Arabinogalactan Structures of Repetitive Serine-Hydroxyproline Glycomodule Expressed by Arabidopsis Cell Suspension Cultures.

Arabinogalactan-proteins (AGPs) are members of the hydroxyproline-rich glycoprotein (HRGP) superfamily. They are heavily glycosylated with arabinogalactans, which are usually composed of a β-1,3-linked galactan backbone with 6--linked galactosyl, oligo-1,6-galactosyl, or 1,6-galactan side chains that are further decorated with arabinosyl, glucuronosyl, rhamnosyl, and/or fucosyl residues. Here, our work with Hyp--polysaccharides isolated from (Ser-Hyp)-EGFP (enhanced green fluorescent protein) fusion glycoproteins overexpressed in transgenic Arabidopsis suspension culture is consistent with the common structural features of AGPs isolated from tobacco.

A Molecular Pinball Machine of the Plasma Membrane Regulates Plant Growth-A New Paradigm.

Novel molecular pinball machines of the plasma membrane control cytosolic Ca levels that regulate plant metabolism. The essential components involve: 1. an auxin-activated proton pump; 2.

Phyllotaxis Turns Over a New Leaf-A New Hypothesis.

Phyllotaxis describes the periodic arrangement of plant organs most conspicuously floral. Oscillators generally underlie periodic phenomena. A hypothetical algorithm generates phyllotaxis regulated by the Hechtian growth oscillator of the stem apical meristem (SAM) protoderm.

The Role of the Primary Cell Wall in Plant Morphogenesis.

Morphogenesis remains a riddle, wrapped in a mystery, inside an enigma. It remains a formidable problem viewed from many different perspectives of morphology, genetics, and computational modelling. We propose a biochemical reductionist approach that shows how both internal and external physical forces contribute to plant morphogenesis via mechanical stress⁻strain transduction from the primary cell wall tethered to the plasma membrane by a specific arabinogalactan protein (AGP).

Intermolecular interactions between glycomodules of plant cell wall arabinogalactan-proteins and extensins.

Hydroxyproline-rich glycoproteins (HRGPs) are a unique component of plant cell walls, undergoing extensive posttranslational modification such as proline hydroxylation and hydroxyproline-O-glycosylation. Arabinogalactan proteins (AGPs) and extensins are major members of the HRGP superfamily. AGPs have repetitive AlaHyp, SerHyp, and ThrHyp peptides, the Hyp residues being glycosylated with large type II arabinogalactan polysaccharides, while extensins contain characteristic SerHyp and SerHyp motifs with arabinosylated (1-4 residues) Hyp.

Pollen tube growth and guidance: Occam's razor sharpened on a molecular arabinogalactan glycoprotein Rosetta Stone.

Occam's Razor suggests a new model of pollen tube tip growth based on a novel Hechtian oscillator that integrates a periplasmic arabinogalactan glycoprotein-calcium (AGP-Ca ) capacitor with tip-localized AGPs as the source of tip-focussed cytosolic Ca oscillations: Hechtian adhesion between the plasma membrane and the cell wall of the growing tip acts as a piconewton force transducer that couples the internal stress of a rapidly growing wall to the plasma membrane. Such Hechtian transduction opens stretch-activated Ca channels and activates H -ATPase proton pump efflux that dissociates periplasmic AGP-Ca resulting in a Ca influx that activates exocytosis of wall precursors. Thus, a highly simplified pectic primary cell wall regulates its own synthesis by a Hechtian growth oscillator that regulates overall tip growth.

Arabinosylation Plays a Crucial Role in Extensin Cross-linking In Vitro.

Extensins (EXTs) are hydroxyproline-rich glycoproteins (HRGPs) that are structural components of the plant primary cell wall. They are basic proteins and are highly glycosylated with carbohydrate accounting for >50% of their dry weight. Carbohydrate occurs as monogalactosyl serine and arabinosyl hydroxyproline, with arabinosides ranging in size from ~1 to 4 or 5 residues.

Identification of the Abundant Hydroxyproline-Rich Glycoproteins in the Root Walls of Wild-Type Arabidopsis, an ext3 Mutant Line, and Its Phenotypic Revertant.

