on binding properties of the ‘Wabelled synthetic peptide 2438.
I. INTRODUCTION The family of IFN tl is a group of related proteins manifesting multiple biological activities [I]. Numerous structure-functional studies of IFN molecules revealed several areas associated with different biological activities [Z--4]. Synthetic peptides representing fragments of IFN polypeptide chains are a good tool for identification of functionally important regions of these macromolecules. A study of a synthetic peptide corresponding to the 131-138 region of the human IFN a-2 moiecuie demonstrated its biological activity and specific binding to surface elements of mouse thymocytes. The binding of the labelled peptide 13!-I 38 to the cells was impaired by IFN a-2 and thymosin OL[5]. In an earlier work, we have slsown that a synthetic peptide, 2438. corresponding to tlie amino acid residues 124-138 of the human IFN a-2 polypeptide chain, inhibited proliferation of human blood leukocytes [6] but did not affect the activity of NK-cells (our unpublished data) and, in contrast to native IFN, this peptidc had no antiviral activity [7].
Ahhreei~trior~s: ConA,
concanavalin A; IFN, in1erfcron; PBMC, pcriphcral blood mononuclear cells; pcp1idc 2438, a syn1hclic pcpridc rcprcscnting \hc IFN a-2 amicc acid rcsihcs 1X-138.
Cctrrrspotrclctrw r!r!!!rcss: A.V. Danilkovich, Dcparm~enl Physiology and Immunology. Biological F;iculty, Moscow vcrsily, Lenin’s Hill, Moscow 119899. USSR.
70
Human; Lymphocylc
Cellular S1a1c Uni-
of
2. MATERIALS
AND METHODS
The pcp1ides corresponding to the amino acid residues 124-138 (2338), 124-1~4 (2444). 129-138 (2938) and 129-144 (2944) OT 1hc human IFN a-2 molcculc wcrc synlhcsizcd as described [7].
2438 2438 was iabcicd by solid phasc oxidalion usin& Na[‘z51] (0.5 rnCi: Amcrsham Corp.)asdcscribcd on a column with Scphadcx G-IO (Pharmacia). of the labcllcd pcp1idc was 2 x IO” cpm&g.
2.1. lodittutiarr It/ pqttirle
50 jrg oi pcplide lodogcn (Sigma)and [8], and gel-iihra1cd The specific aclivity
PB,SK were ob1;:incd from blood of hcal1hy donors according lo Doyum [9]. Adherent cells wcrc rcmovcd by incubation on piaslic Peiri dishes l-or I II ~II 37°C in an almosphcrc of 5% CO:. A suspension or lymphocytes US divided into adhcren1 and non-adbcrcn1 subsets on a nylon wool column [IO]. A non-adhcrcn1 fKICliOfl WIS clutcd wilh RPM1 1640 medium supplemcn1cd wi1h 5% heat-imlctivatcd recta!x!f strum. This tixlion conlaincd T-lymphocy1cs and null cells with small amounts or monocytcs and B-lymphocytes. 2.4. Clrlrrrra co11dirirttr,s PBMC (2 x IOh ccllslml) wcrc cultured in RPMI 1640 medium (Sigma) supplcmcn1cd with 5% hca1-inactiva1ed Te1al calf serum (Sigma), 100 mM L-glulamine, 50,@ml gentamicin. When ncccssary, concanavalin A (ConA) (2/@nl) or IFN a.2 (I000 IWml) wcrc added 10 1hc cul1urc medium. Alicr culturing with Conit or IL2 I-or 20 h in a humid atmosphcrc or 5% CO: 81 37OC 1hc cells were harvencd, w;lshcd 1hrcc limes with fresh Incdium and used in binding ~SSZI~S.
