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

In Strongylocentrotus purpuratus the hyalins are a set of three to four rather large glycoproteins (hereafter referred to as ʹhyalinʹ), which are the major constituents of the hyaline layer, the developing sea urchin embryoʹs 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 shown this finding 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 hyalinʹs effects on the adhesion of the two components of the interaction. Here we have used an elegant direct assay that avoids the limitations, in which we microdissected the two components of the adhesive interaction and tested their re-adhesion 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 0.2 M 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 hyalinʹs specific adhesive function. This first step is important 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.