Extensins are members of the cell wall hydroxyproline-rich glycoprotein (HRGP) superfamily that form covalently cross-linked networks in primary cell walls. A knockout mutation in EXT3 (AT1G21310), the gene coding EXTENSIN 3 (EXT3) in Arabidopsis Landsberg erecta resulted in a lethal phenotype, although about 20% of the knockout plants have an apparently normal phenotype (ANP). In this study the root cell wall HRGP components of wild-type, ANP and the ext3 mutant seedlings were characterized by peptide fractionation of trypsin digested anhydrous hydrogen fluoride deglycosylated wall residues and by sequencing using LC-MS/MS.

Changes in cell wall properties coincide with overexpression of extensin fusion proteins in suspension cultured tobacco cells.

Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin.

Identification of the pI 4.6 extensin peroxidase from Lycopersicon esculentum using proteomics and reverse-genomics.

The regulation of plant cell growth and early defense response involves the insolubilization of hydroxyproline-rich glycoproteins (HRGPs), such as extensin, in the primary cell wall. In tomato (Lycopersicon esculentum), insolubilization occurs by the formation of tyrosyl-crosslinks catalyzed specifically by the pI 4.6 extensin peroxidase (EP).

Self-rescue of an EXTENSIN mutant reveals alternative gene expression programs and candidate proteins for new cell wall assembly in Arabidopsis.

Plants encode a poorly understood superfamily of developmentally expressed cell wall hydroxyproline-rich glycoproteins (HRGPs). One, EXTENSIN3 (EXT3) of the 168 putative HRGPs, is critical in the first steps of new wall assembly, demonstrated by broken and misplaced walls in its lethal homozygous mutant. Here we report the findings of phenotypic (not genotypic) revertants of the ext3 mutant and in-depth analysis including microarray and qRT-PCR (polymerase chain reaction).

Functional identification of a hydroxyproline-o-galactosyltransferase specific for arabinogalactan protein biosynthesis in Arabidopsis.

Although plants contain substantial amounts of arabinogalactan proteins (AGPs), the enzymes responsible for AGP glycosylation are largely unknown. Bioinformatics indicated that AGP galactosyltransferases (GALTs) are members of the carbohydrate-active enzyme glycosyltransferase (GT) 31 family (CAZy GT31) involved in N- and O-glycosylation. Six Arabidopsis GT31 members were expressed in Pichia pastoris and tested for enzyme activity.

An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein.

Plant cell walls are comprised largely of the polysaccharides cellulose, hemicellulose, and pectin, along with ∼10% protein and up to 40% lignin. These wall polymers interact covalently and noncovalently to form the functional cell wall. Characterized cross-links in the wall include covalent linkages between wall glycoprotein extensins between rhamnogalacturonan II monomer domains and between polysaccharides and lignin phenolic residues.

A novel plant cell bioproduction platform for high-yield secretion of recombinant proteins.

Plant cell suspension culture integrates the merits of whole-plant systems with those of microbial fermentation and mammalian cell culture, and has been recognized as a promising alternative biosynthetic platform for valuable proteins. However, the low protein productivity dilemma has been the bottleneck toward commercializing this technology. Here, we describe a new technology, termed hydroxyproline (Hyp)-Glyco technology, that dramatically increases the yield of secreted recombinant proteins from cultured plant cells by expressing them as fusions with a novel glycomodule tag comprising an Hyp-rich repetitive peptide (HypRP) backbone that is subsequently glycosylated through the Hyp residues.

Enhanced accumulation of secreted human growth hormone by transgenic tobacco cells correlates with the introduction of an N-glycosylation site.

Extracellular secretion of recombinant proteins from plant cell suspension culture will simplify the protein purification procedure and greatly reduce the production cost. Our early work indicated that presence of hydroxyproline-O-glycosylation at the C- or N-terminus of the target protein boosted the secreted yields in the culture medium. Inspired by early successes, we tested the possibility of introducing an N-glycosylation site to facilitate the secretion of human growth hormone (hGH) from cultured tobacco cells.

Structural proteins of the primary cell wall: extraction, purification, and analysis.

Structural proteins of the primary cell wall present unusual but interesting problems for structural biologists in particular and plant biologists in general. As structure is the key to function; then the biochemical isolation of these glycoproteins for further study is paramount. Here, we detail the "classical" method for isolating soluble extensin monomers by elution of monomeric precursors to network extensin from tissue cultures.

Identification and characterization of in vitro galactosyltransferase activities involved in arabinogalactan-protein glycosylation in tobacco and Arabidopsis.

