RESUMEN
Novel molecular pinball machines of the plasma membrane control cytosolic Ca2+ levels that regulate plant metabolism. The essential components involve: 1. an auxin-activated proton pump; 2. arabinogalactan glycoproteins (AGPs); 3. Ca2+ channels; 4. auxin-efflux "PIN" proteins. Typical pinball machines release pinballs that trigger various sound and visual effects. However, in plants, "proton pinballs" eject Ca2+ bound by paired glucuronic acid residues of numerous glycomodules in periplasmic AGP-Ca2+. Freed Ca2+ ions flow down the electrostatic gradient through open Ca2+ channels into the cytosol, thus activating numerous Ca2+-dependent activities. Clearly, cytosolic Ca2+ levels depend on the activity of the proton pump, the state of Ca2+ channels and the size of the periplasmic AGP-Ca2+ capacitor; proton pump activation is a major regulatory focal point tightly controlled by the supply of auxin. Auxin efflux carriers conveniently known as "PIN" proteins (null mutants are pin-shaped) pump auxin from cell to cell. Mechanosensitive Ca2+ channels and their activation by reactive oxygen species (ROS) are yet another factor regulating cytosolic Ca2+. Cell expansion also triggers proton pump/pinball activity by the mechanotransduction of wall stress via Hechtian adhesion, thus forming a Hechtian oscillator that underlies cycles of wall plasticity and oscillatory growth. Finally, the Ca2+ homeostasis of plants depends on cell surface external storage as a source of dynamic Ca2+, unlike the internal ER storage source of animals, where the added regulatory complexities ranging from vitamin D to parathormone contrast with the elegant simplicity of plant life. This paper summarizes a sixty-year Odyssey.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Membrana Celular/metabolismo , Mecanotransducción Celular , Proteínas de Transporte de Membrana/metabolismo , Mucoproteínas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas/metabolismo , Canales de Calcio/metabolismo , Galactanos/metabolismo , Ácidos Indolacéticos/metabolismo , Desarrollo de la Planta , Proteínas de Plantas/metabolismo , Bombas de Protones/metabolismo , Especies Reactivas de Oxígeno , Estrés MecánicoRESUMEN
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 oscillator integrates biochemical and mechanical force that regulate morphogenetic gradients of three ionic species, auxin, protons and Ca2+. Hechtian adhesion between cell wall and plasma membrane transduces wall stress that opens Ca2+ channels and reorients auxin efflux "PIN" proteins; they control the auxin-activated proton pump that dissociates Ca2+ bound by periplasmic arabinogalactan proteins (AGP-Ca2+) hence the source of cytosolic Ca2+ waves that activate exocytosis of wall precursors, AGPs and PIN proteins essential for morphogenesis. This novel approach identifies the critical determinants of an algorithm that generates phyllotaxis spiral and Fibonaccian symmetry: these determinants in order of their relative contribution are: (1) size of the apical meristem and the AGP-Ca2+ capacitor; (2) proton pump activity; (3) auxin efflux proteins; (4) Ca2+ channel activity; (5) Hechtian adhesion that mediates the cell wall stress vector. Arguably, AGPs and the AGP-Ca2+ capacitor plays a decisive role in phyllotaxis periodicity and its evolutionary origins.
Asunto(s)
Señalización del Calcio , Ácidos Indolacéticos/metabolismo , Desarrollo de la Planta , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/metabolismo , Calcio/metabolismo , Pared Celular/metabolismo , Magnoliopsida/crecimiento & desarrollo , Magnoliopsida/metabolismo , Mucoproteínas/metabolismo , Periodicidad , Plantas/metabolismoRESUMEN
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 stressâ»strain transduction from the primary cell wall tethered to the plasma membrane by a specific arabinogalactan protein (AGP). The resulting stress vector, with direction defined by Hechtian adhesion sites, has a magnitude of a few piconewtons amplified by a hypothetical Hechtian growth oscillator. This paradigm shift involves stress-activated plasma membrane Ca2+ channels and auxin-activated Hâº-ATPase. The proton pump dissociates periplasmic AGP-glycomodules that bind Ca2+. Thus, as the immediate source of cytosolic Ca2+, an AGP-Ca2+ capacitor directs the vectorial exocytosis of cell wall precursors and auxin efflux (PIN) proteins. In toto, these components comprise the Hechtian oscillator and also the gravisensor. Thus, interdependent auxin and Ca2+ morphogen gradients account for the predominance of AGPs. The size and location of a cell surface AGP-Ca2+ capacitor is essential to differentiation and explains AGP correlation with all stages of morphogenetic patterning from embryogenesis to root and shoot. Finally, the evolutionary origins of the Hechtian oscillator in the unicellular Chlorophycean algae reflect the ubiquitous role of chemiosmotic proton pumps that preceded DNA at the dawn of life.
