Itin C, Roche A C, Monsigny M, Hauri H P
Department of Pharmacology, University of Basel, Switzerland.
Mol Biol Cell. 1996 Mar;7(3):483-93. doi: 10.1091/mbc.7.3.483.
Based on sequence homologies with leguminous lectins, the intermediate compartment marker ERGIC-53 was proposed to be a member of a putative new class of animal lectins associated with the secretory pathway. Independent, a promyelocytic protein, MR60, was purified by mannose-column chromatography, and a cDNA was isolated that matched MR60 peptide sequences. This cDNA was identical to that of ERGIC-53 and homologies with the animal lectin family of the galectins were noticed. Not all peptide sequences of MR60, however, were found in ERGIC-53, raising the possibility that another protein associated with ERGIC-53 may possess the lectin activity. Here, we provide the first direct evidence for a lectin function of ERGIC-53. Overexpressed ERGIC-53 binds to a mannose column in a calcium-dependent manner and also co-stains with mannosylated neoglycoprotein in a morphological binding assay. By using a sequential elution protocol we show that ERGIC-53 has selectivity for mannose and low affinity for glucose and GlcNAc, but no affinity for galactose. To experimentally address the putative homology of ERGIC-53 to leguminous lectins, a highly conserved protein family with an invariant asparagine essential for carbohydrate binding, we substituted the corresponding asparagine in ERGIC-53. This mutation, as well as a mutation affecting a second site in the putative carbohydrate recognition domain, abolished mannose-column binding and co-staining with mannosylated neoglycoprotein. These findings establish ERGIC-53 as a lectin and provide functional evidence for its relationship to leguminous lectins. Based on its monosaccharide specificity, domain organization, and recycling properties, we propose ERGIC-53 to function as a sorting receptor for glyco-proteins in the early secretory pathway.
基于与豆科植物凝集素的序列同源性，中间区室标记物ERGIC-53被认为是与分泌途径相关的一类假定新动物凝集素的成员。另外，一种早幼粒细胞蛋白MR60通过甘露糖柱层析法被纯化，并分离出一个与MR60肽序列匹配的cDNA。该cDNA与ERGIC-53的cDNA相同，且发现其与半乳糖凝集素动物凝集素家族存在同源性。然而，并非所有MR60的肽序列都存在于ERGIC-53中，这增加了一种可能性，即与ERGIC-53相关的另一种蛋白质可能具有凝集素活性。在此，我们提供了ERGIC-53具有凝集素功能的首个直接证据。过表达的ERGIC-53以钙依赖的方式与甘露糖柱结合，并且在形态学结合试验中也与甘露糖基化新糖蛋白共染色。通过使用连续洗脱方案，我们表明ERGIC-53对甘露糖具有选择性，对葡萄糖和GlcNAc具有低亲和力，但对半乳糖没有亲和力。为了通过实验研究ERGIC-53与豆科植物凝集素的假定同源性（豆科植物凝集素是一个高度保守的蛋白质家族，其具有对碳水化合物结合至关重要的不变天冬酰胺），我们替换了ERGIC-53中相应的天冬酰胺。这种突变以及影响假定碳水化合物识别域中第二个位点的突变，消除了甘露糖柱结合以及与甘露糖基化新糖蛋白的共染色。这些发现确立了ERGIC-53作为一种凝集素，并为其与豆科植物凝集素的关系提供了功能证据。基于其单糖特异性、结构域组织和循环特性，我们提出ERGIC-53在早期分泌途径中作为糖蛋白的分选受体发挥作用。
