Winkelhake J L, Kusumi A, McKean L, Mandy W J
J Biol Chem. 1984 Feb 25;259(4):2171-8.
IgG anti-hapten antibodies were purified from the sera of rabbits homozygous for allotypic determinants d11 and d12 in the constant region of the heavy chain. Correlative with this determinant is the absence (d11) or presence (d12) of an oligosaccharide chain just below the hinge region of the IgG molecule. Both d11 and d12 molecules contain a complex heterosaccharide chain located near the carboxyl terminus of the second constant region domain. The two populations of IgG antibodies were thus selectively labeled with the spin probe Tempamine in their second constant region domains by reductive amination primarily of terminal N-acetylneuraminic acid residues. Chemical and enzymatic cleavages showed about 80% of the attached spin labels were N-acetylneuraminic acid-associated. Analysis of probe adducts by ESR spectrometry showed the presence of slower and faster moving subcomponents. Formation of immune complexes by antigen induces slight but significant restrictions of spin label mobility for both d11 and d12 IgG molecules. This restriction is qualitatively different from that seen in glutaraldehyde-, carbodiimide-, or ethanol-induced aggregates of the same IgG antibodies. The addition of purified complement C1 subcomponent C1q to immune aggregates resulted in marked immobilization of spin labels, the rotational correlation time of which was 30-40 mu s for both d11 and d12 molecules (evaluated by saturation transfer spectroscopy). A similar spin probe immobilizing effect is not seen when C1q binds to chemically aggregated IgG antibodies (which also do not activate C1). A novel model is proposed in which C1q is hypothesized to juxtapose Fc moieties in a discrete fashion required for subsequent C1 activation processes mediated by immune complexes.
IgG抗半抗原抗体是从在重链恒定区具有同种异型决定簇d11和d12的纯合兔血清中纯化得到的。与该决定簇相关的是，在IgG分子铰链区下方，寡糖链的缺失（d11）或存在（d12）。d11和d12分子均在第二个恒定区结构域的羧基末端附近含有一条复杂的杂糖链。因此，通过主要对末端N-乙酰神经氨酸残基进行还原胺化，用自旋探针Tempamine对这两种IgG抗体群体在其第二个恒定区结构域进行了选择性标记。化学和酶切显示，约80%的附着自旋标记与N-乙酰神经氨酸相关。通过电子自旋共振光谱法分析探针加合物，发现存在移动较慢和较快的亚组分。抗原形成免疫复合物会导致d11和d12 IgG分子的自旋标记流动性出现轻微但显著的受限。这种受限在性质上不同于在相同IgG抗体的戊二醛、碳二亚胺或乙醇诱导聚集体中观察到的情况。向免疫聚集体中添加纯化的补体C1亚组分C1q会导致自旋标记显著固定，对于d11和d12分子，其旋转相关时间均为30 - 40微秒（通过饱和转移光谱法评估）。当C1q与化学聚集的IgG抗体结合时（也不激活C1），未观察到类似的自旋探针固定效应。提出了一种新模型，其中假设C1q以免疫复合物介导的后续C1激活过程所需的离散方式并列Fc部分。
