The anti-Sm immune response in autoimmunity and cell biology

Autoimmunity Reviews 2 (2003) 235–240

The anti-Sm immune response in autoimmunity and cell biology Gary W. Zieve*, Permanan R. Khusial Department of Pathology, SUNY Stony Brook, Stony Brook, NY 11794-8691, USA Accepted 15 January 2003

Abstract Anti-Sm antibodies are found in greater than 30% of the patients with systemic lupus erythematosus (SLE) and are diagnostic of SLE. The Sm autoantigens are the small nuclear ribonucleoprotein (snRNP) common core proteins. The seven core proteins, B, D1, D2, D3, E, F and G, shared by a majority of the snRNP particles, form a heptamer ring approximately 20 nm in diameter, with the snRNA passing through the center. The Sm epitopes are distributed on the outside surface of the ring. A repeated proline rich motif with homology to an Epstein bar nuclear antigen in the B protein and a gly–arg–gly motif including a symmetrical dimethylarginine post translational modification in the B, D1 and D3 proteins are major Sm epitopes. The anti-Sm response has features typical of an antigen driven immune response. SnRNP proteins share several characteristics with other autoantigens including their assembly into ribonucleoprotein particles, homologies to known viral proteins, presence of post translational modifications, a high abundance and great stability and the presence of repeated motifs. Current work on the snRNP particles is attempting to identify the features that predispose the common core proteins to become autoantigens in vulnerable individuals. 䊚 2003 Elsevier Science B.V. All rights reserved. Keywords: Sm; snRNPs; Lupus

The anti-Sm autoimmune response, a polyclonal humoral immune response against protein components of the small nuclear ribonucleoprotein (snRNP) particles, is found in greater than 30% of the patients with systemic lupus erythematosus (SLE) and is specific for SLE. It is named after Stephanie Smith, the first patient in whom this activity was identified. Although the anti-Sm antibodies are not correlated with clinical symptoms, as much as 20% of the immunoglobulins in a SLE patient may bind Sm epitopes w1x. The Sm antibodies are powerful tools for diagnosing SLE and *Corresponding author. Fax: q1-631-444-3424. E-mail address: [email protected] (G.W. Zieve).

valuable reagents for studying the snRNP particles which are essential cofactors for pre-mRNA splicing in the cell nucleus. 1. Detection of anti-Sm antibodies Anti-Sm antibodies and antibodies against double stranded DNA are common anti-nuclear antibodies (ANAs) found in patients with SLE. The presence of ANAs is one of the 11 parameters used to identify SLE w2x. The presence of at least 4 of the 11 SLE parameters suggests a diagnosis of SLE. The clinical identification of the anti-Sm antibodies and other ANAs usually begins with

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Fig. 1. Model structures of the U1 snRNP and the snRNP common core proteins. A cartoon of the U1 snRNP (panel a) illustrates the snRNA, U1 specific proteins and the snRNP common core proteins. A ribbon diagram (panel b) and space filling model (panel c) of the snRNP common core are also shown w5x.

immunofluorescence staining to identify antibodies that recognize nuclear antigens. This is typically done by indirect immunofluorescent staining of the human larynx carcinoma cell line HEp2 (ATCC CCL-32) with serial dilutions of patient serum. If a distinct nuclear staining pattern is observed in serum dilutions of 1:160 or greater this is classified as a significant ANA response. A coarse speckled pattern, of 10–20 bright staining foci on a diffuse background, is observed with anti-Sm antibodies. This represents the distribution of the snRNP particles in the non-chromosomal regions of the nucleus, or nucleoplasm. However, sera often contain multiple activities and the specific antibody response is determined by analyzing the antibody activity against purified antigens in an ELISA assay. Several companies make ELISA plates with purified snRNPs (Sm), U1 snRNP (nRNP) and other specific antigens immobilized on plastic to detect the presence of a specific antibodies. 2. The snRNP particles The U1–U12 snRNP particles are designated by their snRNA components which range in size from 80 to 260 nucleotides. U1–U6 are the most abundant snRNP particles and U1 and U2 are present

