Vyakarnam A, Dagher S F, Wang J L, Patterson R J
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Mol Cell Biol. 1997 Aug;17(8):4730-7. doi: 10.1128/MCB.17.8.4730.
Galectins are a family of beta-galactoside-binding proteins that contain characteristic amino acid sequences in the carbohydrate recognition domain (CRD) of the polypeptide. The polypeptide of galectin-1 contains a single domain, the CRD. The polypeptide of galectin-3 has two domains, a carboxyl-terminal CRD fused onto a proline- and glycine-rich amino-terminal domain. In previous studies, we showed that galectin-3 is a required factor in the splicing of nuclear pre-mRNA, assayed in a cell-free system. We now document that (i) nuclear extracts derived from HeLa cells contain both galectins-1 and -3; (ii) depletion of both galectins from the nuclear extract either by lactose affinity adsorption or by double-antibody adsorption results in a concomitant loss of splicing activity; (iii) depletion of either galectin-1 or galectin-3 by specific antibody adsorption fails to remove all of the splicing activity, and the residual splicing activity is still saccharide inhibitable; (iv) either galectin-1 or galectin-3 alone is sufficient to reconstitute, at least partially, the splicing activity of nuclear extracts depleted of both galectins; and (v) although the carbohydrate recognition domain of galectin-3 (or galectin-1) is sufficient to restore splicing activity to a galectin-depleted nuclear extract, the concentration required for reconstitution is greater than that of the full-length galectin-3 polypeptide. Consistent with these functional results, double-immunofluorescence analyses show that within the nucleus, galectin-3 colocalizes with the speckled structures observed with splicing factor SC35. Similar results are also obtained with galectin-1, although in this case, there are areas of galectin-1 devoid of SC35 and vice versa. Thus, nuclear galectins exhibit functional redundancy in their splicing activity and partition, at least partially, in the nucleoplasm with another known splicing factor.
半乳糖凝集素是一类β-半乳糖苷结合蛋白，其多肽的碳水化合物识别结构域（CRD）中含有特征性氨基酸序列。半乳糖凝集素-1的多肽含有一个单一结构域，即CRD。半乳糖凝集素-3的多肽有两个结构域，一个羧基末端CRD融合到富含脯氨酸和甘氨酸的氨基末端结构域上。在先前的研究中，我们表明半乳糖凝集素-3是无细胞体系中检测到的核前体mRNA剪接所必需的因子。我们现在证明：（i）源自HeLa细胞的核提取物中同时含有半乳糖凝集素-1和-3；（ii）通过乳糖亲和吸附或双抗体吸附从核提取物中去除这两种半乳糖凝集素会导致剪接活性同时丧失；（iii）通过特异性抗体吸附去除半乳糖凝集素-1或半乳糖凝集素-3中的任何一种都不能消除所有剪接活性，剩余的剪接活性仍可被糖类抑制；（iv）单独的半乳糖凝集素-1或半乳糖凝集素-3都足以至少部分地重建去除了两种半乳糖凝集素的核提取物的剪接活性；（v）尽管半乳糖凝集素-3（或半乳糖凝集素-1）的碳水化合物识别结构域足以将剪接活性恢复到去除半乳糖凝集素的核提取物中，但重建所需的浓度高于全长半乳糖凝集素-3多肽的浓度。与这些功能结果一致，双免疫荧光分析表明，在细胞核内，半乳糖凝集素-3与剪接因子SC35观察到的斑点状结构共定位。半乳糖凝集素-1也得到了类似的结果，不过在这种情况下，存在不含SC35的半乳糖凝集素-1区域，反之亦然。因此，核半乳糖凝集素在其剪接活性方面表现出功能冗余，并且至少部分地与另一种已知的剪接因子在核质中共分布。