Asunto(s)
Pared Celular/metabolismo , Morfogénesis , Células Vegetales/fisiología , Desarrollo de la Planta , Evolución Biológica , Calcio/metabolismo , Mecanotransducción Celular , Proteínas de Plantas/metabolismo , Raíces de Plantas/embriología , Raíces de Plantas/fisiología , Transducción de SeñalRESUMEN
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 SerHyp4 and SerHyp2 motifs with arabinosylated (1-4 residues) Hyp. Although they are less than ten percent in all wall materials, AGPs and extensins play important roles in all aspects of plant growth and development. The detailed mechanisms of their functions are still under investigation. However, many of the functions may be attributed to their adhesive properties. Here, we used a forced unbinding technique to measure relative adhesive potential of the well characterized (AlaHyp)51 and (SerHyp4)18 glycomodules representing AGPs and extensins, respectively. In the presence of different wall ions such as protons, Ca2+, and boron, the glycomodules exhibited different adhesive patterns, suggesting that the wall ion-regulated intermolecular interactions/adhesions between AGPs and/or extensins may be involved in maintaining wall-plasma membrane integrity during wall loosening processes such as wall elongation or expansion. This research applies a biophysical approach to understand the biological function of plant cell wall glycoproteins.
RESUMEN
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-Ca2+ ) capacitor with tip-localized AGPs as the source of tip-focussed cytosolic Ca2+ 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 Ca2+ channels and activates H+ -ATPase proton pump efflux that dissociates periplasmic AGP-Ca2+ resulting in a Ca2+ 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. By analogy with the three cryptic inscriptions of the classical Rosetta Stone, the Hechtian Hypothesis translates classical AGP function as a Ca2+ capacitor, pollen tube guide and wall plasticizer into a simple but widely applicable model of tip growth. Even wider ramifications of the Hechtian oscillator may implicate AGPs in osmosensing or gravisensing and other tropisms, leading us yet further towards the Holy Grail of plant growth.
Asunto(s)
Galactanos/metabolismo , Glicoproteínas/metabolismo , Modelos Biológicos , Tubo Polínico/crecimiento & desarrollo , Membrana Celular/metabolismo , Galactanos/química , Glicoproteínas/química , Mecanotransducción CelularRESUMEN
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. Proposed functions of EXT arabinosylation include stabilizing the polyproline II helix structure and facilitating EXT cross-linking. Here, the involvement of arabinosylation in EXT cross-linking was investigated by assaying the initial cross-linking rate and degree of cross-linking of partially or fully de-arabinosylated EXTs using an in vitro cross-linking assay followed by gel permeation chromatography. Our results indicate that EXT arabinosylation is required for EXT cross-linking in vitro and the fourth arabinosyl residue in the tetraarabinoside chain, which is uniquely α-linked, may determine the initial cross-linking rate. Our results also confirm the conserved structure of the oligoarabinosides across species, indicating an evolutionary significance for EXT arabinosylation.
RESUMEN
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. Several HRGPs, including EXT3, were identified in the wild-type root walls but not in walls of the ANP and lethal mutant. Indeed the ANP walls and walls of mutants displaying the lethal phenotype possessed HRGPs, but the profiles suggest that changes in the amount and perhaps type may account for the corresponding phenotypes.
RESUMEN
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. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.
