Heparan sulfates (HS) are long, linear polysaccharides with a high degree of variability. They bind to a vast number of proteins such as growth factors and cytokines, and these interactions are likely to be mediated by specific HS domains. To investigate the structural diversity and topological distribution of HS domains in tissues, we selected a panel of 10 unique anti-HS antibodies using phage display technology. All 10 antibodies recognize a specific HS epitope as demonstrated by enzyme-linked immunosorbent assay using defined synthetic HS oligosaccharides, modified HS/ heparin molecules, and HS isolated from a variety of organs. The chemical groups involved in the epitopes could be indicated and the position of sulfate groups is of major importance. All HS epitopes have a defined tissue distribution as shown by immunohistochemistry using rat organs. Taken together, the data show that in vivo, a large number of defined HS epitopes exist that do not occur randomly but are tightly, topologically regulated.Heparan sulfates (HS), 1 a class of glycosaminoglycans, are long linear complex polysaccharides covalently bound to a protein core. They have a ubiquitous distribution in the extracellular matrix and on cell surfaces and have been implicated in many basic biological phenomena such as cell migration, adhesion, and differentiation. They play a role in such diverse processes as growth factor/cytokine handling, enzyme regulation, lipid metabolism, blood coagulation, and viral entry (1-4). This involvement is mediated by the interactions of HS with a vast number of proteins such as growth factors/cytokines, enzymes, protease inhibitors, extracellular matrix molecules, and viral coat proteins (5, 6). The large number of interactions suggests an extensive structural variation within HS. Chemical analysis of HS-derived disaccharides indeed indicates a large structural diversity (7-9), which is brought about by specific chain modifications during HS biosynthesis. The importance of defined monosaccharide sequences for specific interactions with proteins has been demonstrated for the binding and activation of antithrombin III by HS/heparin (10, 11). In addition, specific structural requirements for binding to basic fibroblast growth factor and hepatocyte growth factor have been defined (12, 13). These observations indicate that HS modifications do not occur randomly but that a controlled expression of specific domains/sequences in HS exists. To investigate whether a large number of different HS domains indeed occur in tissues, we selected a panel of epitope-specific anti-HS antibodies using phage display technology. Using these antibodies, we chemically and topologically characterized the HS epitopes involved. We chose antibody phage display because it allows for the generation of antibodies against poorly immunogenic molecules such as HS. EXPERIMENTAL PROCEDURES MaterialsA human semisynthetic antibody phage display library (14, now officially named synthetic scFv Library No. 1) was generously provided by Dr G. Winter, C...

The U1‐70K protein is specifically bound to stemloop I of the U1 small nuclear RNA contained in the U1 small nuclear ribonucleoprotein complex (U1 snRNP), which is involved in the splicing of pre‐mRNA. All components of the U1 snRNP complex, including the U1‐70K protein, are important autoantigens in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Here we describe for the first time the selection and characterization of recombinant human anti‐U1‐70K single chain autoantibody fragments (anti‐hU1‐70K scFv) from autoimmune patient‐derived phage display antibody libraries. All scFv specifically recognize parts of the hU1‐70K protein and its apoptotic 40‐kDa cleavage product. In Western blotting assays a number of scFv preferentially recognize the 40‐kDa apoptotic cleavage fragment of the U1‐70K protein, suggesting a possible involvement of this apoptotic cleavage product in the autoimmune response of patients. The germ‐line gene usage of these recombinant autoantibodies was also determined. Using several U1‐70K deletion and point mutants of both human (h) and Drosophila melanogaster (Dm) origin, it was established that the U1‐70K epitope that is recognized by the anti‐hU1‐70K scFv is located within the RNA binding domain.

Abs to U1 RNA are frequently found in patients suffering from systemic lupus erythematosus overlap syndromes and Ab titers correlate with disease activity. We describe the isolation of the first human anti-U1 RNA autoantibodies from a combinatorial IgG library made from the bone marrow of a systemic lupus erythematosus patient. With the use of phage display technology, two anti-U1 RNA single-chain variable fragment (scFv) Abs were selected. Both high affinity anti-U1 RNA Ab fragments (Kd ∼ 1 nM) recognize stem II of U1 RNA and were derived from the same heavy chain gene (VH3–11) and the same λ (3r) light chain gene although somatic mutations, predominantly present in the complementarity-determining regions, are different. Experiments, in which the heavy chain genes of both anti-U1 RNA scFvs were reshuffled with the original light chain repertoire of the patient resulted, after selection on stem loop II, in a large number of RNA-binding Ab fragments. All these stem loop II-specific RNA binding clones used a similar, but not identical, 3r λ light chain. When scFvs were selected from the reshuffled libraries by stem loop IV, representing the other autoantigenic site of U1 RNA, most selected Ab clones did react with stem loop IV, but no longer with stem loop II. The stem loop IV-reactive Ab clones contained different, not 3r-related, light chains. These results point to a major role for the light chain in determining the sequence specificity of these disease-related anti-U1 RNA Abs. The possibility that secondary light chain rearrangements are involved in this autoimmune response is discussed.