Immunology Today, vol. 8, Nos 7and8, 1987
Effectors of the Immune Pesponse(Podack, E.R.,ed.), CRC Press(in press) 14 Cole, J.L., Housley,G.A., Dykrnan,T.R.etal. (1985) Proc. Natl Acad. Sci. USA 82, 859-863 15 Seya,T. etal. (1986)J. Exp. Med. 163, 837-855 16 Yu, G. etal. (1986)J. Clin. Invest. 78, 494-501 17 Medof, M.E., Walter, E.I., Rutgers, J.L. (1987)J. Exp. Med. 165, 848-864 18 Atkinson, J.P.etal. (1987) Fed. Proc. 46, 612 19 Rosse,W.F. and Parker,C.J. (1984) Clin. Haematol. 14, 105--112
20 Pangburn,M.K. etal. (1983)Proc. NatlAcad. Sci. USA 80, 5430-5434 21 Nicholson-Weller,A. et al. ~1983)Proc.Natl Acad. Sci. USA 80, 5066-5070 22 Kinoshita,T. etaL (1985)J. Exp.Med. 162, 75-92 23 Nicholson-Weller,A., Spicer, D.B.and Austen, K.F.(1985) New Engl.J. Med. 312, 1091-1097 24 Medof, M.E., Kinoshita,T., Silber,R. (1985)Proc. NatlAcad. Sci. USA 82, 2980-2984 25 Fries,LF., Friedman,H.M., Cohen,G.H. etal. (1986)J. Imrnunol. 137, 1636-1641
The 65kDa antigen of mycobacteria- a •common baderial protein? The 65 kilodalton antigen of Mycobacterium tuberculosisand M. leprae is a well-characterized, strongly immunogenic protein eliciting antibody and T-cell responses in infected patients. Recent studies have disclosed regions of cross-reactivity between the 65kDa antigen and proteins in many other bacterial species. These include the product of the ams gene in E. coil which b involved in the processing of RNA. Here Douglas Young and his colleagues discuss these observations, the significance of the 65kDa antigen and its possible role in the pathogenesis of mycobacterial and other diseases. The challenge of understanding the mechanisms underlying the immune response to mycobacteria has be..1 taken up by successive generations of scientists and renewed efforts are currently underway to use improved _ _ _ ~ L -- - ! _ Jr . . . . . I • rnutrluu.5 Tur analy$15 of mycobacteriai antigens and for isolation and characterization of T cells. The effective immune response is thought to involve recognition of mycobacterial antigens by helper T lymphocytes, followed by activation of macrophages and perhaps st~muiation or cytotoxic T cells. A key area of research is therefore to identify mycobacterial antigens which are involved in recognition by T cells. A major advance in the identification of mycobacterial protein antigens was accomplished by monoclona! antibodies directed to components from iviycobac~erium tuberculosis and Mycobacterium leprae 1. One of the antigens which was identified by a high proportion of monoclonal antibodies - particularly those raised against M. leprae - has been generally referred to as the '65kDa antigen'. Monoclonal antibodies directed to this antigen frequently bind to bands with multiple molecular weights during western blotting of extracts from M. tuberculosis and M. leprae, although this banding pattern seems to be less marked in the case of more rapidly growing mycobacteria 2. The most likely origin of the multiple banding is that an original protein of about 65 kDa undergoes progressive proteolytic degradation to multiple fragments each containing a subset of the total antibody binding sites of the full length molecule.
MRC Tuberculosisand Related Infections Unit, Royal Postgraduate Medical School, HammersmithHospital, DucaneRoad,London W12 OHS,UK ~9 8 7 . Elsevier Publications, C a m b r i d g e
0167 - 4919/87/$02.00
Douglase. Young,JurajIvanyi,JosephineH. CoxandJonathanR. Lamb Monoclonal antibodies recognizing epitopes situated towards the N terminus of the molecule (for example, Y1.2 - see Fig. 2) show very limited recognition of low molecular weight bands and this would be consistent with an initial site of proteolysis being located near to the N terminus. Antibodies binding in the C terminal, region (e.g. IIIC8) do show extensive 'mui[i-banding. The 65kDa antigen h~s been described as 'cell-wallassociated'2 since much of the antigen is found in the insoluble fraGion remaining after disruption of the mycobacterial cells. Other authors have proposed a 'periplasmic' location for this antigen and have demonstrated release of the molecule into culture supernatants during growth of Mycobacterium boris under conditions of zinc deficiency3. Since the amino acid sequence of the protein (see below) contains no strikingly hydrophobic regions, a close association of such a molecule with the lipid-rich cell wall of mycobacteria seems unlikely. While irnmuno-electron microscopy may be expected to provide definitive information on the location of the 65kDa antigen, an interaction with ribosomes would be consistent with some of the data discussed below. Cloning of the 65kDa antigen
Most of the mycobacterial antigens recognized by monoc!onal antibodies have been expressed from M. leprae and M. tuberculosis genomic libraries using the ~gtl 1 system4.5. Recombinant clones expressing all, or portions, of the 65kDa antigen were found at a particularly high frequency. It has been shown that the gene for the 65kDa protein can be expressed in E. coil regardless of its orientation with respect to promoters on the cloning vector, suggesting that, in contrast with some other mycobacterial enzymes6, the mycobacterial promoter for this gene can be recognized by E. coh7-g. The proteins from M. leprae and M. tuberculosis contain a striking degree of sequence homology, differing in only 2~ amino acid substitutions8.9. From the primary sequence it can be predicted that the 65kDa antigen contains extensive r,-helical secondary structure with no n:ar','ed hydrophobic regions and no cysteine residues.
215
Imrnunoiogy Today, voL 8, Nos 7 and 8, 1987
residue no.
reagent used for identification
N
I _
7~ ~,.~
100 .
P2 antibody II H9
Z00
i ams
"lpq
300 _ q00
antibodiesC!.1 and YI.2 Tcell clone P77
~
antibodyDTC
J~ ,
,
S00- ,=1~,, IbfJ
--I'P6 polyclonal T cells antibodyIII E9 antibody II C8 antibodyT2.3 antibody III C8
F~ 1. Locationof antigenicdeterminantson the 65kDaprotein. The 65kDaantigen of M. lepraeis shown with amino acidsnumberedfrom the which has been shown to be the N terminal residue of the protein in ~ . The ;osition of the antibody t~'ndingsites (hatched boxes) was determined by .qJblibrary mapping8. T-cell determinants (solid boxes) were iden~ed by sublibranj mapping and predictive sequence_ana~r~isI~, Areas ~ n g to syntheticpep6~bscontainingpredicted T
Effectors of the Immune Pesponse(Podack, E.R.,ed.), CRC Press(in press) 14 Cole, J.L., Housley,G.A., Dykrnan,T.R.etal. (1985) Proc. Natl Acad. Sci. USA 82, 859-863 15 Seya,T. etal. (1986)J. Exp. Med. 163, 837-855 16 Yu, G. etal. (1986)J. Clin. Invest. 78, 494-501 17 Medof, M.E., Walter, E.I., Rutgers, J.L. (1987)J. Exp. Med. 165, 848-864 18 Atkinson, J.P.etal. (1987) Fed. Proc. 46, 612 19 Rosse,W.F. and Parker,C.J. (1984) Clin. Haematol. 14, 105--112
20 Pangburn,M.K. etal. (1983)Proc. NatlAcad. Sci. USA 80, 5430-5434 21 Nicholson-Weller,A. et al. ~1983)Proc.Natl Acad. Sci. USA 80, 5066-5070 22 Kinoshita,T. etaL (1985)J. Exp.Med. 162, 75-92 23 Nicholson-Weller,A., Spicer, D.B.and Austen, K.F.(1985) New Engl.J. Med. 312, 1091-1097 24 Medof, M.E., Kinoshita,T., Silber,R. (1985)Proc. NatlAcad. Sci. USA 82, 2980-2984 25 Fries,LF., Friedman,H.M., Cohen,G.H. etal. (1986)J. Imrnunol. 137, 1636-1641
The 65kDa antigen of mycobacteria- a •common baderial protein? The 65 kilodalton antigen of Mycobacterium tuberculosisand M. leprae is a well-characterized, strongly immunogenic protein eliciting antibody and T-cell responses in infected patients. Recent studies have disclosed regions of cross-reactivity between the 65kDa antigen and proteins in many other bacterial species. These include the product of the ams gene in E. coil which b involved in the processing of RNA. Here Douglas Young and his colleagues discuss these observations, the significance of the 65kDa antigen and its possible role in the pathogenesis of mycobacterial and other diseases. The challenge of understanding the mechanisms underlying the immune response to mycobacteria has be..1 taken up by successive generations of scientists and renewed efforts are currently underway to use improved _ _ _ ~ L -- - ! _ Jr . . . . . I • rnutrluu.5 Tur analy$15 of mycobacteriai antigens and for isolation and characterization of T cells. The effective immune response is thought to involve recognition of mycobacterial antigens by helper T lymphocytes, followed by activation of macrophages and perhaps st~muiation or cytotoxic T cells. A key area of research is therefore to identify mycobacterial antigens which are involved in recognition by T cells. A major advance in the identification of mycobacterial protein antigens was accomplished by monoclona! antibodies directed to components from iviycobac~erium tuberculosis and Mycobacterium leprae 1. One of the antigens which was identified by a high proportion of monoclonal antibodies - particularly those raised against M. leprae - has been generally referred to as the '65kDa antigen'. Monoclonal antibodies directed to this antigen frequently bind to bands with multiple molecular weights during western blotting of extracts from M. tuberculosis and M. leprae, although this banding pattern seems to be less marked in the case of more rapidly growing mycobacteria 2. The most likely origin of the multiple banding is that an original protein of about 65 kDa undergoes progressive proteolytic degradation to multiple fragments each containing a subset of the total antibody binding sites of the full length molecule.