Asunto(s)
Pared Celular/metabolismo , Glicoproteínas/genética , Mucoproteínas/genética , Nicotiana/citología , Nicotiana/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Secuencia de Aminoácidos , Secuencia de Bases , Pared Celular/química , Glicoproteínas/análisis , Glicoproteínas/metabolismo , Glicosilación , Datos de Secuencia Molecular , Mucoproteínas/análisis , Mucoproteínas/metabolismo , Proteínas de Plantas/análisis , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/metabolismo , Nicotiana/química , Nicotiana/metabolismo , Regulación hacia ArribaRESUMEN
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). The aim was to identify EXT3 substitute(s), thus gaining a deeper understanding of new wall assembly. The data show differential expression in the ext3 mutant that included 61% (P ≤ 0.05) of the HRGP genes, and ability to self-rescue by reprogramming expression. Independent revertants had reproducible expression networks, largely heritable over the four generations tested, with some genes displaying transgenerational drift towards wild-type expression levels. Genes for nine candidate regulatory proteins as well as eight candidate HRGP building materials and/or facilitators of new wall assembly or maintenance, in the (near) absence of EXT3 expression, were identified. Seven of the HRGP fit the current model of EXT function. In conclusion, the data on phenotype comparisons and on differential expression of the genes-of-focus provide strong evidence that different combinations of HRGPs regulated by alternative gene expression networks, can make functioning cell walls, resulting in (apparently) normal plant growth and development. More broadly, this has implications for interpreting the cause of any mutant phenotype, assigning gene function, and genetically modifying plants for utilitarian purposes.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Pared Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Glicoproteínas/genética , Enfermedades de las Plantas/inmunología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas del Citoesqueleto , Regulación hacia Abajo , Flores/genética , Flores/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Genotipo , Glicoproteínas/metabolismo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Brotes de la Planta/genética , Brotes de la Planta/metabolismo , Plantas Modificadas Genéticamente , Plantones/genética , Plantones/metabolismo , Regulación hacia ArribaRESUMEN
Plant cell walls are comprised largely of the polysaccharides cellulose, hemicellulose, and pectin, along with â¼10% 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. Here, we show that two isoforms of a purified Arabidopsis thaliana arabinogalactan protein (AGP) encoded by hydroxyproline-rich glycoprotein family protein gene At3g45230 are covalently attached to wall matrix hemicellulosic and pectic polysaccharides, with rhamnogalacturonan I (RG I)/homogalacturonan linked to the rhamnosyl residue in the arabinogalactan (AG) of the AGP and with arabinoxylan attached to either a rhamnosyl residue in the RG I domain or directly to an arabinosyl residue in the AG glycan domain. The existence of this wall structure, named ARABINOXYLAN PECTIN ARABINOGALACTAN PROTEIN1 (APAP1), is contrary to prevailing cell wall models that depict separate protein, pectin, and hemicellulose polysaccharide networks. The modified sugar composition and increased extractability of pectin and xylan immunoreactive epitopes in apap1 mutant aerial biomass support a role for the APAP1 proteoglycan in plant wall architecture and function.
Asunto(s)
Arabidopsis/química , Pared Celular/química , Mucoproteínas/química , Pectinas/química , Proteoglicanos/química , Xilanos/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Proteínas de Arabidopsis/metabolismo , Biomasa , Pared Celular/genética , Pared Celular/metabolismo , Epítopos , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Glicoproteínas/metabolismo , Modelos Estructurales , Datos de Secuencia Molecular , Mucoproteínas/genética , Mucoproteínas/inmunología , Mucoproteínas/metabolismo , Mutación , Pectinas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Isoformas de Proteínas , Proteoglicanos/metabolismo , Proteómica , Xilanos/metabolismoRESUMEN
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. The extensive glycosylation of the HypRP tags greatly extends the serum half-life of small therapeutic proteins, such as interferon α2b or human growth hormone, without significantly impairing their bioactivities and the tag greatly enhances solubility.
Asunto(s)
Biotecnología/métodos , Hidroxiprolina/metabolismo , Nicotiana/citología , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Agrobacterium tumefaciens/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Células Cultivadas , Cromatografía Líquida de Alta Presión , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Glicoproteínas/aislamiento & purificación , Glicosilación , Proteínas Fluorescentes Verdes/metabolismo , Interferón-alfa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oligonucleótidos/genética , Plásmidos/genética , Solubilidad , Nicotiana/genética , Transformación Genética/genéticaRESUMEN
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. We also outline an additional approach involving genetic engineering that can potentially yield the complete genomic range of extensins and other hydroxyproline-rich glycoprotein (HRGPs) currently underutilized for biotechnology.
Asunto(s)
Pared Celular/química , Glicoproteínas/química , Mucoproteínas/aislamiento & purificación , Nicotiana/química , Técnicas de Cultivo de Célula , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/aislamiento & purificación , Mucoproteínas/biosíntesis , Proteínas Periplasmáticas/biosíntesis , Proteínas Periplasmáticas/aislamiento & purificación , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Nicotiana/genética , Nicotiana/metabolismo , Transformación GenéticaRESUMEN
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. The Hyp-AGs were isolated from two different arabinogalactan protein fusion glycoproteins expressed in tobacco cells; that is, a 22-residue Hyp-AG and a 20-residue Hyp-AG, both isolated from interferon alpha2b-(Ser-Hyp)(20), and a 14-residue Hyp-AG isolated from (Ala-Hyp)(51)-green fluorescent protein. We used NMR spectroscopy to establish the molecular structure of these Hyp-AGs, which share common features: (i) a galactan main chain composed of two 1-->3 beta-linked trigalactosyl blocks linked by a beta-1-->6 bond; (ii) bifurcated side chains with Ara, Rha, GlcUA, and a Gal 6-linked to Gal-1 and Gal-2 of the main-chain trigalactosyl repeats; (iii) a common side chain structure composed of up to six residues, the largest consisting of an alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->3)-alpha-L-Araf-(1-->3- unit and an alpha-L-Rhap-(1-->4)-beta-D-GlcUAp-(1-->6)-unit, both linked to Gal. The conformational ensemble obtained by using nuclear Overhauser effect data in structure calculations revealed a galactan main chain with a reverse turn involving the beta-1-->6 link between the trigalactosyl blocks, yielding a moderately compact structure stabilized by H-bonds.
