Molecular Basis for Protein-specific Transfer of N-Acetylgalactosamine to N-Linked Glycans by the Glycosyltransferases β1,4-N-Acetylgalactosaminyl Transferase 3 (β4GalNAc-T3) and β4GalNAc-T4
Molecular Basis for Protein-specific Transfer of N-Acetylgalactosamine to N-Linked Glycans by the Glycosyltransferases β1,4-N-Acetylgalactosaminyl Transferase 3 (β4GalNAc-T3) and β4GalNAc-T4
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DOI：
10.1074/jbc.m112.371567
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发表时间：
2012-08-17
影响因子：
4.8
通讯作者：
Baenziger, Jacques U.
中科院分区：
文献类型：
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作者：
Fiete, Dorothy;Beranek, Mary;Baenziger, Jacques U.
两种密切相关的β 1，4-N-乙酰氨基半乳糖转移酶β 4GalNAc-T3和β 4GalNAc-T4被认为是β 1，4-连接的GalNAc与许多糖蛋白（包括糖蛋白激素促黄体激素和碳酸酐酶-6（CA 6））上的Asn连接寡糖的蛋白特异性加成的原因。我们已经利用β 4GalNAc-T3和β 4GalNAc-T4的可溶性分泌形式来定义蛋白质特异性GalNAc体外转移至由Gaussia荧光素酶和糖蛋白底物组成的嵌合底物的基础。通过β 4GalNAc-T3和β 4GalNAc-T4将GalNAc转移至末端GlcNAc是二价阳离子依赖性的。在0.5和1.0 mM MnCl 2之间，GalNAc向含有肽识别决定簇的糖蛋白受体的转移最大;然而，MnCl 2浓度高于1 mM和阴离子浓度高于15 mM时，转移受到越来越多的抑制。相反，GalNAc转移至单糖受体N-乙酰葡糖胺-β-对硝基苯酚（GlcNAc β-pNP）不受MnCl 2或阴离子浓度的抑制，这些浓度将抑制转移至糖蛋白受体> 90%。这一发现表明，与底物中的肽识别决定簇的相互作用对阴离子浓度敏感。β 4GalNAc-T3和β 4GalNAc-T4具有相似但不同的特异性，导致半乳糖人绒毛膜促性腺激素将GalNAc转移到嵌合糖蛋白底物的IC 50相差42倍，β 4GalNAc-T3为29 nM，β 4GalNAc-T4为1.2 μ M。我们的体外分析表明，肽决定簇和寡糖受体的酶识别是独立的事件。
Two closely related beta 1,4-N-acetylgalactosaminyltransferases, beta 4GalNAc-T3 and beta 4GalNAc-T4, are thought to account for the protein-specific addition of beta 1,4-linked GalNAc to Asn-linked oligosaccharides on a number of glycoproteins including the glycoprotein hormone luteinizing hormone and carbonic anhydrase-6 (CA6). We have utilized soluble, secreted forms of beta 4GalNAc-T3 and beta 4GalNAc-T4 to define the basis for protein-specific GalNAc transfer in vitro to chimeric substrates consisting of Gaussia luciferase followed by a glycoprotein substrate. Transfer of GalNAc by beta 4GalNAc-T3 and beta 4GalNAc-T4 to terminal GlcNAc is divalent cation-dependent. Transfer of GalNAc to glycoprotein acceptors that contain a peptide recognition determinant is maximal between 0.5 and 1.0 mM MnCl2; however, transfer is increasingly inhibited by concentrations of MnCl2 above 1 mM and by anion concentrations above 15 mM. In contrast, transfer of GalNAc to the simple sugar acceptor N-acetylglucosamine-beta-p-nitrophenol (GlcNAc beta-pNP) is not inhibited by concentrations of MnCl2 or anions that would inhibit transfer to glycoprotein acceptors by >90%. This finding indicates that interaction with the peptide recognition determinant in the substrate is sensitive to the anion concentration. beta 4GalNAc-T3 and beta 4GalNAc-T4 have similar but distinct specificities, resulting in a 42-fold difference in the IC50 for transfer of GalNAc to chimeric glycoprotein substrates by agalacto human chorionic gonadotropin, comprising 29 nM for beta 4GalNAc-T3 and 1.2 mu M for beta 4GalNAc-T4. Our in vitro analysis indicates that enzymatic recognition of the peptide determinant and the oligosaccharide acceptor are independent events.