Identification of tyrosylprotein sulfotransferase in Arabidopsis.

Tyrosine sulfation is a posttranslational modification common in peptides and proteins synthesized by the secretory pathway in most eukaryotes. In plants, this modification is critical for the biological activities of a subset of peptide hormones such as PSK and PSY1. In animals, tyrosine sulfation is catalyzed by Golgi-localized type II transmembrane proteins called tyrosylprotein sulfotransferases (TPSTs). However, no orthologs of animal TPST genes have been found in plants, suggesting that plants have evolved plant-specific TPSTs structurally distinct from their animal counterparts. To investigate the mechanisms of tyrosine sulfation in plants, we purified TPST activity from microsomal fractions of Arabidopsis MM2d cells, and identified a 62-kDa protein that specifically interacts with the sulfation motif of PSY1 precursor peptide. This protein is a 500-aa type I transmembrane protein that shows no sequence similarity to animal TPSTs. A recombinant version of this protein expressed in yeast catalyzed tyrosine sulfation of both PSY1 and PSK precursor polypeptide in vitro, indicating that the newly identified protein is indeed an Arabidopsis (At)TPST. AtTPST is expressed throughout the plant body, and the highest levels of expression are in the root apical meristem. A loss-of-function mutant of AtTPST displayed a marked dwarf phenotype accompanied by stunted roots, pale green leaves, reduction in higher order veins, early senescence, and a reduced number of flowers and siliques. Our results indicate that plants and animals independently acquired tyrosine sulfation enzymes through convergent evolution.

Tyrosine-sulfated glycopeptide involved in cellular proliferation and expansion in Arabidopsis.

Posttranslational modification can confer special functions to peptides. Based on exhaustive liquid chromatography mass spectrometry analysis targeting tyrosine-sulfated peptides, we identified an 18-aa tyrosine-sulfated glycopeptide in Arabidopsis cell suspension culture medium. This peptide, which we named PSY1, significantly promotes cellular proliferation and expansion at nanomolar concentrations. PSY1 is widely expressed in various Arabidopsis tissues, including shoot apical meristem, and is highly up-regulated by wounding. Perception of PSY1 depends on At1g72300, which is a leucine-rich repeat receptor kinase (LRR-RK) whose two paralogs are involved in the perception of phytosulfokine (PSK), which is a 5-aa tyrosine-sulfated peptide that primarily promotes cellular proliferation. Multiple loss-of-function mutations in these three paralogous LRR-RKs significantly enhanced phenotypes, compared with single disruptants, suggesting that these LRR-RKs have overlapping functions. Triple mutations in these LRR-RKs resulted in dwarfism because of decreases in cell number and cell size and caused insufficiency in tissue repair after wounding. The present results suggest that this paralogous LRR-RK family integrates growth-promoting signals mediated by two structurally distinct sulfated peptides: PSY1 and PSK.

Ion-selective enrichment of tyrosine-sulfated peptides from complex protein digests.

We have developed a novel procedure for concentrating sulfated peptides, as a front end to mass spectrometric analysis, based on ion-selective interaction of sulfate ions with anion exchangers. Ions with a higher charge and smaller solvated ion radius, such as sulfate ions, have higher retention in an ion exchanger due to their greater degree of coulombic interactions. We tested the effectiveness of this approach for enrichment and identification of sulfated peptides using a tryptic digest of bovine serum albumin spiked with model sulfated peptide (molar ratio 20:1) and using a tryptic digest of bovine fibrinogen. Sulfated peptides are identified by mass spectrometry in which both the molecular ion and its specific fragment ion produced by facile loss of SO(3) are detected. In both experiments, sulfated peptides were strongly retained on the anion exchanger and were eluted by higher concentrations of competing ion with minimal contamination of nonsulfated peptides. Using this procedure, we determined that the 13-amino acid C-terminal peptide of the minor gamma'-chain of bovine fibrinogen contains sulfated tyrosine.