- Email: [email protected]
NK-lysin, structure and function of a novel effector molecule of porcine T and NK cells
Veterinay
immunology and immunopathology
Veterinary Immunology and Immunopathology 54(1996)
12X-126
NK-lysin, structure and function of a novel effector molecule of porcine T and NK cells M. Andersson ‘, H. Gunne a, B. Agerberth a7b,A. Boman a, T. Bergman b, B. Olsson ‘, A. Dagerlind d, H. Wigzell ‘, H. G. Boman a, G. H. Gudmundsson bS* a Depurtment c?f‘Microhiolr)gy.Stockholm University. S-106 91. Stockhdm. Swedrn ’ Depurtmmts ofMalicu1 Biochemistry und Biophysics. Karolinsku Instituter. S-l 71 77. Stockholm. Sweden ’ Microbiology
and Tumour Biology Center, Kordmska Instituter. S-171 77. Stockholm. Sweden d Neuroscience. Karo1in.sX-aInstitutet. S-171 77, Stockholm. Sweden
Abstract NK-lysin (NKL), a 7%residue antimicrobial peptide, was isolated from pig small intestine. Standard methods identified the peptide as basic, with six half-cystine residues in three intrachain disulphide bonds. The sequence showed 33% identity with a part of a putative gene product (NKGS) from activated 1‘ and NK cells. NK-lysin showed antibacterial activity against Escherichia coli and Bacillus megaterium and marked lytic activity against YAC-I, a NK sensitive tumour cell line, while sheep red blood cells were unaffected. The cDNA clone corresponding to NK-lysin has been characterized. We have also analyzed the cell and tissue specific expression and the induction of the gene. A lymphocyte fraction enriched in T and NK cells, stimulated by human interleukin-2 (IL-2). showed a 30-fold increase of the NKL transcript. NK-lysin specific mRNA is also detectable in spleen, bone marrow and colon. Immunostaining showed NKL to be present in different types of lymphocytes. Our results strongly suggest that NK-lysin is involved in the inducible cytotoxicity of T and NK cells. Keywrds:
Antibacterial
peptide; Cytotoxic peptide; cDNA
cloning; IL-2 induction
1. Introduction
Antibacterial peptides are considered to be an important part of the innate immunity of mammals (Boman. 1995) and have been isolated from the small intestine of pig
1 Corresponding
author at: Microbiology
Stockholm. Sweden. Tel: 46-8-7286224;
0165.2427/96/515.00
Copyright 6
/=‘/I SO1652427(96)05677-2
and Tumour
Fax: 46-8-328878:
Biology
Center,
Karolinska
Institutet.
S-17177,
e-mail: [email protected].
1996 Elsevier Science B.V. All rights reserved.
(Agerberth et al.. 1991) and mouse (Selated et al.. 1992). while genes for such peptides have also been found in mouse (Huttner et al.. 1994) and in human (Jones and Bevins. 1992). The intestine is not only an organ with a rich supply of blood vessels. but its epithelium is also a major site for T lymphopotesis and contains the largest collection of T cells in the body (Poussier and Julius. 1994). The cytotoxic T and NK cells. together with neutrophils and macrophages, are the primary effector cells of innate immunity and some (or all) of these cell types contain granules with peptides that are of importance in innate immunity. We now describe the structure and properties of a novel 78-residue peptide. NK-lysin. which most likely is a component of the cytotoxic machinery of T and NK cells. This peptide was isolated from porcine intestinal tissue on the basis of its antibacterial activity. The spectrum of this acttvity is narrow. while a strong lytic activity was observed against an NK-sensitive tumour cell line of murine origin.
