I05
609th MEETING, LEEDS
trypsin- and ribonuclease-sensitive cytoplasmic antigen
(Mattioli & Reichlin, 1974), though more recent studies
suggest it is located in the nucleus, giving a speckled nuclear
staining pattern with sparing of the nucleoli by immunofluorescence (Alspaugh et al., 1976). Th'e antigen has been
shown to be a phosphorylated protein of M, 50000 (Stefano,
1984), which is sensitive to degradation giving polypeptides
of 40000 and 29000 M , (Venables et al., 1983a).
Immunoprecipitation of 32P-labelled cell extracts with
human anti-La sera has shown that the La protein binds a
number of small RNA species (Lerner et al., 1981) including
some Epstein-Barr- and Adenovirally encoded species. All
these RNA species are transcripts of RNA polymerase 111,
which has led to the suggestion that La may be a
transcription factor associated with RN A polymerase 111.
All studies on the structure and function of La have been
performed with human sera. Further studies are hindered
by the presence of other autoantibodies in these sera. Our
aim in producing monoclonal antibodies was to use these
reagents to further characterize the structure and function
of the La ribonucleoprotein particle.
Unlike other autoantibody systems, :such as anti-DNA
and anti-Sm antibodies, there is no naturally occurring
murine model which spontaneously produces autoantibodies to La. To isolate antigen for immunization we used
immunoaffinity chromatography with human anti-La antibodies to purify the antigen from a commercially available
acetone extract of rabbit thymus (Venables et al., 1983b).
This material was also used in an e.1.i.s.a. to detect anti-La
antibody production
Balb/C mice were immunized with MOpg of purified La
at 0,2 and 4 weeks, using complete Freunds adjuvant for the
first, and incomplete for all subsequent injections. Antigen
was administered intramuscularly and intraperitoneally
each time. Spleen cells were fused with PIIS-1 myeloma cells
and supernatants assayed by e.1.i.s.a. ; positive wells were
then cloned by limiting dilution and used to generate ascitic
fluids. Using this procedure three anti-La secreting lines,
designated SWI, SW3 and SW5, were established which
were positive in anti-La e.1.i.s.a. to a dilution of 1 :1000000
and did not react in assays for anti-RNP and anti-Ro antibodies. Immunodiffusion showed that all three monoclonals
were immunoglobulin G2b class antibodies.
On immunoblotting, all three lines recognized a single
45000-M, polypeptide in rabbit thymus extract identical
with that identified by human autoimmune sera. When the
monoclonals were used to probe a number of human and
mouse cell lines all reacted with a 48000-Mr polypeptide in
the human lines Raji, Wi-L2 and Molt-4. In contrast, only
monoclonal SW5 recognized La in mouse cells although
SW3 reacted with an as yet unidentified 56000-Mr polypeptide in all cell types analysed. These results show that the
monoclonals recognize different epitopes on the La protein.
To investigate the relationship of the La antigen to other
polypeptides, the monoclonals were coupled to Sepharose
beads to isolate the La ribonucleoprotein particle. Using
rabbit thymus as an antigen source, this approach has
allowed us to purify the 45000-Mr La polypeptide, but as yet
has not demonstrated co-purification of any other
polypeptides.
Another major use of well-defined monoclonal antinuclear antibodies is in the analysis of the distribution and
function of the proteins bearing the antigens in the cell.
Although La is characteristically described as a nuclear
antigen, several workers report the occurrence of substantial quantities of antigen in cytoplasmic fractions
ascribed to leakage from the nucleus (Habets et al., 1983;
Stefano, 1984). We examined the effects of viral infection
and mitogen-induced transformation on the distribution of
La by immunofluorescence. This showed that all three
monoclonals gave similar staining patterns on Hep-2 cells,
giving the characteristic speckled nuclear fluorescence with
sparing of the nucleoli and little cytoplasmic staining. With
B95-8 and Raji cells, in addition to the characteristic
speckled staining there was an intense staining of the
nucleoli, which was also seen with mitogen-stimulated
human lymphocytes. The alteration in intracellular distribution after activation presumably reflects the binding of
La to the small RNA species associated with RNA
polymerase 111. The results obtained are similar to those
obtained by Deng et al. (1981) using synchronized Wi-L2
cells, where La was shown to be nucleoplasmic in all stages
of the cell cycle except for the late G1 and early S phases
when a pronounced nucleolar staining was seen.
Alspaugh, M. A,, Talal, N . & T a n , E. M. (1976) Arthritis Rheum.
