REGULATION OF VARIOUS CELL BEHAVIORS BY PROTEOGLYCANS THAT INHIBIT CELL-ADHESION,ANTI-ADHESIVE MOLECULES

1) PG-M天然黏附活性机制的研究：糖钙蛋白是一种细胞表面受体，被认为可以将PG-M的抗黏附活性传递给细胞，为了提出糖钙蛋白的分子生物学策略，我们首先尝试分离糖钙蛋白cDNA。用硫酸软骨素-偶联凝胶亲和层析法从培养的人细胞中纯化出的糖钙蛋白的部分氨基酸序列和对各种抗体的免疫反应性表明，糖钙蛋白与膜联蛋白vi完全相同，这一结论使我们能够获得cDNA，因为cDNA已经可用。利用以下三种不同的检测方法进一步研究PG-M的抗粘附活性，证实了上述策略：离心力作用下细胞与纤维连接蛋白包被的u形培养皿的粘附比较；脂质体嵌入整合素与底物的结合活性半变性细胞与底物的粘附。上述结果表明，PG-M可能通过糖小钙蛋白介导的信号转导，仅阻断细胞扩散而不阻断细胞结合。(2) PG-M抗粘附活性对细胞行为的调控作用：完成了鸡、鼠、人PG-M的cDNA分析、northern分析和基因组DNA分析。结果表明，由于硫酸软骨素链数的不同及其组织和发育阶段依赖性的改变，多种形式的PG-M具有不同的抗粘附活性。例如，最大形式的PG-M（链中最丰富的）是在发展的早期阶段发现的。因此，PG-M的抗粘附作用可能涉及特定的机制。

1) Studies on the mechanisms for nati-adhesive activity of PG-M : In order to suggest a molecular biological strategy for glycocalfin which is a cell-surface receptor postulated to transmit the antiadhesive activity of PG-M to cells we first tried to isolate glycocalfin cDNA.Partial amino acid sequences and the immunoreactivity to various antibodies of glycocalfin purified from cultured human cells by the affinity chromatography using chondroitin sulfate-conjugated gels have revealed the complete identity of glycocalfin to annexin VI.This conclusion has enabled us to obtain the cDNA,because the cDNA is already available. Further studies on the anti-adhesive activity of PG-M using the following three different types of assay methods have convinced the above strategy : Comparisons of cell adhesion to fibronectin-coated U-shape dishes under the centrifugal force ; binding activities of liposome-intercalated integrin to substrates ; adhesion of half-denatured cells to substrates. All the results have suggested that PG-M only interrupt cell-spreading but not cell-binding probably by glycocalfin-mediated signal transduction. (2) Regulation of cell behaviors by anti-adhesion activity of PG-M : We have finished the cDNA analysis, northern analysis, and genomic DNA analysis for chicken, mouse, and human PG-Ms. The results have suggested the presence of multiple forms of PG-M with different anti-adhesive activity due to the different chondroitin sulfate chain number and their tissue- and developmental stage-dependent alterations. For example, the largest form of PG-M (the richest in the chain) was found in the early stages of development. It is likely, therefore, that specific mechanisms may be involved in the anti-adhesion of PG-M.