Yan L, Pollock G H, Nagase H, Sarras M P
Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City 66160, USA.
Development. 1995 Jun;121(6):1591-602. doi: 10.1242/dev.121.6.1591.
Hydra extracellular matrix (ECM) is composed of a number of components seen in vertebrate ECM such as laminin, type IV collagen, fibronectin, and heparan sulfate proteoglycan. A number of functional studies have shown that hydra ECM plays an important role in pattern formation and morphogenesis of this simple metazoan. The present study was designed to identify matrix degrading proteinases in hydra and determine their potential function in hydra morphogenesis. Using SDS-PAGE gelatin-zymography, five gelatinolytic bands were identified with relative molecular masses of 67 x 10(3), 51-58 x 10(3) (a triplet) and 25-29 x 10(3), respectively. Inhibition studies indicated that all of these gelatinases were metalloproteinases. Gelatin-zymography indicated that there was a differential distribution of these gelatinases along the longitudinal axis of hydra, with the 67 x 10(3) M(r) gelatinase being concentrated in the body column, while the 51-58 x 10(3) M(r) gelatinase triplet and the 25-29 x 10(3) M(r) gelatinase concentrated in the head region. Purification procedures were successfully developed for the 25-29 x 10(3) M(r) metalloproteinase which has been termed hydra metalloproteinase 1 (HMP1) and which appeared as a single band with a SDS-PAGE mobility of 25.7 x 10(3) M(r). The N-terminal sequence of purified HMP1 indicated that it has structural homology with metalloproteinases that belong to the astacin family. Subsequent cloning and sequencing of cDNA clones confirmed the identification of HMP1 as an astacin-like metalloproteinase. Immunocytochemical studies with antibodies generated against the purified enzyme and to a synthetic peptide indicated that HMP1 was localized to the ECM of tentacles. Functional studies were performed in which purified HMP1, anti-HMP1 IgG, or suspected substrates of HMP1 (e.g. growth factors such as TGF-beta 1) were introduced into the interepithelial compartment of hydra using a 'DMSO loading' procedure. These studies indicated that HMP1 has a functional role during a number of developmental processes such as head regeneration and cell differentiation/transdifferentiation of tentacle battery cells.
水螅细胞外基质（ECM）由多种在脊椎动物ECM中可见的成分组成，如层粘连蛋白、IV型胶原蛋白、纤连蛋白和硫酸乙酰肝素蛋白聚糖。大量功能研究表明，水螅ECM在这种简单后生动物的模式形成和形态发生中发挥着重要作用。本研究旨在鉴定水螅中的基质降解蛋白酶，并确定它们在水螅形态发生中的潜在功能。使用SDS-PAGE明胶酶谱法，鉴定出五条明胶酶解带，相对分子质量分别为67×10³、51 - 58×10³（三重态）和25 - 29×10³。抑制研究表明，所有这些明胶酶都是金属蛋白酶。明胶酶谱显示，这些明胶酶沿水螅纵轴存在差异分布，67×10³ M(r) 的明胶酶集中在体柱中，而51 - 58×10³ M(r) 的明胶酶三重态和25 - 29×10³ M(r) 的明胶酶集中在头部区域。已成功开发出针对25 - 29×10³ M(r) 金属蛋白酶的纯化方法，该酶被称为水螅金属蛋白酶1（HMP1），在SDS-PAGE中迁移率为25.7×10³ M(r) 时呈现为单一带。纯化的HMP1的N端序列表明，它与属于虾红素家族的金属蛋白酶具有结构同源性。随后对cDNA克隆的克隆和测序证实，HMP1被鉴定为一种虾红素样金属蛋白酶。使用针对纯化酶和合成肽产生的抗体进行免疫细胞化学研究表明，HMP1定位于触手的ECM中。进行了功能研究，其中使用“二甲基亚砜加载”程序将纯化的HMP1、抗HMP1 IgG或HMP1的疑似底物（如转化生长因子β1等生长因子）引入水螅的上皮间腔室。这些研究表明，HMP1在许多发育过程中发挥功能作用，如头部再生和触手电池细胞的细胞分化/转分化。
