Journal of Autoimmunity (1999) 12, 43–49 Article No. jaut.1998.0252, available online at http://www.idealibrary.com on Side-chain Specificities and Molecular Modelling of Peptide Determinants for Two Anti-Sm B/B′ Autoantibodies Judith A. James 1,2,3 , Micah T. McClain 1,2,3 , Gerald Koelsch 4 , David G. Williams 5 and John B. Harley 1,2,3 1 Arthritis and Immunology Program, Oklahoma Medical Research Foundation 2 Department of Medicine, Microbiology and Immunology, University of Oklahoma Health Sciences Center 3 Department of Veterans Affairs Medical Center, Oklahoma City, OK 73104, USA 4 Protein Studies, Oklahoma Medical Research Foundation, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA 5 Division of Clinical Immunology, Mathilda and Terence Kennedy Institute of Rheumatology, London W6 7DW, UK Received 24 July 1998 Accepted 20 October 1998 Autoantibodies binding the Sm B and B′ peptides (B/B′) are commonly associated with systemic lupus erythematosus in man and in MRL lpr/lpr mice. The linear antigenic regions of two anti-Sm B/B′ murine monoclonal auto- antibodies have been mapped using overlapping octapeptides. Unique epitopes are identified by each antibody. Monoclonal KSm-5 recognizes the peptide, PPPGMRPP, which is repeated three times in the Sm B polypeptide. KSm 3 preferably binds to two similar, almost neighboring octapeptides, PPPGIRGP and PGIRGPPP. The two monoclonal antibodies do not cross react. These regions of Sm B/B′ are major areas of antigenicity in human sera. Amino acid deletion and substitution in antigenic octapeptides show that binding to the KSm-5 epitope is lost with most modifications. Molecular dynamic modelling suggests that when PPPGMRPP is substituted in the sixth position arginine, KSm/5 binding may be associated with a shared peptide backbone structure rather than charge or hydrophobicity of the substituted amino acid. In contrast, binding of KSm-3 to PPPGIRGP is abolished when the sixth position arginine is substituted by any other amino acid. Substitution at arginine and modelling experiments, therefore, suggest very different mech- anisms of binding. Autoantibodies may bind quite different features of similar peptide structures. © 1999 Academic Press Key words: molecular modelling, monoclonal antibodies, Sm, Sm B/B′, systemic lupus erythematosus Introduction Anti-Sm autoantibodies are frequently associated with rheumatic diseases, specifically systemic lupus erythematosus. These aberrant antibodies are found in a moderate proportion of affected human patients as well as in the MRL lpr/lpr murine model of lupus [1]. These autoantibodies precipitate the U1, U2, U4, U5, and U6 small ribonucleoprotein complexes (snRNP) which are involved in premRNA splicing and are constituents of the spliceosome [2, 3]. Anti-Sm anti- bodies are directed against one or a combination of eight polypeptides: B (26 kDa), B′ (27 kDa), D1, D2, D3 (16, 16.5, 18 kDa), E/F (11 kDa doublet) and G (<10 kDa). The most consistent binding present, how- ever, is with the B, B′ and D1 polypeptides [4, 5]. Two sequences of Sm B/B′ have been reported which vary with the antigen source. Rokeach et al. [6] sequenced an Sm B/B′ cDNA clone isolated from a lymphoblastoid (Raji) cell library which is identical to a partial clone from HeLa cells [7] and an Sm-N sequence found in a human cerebellar library [8]. Another sequence with 93% sequence agreement with the Raji Sm B/B′ sequence has been isolated by van Dam et al. [9] and Ohosone et al. [10] The van Dam group used human (HL-60) cells as the Sm B/B′ antigen source, while Ohosone’s group used an anti- gen from a human thyroid carcinoma library. These two sequences differ in 17 of 240 amino acids; all changed residues result from conservative substi- tutions. Sm B and Sm B′ are known to be very closely related and may be alternate splicing products from a common premRNA [9–11]. In this study overlapping octapeptides of the trans- lated region of Sm B/B′ sequences were synthesized by solid phase peptide synthesis. Antigenicity of each peptide was measured with the monoclonal Correspondence to: John B. Harley, 825 N.E. 13th Street, Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA. 43 0896–8411/99/010043+07 $30.00/0 © 1999 Academic Press