19, 216-222
Deng, J. S.,Takasaki,Y.&Tan, E. M. (1981)J. CeNBiol.91,654660
Habets, W. J., den Brok, J. H., Boerbooms, A. M. von de Putte,
L. B. A. & van Venrooij, W. J. (1983) EMBO J . 2, 1625-1631
Kurata, N . & Tan, E. M. (1976) Arthritis Rheum. 19, 574-580
Lerner, M. R., Boyle, J. A., Hardin, J. A. & Steitz, J. A. (1981)
Science 211, 4 0 W 0 2
Mattioli, M. & Reichlin, M. (1974) Arthritis Rheum. 17, 421-429
Stefano, J. E. (1984) Cell 36, 145-154
Venables, P. J. W., Smith, P. R. & Maini, R. N. (1983~)Clin. Exp.
Immunol. 54, 731-738
Venables, P. J. W., Charles, P. C., Buchanan, R. R. C., Tung, Yi,
Mumford, P. A,, Schrieber, L., Room, G.R. W. & Maini, R. N .
(19836) Arthritis Rheum. 26, 143-155
Monoclonal antibodies and the structure of complement component C9
J . PAUL LUZIO,* PETER JACKSON,*
ANTHONY K . CAMPBELL,t B. PAUL MORGAN?
and KEITH K. STANLEY1
*Department of Clinical Biochemistry, h i v e r s i t y of
Cambridge, Addenbrooke's Hospital, Hills Road. Cambridge
C B 2 2 Q R , U.K . , tDepartment of Medical Biochemistry,
Welsh National School of Medicine, Healh Park, CardifJ
CF4 4 X N , U . K . ,and $European Molecu1,w Biology
Laboratory, Meyerhofstrasse 1. 10.2209, ,!I-6900 Heidelberg,
Federal Republic of Germany
Abbreviations used: MAC. membrane attack complex; SDS,
sodium dodecyl sulphate; cDNA. complementary DNA.
VOl. 13
C9 is a serum protein of M , 71 000 that acts as the final component of the complement MAC and is essential for
maximal rates of cell lysis. It is thought that the MAC is
formed by complement components C5b-8 catalysing polymerization of C 9 to form a hollow protein cylinder inserted
in the plasma membrane of the target cell (Tschopp et al.,
1982). There is controversy about the number of C9 molecules within the MAC, their orientation and the nature of
the membrane lesion caused.
In previous experiments we have shown that a rise in
intracellular free Ca2+concentration is a very early event
after C9 insertion into the MAC, is specific to the
incorporation of C9 and precedes the release of other ions
106
and macromolecules (Luzio et al., 1979; Campbell et al.,
1979, 1981). We have proposed that the initial rise in intracellular free Ca2+concentration mediates non-lytic effects
of complement and also contributes to the specificity of
membrane damage (Campbell & Luzio, 1981 ; Hallett et al.,
1981; Hallett & Campbell, 1982; Richardson & Luzio,
1980).
C9 is clinically important since it is involved in the
prevention of infection and also in autoimmune disease. It is
also a fascinating protein biochemically since it undergoes a
transformation from a hydrophilic serum protein to an
amphiphilic integral membrane protein in the MAC.
Monoclonal antibodies were prepared to investigate its
structure, fate on binding to the target membrane and
clinical function.
Five mouse monoclonal antibodies to human C9 were
prepared by standard methods (Galfre & Milstein, 1981) as
described by Morgan et al. (1983~)and coded as follows:
C9-8 (affinity 0.1 x 1 0 9 ~ - ’ ) ;C9-34 (0.3 x 1 0 9 ~ - ’ ) ;C9-36
(3.1 x 1 0 9 ~ - l )c9-42
;
(1.6 x 1 0 9 ~ - l ) c9-47
;
(2.6 x 1 0 9 ~ - 1 ) .
Epitope analysis showed that at least four distinct antigenic
sites on C9 were detected by these monoclonal antibodies
with C9-36 and C9-47 binding to the same or closely related
antigenic sites.
Different monoclonal antibodies were used to purify C9
(C9-8; Morgan et a/., 1983a), for immunoradiometric assay
(C9-34 and C9-47; Morgan et al., 19836, 1984a), for
immunofluorescence and to show internalization of cellsurface-bound C9. Immunolocalization of C9 on muscle
biopsy sections from patients with myositis by indirect
immunofluorescence with antibody C9-47 has shown the
presence of C9 on non-necrotic as well as necrotic muscle
fibres, suggesting a primary role of complement in the
pathogenesis of muscle fibre necrosis in myositis (Morgan et
ai., 19846). Measurement of the binding of antibody C9-47
to C9 inserted into the cell surface of rat polymorphonuclear
leucocytes has also been used to demonstrate internalization
of the MAC, which may be an important mechanism for the
recovery of nucleated cells from complement attack
(Morgan et al., 1984~).
