Bisecting GlcNAc Residues on Laminin-332 Down-regulate Galectin-3-dependent Keratinocyte Motility
Bisecting GlcNAc Residues on Laminin-332 Down-regulate Galectin-3-dependent Keratinocyte Motility
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DOI：
10.1074/jbc.m109.038836
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发表时间：
2010-01-29
影响因子：
4.8
通讯作者：
Gu, Jianguo
中科院分区：
文献类型：
--
作者：
Kariya, Yoshinobu;Kawamura, Chihiro;Gu, Jianguo
层粘连蛋白-332（Lm 332;以前的层粘连蛋白-5）是皮肤中的基底膜蛋白，其促进伤口愈合和癌症侵袭中的细胞运动。在先前的研究中，我们报道了由N-乙酰葡糖胺基转移酶III（GnT-III）催化的将二等分GlcNAc引入Lm 332（GnT-III-Lm 332）减少了细胞迁移（卡里耶，Y.，卡托河，巴西-地Itoh，S.，Fukuda，T.，Shibukawa，Y.，Sanzen，N.，Sekiguchi，K.，韦达，Y.，川崎，N.，和Gu，J.（2008）J.Biol.Chem.283，3303633045）。然而，GnT-III-Lm 332抑制Lm 332正常生物学功能的潜在分子机制仍有待阐明。在这项研究中，我们表明，半乳糖凝集素-3，这是一种β-半乳糖苷结合蛋白，强烈结合到未修饰的Lm 332，但不GnT-III-Lm 332和半乳糖凝集素-3的结合被乳糖完全阻断。外源性半乳糖凝集素-3显着提高角质形成细胞的细胞运动性对对照Lm 332，但不对GnT-III-Lm 332。针对半乳糖凝集素-3的功能性阻断抗体抑制Lm 332诱导的α 3 β 1和α 6 β 4整联蛋白聚集和局部接触形成。免疫共沉淀显示，半乳糖凝集素-3与β 4整联蛋白和表皮生长因子受体，从而交联这两个分子。该协会被抑制的存在下的乳糖或GnT-III的表达。此外，半乳糖凝集素-3一贯增强ERK激活。总之，这项研究的结果是第一个明确确定的分子机制负责的GnT-III细胞外基质整合素介导的细胞粘附，迁移和信号转导的抑制作用。本文提出的研究结果阐明了N-糖基化介导的细胞表面超分子复合物形成的重要性。
Laminin-332 (Lm332; formerly laminin-5) is a basement membrane protein in the skin, which promotes cell motility in wound healing and cancer invasion. In a previous study, we reported that the introduction of bisecting GlcNAc into Lm332 (GnT-III-Lm332), catalyzed by N-acetylglucosaminyltransferase III (GnT-III), reduced cell migration (Kariya, Y., Kato, R., Itoh, S., Fukuda, T., Shibukawa, Y., Sanzen, N., Sekiguchi, K., Wada, Y., Kawasaki, N., and Gu, J. (2008) J. Biol. Chem. 283, 3303633045). However, the underlying molecular mechanism by which GnT-III-Lm332 suppresses the normal biological functions of Lm332 remains to be elucidated. In this study, we show that galectin-3, which is a beta-galactoside-binding protein, strongly bound to unmodified Lm332 but not to GnT-III-Lm332 and that binding of galectin-3 was completely blocked by lactose. Exogenous galectin-3 significantly enhanced keratinocyte cell motility on control Lm332 but not on GnT-III-Lm332. A functional blocking antibody against galectin-3 inhibited Lm332-induced alpha 3 beta 1 and alpha 6 beta 4 integrin clustering and focal contact formation. Co-immunoprecipitation revealed that galectin-3 associated with both beta 4 integrin and epidermal growth factor receptor, thereby cross-linking the two molecules. The associations were inhibited by either the presence of lactose or expression of GnT-III. Moreover, galectin-3 consistently enhanced ERK activation. Taken together, the results of this study are the first to clearly identify the molecular mechanism responsible for the inhibitory effects of GnT-III on extracellular matrix-integrin-meditated cell adhesion, migration, and signal transduction. The findings presented herein shed light on the importance of N-glycosylation-mediated supramolecular complex formation on the cell surface.