Simple Novel Assays in Glycobiology

Abstract

In response to an invitation from this journal, I am providing this mini-review of recent work from the Oppenheimer lab. Over the past 4 decades we have developed many assays that help us examine the role of specifc glycans in cellular interactions. Recently we have used two model systems for this work, the sea urchin embryo and yeast (Saccharomyces cerevisiae). We have developed two assays using the sea urchin embryo. One involves a microplate method where living sea urchin embryos are incubated with specific glycans or glycosidases. A specific set of cellular interactions, development of the primitive gut…archenteron, is examined over time in the presence and absence of the sugars or enzymes in living embryos. L-rhamnose and polyglucans have been identified as playing a role in mediating these cellular interactions. The second assay involves microdissection of the primitive gut away from the blastocoel roof to which it adheres.Using independently characterized glycosidases, we showed that polyglucans appear to mediate this cellular interaction. In the second system using yeast, we examine yeast disaggregation from lectin-derivatized agarose beads in the presence and absence of specific glycans using a quantitative, kinetic graphic profile assay.We found that D-melezitose was the best adhesion inhibitor and may be therapeutically useful in anti-adhesion venues of pathogen binding to cells and in cancer cell binding. The Sea Urchin, NIH Designated Model System Microplate assayWe have used the sea urchin embryo in our glycobiology research for over 4 decades. The sea urchin has been designated by NIH as a model system for understanding mechanisms of importance in human health and disease [1]. 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]. The developing gut is a simple model for investigating cellular interactions. Because molecules easily enter the interior of sea urchin embryos [6], we are able to precisely quantify the effects of the added reagents on specific cellular interactions. Using these methods we have developed statistically evaluated, quantitative, kinetic graphic profiles of the effects of glycans and glycosidases on the development of the sea urchin archenteron, a model set of cellular interactions, and found that L-rhamnose and polyglucans appear to be involved in these cellular interactions. Of importance is that we independently characterize enzymes used in our studies and test for unit activity and contaminants.Assay scheme: Grow sea urchin embryos to 24 hrs or 32 hrs post fertilization in normal artificial sea water. In 96 well micropl...