Volume
793,
number
FEBSLEI-I-ERS
1.2.3
December
I991
I20
100
80
00
10
PO
0
Fig. I. Scalchard
plol I’or the binding IO
or 111~ ‘zsl-labcllcd normal PBhlC.
pcplidc
2438
PBMC or scparaicd T-lymphocytes (2 x lO”cclls!tubc) wcrc-incuba[cd \vith the labcllcd pcplidc ‘$38 41 conccnlralions of IO-“-IO-” M for 40 min al -i’C in 100/A of 199 medium (S:rva) supplcmcnl-d with 2% bovine swum albumin (Sigma). Aficr IIIC incubation, I~C cells wcrc wshcd by ccnwifualion ~lrrough 10% sucrose gradient as dcscribcd [I 11. and IIIC all-bound radioxtivity was measured in D Rack-Bela y.counwr (I_KB. Swdcn). In compchtiw binding assays PBMC wcrc i:!rub;ltcd with IO-” M or rhc labcllcd pcptidc 2438 in the prcs:nce 01 IOO-fold mo!;ir CXCC’SS01’ unlabcllcd IFN a-2. synthciic pcplidcs or cholera toxin (Calbiochcm).
3. RESULTS
AND DISCUSSlOX
We analysed the binding of the labelled synthetic peptide 2438 to normal PBMC and T-lymphocytes, Fig. I presents a Scatcbard plot which is obviously curvilinear. As judged from the ScaLchard analysis of the binding to normal PBMC. there arc two distinct binding sites characuzrized by different dissociation const.ant values (K,,, = I.2 x IO-” M. and Kdz= I.2 x IO-‘” M, for ‘high’ and ‘low’ affinity sites. respectively). The number of bigh- and low-affinity binding sites for PBMC is 2.01 x 10’ and I. 12 x IO’ per cell, respectively. As expected, the unlabclled pcptide 2438 effectively competes with the labellcd one for binding to PBMC. About 20-350/o of the peptide was estimated to bind nonspecifically (Fig. 2). Qualitatively and quantitatively similar results were obtained with separated T-lymphocytes (data not shown). The results shown in Figs. I and 2 suggest that the peptide 2438 can specifically bind to human blood Icukocytes. According to Doolcy et al. [i?], Scatchard plots for the binding of IFN a-2 to normal PBMC arc also curvilinear. Bearing in mind this analogy, we examined the ability of IFN a-2, cholera toxin and a number of IFNderived pcptides of the 124-144 region to compete with
Fig. 2. Compclition bclwccn the “‘I-labclled and unlabcllcd pcptidc 2438 for bindin& IO PBMC. Concentration of the “‘I-labcllcd pcptidc is IO-” M.
the peptide 2438 for PBMC binding sites. All of these molecules are known to contain amino acid tracks of high local homology [ 131.The results of the competition experiments (Fig. 3) clearly demonstrate that neither the whole IFN a-2 molecule nor the aforementioned pep tides nor cholera toxin interfered with the binding of the labelled peptide 2438 to PMBC. The pretreatment of PBMC with ConA was found earlier to colnpletely block biological effects of both 1FN a and the peptide 2438 (our unpublished data). On the other hand, the expression of the IFN-receptor is inhibited by pre-incubalion of cells with IFN [14411]1 Therefore we characterized the effects of ConA and IFN on the peptide 2438 binding. To this end, prior lo binding measurements. PBMC were pre-incubated for 20 h witb 2 pg/rnl ConA or 1000 lU/ml recombinant IFN a-2. In both cases, the binding of the peptide 2438 was unaffected (data not shown). 120
Speci!ic
r-
(96)
5ncing
LOO p
-i7lI-l 1
[’ I (15
00 :
00
T'+
L’
40 k I 20 i
rl o! JT_L C
! -LLL I
’ I
I 2430
z
!944
2930
IFN
Fig. 3. Efl’ccts or dil’fercnt competitors, added al R 100.fold molar excess. on binding or the ‘2V-labcllcd pcplidc 2438 IO PBMC. Compclilors: 2438.2444 and 2944 arc pcptidcs corresponding IO I~C 1X-138, IX-!44 and 129-144 amino acid scqucnccs 01’ lhc human IFN a-2 molcculc; IFN, human intcrfcron a-2: CT, cholera toxin; C. control. wilhoul COmpclilOis.
