Yu H, Sipes J M, Cashel J, Bakos M A, Goldblum R M, Roberts D D
Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-1500, USA.
Arch Biochem Biophys. 1995 Oct 1;322(2):299-305. doi: 10.1006/abbi.1995.1467.
Binding of the mouse IgM antibody 6C4 is lost after treatment of human free secretory component with peptide N-glycosidase F (Bakos et al. (1991) J. Immunol. 146, 162-168) or periodate, suggesting that asparagine-linked oligosaccharides contain the epitope recognized by this antibody. Inhibition of antibody binding to free secretory component by milk oligosaccharides established that lacto-N-tetraose is the minimum structure recognized by the antibody, but larger oligosaccharides with terminal Gal beta 1-3GlcNAc sequences bind with much higher affinity. Antibody binding is enhanced by substitution with the Lewis Fuc alpha 1-4 and is inhibited by Fuc alpha 1-2Gal substitution. Free secretory component, however, does not bind other antibodies that recognize Le(a) or Leb oligosaccharides, and binding is lost after digestion with a beta-galactosidase that cleaves Gal beta 1-3 linkages but not after digestion with alpha-L-fucosidase. Therefore, the major epitope recognized by 6C4 on free secretory component is probably not an asparagine-linked Le(a) oligosaccharide. The antibody also binds to human milk lactoferrin, some human mucins, and lacto-series glycolipids including III4 alpha Fuc-lactotetraosyl ceramide and lactotetraosyl ceramide. Based on affinity chromatography of oligosaccharides released from free secretory component, the epitope recognized by antibody 6C4 is present on approximately 3.5% of the asparagine-linked oligosaccharides.
用肽 N-糖苷酶 F（Bakos 等人，(1991) J. Immunol. 146, 162 - 168）或高碘酸盐处理人游离分泌成分后，小鼠 IgM 抗体 6C4 的结合能力丧失，这表明天冬酰胺连接的寡糖包含该抗体识别的表位。乳寡糖对抗体与游离分泌成分结合的抑制作用表明，乳糖-N-四糖是该抗体识别的最小结构，但具有末端 Galβ1-3GlcNAc 序列的较大寡糖结合亲和力更高。用 Lewis Fucα1-4 取代可增强抗体结合，而 Fucα1-2Gal 取代则抑制抗体结合。然而，游离分泌成分不与其他识别 Le(a) 或 Leb 寡糖的抗体结合，在用切割 Galβ1-3 键的β-半乳糖苷酶消化后结合能力丧失，但用α-L-岩藻糖苷酶消化后结合能力未丧失。因此，6C4 在游离分泌成分上识别的主要表位可能不是天冬酰胺连接的 Le(a) 寡糖。该抗体还与人乳铁蛋白、一些人粘蛋白以及包括 III4αFuc-乳糖四糖神经酰胺和乳糖四糖神经酰胺在内的乳糖系列糖脂结合。基于从游离分泌成分释放的寡糖的亲和层析，抗体 6C4 识别的表位存在于约 3.5% 的天冬酰胺连接的寡糖上。
