Marková Vladimíra, Smetana Karel, Jeníková Gabriela, Láchová Jitka, Krejciríková Veronika, Poplstein Martin, Fábry Milan, Brynda Jirí, Alvarez Richard A, Cummings Richard D, Maly Petr
Laboratory of Molecular Glycobiology, Department of Recombinant Expression and Structural Biology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic.
Int J Mol Med. 2006 Jul;18(1):65-76.
Galectin-4 and its homologue galectin-6 are members of the tandem-repeat subfamily of monomer divalent galectins. Expression of mouse galectin-4 and galectin-6 by RT-PCR using primers designed to distinguish both galectin transcripts indicates that both are expressed in the small intestine, colon, liver, kidney, spleen and heart and P19X1 cells while only galectin-4 is expressed in BW-5147 and 3T3 cell lines. In situ hybridization confirmed the presence of galectin-4/-6 transcripts in the liver and small intestine. Galectin-4 is expressed in spermatozoons and oocytes and its expression during early mouse emryogenesis appears in 8-cell embryos and remains in later stages, as tested by RT-PCR. To study the role of carbohydrate recognition domains (CRDs) in oligosaccharide binding and epitope recognition, we cloned mouse full-length galectin-4 and galectin-6 cDNA and constructed bacterial expression vectors producing histidin-tagged recombinant galectin-4 and its truncated CRD1 and CRD2 forms. Oligosaccharide binding profile for all recombinant forms was assessed using Glycan Array available through the Consortium for Functional Glycomics. Acquired data indicate that mGalectin-4 binds to alpha-GalNAc and alpha-Gal A and B type structures with or without fucose. While the CRD2 domain has a high specificity and affinity for A type-2 alpha-GalNAc structures, the CRD1 domain has a broader specificity in correlation to the total binding profile. These data suggest that CRD2 might be the dominant binding domain of mouse galectin-4. Mapping of epitopes reactive for biotinylated his-tagged CRD1, CRD2 and mGalectin-4 performed on mouse cryosections showed that all three forms bind to alveolar macrophages, macrophages of red pulp of the spleen and proximal tubuli of the kidney and this binding was inhibited by 5 mM lactose. Interestingly, mGalectin-4, but not CRD forms, binds to the suprabasal layer of squamous epithelium of the tongue, suggesting that the link region also plays an important role in ligand recognition.
半乳糖凝集素-4及其同系物半乳糖凝集素-6是单体二价半乳糖凝集素串联重复亚家族的成员。使用设计用于区分两种半乳糖凝集素转录本的引物，通过逆转录聚合酶链反应（RT-PCR）检测小鼠半乳糖凝集素-4和半乳糖凝集素-6的表达，结果表明二者在小肠、结肠、肝脏、肾脏、脾脏、心脏和P19X1细胞中均有表达，而仅半乳糖凝集素-4在BW-5147和3T3细胞系中表达。原位杂交证实肝脏和小肠中存在半乳糖凝集素-4/-6转录本。通过RT-PCR检测发现，半乳糖凝集素-4在精子和卵母细胞中表达，并且在小鼠早期胚胎发育过程中，其表达始于8细胞胚胎，并在后期阶段持续存在。为了研究碳水化合物识别结构域（CRD）在寡糖结合和表位识别中的作用，我们克隆了小鼠全长半乳糖凝集素-4和半乳糖凝集素-6的cDNA，并构建了产生组氨酸标签重组半乳糖凝集素-4及其截短的CRD1和CRD2形式的细菌表达载体。使用通过功能糖组学协会获得的糖阵列评估所有重组形式的寡糖结合谱。获得的数据表明，小鼠半乳糖凝集素-4与含或不含岩藻糖的α-GalNAc以及α-Gal A和B型结构结合。虽然CRD2结构域对A-2型α-GalNAc结构具有高特异性和亲和力，但CRD1结构域与总结合谱相关的特异性更广泛。这些数据表明CRD2可能是小鼠半乳糖凝集素-4的主要结合结构域。对小鼠冷冻切片上生物素化的组氨酸标签CRD1、CRD2和小鼠半乳糖凝集素-4的反应性表位进行定位，结果显示所有三种形式均与肺泡巨噬细胞、脾红髓巨噬细胞和肾近端小管结合，并且这种结合被5 mM乳糖抑制。有趣的是，小鼠半乳糖凝集素-4而非CRD形式与舌鳞状上皮的基底层上层结合，这表明连接区域在配体识别中也起重要作用。