71
Volume 795. number 1.2.3
FEBS LETTERS
These findings suggest that (i) non-responsiveness of ConA-activated PBMC to the peptide 2438 seems not to be associated with the lack of appropriate binding sites on the cell membrane. and (ii) prctrcatment of PBMC with IFN or ConA does not influence the binding of the peptide to the cells. Although the present results may be interpreted as an indication that the peptide 2438 binds to membrane targets different from those for IFN 01-3.this conclusion needs additional support. The question of identity of ccl1 surface binding sites for IFN a-2 and the peptide 2438 remains open and awaits further investigation. REFERENCES [I]
Balkwill. F.R. and Burke. F. (1989) Immunol. Toduy 10. 299304. [2] Rchbcrp. E.. Keldcr. B.. Hoal, E.G. and Pcstka. S. (:982) J. Biol. Chem. 257. 11497-I 1502. [3] Cebrian, M.. Yague. E.. Dc Landazuri. M.O.. Rodiguez-Mow, bl.. Fresno. M.. Pezzi. N.. UiNIIardreS. S. and Sanchez-MadAd. F. (1987) J. Immunol. 138. 484-490. [-I] Fish. E.S.. Bancrgec. K. and Srcbbing. N.J. (1989) J. IntcrTcron Res. 9. 97-l 14.
72
December 199I
[j] Zav’yalov. VP.. Navolotsknya, E.V.. Abramov. V.M.. Galactionav. V.G.. Isacv. IS.. Kaurov. O.A.. Kozhich. A.T.. Maiorov. V.A.. Prusakov. A.N.. Vilsilenkc. R.N. and Volodina, E.Yu. (1991) FEBS Lctt. 278. 187-185. [6! Danilkovich. A.V.. Frczc. K.V.. Shcvalicr. A.F., Samukov. V.V.. Kirkin. A.F. and Guscv. M.V. (1991) Immunol. Lctt. (in press). 171 Shcvalicr. A.F.. Samokov. V.V., Ofiwrov, V.I., Kalashnikov. V.. Mizcnko. G.A. and Kolokollsov. A.A. (1990) Bioorg. Khim. (USSR) 16.916-925. [8] Salacinski. R.R.P.. McLean. C.. Skycs. J.E.C.. Clcmcnt-Jones. W. and Lowry, P.J. (1981) Anal. Biochcm. 117. 136-146. [9J Boyum. A. (1968) Stand. J. Clin. Insesl. 21. suppi. 97. [IO] Aman, I’., Ehlin-Henriksson. B.. Klein. G. (1984) J. Esp. Med. 159. XX. [I I] Lnngcr. J.A. and Pcstka. S. (1986) Mclhods Enzymol. 119. 305 312. [I?] Doolcy, J.B.. Vergalla. J.. Hoofnag1c.J.H.. Zoon. KC.. Munson. P.J. and Jones. E.A. (1989) J. Lab. Clin. Med. I 13. 613-63 I. [I31 Shal%rman. A.. Vclan. B.. Cohen. S.. Leimcr. M. and Grosfeld. Ii. (1987) .I. Biol. Chcm. 262. 6227-6237. [l4] Branca. A.A. and Baglioni, C. (1982) J. Biol. Chcm. 257. 1319713100. [IS] Zoon, K.C.. Zur Ncddcn. D.. Hu. R. and Arnhcitcr, H. (1986) J. Biol. Chcm. 261. 4993-4996.
FEBS 10509 Volume 295. number 12.3. 70-71 0 1991 Federa1ion of European Biochemical Socic1ics 00145793i9l/S3.50
Interaction
of a synthetic peptide of the interferon a-2 C-terminal part with human blood leukocytes Binding to peripheral blood mononuclear
A.V. .i)anilkovitch’,
A.I. Kharitonenkov’, K.V. Freze’, A.F. Shevalier’, A.F. Kirkin’ and M.V. Gusev’
Received 30 September
cells
0-V. Kolosova’, T.V. Bulargina’,
199 I
the 124-138 amino acid scquencc ol11lc human intcrfcron a-_7 (IFN a-2) n~olcculc, which is known IO possessIFN-like antiproliferative activity. specifically binds 10 human blood leukocytes. Scalchard plots rcvcal 1~0 ditTcrcn1 K,, V~IUCS, for the ‘low’ and ‘high’ arfiniiy binding. The inlcrac1ioo of the “51-labcllcd peplid, n 2438 with ~hecclls is no1 impnircd by human IFN a-2 or chokrd
A biologically aclivc syn1hctic pcptidc. 2438. representing
tosin.