in approximately 1=106 copies per nucleus. The U1, U2, U4 and U5 snRNPs share the set of seven common core proteins, B, D1, D2, D3, E, F and G which are the Sm antigens and are designated the Sm class of snRNPs. In addition to the common core proteins each particle has several snRNP specific proteins (Fig. 1a). The anti-nRNP autoimmune response typical of mixed connective tissue disease is directed against the U1 specific proteins. The four snRNP particles, U1, U2, U4y U6 and U5 are essential cofactors for pre-mRNA splicing and assist in the removal of introns from pre-mRNA w3,4x. The seven core proteins B, D1, D2, D3, E, F and G range in size from 13 to 28 kDa and with the exception of F, are quite basic w4x (Table 1Scheme 1). The proteins are highly conserved in eukaryotes. All the core proteins are homogenous and are expressed from a single gene except for the B protein. The B protein has three variants (B, B9 and N) expressed from two closely related genes. However, the significance of the different B proteins is not known. One copy of the seven core proteins associates with each snRNA and forms a ring structure 7 nm in diameter with a 2 nm central hole through which the snRNA passes (Fig. 1b,c) w5x. All the snRNA core proteins share a sequence motif des-

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ignated the Sm motif of 31 amino acids (Sm1) separated by approximately 10–20 amino acids from a second conserved 13 amino acid (Sm2) segment. This motif forms a five stranded antiparallel beta sheet with the core proteins interacting with their neighbors through an anti-parallel b4–b5 pairing (Table 1). The Sm domains are tilted at an angle and resemble the fan blades on a turbine w6x. The snRNP particles are found throughout the nucleus in both a dispersed distribution and clustered in speckles. The dispersed particles are actively involved in the slicing of newly transcribed pre-mRNA and the denser speckles are stored pools of particles. The snRNAs are extremely stable, with half lives of greater than 48 h and they cycle continually between the active and stored pools. During mitosis the snRNPs distribute throughout the cytoplasm after the nuclear envelope breaks down in prophase, and then return to the reforming nuclei in telophase and early G1 w3 x . The Sm class of snRNP particles assemble in the cytoplasm. The snRNAs are transcribed by RNA polymerase II in the nucleus and are transported into the cytoplasm immediately after transcription where they assemble with the snRNP core proteins before returning to the nucleus w4x. The Sm class of snRNAs share a conserved Sm RNA sequence motif (Pu A Un G Pu, ns4–6, Puspurine), in a single stranded region of the RNA, which directs assembly with the snRNP core proteins w7x. The snRNAs undergo several post transcriptional modifications in the cytoplasm including cap hypermethylation, base modifications and 39 shortening. The newly synthesized snRNP core proteins are stored in the cytoplasm in three RNA-free subcore complexes of (1) a pentamer of (D1, D2, E, F and G) at 6S complex (2) B, D3, D1 and a 70 kDa protein recently identified as a type 5 arginine methyltransferase (PRMT5) at 20S and (3) apparently homogenous B protein at 2S–5S complexes. SnRNP core particle assembly occurs by initial binding of the 6S complex to the snRNA followed by addition of the B and D3 proteins w4,8x. The nuclear localization signal is a combination of the

2,2,7 m3G cap and determinants on the assembled core particle. Most snRNP specific proteins assemble with the particles after they enter the nucleus. The PRMT5 methyltransferase in the 20S cytoplasmic pre-snRNP complex generates the sDMA post translational modification on arginines found in GRG motifs (gly–arg–gly) in the C terminus of the D1, D3 and B snRNP core proteins (Table 1) w9,10x. There are approximately 13 GRG motifs in D1, 7 in D3 and 6 in B w11x. The sDMA modified GRG forms a motif that interacts with the survival of motor neuron protein. This protein, which is defective in the disease spinal muscular atrophy, is a hypothetical chaperone that helps localize and transport the snRNP particles w12x.

3. The anti-Sm immune response

The anti-Sm autoimmune response is directed against multiple epitopes on the snRNP common core proteins. The B protein is the major antigen followed by the D1 and D2 proteins. The anti-Sm immune response displays the characteristics of a typical antigen driven response. The T cell epitopes supporting the response are specific to the snRNP core proteins and the anti-Sm Abs show evidence of affinity maturation w13,14x. Because the snRNP particles is a large multi-protein complex, the B and T cell Sm epitopes can reside on different proteins in the complex. A single T cell epitope generated from one protein in the snRNP particle, can support the production of a large family of antibodies that recognize multiple determinants on the snRNP particles. The process in which a single initiating antibody response can then expand to a large polyclonal antibody response against the snRNP particles, as seen with the anti-Sm response, has been called epitope spreading w15x. Hoffman and co-workers recently identified the T cell epitopes in the anti-Sm response as regions of the B and D1 protein that lie within the Sm1 and Sm2 protein motifs w14x. These motifs are likely to be on the inside of the mature snRNP

