MONOCLONAL-ANTIBODIES THAT INHIBIT BINDING OF PROPOLYPEPTIDE OF VONWILLEBRAND-FACTOR TO COLLAGEN - LOCALIZATION OF EPITOPES
MONOCLONAL-ANTIBODIES THAT INHIBIT BINDING OF PROPOLYPEPTIDE OF VONWILLEBRAND-FACTOR TO COLLAGEN - LOCALIZATION OF EPITOPES
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DOI：
10.1111/j.1432-1033.1991.tb15864.x
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发表时间：
1991-03-28
期刊：
影响因子：
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通讯作者：
SAITO, Y
中科院分区：
文献类型：
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作者：
FUJISAWA, T;TAKAGI, J;SAITO, Y
我们以前报道过，牛血管性血友病因子（pp-vWF）的多肽原结合到I型胶原蛋白。 为了确定pp-vWF的胶原结合位点，我们产生了针对牛pp-vWF的单克隆抗体（mAb）。 一种称为TC 8的mAb非常强烈地抑制pp-vWF与I型胶原的结合;另外三种称为TC 2、TC 6和TC 7的mAb表现出中度抑制。 mAb与完整pp-vWF结合的竞争表明，TC 8的表位在结构上独立于TC 6和TC 7。 为了直接确定每个mAb在牛pp-vWF分子上的表位位置，我们通过免疫印迹法检测了mAb对通过赖氨酰内肽酶消化获得的肽片段的反应性。 TC 2和TC 8识别分子量为21 kDa的片段，而TC 6和TC 7识别分子量为18 kDa的不同片段。 将这两个片段纯化至均一，并测定其N-末端氨基酸序列。 将这些序列与人pp-vWF的序列进行比较，这些片段在一级结构中的位置估计为21-kDa片段的Phe 570-Lys 682和18-kDa片段的Glu 281-Lys 375。 这些数据表明，pp-vWF含有至少两个胶原结合位点，位于Phe 570-Lys 682和Glu 281-Lys 375之间的区域内或附近。
We reported previously that bovine propolypeptide of von Willebrand factor (pp-vWF) binds to type I collagen. To determine the collagen-binding sites of pp-vWF we generated monoclonal antibodies (mAbs) against bovine pp-vWF. One mAb, designated TC8, very strongly inhibited the binding of pp-vWF to type I collagen; three other mAbs, designated TC2, TC6 and TC7, exhibited moderate inhibition. Competition between the mAbs for binding to intact pp-vWF revealed that the epitope for TC8 was structurally independent of that for TC6 and TC7. To determine directly the location of the epitope for each mAb on the bovine pp-vWF molecule, we tested the reactivity of mAbs by immunoblotting toward peptide fragments obtained by digestion with lysylendopeptidase. TC2 and TC8 recognized a fragment of mass 21 kDa, while TC6 and TC7 recognized a distinct fragment of 18 kDa. These two fragments were purified to homogeneity and their N-terminal amino acid sequences were determined. Comparing these sequences with the sequence of human pp-vWF, the locations of these fragments in the primary structure were estimated to be Phe570-Lys682 for the 21-kDa fragment and Glu281-Lys375 for the 18-kDa fragment. These data suggest that pp-vWF contains at least two collagen-binding sites which lie within or close to the regions between Phe570-Lys682 and Glu281-Lys375.