IFS a-2: Synlhctic pcp1ide; Specific binding:
In this study, we report on binding properties of the ‘Wabelled synthetic peptide 2438.
I. INTRODUCTION The family of IFN tl is a group of related proteins manifesting multiple biological activities [I]. Numerous structure-functional studies of IFN molecules revealed several areas associated with different biological activities [Z--4]. Synthetic peptides representing fragments of IFN polypeptide chains are a good tool for identification of functionally important regions of these macromolecules. A study of a synthetic peptide corresponding to the 131-138 region of the human IFN a-2 moiecuie demonstrated its biological activity and specific binding to surface elements of mouse thymocytes. The binding of the labelled peptide 13!-I 38 to the cells was impaired by IFN a-2 and thymosin OL[5]. In an earlier work, we have slsown that a synthetic peptide, 2438. corresponding to tlie amino acid residues 124-138 of the human IFN a-2 polypeptide chain, inhibited proliferation of human blood leukocytes [6] but did not affect the activity of NK-cells (our unpublished data) and, in contrast to native IFN, this peptidc had no antiviral activity [7].
Ahhreei~trior~s: ConA,
concanavalin A; IFN, in1erfcron; PBMC, pcriphcral blood mononuclear cells; pcp1idc 2438, a syn1hclic pcpridc rcprcscnting \hc IFN a-2 amicc acid rcsihcs 1X-138.
Cctrrrspotrclctrw r!r!!!rcss: A.V. Danilkovich, Dcparm~enl Physiology and Immunology. Biological F;iculty, Moscow vcrsily, Lenin’s Hill, Moscow 119899. USSR.
70
Human; Lymphocylc
Cellular S1a1c Uni-
of
2. MATERIALS
AND METHODS
The pcp1ides corresponding to the amino acid residues 124-138 (2338), 124-1~4 (2444). 129-138 (2938) and 129-144 (2944) OT 1hc human IFN a-2 molcculc wcrc synlhcsizcd as described [7].
2438 2438 was iabcicd by solid phasc oxidalion usin& Na[‘z51] (0.5 rnCi: Amcrsham Corp.)asdcscribcd on a column with Scphadcx G-IO (Pharmacia). of the labcllcd pcp1idc was 2 x IO” cpm&g.
2.1. lodittutiarr It/ pqttirle
50 jrg oi pcplide lodogcn (Sigma)and [8], and gel-iihra1cd The specific aclivity
PB,SK were ob1;:incd from blood of hcal1hy donors according lo Doyum [9]. Adherent cells wcrc rcmovcd by incubation on piaslic Peiri dishes l-or I II ~II 37°C in an almosphcrc of 5% CO:. A suspension or lymphocytes US divided into adhcren1 and non-adbcrcn1 subsets on a nylon wool column [IO]. A non-adhcrcn1 fKICliOfl WIS clutcd wilh RPM1 1640 medium supplemcn1cd wi1h 5% heat-imlctivatcd recta!x!f strum. This tixlion conlaincd T-lymphocy1cs and null cells with small amounts or monocytcs and B-lymphocytes. 2.4. Clrlrrrra co11dirirttr,s PBMC (2 x IOh ccllslml) wcrc cultured in RPMI 1640 medium (Sigma) supplcmcn1cd with 5% hca1-inactiva1ed Te1al calf serum (Sigma), 100 mM L-glulamine, 50,@ml gentamicin. When ncccssary, concanavalin A (ConA) (2/@nl) or IFN a.2 (I000 IWml) wcrc added 10 1hc cul1urc medium. Alicr culturing with Conit or IL2 I-or 20 h in a humid atmosphcrc or 5% CO: 81 37OC 1hc cells were harvencd, w;lshcd 1hrcc limes with fresh Incdium and used in binding ~SSZI~S.
