N-Glycosylation of Laminin-332 Regulates Its Biological Functions A NOVEL FUNCTION OF THE BISECTING GlcNAc
N-Glycosylation of Laminin-332 Regulates Its Biological Functions A NOVEL FUNCTION OF THE BISECTING GlcNAc
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DOI：
10.1074/jbc.m804526200
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发表时间：
2008-11-28
影响因子：
4.8
通讯作者：
Gu, Jianguo
中科院分区：
文献类型：
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作者：
Kariya, Yoshinobu;Kato, Rika;Gu, Jianguo
层粘连蛋白-332（Lm 332）是一种大的异源三聚体糖蛋白，其已被鉴定为散射因子、癌症侵袭的调节剂以及皮肤的突出基底膜组分。过去的研究已经确定了Lm 332的功能结构域，并揭示了其活性与其亚基加工之间的关系。然而，关于N-糖基化对Lm 332活性的影响的信息很少。在一些癌细胞中，由N-乙酰葡糖胺转移酶V（GnT-V）催化的β 1，6-GlcNAc的增加与癌细胞运动性的促进有关。相比之下，由N-乙酰葡糖胺基转移酶III（GnT-III）催化的二等分GlcNAc抑制分支酶（例如GnT-V）的进一步加工和N-聚糖的延伸。为了检测这些N-糖基化对Lm 332活性的影响，我们从GnT-III-和GnT-V-过表达的MKN 45细胞的条件培养基中纯化了Lm 332。凝集素印迹和质谱分析显示，含有二等分GlcNAc和β 1，6-GlcNAc结构的N-聚糖分别在从GnT-III过表达（GnT-III-Lm 332）和GnT-V过表达（GnT-V-Lm 332）细胞纯化的Lm 332上强烈表达。有趣的是，与对照Lm 332和GnT-V-Lm 332相比，GnT-III-Lm 332的细胞粘附活性明显降低。此外，将二等分GlcNAc引入Lm 332导致其细胞散射和迁移活性降低。减弱的活性最有可能来自受损的α 3 β 1整合素聚集和由此产生的粘着斑形成。综上所述，我们的结果首次清楚地表明，N-糖基化可以调节Lm 332的生物学功能。这一发现可能为癌症引入一种新的治疗策略。
Laminin-332 (Lm332) is a large heterotrimeric glycoprotein that has been identified as a scattering factor, a regulator of cancer invasion as well as a prominent basement membrane component of the skin. Past studies have identified the functional domains of Lm332 and revealed the relationships between its activities and the processing of its subunits. However, there is little information available concerning the effects of N-glycosylation on Lm332 activities. In some cancer cells, an increase of beta 1,6-GlcNAc catalyzed by N-acetylglucosaminyltransferase V (GnT-V) is related to the promotion of cancer cell motility. By contrast, bisecting GlcNAc catalyzed by N-acetylglucosaminyltransferase III (GnT-III) suppresses the further processing with branching enzymes, such as GnT-V, and the elongation of N-glycans. To examine the effects of those N-glycosylations to Lm332 on its activities, we purified Lm332s from the conditioned media of GnT-III- and GnT-V-overexpressing MKN45 cells. Lectin blotting and mass spectrometry analyses revealed that N-glycans containing the bisecting GlcNAc and beta 1,6-GlcNAc structures were strongly expressed on Lm332 purified from GnT-III-overexpressing (GnT-III-Lm332) and GnT-V-overexpressing (GnT-V-Lm332) cells, respectively. Interestingly, the cell adhesion activity of GnT-III-Lm332 was apparently decreased compared with those of control Lm332 and GnT-V-Lm332. In addition, the introduction of bisecting GlcNAc to Lm332 resulted in a decrease in its cell scattering and migration activities. The weakened activities were most likely derived from the impaired alpha 3 beta 1 integrin clustering and resultant focal adhesion formation. Taken together, our results clearly demonstrate for the first time that N-glycosylation may regulate the biological function of Lm332. This finding could introduce a new therapeutic strategy for cancer.