凝血因子V(FV)以两种形式循环,FV1和FV2,具有稍微不同的分子量和磷脂结合性质。目的是确定这种异质性是否归因于Asn(2181)的糖基化程度。FVa1和FVa2被分离并用内切糖苷酶PNGaseF消化。FVa2轻链含有两个N-连接的碳水化合物,而FVa1包含三个。野生型FV和三个突变体,Asn(2181)Gln,Ser(2183)Thr,和Ser(2183)Ala,在COS1细胞中表达,被凝血酶激活,用Western印迹分析.野生型FVa包含71kDa-74kDa双峰,而Asn(2181)Gln和Ser(2183)Ala突变体仅含有71kDa轻链。相比之下,Ser(2183)Thr突变体产生74kDa轻链。这表明Asn-X-Ser/Thr共有结构中的第三个位置影响糖基化效率,Thr与比Ser更高的糖基化程度相关。Ser(2183)Thr突变体FVa在功能上与血浆纯化的FVa1没有区别,而Asn(2181)Gln和Ser(2183)Ala突变体的行为与FVa2相似。因此,Asn(2181)处的碳水化合物损害了FVa与磷脂膜之间的相互作用,与C2结构域的三维模型的结构分析和拟议的磷脂结合位点的位置一致的解释。总之,我们表明,FV1-FV2异质性是由Asn(2181)的差异糖基化引起的,该糖基化与糖基化共有序列中第三个位置的Ser而不是Thr的存在有关。
Coagulation factor V (FV) circulates in two forms, FV1 and FV2, having slightly different molecular masses and phospholipid-binding properties. The aim was to determine whether this heterogeneity is due to the degree of glycosylation of Asn(2181). FVa1 and FVa2 were isolated and digested with endoglycosidase PNGase F. As judged by Western blotting, the FVa2 light chain contained two N-linked carbohydrates, whereas FVa1 contained three. Wild-type FV and three mutants, Asn(2181)Gln, Ser(2183)Thr, and Ser(2183)Ala, were expressed in COS1 cells, activated by thrombin, and analyzed by Western blotting. Wild-type FVa contained the 71 kDa-74 kDa doublet, whereas the Asn(2181)Gln and Ser(2183)Ala mutants contained only the 71 kDa light chain. In contrast, the Ser(2183)Thr mutant gave a 74 kDa light chain. This demonstrated that the third position in the Asn-X-Ser/Thr
consensus affects glycosylation efficiency, Thr being associated with a higher degree of glycosylation than Ser. The Ser(2183)Thr mutant FVa was functionally indistinguishable from plasma-purified FVa1, whereas Asn(2181)Gln and Ser(2183)Ala mutants behaved like FVa2. Thus, the carbohydrate at Asn(2181) impaired the interaction between FVa and the phospholipid membrane, an interpretation consistent with a structural analysis of a three-dimensional model of the C2 domain and the position of a proposed phospholipid-binding site. In conclusion, we show that the FV1-FV2 heterogeneity is caused by differential glycosylation of Asn(2181) related to the presence of a Ser rather than a Thr at the third position in the
consensus sequence of glycosylation.