Laboratório de Bioquímica e Biologia Celular de Glicoconjugados, Hospital Universitário Clementino Fraga Filho and Programa de Glicobiologia, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
BMC Biochem. 2011 May 29;12:29. doi: 10.1186/1471-2091-12-29.
Previously, we have reported the presence of highly sulfated dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra) and Stolidobranchia (Halocynthia pyriformis and Styela plicata). Despite the identical disaccharide backbone, consisting of [→4IdoA(2S)β-1→3GalNAcβ-1→], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for heparin cofactor II activity. As a consequence, 2,4- and 2,6-sulfated dermatans have high and low heparin cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi) and Phlebobranchia (Ciona intestinalis), aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how heparin cofactor II activity responds to dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units.
Disaccharide analyses indicated a high content of disulfated disaccharide units in the dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated disaccharides are found in dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated disaccharides whereas dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated disaccharides, suggesting that the biosynthesis of dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [→4IdoA(2-Sulfate)β-1→3GalNAcβ-1→] modulate heparin cofactor II activity of dermatan sulfate polymers. Thus, high and low heparin cofactor II stimulating activity is observed in 2,4-sulfated dermatan sulfates and 2,6-sulfated dermatan sulfates, respectively, confirming the clear correlation between the anticoagulant activities of dermatan sulfates and the presence of 2,4-sulfated units.
Our results indicate that in ascidian dermatan sulfates the position of sulfation on the GalNAc in the disaccharide [→4IdoA(2S)β-1→3GalNAcβ-1→] is directly related to the taxon and that the 6-O sulfation is a novelty apparently restricted to the Phlebobranchia. We also show that the increased content of [→4IdoA(2S)β-1→3GalNAc(4S)β-1→] disaccharide units in dermatan sulfates from Stolidobranchia accounts for the increased heparin cofactor II stimulating activity.
此前，我们已经报道过在 Phlebobranchia 目（Phallusia nigra）和 Stolidobranchia 目（Halocynthia pyriformis 和 Styela plicata）的独居尾索动物中存在高度硫酸化的皮肤素。尽管二糖骨架相同，均由 [→4IdoA(2S)β-1→3GalNAcβ-1→] 组成，但这些聚合物在 N-乙酰半乳糖胺上的硫酸化位置不同，可能发生在碳 4 或 6 位。我们已经表明，位置而不是硫酸化程度对肝素辅因子 II 活性很重要。因此，2,4-和 2,6-硫酸化的皮肤素有高和低的肝素辅因子 II 活性，分别。在本研究中，我们将尾索动物皮肤素硫酸酯的二糖分析扩展到 Stolidobranchia 目（Herdmania pallida、Halocynthia roretzi）和 Phlebobranchia 目（Ciona intestinalis）的其他物种，旨在研究硫酸化在被囊动物中是如何进化的。此外，我们分析了肝素辅因子 II 活性对含有不同比例 2,6-或 2,4-双硫酸化单位的皮肤素硫酸酯的反应。
二糖分析表明，这两个目皮肤素硫酸酯中二硫酸化二糖单元含量较高。然而，硫酸化程度从 Stolidobranchia 目降低到 Phlebobranchia 目。虽然 76%的 stolidobranch 尾索动物皮肤素硫酸酯中二糖单元是双硫酸化的，但 53%的二硫酸化二糖存在于 phlebobranch 尾索动物皮肤素硫酸酯中。除了硫酸化程度的显著差异外，phlebobranch 尾索动物皮肤素硫酸酯主要含有 2,6-硫酸化二糖，而 stolidobranch 尾索动物皮肤素硫酸酯主要含有 2,4-硫酸化二糖，这表明在被囊动物进化过程中，皮肤素硫酸酯的生物合成可能受到不同的调控。N-乙酰半乳糖胺上硫酸化位置的变化在二糖 [→4IdoA(2-Sulfate)β-1→3GalNAcβ-1→] 中调节皮肤素硫酸酯聚合物的肝素辅因子 II 活性。因此，在 2,4-硫酸化皮肤素硫酸酯和 2,6-硫酸化皮肤素硫酸酯中分别观察到高和低的肝素辅因子 II 刺激活性，证实了皮肤素硫酸酯的抗凝活性与 2,4-硫酸化单位的存在之间存在明显的相关性。
我们的结果表明，在尾索动物皮肤素硫酸酯中，二糖 [→4IdoA(2S)β-1→3GalNAcβ-1→] 中 GalNAc 上的硫酸化位置与分类群直接相关，而 6-O 硫酸化是一种显然仅限于 Phlebobranchia 的新特征。我们还表明，Stolidobranchia 目皮肤素硫酸酯中二糖单元 [→4IdoA(2S)β-1→3GalNAc(4S)β-1→] 含量的增加是肝素辅因子 II 刺激活性增加的原因。
