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

Ranganathan, Lavanya (2004) Purification and characterization of major gelatin-cleavage activities in the apically located extracellular matrix of the sea urchin embryo. Masters thesis, Memorial University of Newfoundland.

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Abstract

The hyaline layer is an apically located extracellular matrix that surrounds the sea urchin embryo from the time of fertilization until metamorphosis occurs. Gelatin-cleavage activities were absent from freshly prepared hyaline layers but a dynamic pattern of activities appeared when layers were incubated at 15°C or 37°C in UV-irradiated and millipore-filtered sea water. Three major gelatin cleavage activities at 55-, 41-, and 32 kDa were induced. The 55 kDa species was identified as the major activity at 37°C, while the 41- and 32 kDa activities were the minor species. In contrast, both the 41- and 32 kDa activities were the major species at 15°C, the ambient temperature for Strongylocentrotus purpuratus. The 55 kDa and other higher molecular weight species appeared only after 92 hours of incubation at 15°C. Interestingly, the gelatin-cleavage activities were absent when the hyaline layers were incubated at 37°C in millipore-filtered sea water containing 10 mM benzamidine hydrochloride, a reversible serine protease inhibitor. The minimum ionic requirements for the 15°C induction of the three activities were determined: both the 55- and 41 kDa species required 300 mM NaCl, while the 32 kDa activity required both 3 mM CaCl₂ and 200 mM NaCl. However, none of these activities required MgCl₂ for their induction. -- The 55-, 41- and 32 kDa gelatinase activities could be dissociated from the hyaline layers when incubated in 50 mM Tris-HCI, pH 8.0 containing 5 mM EDTA for 24 hours at 37°C. All three species were purified using gel filtration chromatography. In both qualitative and quantitative assays, the 55 kDa gelatinase activity was inhibited by 1,10 phenanthroline (a Zn²⁺ - specific chelator), ethylenediamine tetraacetic acid or ethylenebis (oxyethylenenitrilo) acetic acid. In the presence of 2 mM 1, 1 0-phenanthroline, the 55 kDa species was inhibited 93.1 %. Variations in the percent inhibition of the 55 kDa activity, in the range of 33-1 00% were observed in the presence of 5 mM ethylenebis (oxyethylenenitrilo) acetic acid. The quantitative gelatinase assay demonstrated that 10 mM CaCl2 stimulated 58.1% of the EGTA-inhibited (33%) 55 kDa species, which resulted in complete recovery of the activity. On the other hand, only 28.2% activation of the EGTA-inhibited (33%) 55 kDa was noticed in the presence of 50 mM, which recovered up to 85% of the total activity. The calcium reactivation of EGTA-inhibited (100%) 55 kDa occurred with an apparent dissociation constant of 1.2 mM. Since the hyaline layer is in direct contact with sea water, which contains 10 mM CaCl₂, 50 mM MgCl₂ and 500 mM NaCl, the effects of MgCl₂ and NaCl were also studied. Our results show that 85% of the total activity of the 33% EGTA-inhibited 55 kDa was reconstituted in the presence of 50 mM MgCl₂. The 55 kDa gelatin-cleavage activity was inhibited 57.6% in the presence of 500 mM NaCl. The NaCl-dependent inhibition suggests a possible mechanism for regulating the 55 kDa gelatinase activity. -- Developmental substrate gel analysis was performed using embryos of various developmental stages. The 55 kDa species comigrated with a gelatin cleavage activity present in 45 hours-old embryos and persisted until 93 hours after fertilization. This result confirms the presence of the 55 kDa species in the sea urchin embryo. We conclude that the sea urchin ECM, the hyaline layer has several gelatin-cleavage activities including a 55 kDa species, which is a Zn²⁺- and Ca²⁺ dependent, gelatin-specific endopeptidases that belongs to the matrix metalloproteinase class of extracellular matrix remodeling enzymes. Our results suggest that the 55 kDa is secreted as an inactive precursor, which is activated by one or more serine proteinase activity following incubation in MFSW at 15°C or 37°C. Our data indicate that the 55 kDa species might be the precursor of the 41 kDa gelatinase activity.

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