Di-isodityrosine is the intermolecular cross-link of isodityrosine-rich extensin analogs cross-linked in vitro
Di-isodityrosine is the intermolecular cross-link of isodityrosine-rich extensin analogs cross-linked in vitro
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DOI：
10.1074/jbc.m408396200
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发表时间：
2004-12-31
影响因子：
4.8
通讯作者：
Kieliszewski, MJ
中科院分区：
文献类型：
--
作者：
Held, MA;Tan, L;Kieliszewski, MJ
伸展蛋白是细胞壁富含羟脯氨酸的糖蛋白，其形成共价网络，所述共价网络由一种或多种伸展蛋白过氧化物酶催化交联中的酪氨酰和赖氨酰残基参与。精确的交联仍然是化学鉴定的muro网络组件和在体外产生的天然伸展蛋白单体作为底物的酶的产品。然而，一些延伸蛋白单体在其假定的交联基序内包含使交联鉴定复杂化的变化。其他更简单的伸展蛋白是分离不稳定的，包括普遍存在的P3型伸展蛋白，其主要重复基序Ser（Hyp）（4）-Ser-Hyp-Ser-（Hyp）（4）-Tyr-Tyr-Tyr-Lys是特别令人感兴趣的，尤其是因为其Tyr-Tyr-Tyr分子内异酪氨酸交联基序也是进一步分子间交联以形成二-异酪氨酸的推定候选物。因此，我们设计了一组延伸蛋白类似物，编码P3基序的串联重复，包括Tyr -> Phe和Lys -> Leu变异。这些P3类似物在烟草细胞中的表达产生了几乎所有Pro残基羟基化和随后阿拉伯糖基化的糖蛋白，并且可能具有半乳糖基化的Ser残基。这与早期从细胞壁分离的P3糖肽的分析和Hyp邻接假说的预测一致。富含酪氨酸的P3类似物还含有在体内形成的异酪氨酸。值得注意的是，这些含异酪氨酸的类似物在体外通过伸展蛋白过氧化物酶进一步交联，形成四酪氨酸分子间交联氨基酸二异酪氨酸。这是第一次确定的分子间交联氨基酸的伸展蛋白模块，并证实了早期的建议，即二异酪氨酸代表一种机制，在muro的交联伸展蛋白。
Extensins are cell wall hydroxyproline-rich glycoproteins that form covalent networks putatively involving tyrosyl and lysyl residues in cross-links catalyzed by one or more extensin peroxidases. The precise cross-links remain to be chemically identified both as network components in muro and as enzymic products generated in vitro with native extensin monomers as substrates. However, some extensin monomers contain variations within their putative cross-linking motifs that complicate cross-link identification. Other simpler extensins are recalcitrant to isolation including the ubiquitous P3-type extensin whose major repetitive motif, Ser(Hyp)(4)-Ser-Hyp-Ser-(Hyp)(4)-Tyr-Tyr-Tyr-Lys, is of particular interest, not least because its Tyr-Tyr-Tyr intramolecular isodityrosine cross-link motifs are also putative candidates for further intermolecular cross-linking to form di-isodityrosine. Therefore, we designed a set of extensin analogs encoding tandem repeats of the P3 motif, including Tyr --> Phe and Lys --> Leu variations. Expression of these P3 analogs in Nicotiana tabacum cells yielded glycoproteins with virtually all Pro residues hydroxylated and subsequently arabinosylated and with likely galactosylated Ser residues. This was consistent with earlier analyses of P3 glycopeptides isolated from cell wall digests and the predictions of the Hyp contiguity hypothesis. The tyrosine-rich P3 analogs also contained isodityrosine, formed in vivo. Significantly, these isodityrosine-containing analogs were further crosslinked in vitro by an extensin peroxidase to form the tetra-tyrosine intermolecular cross-link amino acid di-isodityrosine. This is the first identification of an intermolecular cross-link amino acid in an extensin module and corroborates earlier suggestions that di-isodityrosine represents one mechanism for cross-linking extensins in muro.