Thesis

Specific glycosidase effects on glucans involved in adhesion of microdissected sea urchin embryo structures

A variety of models of cell adhesion have been proposed by previous investigators; each depended heavily on details of the biochemistry of cell surface sugars. In this study, Lytechinus pictus sea urchin embryos were used to study the molecular mechanisms involved in cellular interaction. The interaction between the tip of the archenteron and the blastocoel roof during gastrulation in sea urchin embryos has been of interest to developmental biologists for over a century. This interaction is fundamental to morphogenesis. An understanding of the molecular mechanisms of the interaction between the archenteron and the blastocoel roof will be of major importance to the field of cell adhesion. These findings will provide a better understanding of what goes wrong in abnormal adhesive interactions, which may lead to diseases such as cancer. This is why the National Institute of Health (NIH) has designated the sea urchin embryo as a model system for studying basic mechanisms in human heath and disease. Here, we examined the effects of two glycosidases (sugar cleaving enzymes) on the surface of the sea urchin embryo cells. The enzymatic activities of two commercially prepared glycosidases were verified by direct chemical assays on known substrates as well as live and fixed sea urchin material to determine the interaction of the tip of the archenteron and the blastocoel roof. SDS-PAGE was carried out to measure possible protease contamination and the purity of the glycosidases. Azocoll test was also performed in order to check for cryptic protease activity. The results suggested there was no detectable smearing or degradation of the substrate and only minor levels of contaminating protease activity. In order to find out if sea urchin embryos have exposed glycans that can be degraded by α-amylase or α-glucosidase live and fixed embryos were disrupted and were used as substrates for these enzymes. A colorimetric assay with 3,5-dinitrosalicylic acid was used to measure the amount of maltose released. α-amylase enzyme did not release detectable amounts of maltose from embryos exposed to formaldehyde for long periods of fixation, but did release small amounts of maltose from live embryos or embryos fixed for shorter terms (1 day). These results suggest that the enzyme we use has specific sites of cleavage that may become inaccessible during long-term formaldehyde fixation. α-glucosidase released a small amount of maltose from the substrate sea urchin. To confirm that the sea urchin embryos do not release inhibitors of α-amylase and other glycosidases, mixed reactions were also conducted in which the defined substrates were tested in the presence of long-term fixed sea urchin embryos. The results suggested that there was no evidence of inhibition of these enzymes. I incubated glycosidases (active and denatured) on microdissected archenteron and blastocoel roofs in adhesion assays to learn if they directly inhibited the adhesive interaction. α-amylase had no effect, while α-glucosidase only substantially inhibited adhesion after 12 hours incubation. While glycans have been implicated in mediating this cellular interaction, this is the first study that directly implicates polyglucans with exposed 1,4 terminal ends in the adhesion of the archenteron tip and blastocoel roof.

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