Identification and characterization of gelatin-cleavage activities in the apically located extracellular matrix of the sea urchin embryo

Abstract

We have identified and partially characterized several gelatinase activities associated with the sea urchin extraembryonic matrix, the hyaline layer. A previously identified 41-kDa collagenase/gelatinase activity was generally not found to be associated with isolated hyaline layers but was dissociated from the surface of 1-h-old embryos in the absence of Ca2+ and Mg2+. While hyaline layers, freshly prepared from 1-h-old embryos, were devoid of any associated gelatinase activities, upon storage at 4 degrees C for 4 days, a number of gelatin-cleavage activities appeared. Comparative analysis of these activities with the 41-kDa collagenase/gelatinase revealed that all species were inhibited by ethylenediamine tetraacetic acid but were refractory to inhibition with the serine protease inhibitors, phenylmethyl sulfonyl fluoride and benzamidine. In contrast, the largely Zn2+ specific chelator 1,10-phenanthroline had markedly different effects on the gelatinase activities. While several of the storage-induced, hyaline-layer-associated gelatinase activities were inhibited, the 41-kDa collagenase/gelatinase was refractory to inhibition as was a second gelatinase species with an apparent molecular mass of 45 kDa. We also examined the effects of a series of divalent metal ions on the gelatin-cleavage activities. In both qualitative and quantitative assays, Ca2+ was the most effective activator while Mn2+, Cu2+, Cd2+, and Zn2+ were all inhibitory. In contrast, Mg2+ had a minimal inhibitory effect on storage-induced gelatinase activities but significantly inhibited the 41-kDa collagenase/gelatinase. These results identify several distinct gelatin-cleavage activities associated with the sea urchin extraembryonic hyaline layer and point to diversity in the biochemical nature of these species.

Effects of calcium and magnesium on a 41-kDa serine-dependent protease possessing collagen-cleavage activity

“…As a control, we fractionated several Zn 2+ ‐dependent gelatinase activities, isolated from sea urchin hyaline layers (lanes 1, 2, 4, and 5) alongside the 41‐kDa collagenase/gelatinase (lanes 3 and 6). We have previously shown that the cleavage activities in lanes 1 and 2 were Zn 2+ ‐dependent [Flood et al, 2000] and, as expected, these activities were substantially reduced in the presence of 1,10‐phenanthroline (lanes 4 and 5). Interestingly, 1,10‐phenanthroline appeared to activate the 41‐kDa collagenase/gelatinase (compare lanes 3 and 6).…”

Zymogen activation and characterization of a major gelatin‐cleavage activity localized to the sea urchin extraembryonic matrix

“…Using the sea urchin as a model, we and others have identified gelatin and collagen cleaving activities in this embryo [Quigley et al, 1993;Vafa and Nishioka, 1995;Mayne and Robinson, 1996]. The study reported here follows from an earlier investigation which showed that newly isolated hyaline layers were devoid of gelatin-cleavage activities [Flood et al, 2000].…”

“…However, no gelatin-cleavage activities were found in layers freshly prepared from 1-h-old-embryos [Flood et al, 2000]. In contrast, several gelatincleavage activities appeared when layers were incubated in MFSW at 158C, the ambient temperature for Strongylocentrotus purpuratus (Fig.…”

Collagenolytic activity in tissue extract of parborlasia corrugatusfrom antarctic region

“…According to the literature in tissues of marine invertebrates was found a number of hydrolytic enzymes which take part in several fundamental processes, such as extracellular matrix remodeling, embryonic development, cell growth and differentiation and also in defense mechanisms thus highlightening their intriguing and unexpected functional importance in invertebrate life (Flood et al, 2000;Ghamari et al, 2014;Salamonea, 2012;Songklanakarin, 2008). …”