Single chain antibodies that recognize the N-glycosylation site.
We aimed to identify antibodies that can recognize the Asn-Xaa-Ser/Thr(NXS/T) N-glycosylation site that guides oligosaccharyltransferase (OT) activity. We used synthetic Asn-Cys-Ser/Thr(NCS/T) tripeptides conjugated to bovine serum albumin to isolate single chain antibody fragments of a variable region (scFv) from the Griffin 1 phage antibody library. Although Ser and Thr have different side chains, the scFv proteins thus isolated bound to both NCS and NCT with Kd values of the order of 10(-6) M and accepted the substitution of the Cys residue with various amino acids, including Ala, Gly, and Val. However, these proteins recognized neither Asn-Pro-Ser/Thr nor non-NXS/T tripeptides. The scFv proteins recognized NCS/T and N-glycosylation site of mutant yeast protein disulfide isomerase when they were in their native but not denatured state. These results indicate that antibody recognition of the NXS/T motif is conformation dependent and suggest that NXS/T spontaneously adopts a specific conformation that is necessary for antibody recognition. These features are likely to correlate with the known binding specificity of OT.
Kikuchi
Masakazu
M
Department of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan. kikuchi@se.ritsumei.ac.jp
Kataoka
Mizuho
M
Kojima
Takehiro
T
Horibe
Tomohisa
T
Fujieda
Kanae
K
Kimura
Taiji
T
Tanaka
Toshiki
T
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000378:metabolism; D001216:Asparagine; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D019151:Peptide Library; D012694:Serine; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013912:Threonine
D000906:Antibodies; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; D009842:Oligopeptides; D019151:Peptide Library; D012710:Serum Albumin, Bovine; D013912:Threonine; D012694:Serine; D001216:Asparagine; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase