期刊
JOURNAL OF BIOCHEMISTRY
卷 157, 期 4, 页码 211-216出版社
OXFORD UNIV PRESS
DOI: 10.1093/jb/mvu071
关键词
alpha 2,6-sialic acid; immunohistochemistry; Sambucus sieboldiana agglutinin (SSA) lectin; transferrin
资金
- Ministry of Health, Labor, and Welfare of Japan [2011-Nanchi-Ippan-018, 2014-Nanchi-Ippan-052]
- Japan Science and Technology Agency [AS221Z00232F, AS231Z01053, 241FT0255, 149]
- Ministry of Education, Culture, Sports, Science, and Technology of Japan [23110002, 23590367]
- Grants-in-Aid for Scientific Research [25460054, 15K18496, 26110724, 23590367] Funding Source: KAKEN
We previously found that a lectin, Sambucus sieboldiana agglutinin (SSA), bound to alpha 2,6-sialylated glycan epitopes on transferrin and inhibited anti-transferrin antibody binding to the antigen in ELISA (SSA inhibition). Here we report that SSA inhibition is applicable to immunohistochemistry, localizing alpha 2,6-sialylated transferrin in the liver. Immunohistochemistry using anti-transferrin polyclonal antibody revealed that transferrin was detected in hepatocytes near interlobular veins. Addition of SSA lectin markedly attenuated the staining. Sialidase treatment of a liver section abolished SSA binding and concomitantly cancelled SSA inhibition, suggesting that SSA binding to glycan epitopes on the section was essential for the inhibition. To examine the importance of proximity between antigen epitopes and SSA-binding (glycosylation) sites, we prepared two anti-peptide antibodies against partial amino acid sequences of transferrin. One antibody (Tf-596Ab) is against a peptide sequence, Cys596-Ala614, which is proximal to N-glycosylation sites (Asn-432 and Asn-630). The other (Tf-120Ab) is against a peptide sequence, Val120-Cys137, distal to the sites. The staining signals of Tf-596Ab were reduced by the addition of SSA, whereas those of Tf-120Ab were reduced only a little. This result suggests that proximity of the antigen epitope to SSA binding sites is critical for SSA inhibition in immunohistochemistry.
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