Cytokeratin peptide SFGSGFGGGY mimics N-acetyl-beta-D-glucosamine in reaction with antibodies and lectins, and induces in vivo anti-carbohydrate antibody response.
Recently, we found that human anti-GlcNAc mAbs reacted with keratin from human skin and recognized specific peptide epitopes of human cytokeratin 14. Our data demonstrate that anti-keratin Ab responses in mice might be driven by Ags bearing terminal O-linked GlcNAc residues. To determine if mouse anti-GlcNAc mAbs recognized peptide epitopes of keratin, several mouse anti-GlcNAc mAbs were reacted with a panel of overlapping synthetic decapeptides of the entire amino acid sequence of human cytokeratin 14 in the ELISA. Results of the ELISA demonstrated that three mAbs, HGAC 54, HGAC 78, and 101.4.1, expressed maximal binding activity to keratin peptide B1 with the amino acid sequence SFGSGFGGGY. In addition, we found that mouse anti-cytokeratin 14 mAb CKB-1 recognized the same peptide and expressed anti-GlcNAc activity as well. Keratin peptide B1 was shown to react not only with anti-GlcNAc mAbs but also with several lectins such as wheat germ agglutinin, Datura stramonium lectin, Lycopersicon esculentum lectin, Solanum tuberosum lectin, and lectin from Wisteria floribunda. Reaction of the lectins with solid-phase keratin peptide B1 was inhibited by soluble GlcNAc and was not inhibited by glucose demonstrating specificity of binding. Using a panel of 24 synthetic keratin B1 peptides, each with a single amino acid substitution, we found that aromatic-aromatic and hydrophobic interactions were the major driving forces in the stabilization of the peptide-protein complexes. Finally, we demonstrated that immunization of BALB/c mice with peptide B1 conjugated to BSA induced an anti-GlcNAc Ab response. Results of our experiments indicated that certain peptides may express functional activity similar to GlcNAc and induce in vivo anti-carbohydrate Ab responses.
Shikhman
A R
AR
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City 73104.
Greenspan
N S
NS
Cunningham
M W
MW
D000117:Acetylglucosamine / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000917:Antibody Formation; D002241:Carbohydrates / Q000276:immunology; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007633:Keratins / Q000276:immunology*; D037102:Lectins / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D000939:Epitopes; D037102:Lectins; D010446:Peptide Fragments; C090852:cytokeratin peptide SFGSGFGGGY; D007633:Keratins; D000117:Acetylglucosamine