Peters J H, Trevithick J E, Johnson P, Hynes R O
Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge 02139, USA.
Cell Adhes Commun. 1995 Feb;3(1):67-89. doi: 10.3109/15419069509081278.
Previous descriptions of the expression and distribution of the alternatively spliced EIIIB segment of fibronectin (FN) relied upon an antibody which, on subsequent testing, was shown not to recognize this segment directly. This raises concerns regarding the reliability of all such previous descriptions. In order to prepare reagents directly reactive with this segment, we raised polyclonal antibodies to two different bacterial fusion proteins containing intact EIIIB segments, and to a synthetic 36 amino acid peptide from the center of the EIIIB segment. Antibodies raised to each of these three immunogens recognized fusion proteins containing the EIIIB segment, but failed to recognize full length EIIIB+ FNs produced by mammalian cells, suggesting that oligosaccharide linked to Asn1359 within the EIIIB segment, or potentially to other residues in FN, might interfere with antibody recognition of this segment. Consistent with this hypothesis, N-deglycosylation of recombinant full and partial length EIIIB+ FNs permitted their specific recognition by the anti-fusion protein (but not anti-peptide) antibodies. Using anti-fusion protein antibodies coupled with deglycosylation procedures, we provide a series of new results relevant to the functions of the EIIIB segment: 1) Endogenously synthesized EIIIB+ FN is incorporated into the extracellular matrix of cultured fibroblasts, where it appears by immunofluorescence microscopy and radioimmunoprecipitation analyses to have a distribution very similar to both EIIIA+ forms and the total pool of FNs. 2) No reproducible changes can be shown to occur in the extent of synthesis or matrix incorporation of EIIIB+ FNs upon cellular transformation. 3) Low levels of EIIIB+ FN are normally present in blood plasma and consequently also in purified preparations of plasma FN. 4) EIIIB+ FN is present in blood platelets, where it constitutes a minor fraction of total platelet FN, yet is greater than 4-fold enriched relative to plasma FN. 5) EIIIB+ FN is synthesized by first passage cultured endothelial cells, suggesting that the endothelium could constitute a source for this FN isoform in the circulating blood. The antibodies and methods used in this study constitute the first direct assays of EIIIB+ FN protein expression and are applicable to a variety of species.
先前关于纤连蛋白（FN）可变剪接的EIIIB片段的表达和分布的描述依赖于一种抗体，后续测试表明该抗体不能直接识别此片段。这引发了对所有此类先前描述可靠性的担忧。为了制备与该片段直接反应的试剂，我们针对两种不同的含有完整EIIIB片段的细菌融合蛋白以及EIIIB片段中心的一个36个氨基酸的合成肽制备了多克隆抗体。针对这三种免疫原中的每一种产生的抗体都能识别含有EIIIB片段的融合蛋白，但不能识别哺乳动物细胞产生的全长EIIIB + FN，这表明与EIIIB片段内的Asn1359相连的寡糖，或可能与FN中的其他残基相连的寡糖，可能会干扰抗体对该片段的识别。与该假设一致，重组全长和部分长度的EIIIB + FN的N-去糖基化使其能够被抗融合蛋白（而非抗肽）抗体特异性识别。使用抗融合蛋白抗体并结合去糖基化程序，我们提供了一系列与EIIIB片段功能相关的新结果：1）内源性合成的EIIIB + FN被整合到培养的成纤维细胞的细胞外基质中，通过免疫荧光显微镜和放射免疫沉淀分析显示其分布与EIIIA +形式和FN的总库非常相似。2）细胞转化后，EIIIB + FN的合成程度或基质整合程度未显示出可重复的变化。3）血浆中通常存在低水平的EIIIB + FN，因此在纯化的血浆FN制剂中也存在。4）EIIIB + FN存在于血小板中，它占血小板总FN的一小部分，但相对于血浆FN富集超过4倍。5）首次传代培养的内皮细胞合成EIIIB + FN，这表明内皮可能是循环血液中这种FN异构体的来源。本研究中使用的抗体和方法构成了对EIIIB + FN蛋白表达的首次直接检测，适用于多种物种。
