Department of Plant Biology, University of Georgia, Athens, GA 30602, USA.
Department of Chemistry, University of Georgia, Athens, GA 30602, USA.
Plant Cell Physiol. 2021 Dec 3;62(10):1589-1602. doi: 10.1093/pcp/pcab113.
Xyloglucan is the most abundant hemicellulose in the primary cell walls of dicots. Dicot xyloglucan is the XXXG type consisting of repeating units of three consecutive xylosylated Glc residues followed by one unsubstituted Glc. Its xylosylation is catalyzed by xyloglucan 6-xylosyltransferases (XXTs) and there exist five XXTs (AtXXT1-5) in Arabidopsis. While AtXXT1 and AtXXT2 have been shown to add the first two Xyl residues in the XXXG repeat, which XXTs are responsible for the addition of the third Xyl residue remains elusive although AtXXT5 was a proposed candidate. In this report, we generated recombinant proteins of all five Arabidopsis XXTs and one rice XXT (OsXXT1) in the mammalian HEK293 cells and investigated their ability to sequentially xylosylate Glc residues to generate the XXXG xylosylation pattern. We found that like AtXXT1/2, AtXXT4 and OsXXT1 could efficiently xylosylate the cellohexaose (G6) acceptor to produce mono- and di-xylosylated G6, whereas AtXXT5 was only barely capable of adding one Xyl onto G6. When AtXXT1-catalyzed products were used as acceptors, AtXXT1/2/4 and OsXXT1, but not AtXXT5, were able to xylosylate additional Glc residues to generate tri- and tetra-xylosylated G6. Further characterization of the tri- and tetra-xylosylated G6 revealed that they had the sequence of GXXXGG and GXXXXG with three and four consecutive xylosylated Glc residues, respectively. In addition, we have found that although tri-xylosylation occurred on G6, cello-oligomers with a degree of polymerization of 3 to 5 could only be mono- and di-xylosylated. Together, these results indicate that each of AtXXT1/2/4 and OsXXT1 is capable of sequentially adding Xyl onto three contiguous Glc residues to generate the XXXG xylosylation pattern and these findings provide new insight into the biochemical mechanism underlying xyloglucan biosynthesis.
木葡聚糖是双子叶植物初生细胞壁中最丰富的半纤维素。双子叶植物木葡聚糖是 XXXG 型，由三个连续的木糖化 Glc 残基组成，随后是一个未取代的 Glc。其木糖基化由木葡聚糖 6-木糖基转移酶（XXT）催化，在拟南芥中存在 5 种 XXT（AtXXT1-5）。虽然 AtXXT1 和 AtXXT2 已被证明在 XXXG 重复中添加前两个 Xyl 残基，但负责添加第三个 Xyl 残基的 XXT 仍不清楚，尽管 AtXXT5 是一个被提议的候选者。在本报告中，我们在哺乳动物 HEK293 细胞中生成了所有 5 种拟南芥 XXT 和 1 种水稻 XXT（OsXXT1）的重组蛋白，并研究了它们将 Glc 残基顺序木糖基化生成 XXXG 木糖基化模式的能力。我们发现，与 AtXXT1/2 一样，AtXXT4 和 OsXXT1 可以有效地木糖基化纤六糖（G6）受体，生成单和二木糖基化的 G6，而 AtXXT5 几乎无法在 G6 上添加一个 Xyl。当使用 AtXXT1 催化的产物作为受体时，AtXXT1/2/4 和 OsXXT1，但不是 AtXXT5，能够在额外的 Glc 残基上进行木糖基化，生成三和四木糖基化的 G6。对三木糖基化和四木糖基化的 G6 的进一步表征表明，它们具有 GXXXGG 和 GXXXXG 的序列，分别具有三个和四个连续的木糖化 Glc 残基。此外，我们发现，尽管三木糖基化发生在 G6 上，但聚合度为 3 到 5 的纤寡糖只能单和二木糖基化。总之，这些结果表明，AtXXT1/2/4 和 OsXXT1 中的每一种都能够将 Xyl 顺序添加到三个连续的 Glc 残基上，生成 XXXG 木糖基化模式，这些发现为木葡聚糖生物合成的生化机制提供了新的见解。
