Wright A, Morrison S L
Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024.
J Exp Med. 1994 Sep 1;180(3):1087-96. doi: 10.1084/jem.180.3.1087.
Immunoglobulin G (IgG) molecules are glycosylated in CH2 at Asn297; the N-linked carbohydrates attached there have been shown to contribute to antibody (Ab) stability and various effector functions. The carbohydrate attached to the IgG constant region is a complex biantennary structure. Alterations in the structure of oligosaccharide have been associated with human diseases such as rheumatoid arthritis and osteoarthritis. To study the effects of altered carbohydrate structure on Ab effector function, we have used gene transfection techniques to produce mouse-human chimeric IgG1 Abs in the Chinese hamster ovary (CHO) cell line Lec 1, which is incapable of processing the high-mannose intermediate through the terminal glycosylation steps. We also produced IgG1 Abs in Pro-5, the wild-type CHO cell line that is the parent of Lec 1. The Pro-5-produced Ab (IgG1-Pro-5) was similar to IgG1-My 1, a myeloma-produced IgG1 Ab of the same specificity, in its biologic properties such as serum half-life, ability to effect complement-mediated cytolysis, and affinity for Fc gamma RI. Although the Lec 1-produced Ab, IgG1-Lec 1, was properly assembled and retained antigen specificity, it was incapable of complement-mediated hemolysis and was substantially deficient in complement consumption, C1q binding, and C1 activation. IgG1-Lec 1 also showed reduced but significant affinity for Fc gamma R1 receptors. The in vivo half-life of IgG1-Lec 1 was shorter than that of either the myeloma- or Pro-5-produced counterpart, with more being cleared during the alpha-phase and with more rapid clearance during the beta-phase. Clearance of IgG1-Lec 1 could be inhibited by the administration of yeast-derived mannan. Thus the uptake of IgG1-Lec 1 appears to be accelerated by the presence of terminally mannosylated oligosaccharide. Therefore, certain Ab functions as well as the in vivo fate of the protein are dramatically affected by altered carbohydrate structure. Expression of Igs in cell lines with defined glycosylation mutations is shown to be a useful technique for investigating the contribution of carbohydrate structure to Ab function.
免疫球蛋白G（IgG）分子在CH2区域的Asn297位点进行糖基化；已证明连接在该位点的N-连接碳水化合物有助于抗体（Ab）的稳定性和各种效应功能。连接到IgG恒定区的碳水化合物是一种复杂的双天线结构。寡糖结构的改变与类风湿性关节炎和骨关节炎等人类疾病有关。为了研究碳水化合物结构改变对Ab效应功能的影响，我们使用基因转染技术在中国仓鼠卵巢（CHO）细胞系Lec 1中产生小鼠-人嵌合IgG1抗体，该细胞系无法通过末端糖基化步骤加工高甘露糖中间体。我们还在Pro-5中产生了IgG1抗体，Pro-5是Lec 1的野生型CHO亲本细胞系。Pro-5产生的抗体（IgG1-Pro-5）在生物学特性如血清半衰期、补体介导的细胞溶解能力以及对FcγRI的亲和力方面，与IgG1-My 1（一种骨髓瘤产生的具有相同特异性的IgG1抗体）相似。尽管Lec 1产生的抗体IgG1-Lec 1正确组装并保留了抗原特异性，但它无法介导补体溶血，并且在补体消耗、C1q结合和C1激活方面存在严重缺陷。IgG1-Lec 1对FcγR1受体的亲和力也降低但仍显著。IgG1-Lec 1的体内半衰期比骨髓瘤或Pro-5产生的对应物短，在α期清除更多，在β期清除更快。给予酵母来源的甘露聚糖可以抑制IgG1-Lec 1的清除。因此，末端甘露糖基化寡糖的存在似乎加速了IgG1-Lec 1的摄取。因此，碳水化合物结构的改变会显著影响某些Ab功能以及蛋白质在体内的命运。在具有特定糖基化突变的细胞系中表达Ig被证明是一种用于研究碳水化合物结构对Ab功能贡献的有用技术。