2. NIL-lysin; peptide characterization
and activity
Gel filtration of a crude peptide fraction obtained from a concentrate of rhemtostabile intestinal polypeptides (CTIP) gave fractions with anti-E. c~li activity. The pooled antibacterial material was lyophilized (600 mg). Further purification included two reverse phase HPLC steps both with ;I linear gradient of acetonitrile. but with different counter-ions. The first step was with trifluoroacetic ac=id. while heptafluorobutyric acid was used in the last step. The change of counter-ion in the last step resolved the antibacterial activity into two main components. NKL and des-Glyl NKL, which were sequenced (Andersson et al.. 10%). Sequence data from intact NK-lysin and fragments generated enzymatically or chemically (by CNBr) gave the entire 7X-residue amino acid sequence. The molecular mass X923.2 f 1 determined by electrospray mass spectrometry is in good agreement with the value of 8924.8 calculated from sequence data (assuming that the half-cystines form three intramolecular S-S bonds). Similarly. total composition after hydrolysis of NKL in the native state and after oxidation by per-formic acid gave results in agreement with the sequence data. Mass spectrometric analysis of des-Gly I NKL gave a molecular mass of 8866.3 + I, or 56.9 mass units less than for NKL. which is close to 57.2. the mass of the missing glycinc restdue. Edman degradation of the two peptides gave identical sequences except for an N-terminal glycine (missing in des-Glyl NK-lysin). The peptide without glycine could originate from :I difference in N-terminal processing or. more likely. from cxopeptitlasc tlepradation of NKI> during isolation. For determination of the disulphide bridges. NK-lysin WIS digested enzymatically and three fragments containing half-cystinr were analyzed by Edman degradation. Results showed in each case two sequences. suggesting a c’ys4Cys76 bond. Cys7-Cys70 bond and Cys35-Cys45 bond. The antibacterial acttvity for NK-lysirt was Investigated on several Gram and Gram bacteria strains. The result only showed good activity against two sensitive strains ( E. cvli D2 I and B. r,rc:yclrcriu~r Bm I I ).
M. Andersson
et tit./
Veterinury
Immunology
und Immunopatholo~y
54 (1996)
123-126
125
Searching the SwissProt data base revealed that the sequence of NK-lysin showed similarities to the C-terminal portions of two almost identical proteins, 519 and NKGS, both predicted but never isolated and with unknown functions. They were deduced from cDNA clones isolated from activated human T and NK cells, respectively (Jongstra et al., 1987; Yabe et al., 1990). The C-terminal region of 519 and NKGS both showed about 33% identity with NK-lysin (using program BESTFIT, GCG). Since some antibacterial peptides are known to be also cytotoxic and peptides related to NK-lysin are expressed in T and NK cells, we investigated the possible involvement of NK-lysin in cytotoxicity. A cell line from mouse (YAC-1) known to be sensitive to the lytic activity of NK cells gave 90% lysis at a concentration of 50 pg NK-lysin ml-‘, while no lytic activity was detected with sheep red cells.
3. NK-lysin;
cloning and localization
The cloning strategy was based on a 3’RACE-PCR (Frohman et al., 1988) using RNA from bone marrow as starting material. This approach gave a fragment containing the coding information for the C-terminal part of the peptide. The fragment was then used as a probe to screen a cDNA library, which we made from porcine bone marrow. The sequence of the isolated clones revealed a homology of the precursor sequences between NK-lysin and the two predicted human proteins NKGS and 519. Furthermore, the clone served as an important tool to analyze the tissue specific expression by Northern blot analyses. We analyzed a lymphocyte fraction enriched in T and NK cells and a part of the fraction was stimulated with human interleukin-2 (IL-2). A 30-fold increase in NK-lysin specific mRNA was seen after this stimulation. In addition, the transcript was shown to be present in small intestine, colon, peripheral blood, lymph nodes and in spleen, tissues known to contain many lymphocytes. Immunostaining with two antibodies, one polyclonal directed to NK-lysin and the other monoclonal directed to cell specific surface proteins (CD2, CD4 and CD8) showed NK-lysin to be present in intracellular granules of CD2+, CD4+ and CD8+ cells. These results are taken to mean that NK-lysin is synthesized in cytotoxic T cells and probably also in NK cells.
