Isolation of pl 4.6 extensin peroxidase from tomato cell suspension cultures and identification of Val—Tyr—Lys as putative intermolecular cross‐link site

Abstract

Extensins and kindred hydroxyproline-rich glycoproteins occur in dicot cell walls mainly as insoluble integral components that may form an intermolecularly cross-linked network interpenetrated by other polymers. Extensins also occur in muro as a small pool of soluble monomeric precursors to network extensin. These precursors were prepared in milligram quantities by salt elution from the surface of intact cells grown as tomato suspension cultures. Based on an FPLC (Superose-6) gel filtration assay of cross-linked extensin oligomers, a pl 4.6 extensin cross-linking peroxidase isozyme was partially purified from the culture growth medium. Purification involved: volume reduction, ultracentrifugation to remove pectin and co-adsorbed cationic peroxidase, followed by chromatography of anionic extensin peroxidase (pl 4.6) on DEAE-Trisacryl and TSK-gel DEAE-5PW columns. With tomato P1 extensin as substrate and 60 microM H2O2 as co-substrate, at 23 degrees pl 4.6 extensin peroxidase gave a Km of 0.22 mM P1 and a Vmax 0f 70 mumol P1 cross-linked min-1mg-1 enzyme, at the optimum pH 5.5. Assayed with 12 different extensins from representative monocots, dicots, and gymnosperms, the pl 4.6 isozyme cross-linked highly selectively, indicating two natural groups: cross-linking or CL-extensins and non-cross-linking or NCL-extensins. CL-extensins contained the X-Hyp-Val-Tyr-Lys motif and were also highly glycosylated. However, the simplest motif common to CL-extensins but absent from NCL-extensins was Val-Tyr-Lys. Thus, peroxidative coupling of extensin monomers and resistance of the resultant oligomers to depolymerization by anhydrous HF suggests that the intermolecular cross-link involves tyrosine or lysine.

Role of the Extensin Superfamily in Primary Cell Wall Architecture

“…Significantly, extensin peroxidase did not cross-link extensins like the maize THRGP (Schnabelrauch et al, 1996), which lacked the putative VYK and YXYK cross-link motifs. Some extensins, like tomato P1, ap parently lacking Idt motifs (Smith et al, 1984(Smith et al, , 1986), were readily cross-linked, suggesting VYK as an intermolecular cross-link.…”

“…Meanwhile, the significance of putative cross-link motifs, VYK and Idt (YXY), became apparent with the availability of FPLC, notably Superose-6 columns that allowed the resolution of extensin monomers, oligomers, and polymers following in vitro enzymatic cross-linking of the monomers (Schnabelrauch et al, 1996), and led to the discovery of a specific extensin peroxidase.…”

Characterization and Expression of Four Proline-Rich Cell Wall Protein Genes in Arabidopsis Encoding Two Distinct Subsets of Multiple Domain Proteins

“…Tyr and Lys are an abundant amino acids in both PRPs and extensins (a second family of Hyp-rich structural cell wall proteins) and have been implicated as the substrate for the peroxidase-mediated insolubilization of PRPs in soybean (Kleis-San Francisco and Tierney, 1990;Bradley et al, 1992;Brisson et al, 1994) and in the cross-linking of extensins within the cell wall of suspension-cultured cells (Brady et al, 1996;Schnabelrauch et al, 1996). An extensinspecific peroxidase has been identified in tomato cell suspension cultures, and the substrate for this enzyme has tentatively been identified as Val-Tyr-Lys.…”

“…An extensinspecific peroxidase has been identified in tomato cell suspension cultures, and the substrate for this enzyme has tentatively been identified as Val-Tyr-Lys. Interestingly, two soybean PRPs containing this motif were not substrates for this enzyme in vitro (Schnabelrauch et al, 1996). Pectin/extensin cross-links have been identified in cotton cell walls (Qi et al, 1995) and are thought to occur through either a 3,6-linked galactan or a ferulated sugar/amino acid cross-link (Keegstra et al, 1973;Brownleader and Dey, 1993).…”

Whole-Genome Comparison of Leucine-Rich Repeat Extensins in Arabidopsis and Rice. A Conserved Family of Cell Wall Proteins Form a Vegetative and a Reproductive Clade,

“…Experiments involving complementation of the lrx1 mutant (Baumberger et al, 2001) by chimeric AtLRX constructs containing different extensin domains will address this question. It is interesting to note that none of the LRXs contains the motif VYK, thought to be important for intermolecular crosslinking (Schnabelrauch et al, 1996). Because protein immobilization in the cell wall was demonstrated for AtLRX1 and ZmPEX1 (Baumberger et al, 2001;Rubinstein et al, 1995b), it is likely that this process is mediated by other motifs than those so far identified in extensins.…”