Jones F S, Hoffman S, Cunningham B A, Edelman G M
The Rockefeller University, New York, NY 10021.
Proc Natl Acad Sci U S A. 1989 Mar;86(6):1905-9. doi: 10.1073/pnas.86.6.1905.
A combination of cDNA sequencing of the complete coding region, protein comparisons, binding site mapping, and electron microscopic imaging has permitted the formulation of a structural model of cytotactin. Cytotactin is a large extracellular matrix glycoprotein that displays a restricted tissue distribution during development. Although there appears to be a single cytotactin gene, multiple cytotactin polypeptides and mRNAs are detected in a variety of tissues. We report here the sequences and relationships of cDNAs that encode the complete amino acid sequences of two cytotactin polypeptides in chicken brain. The translated cDNA sequences agree with those obtained by direct analysis of cytotactin and fragments of the molecule. All regions of the polypeptides appear to be identical except for a 273 amino acid segment found in the larger but not in the smaller. At their amino termini, both polypeptides contain a cysteine-rich segment that probably includes those residues that link monomers into hexamers. This segment is followed by 13 epidermal growth factor-like (EGFL) repeats and then 8 consecutive segments that each resemble the type III repeats found in fibronectin. At their carboxyl termini, the polypeptides are similar to the beta and gamma chains of fibrinogen, including a calcium-binding segment. The additional sequence in the large polypeptide is inserted after the fifth type III repeat and includes three additional type III repeats. On RNA transfer blot analyses, cytotactin cDNA probes detected a 6.4-kilobase (kb) component in both brain and gizzard and larger mRNAs in both tissues, but those in gizzard were larger by about 1 kb than those in brain. A probe specific to the insert did not hybridize to the 6.4-kb mRNA in either tissue but detected the larger mRNAs in both tissues. At least a portion of the insert is thus present in both tissues, but there may be additional inserts in the gizzard mRNAs. The proposed model of cytotactin specifies the orientation of the polypeptides, the localization of interchain disulfide bonds, the structural elements constituting the thin and thick segments (EGFL repeats and type III repeats, respectively), the terminal fibrinogen-like nodular region, and the relative location of the cell-binding region.
通过对完整编码区进行cDNA测序、蛋白质比较、结合位点定位以及电子显微镜成像等方法相结合，已构建出细胞趋触蛋白的结构模型。细胞趋触蛋白是一种大型细胞外基质糖蛋白，在发育过程中其组织分布受限。虽然似乎只有一个细胞趋触蛋白基因，但在多种组织中可检测到多种细胞趋触蛋白多肽和mRNA。我们在此报告了编码鸡脑中两种细胞趋触蛋白多肽完整氨基酸序列的cDNA的序列及相互关系。翻译后的cDNA序列与通过对细胞趋触蛋白及其分子片段进行直接分析所获得的序列一致。除了在较大的多肽中存在而在较小的多肽中不存在的一个273个氨基酸的片段外，两种多肽的所有区域似乎都是相同的。在它们的氨基末端，两种多肽都含有一个富含半胱氨酸的片段，该片段可能包含将单体连接成六聚体所需的那些残基。该片段之后是13个表皮生长因子样（EGFL）重复序列，然后是8个连续的片段，每个片段都类似于纤连蛋白中发现的III型重复序列。在它们的羧基末端，这些多肽类似于纤维蛋白原的β链和γ链，包括一个钙结合片段。较大多肽中的额外序列插入在第五个III型重复序列之后，并且包括另外三个III型重复序列。在RNA转移印迹分析中，细胞趋触蛋白cDNA探针在脑和砂囊中均检测到一个6.4千碱基（kb）的成分以及两种组织中更大的mRNA，但砂囊中的mRNA比脑中的大约大1 kb。一个针对插入片段的探针在两种组织中均未与6.4-kb的mRNA杂交，但检测到了两种组织中更大的mRNA。因此，至少插入片段的一部分存在于两种组织中，但砂囊mRNA中可能存在额外的插入片段。所提出的细胞趋触蛋白模型明确了多肽的方向、链间二硫键的定位、构成薄段和厚段的结构元件（分别为EGFL重复序列和III型重复序列）、末端纤维蛋白原样结节区域以及细胞结合区域的相对位置。