4. Discussion The details of cytotoxic mechanisms have been a matter of extensive research and debate. Two effector mechanism have been suggested for cytotoxic lymphocytes. The first one involves the secretion of membranolytic molecules (perforin and granzymes), initiated after contact with the target cell. The other mechanism is mediated through the APO-l/Fas receptor and triggers apoptosis in the target cell. However, the lytic machinery of cytolytic lymphocytes is probably more diverse than previously assumed (Berke, 1994). In addition to the above mentioned mechanisms we suggest that peptides like NK-lysin are included in the cytotoxic reactions of T and NK cells.
References Agerberth, B.. Lee, J.-Y.. Bergman, T.. Carlquist, M.. Boman, H.G., Mutt, V. and JGmvall. H., 1991. Eur. J Biochem., 202: 849. Andersson. M.. Gunne, H.. Agerberth. B.. Boman. A.. Bergman. T., Sillard, R.. Jijmvall. H., Mutt, V., Olsson B., Wigzell.
H.. Dagerlind, A.. Boman. H.G. and Gudmundsson. G.H..
Be&e. G.. 1994. Ann. Rev. Immunol.. Boman. H.G., Frohman. M.A.. Huttner, K.M..
1995. Annu. Rev. Immunol.. Dush, M.K.
1988. Proc. Natl. Acad. Sci. USA, X5: 8998.
Selsted, M.E. and Ouellette, A.J., 1994. Genomlcs, 1992. J. Biol. Chem.. 267
19: 448.
23216.
Jongstra. J., Schall. T.J.. Dyer. B.J., Clayberper. C.. Jorgensen. J., Davis, M.M. Exp. Med.,
and Krensky. A.M.,
165: 601.
Poussier, P. and Julius. M., Selsted. M.E.,
J., IS: 1615.
13,6 I.
and Martin, G.R.,
Jones, D.E. and Bevins. C.L..
1995. EMBO
I?: 735.
1994, Annu. Rev. Immunol..
Miller, S.I., Henschen, A.H. and Ouellrtte.
Yabe. T.. McSherry.
17 521. .A.J., 1992. 1. Cell. Biol.,
C.. Bach, F.H. and Houchins, J.P.. 1990. J. Exp. Med.,
I IX. 979.
172. 1159.
1987. J
immunology and immunopathology
Veterinary Immunology and Immunopathology 54(1996)
12X-126
NK-lysin, structure and function of a novel effector molecule of porcine T and NK cells M. Andersson ‘, H. Gunne a, B. Agerberth a7b,A. Boman a, T. Bergman b, B. Olsson ‘, A. Dagerlind d, H. Wigzell ‘, H. G. Boman a, G. H. Gudmundsson bS* a Depurtment c?f‘Microhiolr)gy.Stockholm University. S-106 91. Stockhdm. Swedrn ’ Depurtmmts ofMalicu1 Biochemistry und Biophysics. Karolinsku Instituter. S-l 71 77. Stockholm. Sweden ’ Microbiology
and Tumour Biology Center, Kordmska Instituter. S-171 77. Stockholm. Sweden d Neuroscience. Karo1in.sX-aInstitutet. S-171 77, Stockholm. Sweden
Abstract NK-lysin (NKL), a 7%residue antimicrobial peptide, was isolated from pig small intestine. Standard methods identified the peptide as basic, with six half-cystine residues in three intrachain disulphide bonds. The sequence showed 33% identity with a part of a putative gene product (NKGS) from activated 1‘ and NK cells. NK-lysin showed antibacterial activity against Escherichia coli and Bacillus megaterium and marked lytic activity against YAC-I, a NK sensitive tumour cell line, while sheep red blood cells were unaffected. The cDNA clone corresponding to NK-lysin has been characterized. We have also analyzed the cell and tissue specific expression and the induction of the gene. A lymphocyte fraction enriched in T and NK cells, stimulated by human interleukin-2 (IL-2). showed a 30-fold increase of the NKL transcript. NK-lysin specific mRNA is also detectable in spleen, bone marrow and colon. Immunostaining showed NKL to be present in different types of lymphocytes. Our results strongly suggest that NK-lysin is involved in the inducible cytotoxicity of T and NK cells. Keywrds:
Antibacterial
peptide; Cytotoxic peptide; cDNA
cloning; IL-2 induction
1. Introduction
Antibacterial peptides are considered to be an important part of the innate immunity of mammals (Boman. 1995) and have been isolated from the small intestine of pig
1 Corresponding
author at: Microbiology
Stockholm. Sweden. Tel: 46-8-7286224;
0165.2427/96/515.00
Copyright 6
/=‘/I SO1652427(96)05677-2
and Tumour
Fax: 46-8-328878:
Biology
Center,
Karolinska
Institutet.
