Extracellular Sunflower Proteins: Evidence on Non-classical Secretion of a Jacalin-Related Lectin

Abstract

Extracellular proteins from sunflower seedlings were analyzed by electrophoresis followed by peptide mass fingerprinting. Tentative identification revealed novel proteins for this crop. A significant number of those proteins were not expected to be extracellular because they lacked the typical signal peptide responsible for secretion. In silico analysis showed that some members of this group presented the characteristic disordered structures of certain non-classical and leaderless mammalian secretory proteins. Among these proteins, a putative jacalin-related lectin (Helja) with a mannose binding domain was further isolated from extracellular fluids by mannose-affinity chromatography, thus validating its identification. Besides, immunolocalization assays confirmed its extracellular location. These results showed that a lectin, not predicted to be secreted in strict requirement of the N-terminal signal peptide, occurs in a sunflower extracellular compartment. The implications of this finding are discussed.

The Thioredoxin GbNRX1 Plays a Crucial Role in Homeostasis of Apoplastic Reactive Oxygen Species in Response to Verticillium dahliae Infection in Cotton

“…However, 31 of the identified proteins are SecretomeP-positive, suggesting unconventional protein secretion (Nickel and Rabouille, 2009;Pinedo et al, 2012). Similar results were reported from other studies of apoplastic proteins (Fernández et al, 2012;Kim et al, 2013;Wang et al, 2013).…”

Apoplastic exosome-like vesicles: A new way of protein secretion in plants?

“…We have previously deduced that Helja was a leaderless protein, 6 which was confirmed by the analysis of its genomic sequence. In fact, the complete gene sequence from Helianthus annuus Helja available at the Sunflower Genome Project 18 (scaffold 12-HO_S039609, gene HA412r201104S039609.1) revealed the absence of a signal peptide in the predicted protein.…”

“…Among them we have detected a jacalinrelated lectin (Helja), whose extracellular localization was confirmed by immunolocalization experiments. 6 Extracellular proteins lacking a predicted signal peptide, known as leaderless or non classically secreted, have also been systematically observed in apoplastic proteomes from Brassica, 7 Arabidopsis 8-10 and rice, 11 among others. Apart from the increasing repertoire of leaderless proteins in extracellular plant fractions, the question is if there is any evidence on exosome-dependent protein secretion.…”

“…A purified fraction of Helja was obtained from sunflower extracellular fluids (EF) collected from 10 g of seeds as previously described 20 and subsequent purification on a D-mannose-agarose chromatography. 6 Twenty micrograms of the mannose-eluted fraction (Helja) was analyzed in a 12% SDS-PAGE and sequentially stained with GelCode Glycoprotein dye (Pierce) (A) and Coomassie brilliant blue (B). Radish peroxidase was used as positive glycosylation control (+) and soybean trypsin inhibitor as a negative control (-).…”

Unconventional Transport Routes of Soluble and Membrane Proteins and Their Role in Developmental Biology

“…In plants, the situation is different because there are many putative leaderless secretory proteins but very few of them have been experimentally demonstrated to localize to the apoplast and their physiological role determined [4]. The only exception now is Helja, a mannose-specific jacalin-related lectin in sunflower seedlings [16]. …”