Peña M, Williams C, Pfeiler E
Department of Chemistry and Biochemistry, Arizona State University, Tempe 85287, USA.
Carbohydr Res. 1998 Jun;309(1):117-24. doi: 10.1016/s0008-6215(98)00128-1.
Structural details of keratan sulfate (KS) glycosaminoglycan, isolated from early-metamorphosing larvae (leptocephali) of bonefish (Albula sp.), are described. Bonefish KS was analyzed by first hydrolyzing the purified compound with KS endo-beta-galactosidase (keratanase) from Pseudomonas spp., and then examining the resulting oligosaccharides with reversed-phase high-performance liquid chromatography (HPLC) and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy at 400 MHz. Spectral analyses were performed by COSY and HMQC. The results showed that a single oligosaccharide was produced whose structure is consistent with that of a tetrasaccharide containing two, beta-linked, N-acetyllactosamine units. Enzymic evidence indicated that the internal galactose of the tetrasaccharide was O-sulfated at C-6, and that the reducing-end galactose was unsulfated. Spectral data for C-1 of the two galactose residues were consistent with the proposed sulfation pattern. In addition, spectral evidence confirmed that a C-6 on one of the sugars was sulfated: this sulfate was tentatively assigned to the internal galactose. Chemical studies have shown that an additional sulfate group is present, but its assignment could not be confirmed, owing to the complexity of the spectral data. The known specificities of keratanase, and the production of a single tetrasaccharide, however, require that the additional sulfate reside on C-6 of either of the two available N-acetylglucosamine (GlcNAc) moieties, and that it cannot alternate between the two. The inability of beta-N-acetylglucosaminidase from beef kidney to liberate GlcNAc from the tetrasaccharide provided preliminary support for the view that this sulfate is located on the nonreducing-end GlcNAc. We conclude that the native, high molecular weight (M(r) = 55,000) KS polymer from bonefish larvae consists of a disulfated disaccharide alternating with an unsulfated disaccharide in the adjacent N-acetyllactosamine unit, with this pattern repeating itself in a regular fashion along most, or all, of the chain. This structure could provide an explanation for the ability of bonefish KS chains to self-associate into dimers. Although the N-acetyllactosamine repeat is characteristics of KS in general, the sulfation pattern is different from that postulated for the well-characterized KS chains of lower molecular weight obtained from mammalian cornea and cartilage. An additional difference was the inability to demonstrate sialic acid in bonefish KS.
描述了从骨鱼（Albula sp.）早期变态幼虫（柳叶鳗）中分离出的硫酸角质素（KS）糖胺聚糖的结构细节。对骨鱼KS进行分析时，首先用来自假单胞菌属的KS内切β-半乳糖苷酶（角质素酶）水解纯化后的化合物，然后用反相高效液相色谱（HPLC）以及400 MHz的1H和13C核磁共振（NMR）光谱检查所得的寡糖。通过COSY和HMQC进行光谱分析。结果表明产生了一种单一的寡糖，其结构与含有两个β-连接的N-乙酰乳糖胺单元的四糖一致。酶学证据表明四糖的内部半乳糖在C-6位被O-硫酸化，而还原端半乳糖未被硫酸化。两个半乳糖残基C-1的光谱数据与所提出的硫酸化模式一致。此外，光谱证据证实其中一个糖的C-6位被硫酸化：该硫酸盐暂定为内部半乳糖。化学研究表明存在一个额外的硫酸基团，但由于光谱数据复杂，其归属无法得到证实。然而，角质素酶已知的特异性以及单一四糖的产生，要求额外的硫酸盐位于两个可用的N-乙酰葡糖胺（GlcNAc）部分中任一个的C-6位，并且它不能在两者之间交替。牛肾β-N-乙酰葡糖胺酶无法从四糖中释放出GlcNAc，这为该硫酸盐位于非还原端GlcNAc上的观点提供了初步支持。我们得出结论，来自骨鱼幼虫的天然高分子量（M(r)=55,000）KS聚合物由一个二硫酸化二糖与相邻N-乙酰乳糖胺单元中的一个未硫酸化二糖交替组成，这种模式沿着大部分或全部链以规则方式重复。这种结构可以解释骨鱼KS链自缔合成二聚体的能力。虽然N-乙酰乳糖胺重复序列通常是KS的特征，但硫酸化模式与从哺乳动物角膜和软骨获得的已充分表征的低分子量KS链所假定的模式不同。另一个差异是在骨鱼KS中无法检测到唾液酸。