S-17177,
e-mail: [email protected].
1996 Elsevier Science B.V. All rights reserved.
(Agerberth et al.. 1991) and mouse (Selated et al.. 1992). while genes for such peptides have also been found in mouse (Huttner et al.. 1994) and in human (Jones and Bevins. 1992). The intestine is not only an organ with a rich supply of blood vessels. but its epithelium is also a major site for T lymphopotesis and contains the largest collection of T cells in the body (Poussier and Julius. 1994). The cytotoxic T and NK cells. together with neutrophils and macrophages, are the primary effector cells of innate immunity and some (or all) of these cell types contain granules with peptides that are of importance in innate immunity. We now describe the structure and properties of a novel 78-residue peptide. NK-lysin. which most likely is a component of the cytotoxic machinery of T and NK cells. This peptide was isolated from porcine intestinal tissue on the basis of its antibacterial activity. The spectrum of this acttvity is narrow. while a strong lytic activity was observed against an NK-sensitive tumour cell line of murine origin.
2. NIL-lysin; peptide characterization
and activity
Gel filtration of a crude peptide fraction obtained from a concentrate of rhemtostabile intestinal polypeptides (CTIP) gave fractions with anti-E. c~li activity. The pooled antibacterial material was lyophilized (600 mg). Further purification included two reverse phase HPLC steps both with ;I linear gradient of acetonitrile. but with different counter-ions. The first step was with trifluoroacetic ac=id. while heptafluorobutyric acid was used in the last step. The change of counter-ion in the last step resolved the antibacterial activity into two main components. NKL and des-Glyl NKL, which were sequenced (Andersson et al.. 10%). Sequence data from intact NK-lysin and fragments generated enzymatically or chemically (by CNBr) gave the entire 7X-residue amino acid sequence. The molecular mass X923.2 f 1 determined by electrospray mass spectrometry is in good agreement with the value of 8924.8 calculated from sequence data (assuming that the half-cystines form three intramolecular S-S bonds). Similarly. total composition after hydrolysis of NKL in the native state and after oxidation by per-formic acid gave results in agreement with the sequence data. Mass spectrometric analysis of des-Gly I NKL gave a molecular mass of 8866.3 + I, or 56.9 mass units less than for NKL. which is close to 57.2. the mass of the missing glycinc restdue. Edman degradation of the two peptides gave identical sequences except for an N-terminal glycine (missing in des-Glyl NK-lysin). The peptide without glycine could originate from :I difference in N-terminal processing or. more likely. from cxopeptitlasc tlepradation of NKI> during isolation. For determination of the disulphide bridges. NK-lysin WIS digested enzymatically and three fragments containing half-cystinr were analyzed by Edman degradation. Results showed in each case two sequences. suggesting a c’ys4Cys76 bond. Cys7-Cys70 bond and Cys35-Cys45 bond. The antibacterial acttvity for NK-lysirt was Investigated on several Gram and Gram bacteria strains. The result only showed good activity against two sensitive strains ( E. cvli D2 I and B. r,rc:yclrcriu~r Bm I I ).