Volume
793,
number
FEBSLEI-I-ERS
1.2.3
December
I991
I20
100
80
00
10
PO
0
Fig. I. Scalchard
plol I’or the binding IO
or 111~ ‘zsl-labcllcd normal PBhlC.
pcplidc
2438
PBMC or scparaicd T-lymphocytes (2 x lO”cclls!tubc) wcrc-incuba[cd \vith the labcllcd pcplidc ‘$38 41 conccnlralions of IO-“-IO-” M for 40 min al -i’C in 100/A of 199 medium (S:rva) supplcmcnl-d with 2% bovine swum albumin (Sigma). Aficr IIIC incubation, I~C cells wcrc wshcd by ccnwifualion ~lrrough 10% sucrose gradient as dcscribcd [I 11. and IIIC all-bound radioxtivity was measured in D Rack-Bela y.counwr (I_KB. Swdcn). In compchtiw binding assays PBMC wcrc i:!rub;ltcd with IO-” M or rhc labcllcd pcptidc 2438 in the prcs:nce 01 IOO-fold mo!;ir CXCC’SS01’ unlabcllcd IFN a-2. synthciic pcplidcs or cholera toxin (Calbiochcm).
3. RESULTS
AND DISCUSSlOX
We analysed the binding of the labelled synthetic peptide 2438 to normal PBMC and T-lymphocytes, Fig. I presents a Scatcbard plot which is obviously curvilinear. As judged from the ScaLchard analysis of the binding to normal PBMC. there arc two distinct binding sites characuzrized by different dissociation const.ant values (K,,, = I.2 x IO-” M. and Kdz= I.2 x IO-‘” M, for ‘high’ and ‘low’ affinity sites. respectively). The number of bigh- and low-affinity binding sites for PBMC is 2.01 x 10’ and I. 12 x IO’ per cell, respectively. As expected, the unlabclled pcptide 2438 effectively competes with the labellcd one for binding to PBMC. About 20-350/o of the peptide was estimated to bind nonspecifically (Fig. 2). Qualitatively and quantitatively similar results were obtained with separated T-lymphocytes (data not shown). The results shown in Figs. I and 2 suggest that the peptide 2438 can specifically bind to human blood Icukocytes. According to Doolcy et al. [i?], Scatchard plots for the binding of IFN a-2 to normal PBMC arc also curvilinear. Bearing in mind this analogy, we examined the ability of IFN a-2, cholera toxin and a number of IFNderived pcptides of the 124-144 region to compete with
Fig. 2. Compclition bclwccn the “‘I-labclled and unlabcllcd pcptidc 2438 for bindin& IO PBMC. Concentration of the “‘I-labcllcd pcptidc is IO-” M.
the peptide 2438 for PBMC binding sites. All of these molecules are known to contain amino acid tracks of high local homology [ 131.The results of the competition experiments (Fig. 3) clearly demonstrate that neither the whole IFN a-2 molecule nor the aforementioned pep tides nor cholera toxin interfered with the binding of the labelled peptide 2438 to PMBC. The pretreatment of PBMC with ConA was found earlier to colnpletely block biological effects of both 1FN a and the peptide 2438 (our unpublished data). On the other hand, the expression of the IFN-receptor is inhibited by pre-incubalion of cells with IFN [14411]1 Therefore we characterized the effects of ConA and IFN on the peptide 2438 binding. To this end, prior lo binding measurements. PBMC were pre-incubated for 20 h witb 2 pg/rnl ConA or 1000 lU/ml recombinant IFN a-2. In both cases, the binding of the peptide 2438 was unaffected (data not shown). 120
Speci!ic
r-
(96)
5ncing
LOO p
-i7lI-l 1
[’ I (15
00 :
00
T'+
L’
40 k I 20 i
rl o! JT_L C
! -LLL I
’ I
I 2430
z
!944
2930
IFN
Fig. 3. Efl’ccts or dil’fercnt competitors, added al R 100.fold molar excess. on binding or the ‘2V-labcllcd pcplidc 2438 IO PBMC. Compclilors: 2438.2444 and 2944 arc pcptidcs corresponding IO I~C 1X-138, IX-!44 and 129-144 amino acid scqucnccs 01’ lhc human IFN a-2 molcculc; IFN, human intcrfcron a-2: CT, cholera toxin; C. control. wilhoul COmpclilOis.
