Ghazarian Haike, Coyle-Thompson Catherine, Dalrymple William, Hutchins-Carroll Virginia, Metzenberg Stan, Razinia Ziba, Carroll Edward J, Oppenheimer Steven B
Department of Biology and Center for Cancer and Developmental Biology, California State University, Northridge, Northridge, CA 91330-8303, USA.
Zygote. 2010 Feb;18(1):17-26. doi: 10.1017/S0967199409005498. Epub 2009 Jun 8.
In 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 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 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, 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 degrees 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 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.
在紫海胆中，透明质蛋白是一组三到四种相当大的糖蛋白（以下简称“透明质蛋白”），它们是透明层的主要成分，而透明层是发育中的海胆胚胎的细胞外基质。我们实验室最近的研究表明，透明质蛋白是一种细胞粘附分子，参与海胆胚胎特异性的细胞相互作用。其他实验室表明，它含有2 - 3%的碳水化合物，一个克隆并测序的片段显示出重复结构域（HYR）和非重复区域。对这种分子的兴趣有所增加，因为在细菌、果蝇、蠕虫、小鼠、人类以及海胆等多种生物中都发现了HYR。我们实验室表明，透明质蛋白似乎介导了一种特定的细胞相互作用，这种相互作用已经引起研究人员超过一个世纪的兴趣，即原肠伸长/附着于囊胚腔顶壁。我们通过将透明质蛋白定位在细胞相互作用的两个组分上，并通过使用定量微孔板测定法表明透明质蛋白和抗透明质蛋白抗体阻断细胞相互作用，从而证明了这一发现。然而，微孔板测定法有其局限性，因为它不能直接评估透明质蛋白对相互作用的两个组分粘附的影响。在这里，我们使用了一种巧妙的直接测定法来避免这些局限性，在该方法中，我们显微解剖了粘附相互作用的两个组分，并测试它们彼此重新粘附的情况，从而避免了整个胚胎中可能混淆或干扰结果的因素。使用这两种测定法，我们发现对透明质蛋白进行温和的高碘酸盐处理（在4℃黑暗条件下，于含有0.2 M高碘酸钠的醋酸钠缓冲液中处理6小时至24小时）会消除其阻断细胞相互作用的能力，这表明碳水化合物组分可能参与了透明质蛋白的特异性粘附功能。这第一步对于确定美国国立卫生研究院指定的海胆胚胎模型中一种众所周知的细胞相互作用的分子机制很重要，该系统已经导致发现了许多生理机制，包括那些与人类健康和疾病相关的机制。
