Di-isodityrosine Is the Intermolecular Cross-link of Isodityrosine-rich Extensin Analogs Cross-linked in Vitro

• Published in: The Journal of biological chemistry Vol. 279; no. 53; pp. 55474 - 55482
• Main Authors: Held, Michael A., Tan, Li, Kamyab, Abdolreza, Hare, Michael, Shpak, Elena, Kieliszewski, Marcia J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 31.12.2004; American Society for Biochemistry and Molecular Biology
• Subjects: Agrobacterium tumefaciens - metabolism; Amino Acid Sequence; Amino Acids - chemistry; Base Sequence; Carbohydrates - chemistry; Carrier Proteins; Catalysis; Chromatography; Chromatography, Gel; Chromatography, High Pressure Liquid; Cross-Linking Reagents - pharmacology; Culture Media - metabolism; Extracellular Matrix Proteins - chemistry; Glycoproteins - chemistry; Glycosylation; Green Fluorescent Proteins - metabolism; Hydrofluoric Acid - chemistry; Hydroxyproline - chemistry; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Lysine - chemistry; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Nicotiana - metabolism; Peptides - chemistry; Plant Proteins - chemistry; Plants, Genetically Modified; Plasmids - metabolism; Protein Sorting Signals; Recombinant Fusion Proteins - chemistry; Solanum lycopersicum - metabolism; Time Factors; Tyrosine - analogs & derivatives; Tyrosine - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M408396200

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.