Shafferman A, Velan B, Cohen S, Leitner M, Grosfeld H
J Biol Chem. 1987 May 5;262(13):6227-37.
Bovine interferon alpha C (IFN-alpha C) manifest at least 10(5)-fold lower antiviral activity on human cells than on bovine cells (Velan, B., Cohen, S., Grosfeld, H., Leitner, M., and Shafferman, A. (1985) J. Biol. Chem. 260, 5498-5504). By oligonucleotide site-directed mutagenesis within the coding region for the NH2-terminal 44-residue domain of BoIFN-alpha C, we replaced up to 18 residues by the corresponding HuIFN-alpha J1 residues. (HuIFN-alpha J1 is less than 60% homologous in sequence to BoIFN-alpha C.) The nine different bovine-human-IFN alpha hybrids obtained were compared to BoIFN-alpha C and HuIFN-alpha J1 with respect to their potential to induce an antiviral state, synthesis of 2-5A-synthetase, and their specific binding to human and bovine cells. Relative to BoIFN-alpha C, a gradual increase in biological activities (antiviral or 2-5A-synthetase) of approximately 10-, 10(2)-, 10(3)-, and approximately 10(4)-fold is obtained, depending on the number and positions of the residues substituted. A direct correlation exists between biological response and ability of IFN alpha to bind specifically to human cells. A BoIFN alpha molecule mutated in the 10-44 NH2-terminal domain was obtained which is 15, 8, and 35% as active as HuIFN-alpha J1 on human cells in specific binding, induction of antiviral, and 2-5A-synthetase activities, respectively. We concluded that at least 5 of the 12 residues at positions 10; 21, 22, 24; 27; 31, 34, 35, 37, 40; 42, 43 in the 10-44 NH2-terminal domain are critical for recognition of the human IFN-alpha cell receptor and for biological activity. These residues are found among 10 strictly conserved residues in all reported mammalian IFN alpha S, and they act in a cooperative manner to induce a biological response in human cells. The gap between the extent of improvement in binding capacity of the BoIFN alpha mutants on human cells and the corresponding biological response suggests that the primary signal of binding to the cell receptor is amplified within the cell. On bovine cells, HuIFN-alpha J1 and BoIFN-alpha C also compete for the same receptor, and it seems that at least part of the 10-44 NH2-terminal domain on IFN alpha is also involved in interaction with the bovine IFN alpha cell receptor.
牛α干扰素C（IFN-αC）对人细胞的抗病毒活性比对牛细胞的活性至少低10⁵倍（韦兰，B.，科恩，S.，格罗斯费尔德，H.，莱特纳，M.，以及沙弗曼，A.（1985年）《生物化学杂志》260卷，5498 - 5504页）。通过对牛IFN-αC氨基末端44个残基结构域编码区进行寡核苷酸定点诱变，我们用相应的人IFN-αJ1残基替换了多达18个残基。（人IFN-αJ1与牛IFN-αC的序列同源性小于60%。）将获得的9种不同的牛 - 人α干扰素杂种与牛IFN-αC和人IFN-αJ1在诱导抗病毒状态、2 - 5A合成酶的合成以及它们与人细胞和牛细胞的特异性结合潜力方面进行了比较。相对于牛IFN-αC，根据取代残基的数量和位置，生物活性（抗病毒或2 - 5A合成酶）逐渐增加约1倍、10²倍、10³倍和约10⁴倍。生物反应与α干扰素特异性结合人细胞的能力之间存在直接相关性。获得了一种在10 - 44氨基末端结构域发生突变的牛IFN-α分子，其在人细胞上的特异性结合、抗病毒诱导和2 - 5A合成酶活性分别是人类IFN-αJ1的15%、8%和35%。我们得出结论，在10 - 44氨基末端结构域中位置10；21、22、24；27；31、34、35、37、40；42、43的12个残基中至少有5个对于识别人类IFN-α细胞受体和生物活性至关重要。这些残基存在于所有已报道的哺乳动物IFN-α中10个严格保守的残基之中，并且它们以协同方式在人细胞中诱导生物反应。牛IFN-α突变体在人细胞上结合能力的提高程度与相应生物反应之间的差距表明，与细胞受体结合的初级信号在细胞内被放大。在牛细胞上，人IFN-αJ1和牛IFN-αC也竞争相同的受体，并且似乎IFN-α上10 - 44氨基末端结构域的至少一部分也参与与牛IFN-α细胞受体的相互作用。
