Lund J, Tanaka T, Takahashi N, Sarmay G, Arata Y, Jefferis R
Department of Immunology, University of Birmingham Medical School, Edgbaston, U.K.
Mol Immunol. 1990 Nov;27(11):1145-53. doi: 10.1016/0161-5890(90)90103-7.
Glycosylated chimeric mouse-human anti-NIP IgG3 antibody produced by growth of the J558L mouse B cell plasmacytoma is characterised with respect to the single carbohydrate chain at Asn-297 in the CH2 domain indicating that the mouse cell glycosyl transferases dictate the pattern of glycosylation rather than the human CH region of the heavy chain. Additionally, three unusual alpha-galactose-containing oligosaccharides are reported. Only the Fc region has detectable carbohydrate. Aglycosylated anti-NIP IgG3 antibody has been produced by cell growth in the presence of the antibiotic tunicamycin. Functionally, whilst the glycosylated intact IgG3 interacts with human Fc gamma R111 expressed on human killer (K) cells to trigger antibody-dependent cellular cytotoxicity the aglycosylated intact IgG3 fails to trigger cell lysis, localising the site on IgG for triggering human Fc gamma R111 mediated functions to the CH2 domain. The monomeric aglycosylated trypsin Fc fragment inhibits human Fc gamma R1 recognition by U937 cells 115-fold less well (K50 = 2 microM) than does glycosylated Fc (K50 = 17 nM), confirming that aglycosylation disrupts the site for human Fc gamma R1 within the CH2 domain and indicating that the trypsin Fc fragments reflect the functional properties of the intact IgG glycoforms. Structurally, 1H NMR shows that the absence of carbohydrate at Asn-297 results in a small and localised protein structural change in the vicinity of the reporter group His-268 within the CH2 domain. The site on IgG for triggering human Fc gamma R111 mediated functions is then localised to the vicinity of His-268. The profound impact of aglycosylation on human Fc gamma R1 recognition implies structural disruption of the proposed site for human Fc gamma R1 in the lower hinge region of IgG (residues 234-239), proximal to His-268.
由J558L小鼠B细胞浆细胞瘤生长产生的糖基化嵌合小鼠-人抗NIP IgG3抗体，对CH2结构域中Asn-297处的单条碳水化合物链进行了表征，这表明小鼠细胞糖基转移酶决定了糖基化模式，而非重链的人CH区域。此外，还报道了三种不寻常的含α-半乳糖的寡糖。只有Fc区域有可检测到的碳水化合物。通过在抗生素衣霉素存在下细胞生长产生了无糖基化的抗NIP IgG3抗体。在功能上，糖基化的完整IgG3与人类杀伤（K）细胞上表达的人FcγRIII相互作用以触发抗体依赖性细胞毒性，而无糖基化的完整IgG3则无法触发细胞裂解，从而将IgG上触发人FcγRIII介导功能的位点定位到CH2结构域。单体无糖基化的胰蛋白酶Fc片段抑制U937细胞对人FcγR1的识别，其效果比糖基化Fc（K50 = 17 nM）差115倍（K50 = 2 μM），这证实无糖基化破坏了CH2结构域内人FcγR1的位点，并表明胰蛋白酶Fc片段反映了完整IgG糖型的功能特性。在结构上，1H NMR表明Asn-297处缺乏碳水化合物会导致CH2结构域内报告基团His-268附近出现微小的局部蛋白质结构变化。IgG上触发人FcγRIII介导功能的位点随后被定位到His-268附近。无糖基化对人FcγR1识别的深远影响意味着在IgG的下铰链区（残基234-239）中靠近His-268的人FcγR1假定位点发生了结构破坏。
