Hogsett W E, Quatrano R S
J Cell Biol. 1978 Sep;78(3):866-73. doi: 10.1083/jcb.78.3.866.
Zygotes of the brown alga Fucus distichus L. Powell accumulate a sulfated polysaccharide (fucoidin) in the cell wall at the site of rhizoid formation. Previous work indicated that zygotes grown in seawater minus sulfate do not sulfate the preformed fucan (an unsulfated fucoidin) but form rhizoids. Under these conditions, we determined whether sulfation of the fucan is required for its localization in the rhizoid wall. This was accomplished by developing a specific stain for both the fucan and fucoidin. Using a precipitin assay, we demonstrated in vitro that the lectin ricin (RCA(I)) specifically complexes with both the sulfated and desulfated polysaccharide. No precipitate is observed when either is incubated in 0.1 M D-galactose or when RCA(I) is mixed with laminarin or alginic acid, the other major polysaccharides in Fucus. RCA(I) conjugated with fluorescein isothiocyanate (FITC) is also shown to bind specifically to fucoidin using a filter paper (DE81) assay. When added to zygotes, RCA(I)-FITC binds only to the site of fucoidin localization, i.e., the rhizoid cell wall. However, RCA(I)-FITC is not observed in the rhizoid wall of zygotes grown in the absence of sulfate. This observation is not due to inability of RCA(I)-FITC to bind to the fucan in vivo. Chemically desulfated cell walls that contained fucoidin in the rhizoid wall bind RCA(I)-FITC only in the rhizoid region. Also, the concentration of fucose-containing polymers and polysaccharides that form precipitates with RCA(I) is the same in embryos grown in the presence or absence of sulfate. If sulfate is added back to cultures of zygotes grown without sulfate, fucoidin is detected at the rhizoid tip by RCA(I)-FITC several hours later. These results support the conclusion that the enzymatic sulfation of the fucan is a modification of the polysaccharide required for its localization and/or assembly into a specific region of the cell wall.
褐藻鹿角菜（Fucus distichus L. Powell）的合子在假根形成部位的细胞壁中积累一种硫酸化多糖（岩藻依聚糖）。先前的研究表明，在缺硫酸盐的海水中生长的合子不会将预先形成的岩藻多糖（一种未硫酸化的岩藻依聚糖）硫酸化，但会形成假根。在这些条件下，我们确定了岩藻多糖的硫酸化对于其在假根壁中的定位是否必要。这是通过开发一种针对岩藻多糖和岩藻依聚糖的特异性染色方法来实现的。利用沉淀素测定法，我们在体外证明凝集素蓖麻毒素（RCA(I)）能与硫酸化和去硫酸化的多糖特异性结合。当其中任何一种在0.1 M D -半乳糖中孵育时，或者当RCA(I)与海带多糖或海藻酸（鹿角菜中的其他主要多糖）混合时，均未观察到沉淀。用滤纸（DE81）测定法还表明，与异硫氰酸荧光素（FITC）偶联的RCA(I)能特异性结合岩藻依聚糖。当添加到合子中时，RCA(I)-FITC仅结合到岩藻依聚糖定位的部位，即假根细胞壁。然而，在无硫酸盐条件下生长的合子的假根壁中未观察到RCA(I)-FITC。这一观察结果并非由于RCA(I)-FITC在体内无法结合岩藻多糖。在假根壁中含有岩藻依聚糖的化学去硫酸化细胞壁仅在假根区域结合RCA(I)-FITC。此外，在有或无硫酸盐条件下生长的胚胎中，与RCA(I)形成沉淀的含岩藻糖聚合物和多糖的浓度相同。如果将硫酸盐重新添加到无硫酸盐培养的合子培养物中，数小时后通过RCA(I)-FITC可在假根尖端检测到岩藻依聚糖。这些结果支持这样的结论：岩藻多糖的酶促硫酸化是该多糖在细胞壁特定区域定位和/或组装所必需的一种修饰。