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particle because they are the sites of protein– protein interaction between the snRNP proteins. A variety of approaches have been used over the last decade to identify the B cell epitopes recognized by the anti-Sm antibodies w8,11,16,17x. The proline rich sequence PPPGMRPP which is repeated three times in the C terminus of the B protein is the major Sm epitope in patient sera. This sequence is similar to the PPPGRRP sequence, in the Epstein–Barr nuclear antigen 1 (EBNA-1) in Epstein Barr virus (EBV) and has generated the hypothesis that infection with EBV predisposes vulnerable individuals to develop lupus through a cross reaction of between the two epitopes w18x. Many other Sm epitopes, primarily on the C terminus of the B, D1 and D3 proteins, regions of the proteins that appear on the outside of the snRNP core particle have also been identified w16x. A large library of anti-Sm monoclonal antibodies generated from the MRL mouse has proven to be valuable tools in this work w8,11x. The observation that several anti-Sm mAbs recognize more than one snRNP core protein in immunoblots was the first suggestion that Sm epitopes are shared among the snRNP core proteins. The recent identification of the sDMA modified GRG repeats on the B, D1 and D3 proteins as major epitopes has helped to explain this cross reactivity w11x. However, there must be other factors contributing to these epitopes because the D2 core protein, which lacks sDMA modified GRG repeats is also recognized by some of these antibodies and several epitopes include the GRG motifs and unique flanking sequences. 4. Origins of the anti-Sm response The etiology of lupus is unknown. A large number of genetic factors and several environmental factors are hypothesized as risk factors for the disease w19x. This suggests a variety of different paths can lead to lupus, thus it is a syndrome rather than a specific disease. However, in many lupus patients anti-Sm autoantibodies are generated. This suggests there are some unusual features of the snRNP particles that predispose them to becoming autoantigens in SLE. Comparisons with other autoantigens identify at least seven charac-

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teristics of the snRNP particles that may contribute to them becoming autoantigens. They are (1) the abundance and stability of the particles (2) the highly conserved nature of the snRNP proteins (3) the inclusion of both protein and RNA in the complexes including extensive double stranded RNA (4) localization of the mature particles in the nucleus (5) extensive post translational modification of the snRNP core proteins (6) presence of polyvalent epitopes on the particles and (7) the presence of motifs that mimic viral epitopes. Several studies have suggested that stable and abundant cellular proteins are presented to the immune system during normal cell death. Their availability makes them vulnerable to immune recognition when other factors lead to a loss of tolerance and their continued availability helps perpetuate the immune response once it begins w20x. A recent report suggested protein–nucleic acid complexes may stimulate receptors in dentritic cells that are specifically designed to detect single and double stranded nuclei acids and boost the immune response against the associated proteins w21x. The snRNP proteins are extensively posttranslationally modified by the sDMA modification and other as yet unidentified modifications. Several reports have suggested these modifications predispose proteins to autoimmune recognition w22x. Similarly the snRNP particles have a variety of shared motifs and this polyvalent nature is unknown to increase immunogenicity. Finally, the snRNP B protein has the poly-proline motif that is similar to the motif in the EBNA-1 protein which may predispose people to developing antibodies against the snRNP B protein. In a model system immunization with this motif led to the development of anti-Sm antibodies and there is a strong correlation with EBV virus in patients with anti-Sm antibodies w18x. However, there must be a combination of factors including a vulnerable genotype and viral infection, inflammatory response or stochastic events that initiates the anti-Sm response in vulnerable individuals. Understanding what predisposes the snRNP protein to becoming autoantigens in the anti-Sm response will help identify some of the factors that lead to the development of SLE.

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Take-home messages ● Sm antigens are the seven snRNP common core proteins. ● SnRNP common core proteins form a heptamer ring of approximately 20 nm in diameter. ● Anti-Sm immune response is antigen driven. ● A polyproline motif with homology to Epstein bar nuclear antigen is a major Sm epitope. ● A post translational modification of symmetrical dimethylarginine is a major Sm epitope.

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