Type IIA procollagen amino propeptide is localized in human embryonic tissues
Type IIA procollagen amino propeptide is localized in human embryonic tissues
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DOI：
10.1177/002215549704501104
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发表时间：
1997-11-01
影响因子：
3.2
通讯作者：
Sandell, LJ
中科院分区：
文献类型：
--
作者：
Oganesian, A;Zhu, Y;Sandell, LJ
II型前胶原以其前体mRNA的选择性剪接产生的两种形式合成。选择性剪接结构域，外显子2，编码69个氨基酸的NH2前肽富含半胱氨酸的区域。mRNA表达的研究表明，较长的形式，命名为IIA型前胶原，由软骨祖细胞和各种非软骨组织合成。较短的IIB型前胶原由分化的软骨细胞合成。作为我们研究IIA型前胶原功能的第一步，将对应于外显子2的蛋白结构域作为重组融合蛋白产生，并用于在兔中产生抗体。ELISA、Western blotting和免疫荧光共定位检测结果表明，该抗血清特异性地识别Ⅱ A型前胶原NH_2前肽，并在54天人胎肋骨组织培养液中鉴定出Ⅱ A型前胶原。共聚焦显微镜用于在第50天和第53天的人胚胎中定位IIA型NH2前肽。在发育中的手的指射线中，仅检测到IIA型前胶原mRNA，在细胞外基质中观察到IIA型前胶原NH 2前肽。在下臂和椎体的发育中的长骨的软骨中观察到IIA型前胶原NH 2前肽的存在，即使这些组织在该发育阶段合成IIB型mRNA。IIA型前胶原NH2前肽位于发育中的气管中，气管是一种不经历软骨内骨形成的软骨。IIA型NH 2前肽也定位于已知合成IIA型mRNA的非软骨组织中，如椎间区、软骨膜、脊索鞘和耳泡的神经上皮。在大多数组织中，共定位与抗血清对三螺旋结构域的II型胶原蛋白。与IIA型NH2前肽抗血清的阳性免疫反应性表明，第一次，这种前肽存在于组织中。NH2前肽抗体与胶原分子的三螺旋结构域的共定位表明IIA型前胶原在这些组织的细胞外基质中是完整的。总而言之，这些结果强烈表明，在合成IIA型前胶原mRNA的细胞周围，IIA型前胶原NH2前肽被分泌并沉积到细胞外基质中。根据这些结果，我们预测IIA型前胶原在组织分化中发挥作用，增强其纯粹的建筑功能。
Type II procollagen is synthesized in two forms generated by the alternative splicing of its precursor mRNA. The alternatively spliced domain, exon 2, encodes the 69-amino-acid cysteine-rich region of the NH2 propeptide. Studies of mRNA expression have shown that the longer form, designated Type IIA procollagen, is synthesized by chondroprogenitor cells and various noncartilaginous tissues. The shorter form, Type IIB procollagen, is synthesized by differentiated chondrocytes. As the initial step in our investigations of the function of the Type IIA procollagen, the protein domain corresponding to exon 2 was created as a recombinant fusion protein and used to raise antibodies in rabbits. The resulting antiserum was specific for Type IIA procollagen NH2 propeptide as shown by ELISA, Western blotting, and immunofluorescent co-localization with the triple-helical domain of Type II collagen, Type IIA procollagen was identified in tissue culture medium of 54-day hu man fetal ribs. Confocal microscopy was used to localize the Type IIA NH2 propeptide in Day 50 and 53 human embryos. In the digital rays of the developing hand, where only Type IIA procollagen mRNA was detected, Type IIA procollagen NH2 propeptide was observed in the extracellular matrix, The presence of Type IIA procollagen NH2 propeptide was observed in the cartilage of the developing long bones of the lower arm and vertebral bodies even though these tissues synthesize Type IIB mRNA at this developmental stage. Type IIA procallagen NH2 propeptide was localized in the developing trachea, a cartilage that does not undergo endochondral bone formation. Type IIA NH2 propeptide was also localized in noncartilaginous tissues known to synthesize Type IIA mRNA, such as the intervertebral area, perichondrium, notochordal sheath, and neuroepithelium of the otic vesicle. In most tissues, co-localization with antiserum against the triple-helical domain of Type II collagen was observed. Positive immunoreactivity with the Type IIA NH2 propeptide antiserum indicates, for the first time, that this propeptide is present in the tissue. Co-localization of NH2 propeptide antibodies with the triple-helical domain of the collagen molecule suggests that Type IIA procollagen is intact in the extracellular matrix of these tissues. Taken together, these results strongly suggest that around cells that synthesize Type IIA procollagen mRNA, Type IIA procollagen NH2 propeptide is secreted, and deposited into the extracellular matrix. In light of these results, we predict that Type IIA procollagen plays a role in differentiation of tissues that augments its purely architectural function.