M. Andersson
et tit./
Veterinury
Immunology
und Immunopatholo~y
54 (1996)
123-126
125
Searching the SwissProt data base revealed that the sequence of NK-lysin showed similarities to the C-terminal portions of two almost identical proteins, 519 and NKGS, both predicted but never isolated and with unknown functions. They were deduced from cDNA clones isolated from activated human T and NK cells, respectively (Jongstra et al., 1987; Yabe et al., 1990). The C-terminal region of 519 and NKGS both showed about 33% identity with NK-lysin (using program BESTFIT, GCG). Since some antibacterial peptides are known to be also cytotoxic and peptides related to NK-lysin are expressed in T and NK cells, we investigated the possible involvement of NK-lysin in cytotoxicity. A cell line from mouse (YAC-1) known to be sensitive to the lytic activity of NK cells gave 90% lysis at a concentration of 50 pg NK-lysin ml-‘, while no lytic activity was detected with sheep red cells.
3. NK-lysin;
cloning and localization
The cloning strategy was based on a 3’RACE-PCR (Frohman et al., 1988) using RNA from bone marrow as starting material. This approach gave a fragment containing the coding information for the C-terminal part of the peptide. The fragment was then used as a probe to screen a cDNA library, which we made from porcine bone marrow. The sequence of the isolated clones revealed a homology of the precursor sequences between NK-lysin and the two predicted human proteins NKGS and 519. Furthermore, the clone served as an important tool to analyze the tissue specific expression by Northern blot analyses. We analyzed a lymphocyte fraction enriched in T and NK cells and a part of the fraction was stimulated with human interleukin-2 (IL-2). A 30-fold increase in NK-lysin specific mRNA was seen after this stimulation. In addition, the transcript was shown to be present in small intestine, colon, peripheral blood, lymph nodes and in spleen, tissues known to contain many lymphocytes. Immunostaining with two antibodies, one polyclonal directed to NK-lysin and the other monoclonal directed to cell specific surface proteins (CD2, CD4 and CD8) showed NK-lysin to be present in intracellular granules of CD2+, CD4+ and CD8+ cells. These results are taken to mean that NK-lysin is synthesized in cytotoxic T cells and probably also in NK cells.
4. Discussion The details of cytotoxic mechanisms have been a matter of extensive research and debate. Two effector mechanism have been suggested for cytotoxic lymphocytes. The first one involves the secretion of membranolytic molecules (perforin and granzymes), initiated after contact with the target cell. The other mechanism is mediated through the APO-l/Fas receptor and triggers apoptosis in the target cell. However, the lytic machinery of cytolytic lymphocytes is probably more diverse than previously assumed (Berke, 1994). In addition to the above mentioned mechanisms we suggest that peptides like NK-lysin are included in the cytotoxic reactions of T and NK cells.
References Agerberth, B.. Lee, J.-Y.. Bergman, T.. Carlquist, M.. Boman, H.G., Mutt, V. and JGmvall. H., 1991. Eur. J Biochem., 202: 849. Andersson. M.. Gunne, H.. Agerberth. B.. Boman. A.. Bergman. T., Sillard, R.. Jijmvall. H., Mutt, V., Olsson B., Wigzell.
H.. Dagerlind, A.. Boman. H.G. and Gudmundsson. G.H..
Be&e. G.. 1994. Ann. Rev. Immunol.. Boman. H.G., Frohman. M.A.. Huttner, K.M..
1995. Annu. Rev. Immunol.. Dush, M.K.
1988. Proc. Natl. Acad. Sci. USA, X5: 8998.
Selsted, M.E. and Ouellette, A.J., 1994. Genomlcs, 1992. J. Biol. Chem.. 267
19: 448.
23216.
Jongstra. J., Schall. T.J.. Dyer. B.J., Clayberper. C.. Jorgensen. J., Davis, M.M. Exp. Med.,
and Krensky. A.M.,
165: 601.
Poussier, P. and Julius. M., Selsted. M.E.,
J., IS: 1615.
13,6 I.
and Martin, G.R.,
Jones, D.E. and Bevins. C.L..
1995. EMBO
I?: 735.
1994, Annu. Rev. Immunol..
Miller, S.I., Henschen, A.H. and Ouellrtte.
Yabe. T.. McSherry.
17 521. .A.J., 1992. 1. Cell. Biol.,
C.. Bach, F.H. and Houchins, J.P.. 1990. J. Exp. Med.,
I IX. 979.
172. 1159.
1987. J