A Novel Hydroxyproline-Deficient Arabinogalactan Protein Secreted by Suspension-Cultured Cells of Daucus carota (Purification and Partial Characterization)

Abstract

Arabinogalactan proteins (ACPs) are secreted or membraneassociated glycoproteins that have been operationally defined as binding to ,9-glucosyl Yariv artificial antigen, being rich in arabinose and galactose, and containing high levels of alanine, serine, and hydroxyproline. Using an anti-ACP monoclonal antibody (MAC 207) bound to cyanogen bromide-activated Sepharose 46, we have purified by immunoaffinity chromatography an extracellular ACP from the culture medium of suspension-cultured cells of carrot (Daucus carota). The apparent molecular mass of this highly glycosylated proteoglycan is 70 to 100 kD as judged by sodium dodecyl sulfate-polyacrylamide gels. Although its sugar analysis, j3-glucosyl Yariv binding, and high alanine, serine, and proline content are consistent with it being an AGP, the amino acid composition unexpectedly revealed this molecule to have no detectable hydroxyproline. This suggests that this glycoprotein is not a "classical" ACP, but represents the first example of a new class of hydroxyproline-poor ACPs. Deglycosylation of the ACP with anhydrous hydrogen fluoride revealed that the purified proteoglycan contains probably a single core protein with an apparent molecular mass of 30 kD. Direct visualization of the native ACP in the electron microscope showed ellipsoidal putative ACP monomers, approximately 25 nm by 15 nm, that showed a strong tendency to self assemble into higher-order structures. Upon desiccation, the glycosylated ACP formed paracrystalline arrays visible in the light microscope. Polarized Fourier transform infrared microspectroscopy of these arrays demonstrated a high degree of polarization of the sugar moieties under these conditions. These results put possible constraints on current models of ACP structure; a putative role for these nove1 AGPs as pectin-binding proteins is discussed.

Arabinogalactan-proteins: structure, expression and function

“…Such aggregates were observed in imaging the Hyp-poor carrot AGP and the TTS proteins by electron microscopy [22,26]. These self-associations are consistent with the adhesive nature of AGPs and may relate to the observed cross-linking of AGPs [41].…”

“…For example, negatively charged galacturonic acid residues in pectin could interact (i) indirectly with negatively charged AGPs (i. e. AGPs having glucoronic acid in the polysaccharide side chains) through Ca ++ bridges or (ii) directly with clusters of basic amino acid residues (i. e. positively charged regions) found in some AGPs, such as the carrot Hyp-poor AGP and tomato LeAGP-1. Furthermore, Baldwin and co-workers have provided some support to this idea in their study of the Hyp-poor carrot AGP, which appears to show some degree of Ca ++ -mediated binding of pectin in blotting experiments performed in vitro [22]. However, the observed binding appears weak, and this entire area of molecular interactions with AGPs requires additional, rigorous study.…”

“…Visualization of three different AGPs using transmission electron microscopy provides support for both models. Specifically, the Hyppoor carrot AGP and the TTS proteins appeared spheroidal, whereas an AGP from gum arabic was rodlike [22,26,30]. Moreover, it is also likely that these models will be modified.…”

SALT-OVERLY SENSITIVE5 Mediates Arabidopsis Seed Coat Mucilage Adherence and Organization through Pectins

“…Treatment of cell wall fractions with pectin-degrading enzymes allows for the increased release of AGPs (Immerzeel et al, 2006;Lamport et al, 2006). AGPs have also been shown to bind specifically to pectins in a calcium-dependent manner (Baldwin et al, 1993). Most recently, an AGP has been shown to be covalently linked to rhamnosyl residues of RG I and an arabinoxylan polysaccharide (Tan et al, 2013).…”

Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy

“…In the meantime, the AGPs have been detected in some other botanic adhesives (14)(15)(16)(17). Additionally, the tertiary structure of the AGPs has theoretically been predicted to be spheroidal according to a wattle-blossom model, which was established to describe their 3D structure by analyzing the constituents and flexibility of the AGPs as a whole (24,(29)(30)(31).…”