Hsu H C, Hsu C I, Lin R H, Kao C L, Lin J Y
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan 100, Republic of China.
Biochem J. 1997 Apr 15;323 ( Pt 2)(Pt 2):557-65. doi: 10.1042/bj3230557.
A new fungal immunomodulatory protein (Fip) has been purified from the edible mushroom, Volvariella volvacea, and designated Fip-vvo. Analysis of the purified protein by SDS/PAGE followed by Coomassie Blue staining demonstrated that Fip-vvo is a single polypeptide with an apparent molecular mass of 15 kDa. Periodic acid/Schiff staining showed that this single polypeptide lacks carbohydrates. Using an in vitro bioassay measuring blast-formation stimulatory activity, Fip-vvo was shown to stimulate the maximum proliferation of human peripheral blood lymphocytes at a concentration of 5 microg/ml. Fip-vvo was capable of agglutinating rat red blood cells. Neither haemagglutination nor mitogenic activities were inhibited by mono- or dimeric sugars. In vivo, repeat administration of Fip-vvo greatly reduced the production of BSA-induced Arthus reaction in mice, whereas little effect was observed on the prevention of systemic anaphylaxis reactions. The selectively enhanced transcriptional expression of interleukin (IL)-2, IL-4, interferon-gamma, tumour necrosis factor-alpha, lymphotoxin and IL-2 receptor by Fip-vvo was also demonstrated by reverse transcriptase-PCR. This finding suggests that Fip-vvo exerts its immunomodulatory effects via cytokine regulation. In addition, the complete amino acid sequence of Fip-vvo was obtained by direct protein sequencing. This protein consists of 112 amino acid residues with a blocked N-terminal end and has a calculated molecular mass of 12667 Da not including the N-terminal blocking group. By gel filtration analysis, Fip-vvo exhibited a molecular mass of 26 kDa for the native molecules in PBS. This result indicates that native Fip-vvo is most likely a non-covalently associated homodimeric molecule.
一种新的真菌免疫调节蛋白（Fip）已从食用菌草菇中纯化出来，并命名为Fip-vvo。通过SDS/PAGE分析纯化后的蛋白，然后进行考马斯亮蓝染色，结果表明Fip-vvo是一种表观分子量为15 kDa的单一多肽。过碘酸/希夫染色显示该单一多肽不含碳水化合物。使用体外生物测定法测量 blast-形成刺激活性，结果表明Fip-vvo在浓度为5 μg/ml时能刺激人外周血淋巴细胞的最大增殖。Fip-vvo能够凝集大鼠红细胞。单糖或二糖均不抑制血凝和促有丝分裂活性。在体内，重复给予Fip-vvo可显著降低小鼠中牛血清白蛋白诱导的阿瑟斯反应的产生，而对预防全身过敏反应几乎没有影响。逆转录聚合酶链反应（RT-PCR）也证明了Fip-vvo可选择性增强白细胞介素（IL）-2、IL-4、干扰素-γ、肿瘤坏死因子-α、淋巴毒素和IL-2受体的转录表达。这一发现表明Fip-vvo通过细胞因子调节发挥其免疫调节作用。此外，通过直接蛋白质测序获得了Fip-vvo的完整氨基酸序列。该蛋白由112个氨基酸残基组成，N端封闭，计算分子量为12667 Da（不包括N端封闭基团）。通过凝胶过滤分析，Fip-vvo在PBS中天然分子的分子量为26 kDa。这一结果表明天然Fip-vvo很可能是一种非共价结合的同二聚体分子。
