Aeschlimann D, Paulsson M, Mann K
M. E. Müller Institute for Biomechanics, University of Bern, Switzerland.
J Biol Chem. 1992 Jun 5;267(16):11316-21.
The laminin-nidogen complex, the most abundant noncollagenous component of basement membranes, was recently shown to be a specific substrate for tissue transglutaminase (Aeschlimann, D., and Paulsson, M. (1991) J. Biol. Chem. 266, 15308-15317). Saturation experiments to determine the number of amine acceptor site(s) indicated a single reactive Gln residue in nidogen and none in laminin. Murine nidogen was labeled with [3H]putrescine in the tissue transglutaminase-catalyzed reaction, and two major radioactively labeled fragments, T70 and T40, were isolated after limited trypsin digestion. NH2-terminal sequencing showed that T40 is contained in T70 and corresponds to the rodlike structure of nidogen, made up of epidermal growth factor-like repeats. Three radioactively labeled peptides, obtained by extensive trypsin digestion of reduced and alkylated T40, were sequenced. In all a single residue, Gln726, was found to contain label. Sequencing of additional peptides, obtained after further treatment of the largest radioactively labeled peptide with endoproteinase Asp-N, gave the same result. Gln726 is located in an exposed loop between the second and the third EGF-like repeat in nidogen. This site is also conserved in the human sequence.
层粘连蛋白-巢蛋白复合物是基底膜中最丰富的非胶原蛋白成分，最近被证明是组织转谷氨酰胺酶的特异性底物（埃施利曼，D.，和保尔松，M.（1991年）《生物化学杂志》266，15308 - 15317）。用于确定胺受体位点数量的饱和实验表明，巢蛋白中有一个反应性谷氨酰胺残基，而层粘连蛋白中没有。在组织转谷氨酰胺酶催化的反应中，用[³H]腐胺标记小鼠巢蛋白，经有限的胰蛋白酶消化后，分离出两个主要的放射性标记片段，T70和T40。氨基末端测序表明，T40包含在T70中，对应于巢蛋白的杆状结构，由表皮生长因子样重复序列组成。对还原和烷基化的T40进行广泛的胰蛋白酶消化后得到的三个放射性标记肽段进行了测序。总共发现只有一个残基Gln726含有标记。用天冬氨酸内肽酶-N对最大的放射性标记肽段进一步处理后得到的其他肽段测序，结果相同。Gln726位于巢蛋白中第二个和第三个表皮生长因子样重复序列之间的一个暴露环中。该位点在人类序列中也保守。
