title: Crystallographic analysis of the NNA7 Fab and proposal for the mode of human blood-group recognition.
abstract: The NNA7 Fab antibody fragment recognizes the human N-type blood-group antigen comprised of the N-terminal glycopeptide of glycophorin A (GPA). A mutant form of this Fab fragment, NNA7-G91S, exhibits markedly reduced antigen binding. To provide insight into how these Fab fragments recognize this glycopeptide antigen, the crystal structures of NNA7 and NNA7-G91S were solved and refined to 1.83 and 1.97 A resolution, respectively. Both molecules are composed of the same heavy (H) chain Fd fragment, but each contains a slightly different light (L) chain owing to the G91S substitution. Specifically, the G91S mutation pushes the backbone of the neighboring H chain away from complementarity-determining region 3 (CDR3) of the L-chain variable region, allowing an additional glycerol cryoprotectant molecule to enter the antigen-combining site near the putative location of O-linked glycosylation. Each Fab fragment also possesses a well defined 2-(N-morpholino)ethanesulfonic acid (MES) molecule trapped in its antigen-combining site, as well as a crystallographic symmetry-related molecule comprising an amino-acid sequence that is virtually identical to the N-terminus of GPA. The MES molecule interacts with the H-chain CDR in a manner reminiscent of antibody-carbohydrate complexes. These results suggest a model for recognition of the glycopeptide antigen that accounts for the deleterious effect of the G91S substitution.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
authors.forename: Kefang
authors.initials: K
authors.lastname: Xie
authors.forename: Shuh Chyung
authors.initials: SC
authors.lastname: Song
authors.forename: Steven L
authors.initials: SL
authors.lastname: Spitalnik
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Wedekind
mesh_terms: D017738:Alkanesulfonic Acids / Q000737:chemistry; D000906:Antibodies / Q000737:chemistry; D000941:Antigens / Q000737:chemistry; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry*; D002241:Carbohydrates / Q000737:chemistry; D003451:Cryoprotective Agents / Q000494:pharmacology; D018360:Crystallography, X-Ray / Q000379:methods*; D004583:Electrons; D005690:Galactose / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry; D006021:Glycophorins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D008951:MNSs Blood-Group System; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D009025:Morpholines / Q000737:chemistry; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D017738:Alkanesulfonic Acids; D000906:Antibodies; D000941:Antigens; D001798:Blood Proteins; D002241:Carbohydrates; D003451:Cryoprotective Agents; D006020:Glycopeptides; D006021:Glycophorins; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D008951:MNSs Blood-Group System; D009025:Morpholines; C004550:2-(N-morpholino)ethanesulfonic acid; D005990:Glycerol; D005690:Galactose