71
Volume 795. number 1.2.3
FEBS LETTERS
These findings suggest that (i) non-responsiveness of ConA-activated PBMC to the peptide 2438 seems not to be associated with the lack of appropriate binding sites on the cell membrane. and (ii) prctrcatment of PBMC with IFN or ConA does not influence the binding of the peptide to the cells. Although the present results may be interpreted as an indication that the peptide 2438 binds to membrane targets different from those for IFN 01-3.this conclusion needs additional support. The question of identity of ccl1 surface binding sites for IFN a-2 and the peptide 2438 remains open and awaits further investigation. REFERENCES [I]
Balkwill. F.R. and Burke. F. (1989) Immunol. Toduy 10. 299304. [2] Rchbcrp. E.. Keldcr. B.. Hoal, E.G. and Pcstka. S. (:982) J. Biol. Chem. 257. 11497-I 1502. [3] Cebrian, M.. Yague. E.. Dc Landazuri. M.O.. Rodiguez-Mow, bl.. Fresno. M.. Pezzi. N.. UiNIIardreS. S. and Sanchez-MadAd. F. (1987) J. Immunol. 138. 484-490. [-I] Fish. E.S.. Bancrgec. K. and Srcbbing. N.J. (1989) J. IntcrTcron Res. 9. 97-l 14.
72
December 199I
[j] Zav’yalov. VP.. Navolotsknya, E.V.. Abramov. V.M.. Galactionav. V.G.. Isacv. IS.. Kaurov. O.A.. Kozhich. A.T.. Maiorov. V.A.. Prusakov. A.N.. Vilsilenkc. R.N. and Volodina, E.Yu. (1991) FEBS Lctt. 278. 187-185. [6! Danilkovich. A.V.. Frczc. K.V.. Shcvalicr. A.F., Samukov. V.V.. Kirkin. A.F. and Guscv. M.V. (1991) Immunol. Lctt. (in press). 171 Shcvalicr. A.F.. Samokov. V.V., Ofiwrov, V.I., Kalashnikov. V.. Mizcnko. G.A. and Kolokollsov. A.A. (1990) Bioorg. Khim. (USSR) 16.916-925. [8] Salacinski. R.R.P.. McLean. C.. Skycs. J.E.C.. Clcmcnt-Jones. W. and Lowry, P.J. (1981) Anal. Biochcm. 117. 136-146. [9J Boyum. A. (1968) Stand. J. Clin. Insesl. 21. suppi. 97. [IO] Aman, I’., Ehlin-Henriksson. B.. Klein. G. (1984) J. Esp. Med. 159. XX. [I I] Lnngcr. J.A. and Pcstka. S. (1986) Mclhods Enzymol. 119. 305 312. [I?] Doolcy, J.B.. Vergalla. J.. Hoofnag1c.J.H.. Zoon. KC.. Munson. P.J. and Jones. E.A. (1989) J. Lab. Clin. Med. I 13. 613-63 I. [I31 Shal%rman. A.. Vclan. B.. Cohen. S.. Leimcr. M. and Grosfeld. Ii. (1987) .I. Biol. Chcm. 262. 6227-6237. [l4] Branca. A.A. and Baglioni, C. (1982) J. Biol. Chcm. 257. 1319713100. [IS] Zoon, K.C.. Zur Ncddcn. D.. Hu. R. and Arnhcitcr, H. (1986) J. Biol. Chcm. 261. 4993-4996.