RESUMEN
Heparan sulfate (HS) is a linear polysaccharide with high structural and functional diversity. Detection and localization of HS in tissues can be performed using single chain variable fragment (scFv) antibodies. Although several anti-HS antibodies recognizing different sulfation motifs have been identified, little is known about their interaction with HS. In this study the interaction between the scFv antibody HS4C3 and heparin was investigated. Heparin-binding lysine and arginine residues were identified using a protect and label methodology. Site-directed mutagenesis was applied to further identify critical heparin-binding lysine/arginine residues using immunohistochemical and biochemical assays. In addition, computational docking of a heparin tetrasaccharide towards a 3-D homology model of HS4C3 was applied to identify potential heparin-binding sites. Of the 12 lysine and 15 arginine residues within the HS4C3 antibody, 6 and 9, respectively, were identified as heparin-binding. Most of these residues are located within one of the complementarity determining regions (CDR) or in their proximity. All basic amino acid residues in the CDR3 region of the heavy chain were involved in binding. Computational docking showed a heparin tetrasaccharide close to these regions. Mutagenesis of heparin-binding residues reduced or altered reactivity towards HS and heparin. Identification of heparin-binding arginine and lysine residues in HS4C3 allows for better understanding of the interaction with HS and creates a framework to rationally design antibodies targeting specific HS motifs.
RESUMEN
Heparan sulfate (HS) is a linear polysaccharide with high structural diversity. Different HS epitopes have been detected and localized using single chain variable fragment (scFv) antibodies from a 'single pot' phage display library containing a randomized complementarity determining region of the heavy chain (CDR3). In this study, we created a new library containing anti-HS scFvs that all harbor a dp-38 heavy chain segment where the CDR3 region was engineered to contain the XBBXBX heparin binding consensus site (X = any amino acid, B = R, K or H). The library contained ~1.73 × 106 unique antibodies and was biopanned against HS from several sources. The selected antibodies were sequenced and chemically/immunohistologically characterized. A number of 67 anti-HS scFv antibodies were selected, of which 31 contained a XBBXBX CDR3 sequence. There was a clear preference for glycine at the first and proline at the fourth position of the CDR3. The sequence GZZP(R/K)X (Z = R, K or H, but may also contain N, S, or Q) was unusually overrepresented. Selected antibodies reacted with HS/heparin, but not with other glycosaminoglycans. Antibodies reacted differentially with respect to N-, 2-O, or 6-O-desulfated heparin preparations, and showed distinct topologies of HS epitopes in rat kidney sections. The library may be instrumental in the selection of a large pool of HS epitope-specific antibodies, and - since all antibodies differ only in their 6 amino acid CDR region - may be a tool for a rational design of antibodies recognizing specific HS sulfation patterns.