title: The N14 anti-afamin antibody Fab: a rare VL1 CDR glycosylation, crystallographic re-sequencing, molecular plasticity and conservative versus enthusiastic modelling.
abstract: The monoclonal antibody N14 is used as a detection antibody in ELISA kits for the human glycoprotein afamin, a member of the albumin family, which has recently gained interest in the capture and stabilization of Wnt signalling proteins, and for its role in metabolic syndrome and papillary thyroid carcinoma. As a rare occurrence, the N14 Fab is N-glycosylated at Asn26L at the onset of the VL1 antigen-binding loop, with the α-1-6 core fucosylated complex glycan facing out of the L1 complementarity-determining region. The crystal structures of two non-apparent (pseudo) isomorphous crystals of the N14 Fab were analyzed, which differ significantly in the elbow angles, thereby cautioning against the overinterpretation of domain movements upon antigen binding. In addition, the map quality at 1.9 Å resolution was sufficient to crystallographically re-sequence the variable VLand VHdomains and to detect discrepancies in the hybridoma-derived sequence. Finally, a conservatively refined parsimonious model is presented and its statistics are compared with those from a less conservatively built model that has been modelled more enthusiastically. Improvements to the PDB validation reports affecting ligands, clashscore and buried surface calculations are suggested.
authors.affiliation: Division of Biological Chemistry, Medical University of Innsbruck, Innrain 80, 6020 Innsbruck, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Naschberger
authors.affiliation: Division of Biological Chemistry, Medical University of Innsbruck, Innrain 80, 6020 Innsbruck, Austria.
authors.forename: Barbara G
authors.initials: BG
authors.lastname: Fürnrohr
authors.affiliation: Center for Proteomics, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia.
authors.forename: Tihana
authors.initials: T
authors.lastname: Lenac Rovis
authors.affiliation: Center for Proteomics, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia.
authors.forename: Suzana
authors.initials: S
authors.lastname: Malic
authors.affiliation: Division of Biological Chemistry, Medical University of Innsbruck, Innrain 80, 6020 Innsbruck, Austria.
authors.forename: Klaus
authors.initials: K
authors.lastname: Scheffzek
authors.affiliation: Division of Genetic Epidemiology, Medical University of Innsbruck, Schöpfstrasse 41, 6020 Innsbruck, Austria.
authors.forename: Hans
authors.initials: H
authors.lastname: Dieplinger
authors.affiliation: Division of Genetic Epidemiology, Medical University of Innsbruck, Schöpfstrasse 41, 6020 Innsbruck, Austria.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Rupp
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D000936:Antigen-Antibody Complex; D002352:Carrier Proteins / Q000276:immunology*; D022801:Complementarity Determining Regions; D018360:Crystallography, X-Ray; D006023:Glycoproteins / Q000276:immunology*; D006031:Glycosylation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D012709:Serum Albumin / Q000276:immunology*; D000075462:Serum Albumin, Human
chemical_list: C088246:AFM protein, human; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002352:Carrier Proteins; D022801:Complementarity Determining Regions; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D012709:Serum Albumin; D000075462:Serum Albumin, Human
keywords: accuracy; antibody fragment; elbow angle; flexibility; non-apparent isomorphism; precision; solvent; solvent modelling; variable-chain glycosylation