MRC Tuberculosisand Related Infections Unit, Royal Postgraduate Medical School, HammersmithHospital, DucaneRoad,London W12 OHS,UK ~9 8 7 . Elsevier Publications, C a m b r i d g e
0167 - 4919/87/$02.00
Douglase. Young,JurajIvanyi,JosephineH. CoxandJonathanR. Lamb Monoclonal antibodies recognizing epitopes situated towards the N terminus of the molecule (for example, Y1.2 - see Fig. 2) show very limited recognition of low molecular weight bands and this would be consistent with an initial site of proteolysis being located near to the N terminus. Antibodies binding in the C terminal, region (e.g. IIIC8) do show extensive 'mui[i-banding. The 65kDa antigen h~s been described as 'cell-wallassociated'2 since much of the antigen is found in the insoluble fraGion remaining after disruption of the mycobacterial cells. Other authors have proposed a 'periplasmic' location for this antigen and have demonstrated release of the molecule into culture supernatants during growth of Mycobacterium boris under conditions of zinc deficiency3. Since the amino acid sequence of the protein (see below) contains no strikingly hydrophobic regions, a close association of such a molecule with the lipid-rich cell wall of mycobacteria seems unlikely. While irnmuno-electron microscopy may be expected to provide definitive information on the location of the 65kDa antigen, an interaction with ribosomes would be consistent with some of the data discussed below. Cloning of the 65kDa antigen
Most of the mycobacterial antigens recognized by monoc!onal antibodies have been expressed from M. leprae and M. tuberculosis genomic libraries using the ~gtl 1 system4.5. Recombinant clones expressing all, or portions, of the 65kDa antigen were found at a particularly high frequency. It has been shown that the gene for the 65kDa protein can be expressed in E. coil regardless of its orientation with respect to promoters on the cloning vector, suggesting that, in contrast with some other mycobacterial enzymes6, the mycobacterial promoter for this gene can be recognized by E. coh7-g. The proteins from M. leprae and M. tuberculosis contain a striking degree of sequence homology, differing in only 2~ amino acid substitutions8.9. From the primary sequence it can be predicted that the 65kDa antigen contains extensive r,-helical secondary structure with no n:ar','ed hydrophobic regions and no cysteine residues.
215
Imrnunoiogy Today, voL 8, Nos 7 and 8, 1987
residue no.
reagent used for identification
N
I _
7~ ~,.~
100 .
P2 antibody II H9
Z00
i ams
"lpq
300 _ q00
antibodiesC!.1 and YI.2 Tcell clone P77
~
antibodyDTC
J~ ,
,
S00- ,=1~,, IbfJ
--I'P6 polyclonal T cells antibodyIII E9 antibody II C8 antibodyT2.3 antibody III C8
F~ 1. Locationof antigenicdeterminantson the 65kDaprotein. The 65kDaantigen of M. lepraeis shown with amino acidsnumberedfrom the which has been shown to be the N terminal residue of the protein in ~ . The ;osition of the antibody t~'ndingsites (hatched boxes) was determined by .qJblibrary mapping8. T-cell determinants (solid boxes) were iden~ed by sublibranj mapping and predictive sequence_ana~r~isI~, Areas ~ n g to syntheticpep6~bscontainingpredicted T
