Miyazaki K, Uchiyama K, Imokawa Y, Yoshizato K
Yoshizato MorphoMatrix Project, Exploratory Research for Advanced Technology, Research Development Corporation of Japan, Ibaraki, Japan.
Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6819-24. doi: 10.1073/pnas.93.13.6819.
Matrix metalloproteinases (MMPs) of regenerating urodele limbs have been suggested to play crucial roles in the process of the dedifferentiation of cells in the damaged tissues and the ensuing blastema formation because the activation of MMPs is an early and conspicuous event occurring in the amputated limb. MMP cDNAs were cloned as products of the reverse transcription-PCR from cDNA libraries of newt limbs, and their structures were characterized. Three cDNAs encoding newt MMPs (2D-1, 2D-19, and 2D-24) have been cloned from second day postamputation regenerating limbs, and a cDNA (EB-1) was cloned from early bud-stage regenerating limbs. These cDNAs included the full-length coding regions. The deduced amino acid sequences of 2D-1, 2D-19, 2D-24, and EB-1 had a homology with mammalian MMP9, MMP3/10, MMP3/10, and MMP13, respectively. The basic motif of these newt MMP genes was similar to mammalian counterparts and contained regions encoding a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. However, some unique molecular evolutionary features were also found in the newt MMPs. cDNAs of 2D-19 and 2D-24 contained a specific insertion and deletion, respectively. The insertion of 2D-19 is threonine-rich, similar to the threonine cluster found in the collagenase-like sea urchin hatching enzyme. Northern blot analysis showed that the expression levels of the newt MMPs were dramatically increased after amputation, suggesting that they play an important role(s) in tissue remodeling of the regenerating limb.
再生蝾螈肢体中的基质金属蛋白酶（MMPs）被认为在受损组织细胞去分化及随后的芽基形成过程中发挥关键作用，因为MMPs的激活是截肢肢体中发生的早期且显著的事件。通过逆转录PCR从蝾螈肢体的cDNA文库中克隆出MMP cDNA，并对其结构进行了表征。从截肢后第二天的再生肢体中克隆出三个编码蝾螈MMPs的cDNA（2D - 1、2D - 19和2D - 24），并从早期芽阶段的再生肢体中克隆出一个cDNA（EB - 1）。这些cDNA包含全长编码区。2D - 1、2D - 19、2D - 24和EB - 1推导的氨基酸序列分别与哺乳动物的MMP9、MMP3/10、MMP3/10和MMP13具有同源性。这些蝾螈MMP基因的基本基序与哺乳动物的对应物相似，包含编码假定信号序列、前肽、具有三个锌结合组氨酸残基的活性位点、钙结合结构域、血红素结合蛋白区域和三个关键半胱氨酸残基的区域。然而，在蝾螈MMPs中也发现了一些独特的分子进化特征。2D - 19和2D - 24的cDNA分别包含一个特定的插入和缺失。2D - 19的插入富含苏氨酸，类似于在胶原酶样海胆孵化酶中发现的苏氨酸簇。Northern印迹分析表明，蝾螈MMPs的表达水平在截肢后显著增加，表明它们在再生肢体的组织重塑中起重要作用。