Asunto(s)
Galactanos/química , Galactosa/química , Hidroxiprolina/química , Secuencia de Carbohidratos , Carbohidratos/química , Pared Celular/metabolismo , Glicoproteínas/química , Proteínas Fluorescentes Verdes/metabolismo , Enlace de Hidrógeno , Interferón gamma/metabolismo , Cinética , Espectroscopía de Resonancia Magnética/métodos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
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. Thus, rhGH was expressed with 10 repeats of the AGP glycomodule Ser-Hyp (SO) at the C-terminus (rhGH-(SO)(10)). We also expressed rhGH as an AGP-enhanced green fluorescent protein (EGFP) fusion, designated rhGH-(SO)(10)-EGFP, to assess the cellular distribution of the glycoprotein, which was mainly extracellular. Recombinant hGH-(SO)(10) bound the hGH receptor with an affinity similar to that of a rhGH standard, stimulated the same intracellular signaling pathway as hGH, but possessed an in vivo serum half-life more than sixfold that of the hGH control. Furthermore, rhGH-(SO)(10) gave a 500 fold greater secreted yield than the non-glycosylated control rhGH that was also targeted for secretion. Detailed analysis of the rhGH-(SO)(10) glycans indicated a conserved structure with relatively little microheterogeneity and an average size of 25 monosaccharide residues. These results were consistent with earlier work expressing interferon alpha 2b as an AGP chimera and further demonstrate the feasibility of this approach to the production of long-acting, biologically potent therapeutic proteins by plant cells.
Asunto(s)
Galactanos/química , Hormona de Crecimiento Humana/química , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/farmacocinética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Conformación de Carbohidratos , Secuencia de Carbohidratos , Células Cultivadas , Medios de Cultivo Condicionados/química , Glicosilación , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Semivida , Hormona de Crecimiento Humana/sangre , Hormona de Crecimiento Humana/genética , Hormona de Crecimiento Humana/aislamiento & purificación , Hormona de Crecimiento Humana/farmacocinética , Humanos , Células L , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Transducción de Señal/efectos de los fármacos , Nicotiana/citologíaRESUMEN
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. Elucidation of this code involved a single species, tobacco (Nicotiana tabacum) BY-2 cells. However, recent work suggests species variation, perhaps tissue specific Hyp glycosylation. Thus, the extent to which the Hyp glycosylation code is 'global' needs testing. We compared the ability of distantly related Arabidopsis cell cultures to process putative HRGP glycosylation motifs encoded by synthetic genes. The genes included: repetitive Ser-Pro, Ser-Pro2, Ser-Pro4 and an analog of the tomato arabinogalactan-protein, LeAGP-1DeltaGPI. All were expressed as enhanced green fluorescent protein (EGFP) fusion glycoproteins, designated: AtSO-EGFP (O=Hyp), AtSO2-EGFP, AtSO4-EGFP and AtEGFP-LeAGP-1DeltaGPI, respectively. The Arabidopsis glycosylation patterns were essentially similar to those observed in Nicotiana: non-contiguous Hyp residues in AtSO-EGFP were glycosylated exclusively with arabinogalactan polysaccharides while contiguous Hyp in AtSO2-EGFP and AtSO4-EGFP was exclusively arabinosylated. Mixed contiguous and non-contiguous Hyp residues in AtEGFP-LeAGP-1DeltaGPI were also arabinosylated and arabinogalactosylated consistent with the code. However, slightly more arabinogalactosylated Hyp and less non-glycosylated Hyp in AtEGFP-LeAGP-1DeltaGPI than tobacco NtEGFP-LeAGP-1DeltaGPI suggested Arabidopsis prolyl hydroxylases have a slightly broader specificity. Arabidopsis Hyp-arabinogalactans differed from tobacco in decreased glucuronic acid content and lack of rhamnose. Yields of the EGFP fusion glycoproteins were dramatically higher than targeted EGFP lacking Hyp-glycomodules. This validates earlier suggestions that the glycosylation of proteins facilitates their secretion.
