Ebanks R O, Isenman D E
Department of Biochemistry, University of Toronto, Ontario, Canada.
Mol Immunol. 1996 Feb;33(3):297-309. doi: 10.1016/0161-5890(95)00135-2.
It has long been known that mouse C4 has unusually low hemolytic activity relative to the C4 of other mammalian species (e.g. human and guinea pig), the measurements being done in most cases using a C4-deficient guinea pig serum reagent in a one-step assay with EA. This low activity for mouse C4 previously had been attributed to "technical" difficulties such as lability of the protein during blood collection and partial species incompatibilities with guinea pig components. Recently, we presented evidence for the involvement of human C4 beta-chain residues 455-469, a putatively exposed hydrophilic segment, in contributing to a C5 binding site in the C4b subunit of the classical pathway C5 convertase, C4b3b2a. Given that there were five sequence differences between the human and mouse protein within this segment, we hypothesized that these substitutions may have compromised the C5 convertase subunit activity of mouse C4, thereby resulting in its low hemolytic activity. Using a multi-step hemolytic assay which was totally dependent upon C5 cleavage by the classical pathway, we found that mouse C4 was completely devoid of classical pathway C5 convertase subunit activity. We have been able to rule out the most obvious potential species incompatibilities (e.g. between C4mo and C5gp) as being responsible for this lack of activity. Moreover, we found that the low level of hemolytic activity of mouse C4 measured in the one-step assay can be ascribed totally to C5 cleavage, and subsequent terminal component assembly, by the alternative pathway C5 convertase, (C3b)2Bb. However, the assembly of the latter enzyme complex is dependent upon the presence of C3b molecules deposited initially via the classical pathway C3 convertase in which mouse C4b is a subunit. Finally, whereas conversion of human residues 458RP to the mouse-like sequence PL was sufficient to abrogate classical pathway C5 convertase subunit activity in human C4, the five substitutions which "humanized" the 452-466 segment of mouse C4 (corresponding to human residues 455-469) were on their own insufficient to impart this activity to mouse C4. This implies that, in addition to the 455-469 beta-chain segment of human C4, there are other regions of the molecule contributing to C5 binding which are also non-conserved between human and mouse C4.
长期以来，人们一直知道，相对于其他哺乳动物物种（如人类和豚鼠）的C4，小鼠C4的溶血活性异常低，在大多数情况下，测量是在一步法EA试验中使用C4缺陷型豚鼠血清试剂进行的。小鼠C4的这种低活性以前被归因于“技术”困难，如采血过程中蛋白质的不稳定性以及与豚鼠成分的部分物种不相容性。最近，我们提供了证据表明，人C4β链残基455 - 469（一个假定暴露的亲水性片段）参与了经典途径C5转化酶C4b3b2a的C4b亚基中C5结合位点的形成。鉴于该片段中人和小鼠蛋白质之间存在五个序列差异，我们推测这些替换可能损害了小鼠C4的C5转化酶亚基活性，从而导致其溶血活性较低。使用完全依赖经典途径裂解C5的多步法溶血试验，我们发现小鼠C4完全缺乏经典途径C5转化酶亚基活性。我们已经能够排除最明显的潜在物种不相容性（如C4mo和C5gp之间）是导致这种活性缺乏的原因。此外，我们发现一步法试验中测得的小鼠C4溶血活性水平完全可归因于替代途径C5转化酶（C3b）2Bb对C5的裂解以及随后的末端成分组装。然而，后一种酶复合物的组装取决于最初通过经典途径C3转化酶沉积的C3b分子的存在，其中小鼠C4b是一个亚基。最后，虽然将人残基458RP转换为小鼠样序列PL足以消除人C4中的经典途径C5转化酶亚基活性，但使小鼠C4的452 - 466片段（对应于人残基455 - 469）“人源化”的五个替换自身不足以赋予小鼠C4这种活性。这意味着，除了人C4的455 - 469β链片段外，分子中还有其他有助于C5结合的区域在人和小鼠C4之间也是不保守的。