Arabinogalactan-proteins (AGPs) are highly glycosylated hydroxyproline (Hyp)-rich glycoproteins that are frequently characterized by the presence of [Alanine-Hyp] ([AO]) repetitive units. AGP galactosyltransferase (GalT) activities in tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana) microsomal membranes were studied here with an in vitro GalT reaction system, which used acceptor substrates composed of [AO] repetitive units, specifically, a chemically synthesized [AO](7) acceptor and a transgenically produced and deglycosylated d[AO](51) acceptor. Incorporation of [(14)C]Gal from UDP-[(14)C]Gal into the [AO](7) and d[AO](51) acceptors was observed following HPLC fractionation of the reaction products.

Plant O-hydroxyproline arabinogalactans are composed of repeating trigalactosyl subunits with short bifurcated side chains.

Classical arabinogalactan proteins partially defined by type II O-Hyp-linked arabinogalactans (Hyp-AGs) are structural components of the plant extracellular matrix. Recently we described the structure of a small Hyp-AG putatively based on repetitive trigalactosyl subunits and suggested that AGs are less complex and varied than generally supposed. Here we describe three additional AGs with similar subunits.

Novel fusion proteins of interferon alpha2b cause growth inhibition and induce JAK-STAT signaling in melanoma.

Melanoma is an aggressive form of skin cancer with high occurrence in the United States. Interferon alpha2b (IFNalpha2b/IFNalpha2) has been used in high doses to treat melanoma. However, problems associated with small molecule therapeutics such as with IFN treatment include small molecular size, degradation by serum proteases, and rapid kidney clearance.

Human growth hormone expressed in tobacco cells as an arabinogalactan-protein fusion glycoprotein has a prolonged serum life.

Therapeutic proteins with molecular weights lower than 40 kDa often have short serum half-lives due to their susceptibility to serum proteases and rapid renal clearance. Chemical derivatization, such as PEGylation, or expression as serum albumin fusions increases molecular mass and overcome these problems but at the expense of decreased bioactivity. Here we applied a new method that yields biologically potent recombinant human growth hormone (rhGH) with increased serum half-life when expressed as an arabinogalactan-protein (AGP) in tobacco BY-2 cells.

KDEL-tailed cysteine endopeptidases involved in programmed cell death, intercalation of new cells, and dismantling of extensin scaffolds.

KDEL-tailed cysteine endopeptidases are a group of papain-type peptidases found in senescing tissue undergoing programmed cell death (PCD). Their genes have so far been cloned and analyzed in 12 angiosperms. They are synthesized as proenzymes with a C-terminal KDEL endoplasmatic reticulum retention signal, which is removed with the prosequence to activate enzyme activity.

The O-Hyp glycosylation code in tobacco and Arabidopsis and a proposed role of Hyp-glycans in secretion.

Most aspects of plant growth involve cell surface hydroxyproline (Hyp)-rich glycoproteins (HRGPs) whose properties depend on arabinogalactan polysaccharides and arabinosides that define the molecular surface. Potential glycosylation sites are defined by an O-Hyp glycosylation code: contiguous Hyp directs arabinosylation. Clustered non-contiguous Hyp directs arabinogalactosylation.

Self-assembly of the plant cell wall requires an extensin scaffold.

Cytokinesis partitions the cell by a cleavage furrow in animals but by a new cross wall in plants. How this new wall assembles at the molecular level and connects with the mother cell wall remains unclear. A lethal Arabidopsis embryogenesis mutant designated root-, shoot-, hypocotyl-defective (rsh) provides some clues: RSH encodes extensin AtEXT3, a structural glycoprotein located in the nascent cross wall or "cell plate" and also in mature cell walls.

High-yields and extended serum half-life of human interferon alpha2b expressed in tobacco cells as arabinogalactan-protein fusions.

Therapeutic proteins like human interferon alpha2 generally possess short serum half-lives due to their small size, hence rapid renal clearance, and susceptibility to serum proteases. Chemical derivatization, such as addition of polyethylene glycol (PEG) groups overcomes both problems, but at the expense of greatly decreased bioactivity. We describe a new method that yields biologically potent interferon alpha2b (IFNalpha2) in high yields and with increased serum half-life when expressed as arabinogalactan-protein (AGP) chimeras in cultured tobacco cells.