Held Michael A, Tan Li, Kamyab Abdolreza, Hare Michael, Shpak Elena, Kieliszewski Marcia J
Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA.
J Biol Chem. 2004 Dec 31;279(53):55474-82. doi: 10.1074/jbc.M408396200. Epub 2004 Oct 1.
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, 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 cross-linked 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 inter-molecular cross-link amino acid in an extensin module and corroborates earlier suggestions that di-isodityrosine represents one mechanism for cross-linking extensins in muro.
伸展蛋白是细胞壁中富含羟脯氨酸的糖蛋白，它们形成共价网络，推测在一种或多种伸展蛋白过氧化物酶催化的交联反应中涉及酪氨酰和赖氨酰残基。无论是作为细胞壁内的网络成分，还是以天然伸展蛋白单体为底物在体外产生的酶促产物，精确的交联结构仍有待化学鉴定。然而，一些伸展蛋白单体在其推测的交联基序内存在变异，这使得交联鉴定变得复杂。其他更简单的伸展蛋白难以分离，包括普遍存在的P3型伸展蛋白，其主要重复基序Hyp)(4)-Ser-Hyp-Ser-(Hyp)(4)-Tyr-Tyr-Tyr-Lys特别引人关注，尤其是因为其Tyr-Tyr-Tyr分子内异二酪氨酸交联基序也是进一步分子间交联形成二异二酪氨酸的推测候选者。因此，我们设计了一组编码P3基序串联重复的伸展蛋白类似物，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糖蛋白，几乎所有的脯氨酸残基都被羟基化，随后被阿拉伯糖基化，丝氨酸残基可能被半乳糖基化。这与早期从细胞壁消化物中分离出的P3糖肽的分析以及羟脯氨酸邻接假说的预测一致。富含酪氨酸的P3类似物在体内也含有异二酪氨酸。值得注意的是，这些含异二酪氨酸的类似物在体外被一种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二酪氨酸。这是首次在伸展蛋白模块中鉴定出分子间交联氨基酸，证实了早期的推测，即二异二酪氨酸代表了细胞壁内伸展蛋白交联的一种机制。
