Kuhns W J, Popescu O, Burger M M, Misevic G
Hospital for Sick Children, Toronto, Canada.
J Cell Biochem. 1995 Jan;57(1):71-89. doi: 10.1002/jcb.240570109.
Sulfate is an important component relating to normal proteoglycan secretion and normal motility in the marine sponge, Microciona prolifera. The following alterations were observed in sponge cells when sulfate free artificial sea water was used as the suspension medium: 1) impairment of aggregation, 2) loss of cell movements, 3) a marked reduction in the secretion of the adhesion proteoglycan (AP). Reversal of this effect occurred if sulfate depleted cells were again rotated in sulfate containing artificial sea water. Motility and reaggregation of sulfate deprived cells could be completely restored by purified AP, but only if cells were first pre-conditioned in normal sea water. Comparisons of 35SO4(2-) uptake between normal and sulfate deprived cells which had been treated to reduce preformed secretions showed a marked increase in 35SO4(2-) uptake and incorporation which could be greatly augmented in the presence of Ca2+/Mg2+. Excessive retention of AP in sulfate starved cells demonstrated by immunostaining suggested that AP secretion and cellular motility may be controlled by a sulfate dependent secretogogue or that undersulfated AP itself had developed a secretory defect. SDS-PAGE of Triton treated cellular extracts demonstrated a 116 kDa 35SO4(2-) sulfated band which co-migrated with AP, but only in extracts derived from sulfate starved cells. Western blots prepared from such extracts incubated in the presence of a monoclonal anti-band 3 antibody demonstrated labelling of a single 97 kDa band only in material from sulfate deprived cells. The absence of this component in normal cell extracts indicated that this protein may be involved in facilitated sulfate transport. This study lends support to a heretofore unrecognized role for sulfate in cell motility and secretion.
硫酸盐是与海洋海绵微拟球藻正常蛋白聚糖分泌和正常运动相关的重要成分。当使用无硫酸盐的人工海水作为悬浮介质时,在海绵细胞中观察到以下变化:1)聚集受损;2)细胞运动丧失;3)粘附蛋白聚糖(AP)分泌显著减少。如果将硫酸盐缺乏的细胞再次在含硫酸盐的人工海水中旋转,这种效应会逆转。纯化的AP可以完全恢复硫酸盐剥夺细胞的运动性和再聚集能力,但前提是细胞首先在正常海水中进行预处理。对已处理以减少预先形成的分泌物的正常细胞和硫酸盐剥夺细胞之间的35SO4(2-)摄取进行比较,结果显示35SO4(2-)摄取和掺入显著增加,并且在Ca2+/Mg2+存在的情况下可以大大增强。免疫染色显示硫酸盐饥饿细胞中AP的过度保留,这表明AP分泌和细胞运动性可能受硫酸盐依赖性促分泌素控制,或者硫酸化不足的AP本身出现了分泌缺陷。经Triton处理的细胞提取物的SDS-PAGE显示出一条116 kDa的35SO4(2-)硫酸化条带,它与AP共迁移,但仅在来自硫酸盐饥饿细胞的提取物中出现。用单克隆抗带3抗体孵育此类提取物制备的Western印迹显示,仅在来自硫酸盐剥夺细胞的材料中标记了一条单一的97 kDa条带。正常细胞提取物中不存在该成分,表明该蛋白质可能参与促进硫酸盐转运。这项研究支持了硫酸盐在细胞运动和分泌中迄今未被认识的作用。