Asunto(s)
Arabidopsis/metabolismo , Glicoproteínas/metabolismo , Glicosilación , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Polisacáridos/fisiología , Arabidopsis/citología , Arabidopsis/genética , Pared Celular/química , Pared Celular/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Proteínas Fluorescentes Verdes/genética , Hidroxiprolina/química , Hidroxiprolina/metabolismo , Mucoproteínas/química , Mucoproteínas/genética , Mucoproteínas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Nicotiana/citología , Nicotiana/genéticaRESUMEN
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. Here we report that electron micrographs of rsh mutant cells lacking RSH extensin correspond to a wall phenotype typified by incomplete cross wall assembly. Biochemical characterization of the purified RSH glycoprotein isolated from wild-type Arabidopsis cell cultures confirmed its identity as AtEXT3: a (hydroxy)proline-rich glyco protein comprising 11 identical amphiphilic peptide repeats with a 28-residue periodicity: SOOOOKKHYVYKSOOOOVKHYSOOOVYH (O = Hyp), each repeat containing a hydrophobic isodityrosine cross-link motif (YVY, underlined). Atomic force microscopy of RSH glycoprotein imaged its propensity for self-assembly into a dendritic scaffold. Extensin peroxidase catalyzed in vitro formation of insoluble RSH gels with concomitant tyrosine cross-linking, hence this likelihood in muro. We conclude that self-assembling amphiphiles of lysine-rich RSH extensin form positively charged scaffolds in the cell plate. These react with negatively charged pectin to create an extensin pectate coacervate that may template further orderly deposition of the new cross wall at cytokinesis.
Asunto(s)
Arabidopsis/citología , Pared Celular/ultraestructura , Glicoproteínas/fisiología , Proteínas de Plantas/fisiología , Secuencia de Aminoácidos , Glicoproteínas/química , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Proteínas de Plantas/químicaRESUMEN
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. We previously identified three genes for KDEL-tailed cysteine endopeptidases (AtCEP1, AtCEP2, AtCEP3) in Arabidopsis thaliana. Transgenic plants of A. thaliana expressing ß-glucuronidase (GUS) under the control of the promoters for the three genes were produced and analyzed histochemically. GUS activity was promoter- and tissue-specific GUS activity during seedling, flower, and root development, especially in tissues that collapse during final stages of PCD, and in the course of lateral root formation. KDEL-tailed cysteine endopeptidases are unique in being able to digest the extensins that form the basic scaffold for cell wall formation. The broad substrate specificity is due to the structure of the active site cleft of the KDEL-tailed cysteine endopeptidase that accepts a wide variety of amino acids, including proline and glycosylated hydroxyproline of the hydroxyproline rich glycoproteins of the cell wall.
RESUMEN
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. Thus IFNalpha2-AGPs targeted for secretion typically gave 350-1400-fold greater secreted yields than the non-glycosylated IFNalpha2 control. The purified AGP domain itself was not immunogenic when injected into mice and only mildly so when injected as a fusion glycoprotein. Importantly, the AGP-IFNalpha2 chimeras showed up to a 13-fold increased in vivo serum half-life while the biological activity remained similar to native IFNalpha2. The use of arabinogalactan glycomodules may provide a general approach to the enhanced production of therapeutic proteins by plants.
Asunto(s)
Interferón-alfa/biosíntesis , Interferón-alfa/sangre , Mucoproteínas/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Ingeniería de Proteínas/métodos , Células Cultivadas , Clonación Molecular/métodos , Mejoramiento Genético/métodos , Semivida , Humanos , Interferón alfa-2 , Interferón-alfa/química , Interferón-alfa/genética , Tasa de Depuración Metabólica , Mucoproteínas/química , Mucoproteínas/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas RecombinantesRESUMEN
A series of gene constructs encoding synthetic glycomodule peptides with N-terminal signal sequences and C-terminal green fluorescent proteins were expressed in transgenic Arabidopsis (Arabidopsis thaliana) under the control of the 35S promoter. The synthetic glycomodule peptides were composed of repetitive proline-containing motifs that have been previously found to be substrates for prolyl hydroxylases and subsequent O-glycosylation of the hydroxyproline residues. All of the constructs were secreted in aerial tissues, but not in roots. The amount of hydroxylation and glycosylation of the various constructs varied depending on the tissue. Also, accumulation of the proteins exhibited a high degree of cell-type specificity within various tissues due to posttranscriptional effects. The observations reveal a high level of complexity in the synthesis, secretion, and turnover of the glycoproteins.