%0 Journal Article
%T Identification of heparin-binding amino acid residues in antibody HS4C3 with the potential to design antibodies against heparan sulfate domains.
%A Damen LAA
%A Bui TP
%A van Wessel T
%A Li Y
%A Straten BF
%A Pampiermole R
%A Daamen WF
%A Fernig DG
%A van Kuppevelt TH
%J Glycobiology
%V 34
%N 8
%D 2024 Jun 22
%M 38963938
%F 5.954
%R 10.1093/glycob/cwae046
%X 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.