Four of the monoclonal antibodies can be shown to bind
to C9 when added extracellularly to pigeon erythrocytes
containing MAC (Morgan et al., 19846). Three of these
(C9-36, C9-42 and C9-47) wiil inhibit release of marker
[ 14C]sucrose when added extracellularly to pigeon erythrocyte ‘ghosts’ containing MAC (Morgan et a[., 1984d). One
antibody (C9-34) which will not bind to the MAC when
added extracellularly inhibits complement-stimulated
marker release when re-sealed inside pigeon erythrocyte
‘ghosts’ (Morgan et al., 1984d). This has provided definitive
proof that C9 becomes a transmembrane protein in the
MAC and shows that one of the antigenic sites is expressed
on the inner membrane face after C9 insertion whereas the
others are expressed at the extracellular membrane surface.
Western blotting (Burnette, 1981) of C9 after reduction
and alkylation of the molecule followed by SDS/polyacrylamide-gel electrophoresis and electrophoretic transfer on to
nitrocellulose allowed recognition of C9 only by monoclonal
antibodies C9-42 and C9-47. C9-36 did not react in these
Western blots in spite of its binding to C9 being
indistinguishable from C9-47 by epitope analysis. Chemical
cleavage of C9 by 2-(2-nitrophenylsulphenyl)-3-methyl-3’bromoindolenine (‘BNPS-skatole’) which acts at tryptophan produced several smaller polypeptides detectable by
Coomassie Blue staining after SDS/polyacrylamide-gel
electrophoresis. Electrophoretic transfer of the stained
BIOCHEMICAL SOCIETY TRANSACTIONS
peptides to nitrocellulose (Jackson & Thompson, 1984) and
treatment with monoclonal antibody C9-47 followed by
peroxidase-labelled second antibody stained two smaller
fragments of M , 48000 and 38000. The ability to cleave C9
followed by immunodetection of fragments with monoclonal antibodies should, in combination with functional
inhibition experiments, allow dissection of those parts of
the molecule expressed at different sides of the membrane
after C9 insertion into the target cell.
Monoclonal antibody C9-47 has also been used to detect a
human liver cDNA coding for C9 cloned in a bacterial
expression vector (Stanley & Luzio, 1984). The expression
vector was one of a family, pEX 1-3, derived from a cro-lac Z
gene fusion plasmid, which expresses large quantities of
fusion protein that is insoluble and can be detected using an
immune ‘colony blot’ procedure (Stanley, 1983). Several
cDNA clones coding for C9 were detected with polyclonal
anti-C9 antibodies and one, clone 7, was also detected with
monoclonal antibody C9-47. Clone 7 contains a cDNA
insert of 1350 base pairs, which has been sequenced and
found to contain the C-terminal end of C9. The sequence
was confirmed by comparison with the known amino acid
composition and partial sequence of an a-thrombin cleavage product of C9 (Biesecker et al., 1982). Monoclonal
antibody C9-47 reacts with an epitope at the C-terminal end
of C9; however, examination of the primary sequence in
combination with the immunoblotting of cleavage fragments suggests this may not include the C-terminus itself.
The five monoclonal antibodies characterized, and
others, should prove of further value in analysing the folding
of C9 as it inserts into the MAC.
We thank the M.R.C., EMBO and the Arthritis and Rheumatism Council for support.
Biesecker, G . , Gerard, C . & Hugh, T. E. (1982)J. Biol. Chem 257,
2584-2590
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55-60
Campbell, A. K . , Daw, R. A,, Hallett, M . B. & Luzio, J. P. (1981)
Eiochem. J . 194, 551-560
Galfre, G . & Milstein, C . (1981) Methods Enzymol. 73, 3 4 6
Hallett, M. B. &Campbell, A . K . (1982) Nature(London)295,155158
Hallett, M. B., Luzio, J . P. & Campbell, A. K . (1981) Immunology
44, 569-576
Jackson, P. & Thompson, R. J . (1984) Electrophoresis 5 , 3 5 4 2
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A. K . (1983a) J . fmmunol. Merhods 64, 269-281
Morgan, B. P., Campbell, A. K . , Luzio, J . P. & Siddle, K . (19836)
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Morgan, B. P., Compston, A. &Campbell, A. K . (1984a) Lancet ii,
251-255
Morgan, B. P., Sewry, C. A., Siddle, K . , Luzio, J . P. & Campbell,
A. K . (19846) Immunology 52, 181-188
Morgan, B. P., Campbell, A. K., Luzio, J . P. & Hallett, M. B.
(1984~)Biochem. Sor. Trans. 12, 779
Morgan, B. P . , Luzio, J . P. & Campbell, A . K . (1984d) Biochem.
Biophys. Res. Commun. 118, 616-622
Richardson, P. J . & Luzio, J . P. (1980) Biochem. J . 186, 897-906
Stanley, K . K . (1983) Nucleic Acids Res. 11, 40774092
Stanley, K . K . & Luzio, J . P. (1984) EMBO J . 3 , 1429-1434
Tschopp, J . , Podack, E. R. & Muller-Eberhard, H. J . (1982) Proc.
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1985