title: Generation of antibodies recognizing an aberrant glycoform of human tissue inhibitor of metalloproteinase-1 (TIMP-1) using decoy immunization and phage display.
abstract: Aberrant glycosylation of human tissue inhibitor of metalloproteinase-1 (TIMP-1) by N-acetylglucosaminyltransferase-V (GnT-V) was previously reported to be related to cancer progression. Here, we report on the antibodies recognizing the structural features initiated by an addition of N-linked β(1,6)-N-acetylglucosamine (GlcNAc) branch by GnT-V on TIMP-1. Two glycoforms of TIMP-1, TIMP1-L produced in Lec4 cells without GnT-V activity and TIMP1-B in GnT-V overexpressing transfectant cells, were purified from culture supernatant and used for generation of antibodies. TIMP1-L was injected in the left hind footpad of mice as decoy and TIMP1-B in the right hind footpad as immunogen. Phage-displayed scFv library was constructed from the B cells retrieved from the right popliteal lymph nodes and subjected to panning and screening. Phage ELISA of individual clones revealed the scFv clones with preferential binding activity to TIMP1-B, and they were converted into an scFv-Fc format for further characterization of binding specificity. Glycan binding assay of an antibody, 1-9F, revealed its differential specificity toward an extension of glycan structure initiated with β(1,6)-GlcNAc linkage and terminally decorated with a sialic acid. This study demonstrates feasibility of a new strategy combining decoy immunization with phage display for the efficient generation of antibodies tracking down structural features of different glycoforms.
authors.affiliation: Daejon-KRIBB-Fred Hutchinson Cancer Research Center Research Cooperation Center, Korea Research Institute of Bioscience and Biotechnology, 52 Yuseong, Daejon 305-806, Republic of Korea.
authors.forename: Hyun Joo
authors.initials: HJ
authors.lastname: Ahn
authors.forename: Yong-Sam
authors.initials: YS
authors.lastname: Kim
authors.forename: Chul-Ho
authors.initials: CH
authors.lastname: Lee
authors.forename: Eun-Wie
authors.initials: EW
authors.lastname: Cho
authors.forename: Hyang-Sook
authors.initials: HS
authors.lastname: Yoo
authors.forename: Seung-Ho
authors.initials: SH
authors.lastname: Kim
authors.forename: Jeong-Heon
authors.initials: JH
authors.lastname: Ko
authors.forename: Sang Jick
authors.initials: SJ
authors.lastname: Kim
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D015723:Gene Library; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D007128:Immunoglobulin Fragments; D051379:Mice; D008807:Mice, Inbred BALB C; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry*; D019151:Peptide Library; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000737:chemistry*
chemical_list: D006023:Glycoproteins; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011134:Polysaccharides; D020033:Protein Isoforms; D019715:Tissue Inhibitor of Metalloproteinase-1; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase