Human autoantibodies that react with SRP have been previously described and shown to immunoprecipitate the 7SL SRP RNA component and to react strongly with SRP54 on immunoblots [12
]. Anti-SRP autoantibodies are part of a family of myositis-specific antibodies that are directed primarily against conserved conformational epitopes on cytoplasmic components involved in protein synthesis [10
]. A unique feature of a number of myositis-specific autoantibodies is that they, in contrast to animal antisera raised to the same proteins, have been found to inhibit the functions of their respective autoantigens [11
]. We therefore investigated the effects of anti-SRP autoantibodies on SRP function in vitro
Sera from polymyositis patients containing anti-SRP autoantibodies specifically inhibited the in vitro
translocation of the secretory protein PPL into the ER. Both IgG fractions and Fab fragments had the same effect, suggesting that a wide-ranging steric inhibition was not occurring. We concluded that one or more anti-SRP autoantibodies present in these sera could block the ER targeting function of SRP. Since SRP54 plays a pivotal role in this process [3
], and since anti-SRP positive sera were known to contain autoantibodies recognizing SRP54 [15
], we focused on the antiSRP54-specific reactivity of these autoantisera.
We first mapped the region(s) of SRP54 that the anti-SRP positive human sera recognized and found that all four of the sera tested (4-2, 17-1, 19-1 and 25-1) contained autoantibodies that recognized an epitope(s) located within the SRP54G domain. Both sera 19-1 and 25-1 also contained a distinct reactivity towards the SRP54N domain. The SRP54N and SRP54G domains are primarily involved in binding to the SRP receptor (cf. Figure ), although these regions may indirectly influence signal-sequence binding [34
]. None of the sera recognized epitopes in the SRP54M domain, the region of the protein that binds to ER targeting signals [3
Figure 6 Schematic showing the effect of subdomain-specific autoantibodies on SRP54 function. (a) Autoantibodies against the SRP54G domain inhibit signal-sequence binding. Two conformations for SRP54 have been proposed: one where the signal sequence (S) binding (more ...)
Autoantibodies specific for an N-terminal region of SRP54, including the N-domain and part of the G-domain, were affinity-purified from sera 19-1 and 25-1 and were analysed for their effects upon SRP function. We found that both the affinity-purified material, representing autoantibodies specific for the SRP54N domain, and the unbound fraction, which included the activity recognizing the SRP54G domain, blocked PPL translocation in a cell-free system. Autoantibody binding to both of these regions could therefore block SRP-mediated targeting to the ER.
Protein translocation across the ER membrane requires signal-sequence binding to SRP54, followed by targeting of the SRP/nascent secretory protein/ribosome complex to the ER membrane [3
]. We found that all of the anti-SRP autoantisera interfered with the binding of SRP54 to the PPL signal sequence when this process was examined directly by photo-crosslinking [25
]. While the affinity-purified antibodies specific for the SRP54 N-terminal region had no effect, the unbound antibodies recovered during the purification displayed a strong inhibition of SRP54 binding. We conclude that the binding of SRP54G domain-specific autoantibodies inhibits the ability of SRP to bind to the signal sequence of PPL (Figure ). We cannot, however, formally exclude the possibility that this unbound antibody fraction also contains activities that inhibit the function of other SRP subunits, such as the SRP 68 kDa and SRP 72 kDa proteins [37
Having established that anti-SRP autoantibodies block signal-sequence binding, we investigated their effect upon the SRP-dependent targeting [38
]. To study this process we used the model membrane protein IMC-CAT, which contains a signal-anchor sequence that binds to the SRP54 subunit and, upon SRP receptor-mediated delivery to the ER membrane, is subsequently transferred to the Sec61 translocon for membrane integration [24
] (Figure ). All four anti-SRP sera completely blocked the transfer of nascent IMC-CAT chains from SRP54 to the Sec61 complex, and the affinity-purified, SRP54N domain-specific antibodies displayed the same level of inhibition as the crude sera. Thus, in contrast to the effects upon signal-sequence binding, autoantibodies specific for both the SRP54N and SRP54G domains can block SRP receptor-mediated targeting to the ER membrane (Figure and ). This is entirely consistent with our current understanding of the pivotal role played by the SRP54N and SRP54G domains in the interaction of SRP with the SRP receptor [39
] (Figure ). Whether the effect of the anti-SRP autoantibodies is simply to prevent the authentic binding of SRP54 and the SRP receptor, or whether their binding compromises a specific function such as GTP hydrolysis, remains to be established.