Exogenous hyalin and sea urchin gastrulation. Part IV: a direct adhesion assay – progress in identifying hyalin's active sites

Abstract

SummaryIn Strongylocentrotus purpuratus the hyalins are a set of three to four rather large glycoproteins (hereafter referred to as "hyalin"), which are the major constituents of the hyaline layer, the developing sea urchin embryo's extracellular matrix. Recent research from our laboratories has shown that hyalin is a cell adhesion molecule involved in sea urchin embryo specific cellular interactions. Other laboratories have shown it to consist of 2-3% carbohydrate, and a cloned, sequenced fragment demonstrated repeat domains (HYR) and non-repeat regions. Interest in this molecule has increased because HYR has been identified in organisms as diverse as bacteria, flies, worms, mice and humans, as well as sea urchins. Our laboratories have shown that hyalin appears to mediate a specific cellular interaction that has interested investigators for over a century, archenteron elongation/attachment to the blastocoel roof. We have done this by localizing hyalin on the two components of the cellular interaction and by showing that hyalin and anti-hyalin antibody block the cellular interaction using a quantitative microplate assay. The microplate assay, however, has limitations because it does not directly assess hyalin's effects on the adhesion of the two components of the interaction. Here we have used an elegant direct assay that avoids the limitations, where we microdissected the two components of the adhesive interaction and tested their readhesion to each other, thereby avoiding possible factors in the whole embryos that could confound or confuse results. Using both assays, we found that mild periodate treatment (6 h to 24 h in sodium acetate buffer with 0.2M sodium periodate at 4 °C in the dark) of hyalin eliminates its ability to block the cellular interaction, suggesting that the carbohydrate component(s) may be involved in hyalin's specific adhesive function. This is an important first step in identifying the molecular mechanisms of a well known cellular interaction in the NIH designated sea urchin embryo model, a system that has led to the discovery of scores of physiological mechanisms, including those involved in human health and disease.

Proximate composition of marine invertebrates from tropical coastal waters, with emphasis on the relationship between nitrogen and protein contents

“…The concentration of carbohydrate of H. grisea was 30% lower than that reported by Bechtel et al (2013) for the giant sea cucumber Parastichopus californicus from Alaska (USA). Sea urchins are poor in carbohydrate, but this substance has a significant role in both body coating and fertilization events (Ghazarian et al, 2010). Mol et al (2008) reported a concentration of 2.8% dw of carbohydrate in the roe of the sea urchin Paracentrotus lividus, which represents a carbohydrate content ~50-60% lower than those we found in the bodies of E. brasiliensis and L. variegatus.…”

Use of specific glycosidases to probe cellular interactions in the sea urchin embryo

“…There are four lines of evidence which suggest the presence of an appropriate substrate on the embryo surface. Firstly the work of Evans and Bosmann [21] showed the presence of glycosyltransferase activity in developing sea urchin embryos; secondly Latham et al[11], showed the presence of Con A ligands on the outside embryo surface and on the inner blastocoel surface; thirdly, exogeneously added hyalin that includes glucose residues causes similar developmental pathology to the effects reported here for glycosidases [5], and fourthly, Ghazarian et al [13], showed that periodate-treated hyalin blocked the ability of hyalin to cause developmental pathology. Latham et al [6] and Itza & Mozingo [7] showed that LCASW increased permeability of the embryo epithelium such that proteins could enter the blastocoel and that septate junctions mediating the barrier to permeability were loosened.…”

“…Past studies have implicated glycans and their binding partners in various sea urchin embryo cellular interactions, but definitive evidence for a glycan role(s) has been generally indirect [8, 9, 10, 11, 12, 13]. …”

Achieving monospermy or preventing polyspermy?

“…Fusion starts at the site of sperm entry and then traverses the oocyte in a wave to the antipode taking ~30 seconds to complete. Following the cortical reaction, a tightly adhering protective layer around the oocyte called the hyaline layer is formed, [10][11][12] while the peroxidase hardens the elevated fertilization membrane by cross-linking the tyrosine residues of the vitelline coat aided by hydrogen peroxide. This leads to a net increase in the total surface area of the plasma membrane, which can be observed as a transient increase in length of microvilli, and presumably changes the permeability and exchange properties of the oocyte with the environment.…”