A role for polyglucans in a model sea urchin embryo cellular interaction

Abstract

The enzymatic activities of commercially prepared glycosidases were verified by direct chemical assays using defined substrates and fixed and live sea urchin (Lytechinus pictus) embryos to determine if a model cellular interaction of interest to developmental biologists for over a century (interaction of archenteron tip and roof of the blastocoel) was mediated by glycans. Glycosidases (active and denatured) were incubated with microdissected archenterons and blastocoel roofs in a direct assay to learn if their enzymatic activities could prevent the normal adhesive interaction. Of the five glycosidases tested only β-amylase (an exoglycosidase) immediately inhibited the interaction at relatively low unit activity. α-Amylase (an endoglycosidase) had no measurable effect, while other glycosidases (α-glucosidase, β-glucosidase, β-galactosidase) only substantially inhibited adhesion after a 12-h incubation. We demonstrated that the five glycosidases were active (not inhibited) in the presence of embryo materials, and that cleaved sugars could be detected directly after incubation of some enzymes with the embryos. The biochemical purity of the enzymes was examined using gel electrophoresis under denaturing conditions, and the absence of contaminating proteases was confirmed using Azocoll™ substrate. As we cannot entirely rule out the presence of minor contaminating enzymatic activities, only inhibitions of adhesion after very short incubations with enzyme were considered significant and biologically relevant. Although glycans in indirect experiments have been implicated in mediating the interaction of the tip of the archenteron and roof of the blastocoel, to our knowledge, this is the first study that directly implicates polyglucans with terminal 1,4-linked glucose residues in this adhesive event.

Simple Novel Assays in Glycobiology

“…Then we assess whether or not the gut will attach to the blastocoel roof in the presence or absence of independently characterized contaminant-free glycosidases. In this way we are able to provide convincing evidence for a role of specific glycans in mediating a specific model cellular interaction in a model system [3]. In these experiments, using independently characterized glycosidases, we have found a role for polyglucans in this cellular interaction.…”

“…Because of its transparency and simplicity, the sea urchin is well suited for research in glycobiology. We have developed new assays using the sea urchin embryo to learn about the roles of specific glycans in mediating cellular interactions [2][3][4][5]. The first assay involves culturing sea urchin embryos in 96 well microplates with and without glycans or glycosidases over time and counting the different morphologies of the developing gut (archenteron) in tens of thousands of living embryos [2,4].…”

“…In order to directly identify the roles of specific glycans in mediating a specific cellular interaction in the sea urchin model, we microdissected the developing gut (archenteron) from the blastocoel roof to what it specifically attaches [3]. Then we assess whether or not the gut will attach to the blastocoel roof in the presence or absence of independently characterized contaminant-free glycosidases.…”

Involvement of l(–)-rhamnose in sea urchin gastrulation: a live embryo assay

“…The most important finding is that the current assay has quantitatively been able to identify a specific sugar of interest with great statistical reliability in thousands of living, swimming embryos. The next series of experiments will involve use of α- l -rhamnosidase in similar quantitative assays and also using micro-dissected archenterons and blastocoel roofs (Singh et al ., 2013) to determine if l (–)-rhamnose is directly involved in adhesion between the archenteron and blastocoel roof. This series will help to pinpoint l (–)-rhamnose's mechanism of action.…”

“…The identification of polyglucans has been identified recently as involved in mediation of the adhesive interaction between the tip of the archenteron and the blastocoel roof (Singh et al ., 2013). In the present study, the sugar l (–)-rhamnose is identified as a putative component in the control of archenteron elongation and attachment to the blastocoel roof.…”