WO2008011681A1 - Polysaccharide transferase - Google Patents
Polysaccharide transferase Download PDFInfo
- Publication number
- WO2008011681A1 WO2008011681A1 PCT/AU2007/001045 AU2007001045W WO2008011681A1 WO 2008011681 A1 WO2008011681 A1 WO 2008011681A1 AU 2007001045 W AU2007001045 W AU 2007001045W WO 2008011681 A1 WO2008011681 A1 WO 2008011681A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- polysaccharide
- transferase
- xyloglucan
- acceptor
- donor
- Prior art date
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01074—Glycosaminoglycan galactosyltransferase (2.4.1.74)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01207—Xyloglucan:xyloglucosyl transferase (2.4.1.207)
Definitions
- the present invention relates generally to enzymes which act on polysaccharide substrates. More particularly, the present invention provides isolated or substantially purified polysaccharide transferases.
- Plant cell walls are dynamic structures that are altered during cell division, growth and differentiation to enable cells to adapt to changing functional requirements and to environmental and pathogen-induced challenges.
- the overall strength and flexibility of land plants are determined largely through the collective strength and flexibility of the walls that surround individual cells.
- walls are important for intercellular cohesion and cell- cell communication, and must be selectively permeable to water, nutrients and phytohormones.
- the primary cell w ⁇ alls of vascular plants consist of cellulosic microfibrils that are embedded in a chemically complex matrix consisting mostly of polysaccharides, but also containing structural proteins and enzymes, and phenolic acids.
- Xyloglucans and pectic polysaccharides are the major non-cellulosic polysaccharides of primary walls from dicotyledonous plants, while in the Poales and related commelinoid monocots, including commercially important cereals and grasses, glucuronoarabinoxylans and (l,3;l,4)-[3-D-glucans are the predominant non-cellulosic wall polysaccharides, and levels of pectic polysaccharides, glucomannans and xyloglucans are relatively low.
- wall composition and the fine structures of component polysaccharides vary depending upon the growth phase, cell type, cell position, and local region within the wall.
- lignin is deposited throughout the wall during secondary thickening and, in response to pathogen attack, the rapid formation of a cross-linked protein network, together with the deposition of callose and lignin, can create a physical barrier to invading microorganisms.
- compositions of plant cell walls have been defined in detail, there is little information on the molecular interactions between constituent polysaccharides in the wall.
- manipulation of the major wall polysaccharides via their biosynthesis has also been hampered by a lack of knowledge of the mechanism(s) and control of the biosynthetic steps, coupled with a limited understanding of the physical and chemical interactions between wall components.
- the present invention is predicated, in part, on the isolation and functional characterisation of polysaccharide transferases which are capable of catalysing the formation of a covalent bond between a donor polysaccharide and an acceptor polysaccharide.
- a polysaccharide transferase purified from barley seedlings can catalyze the formation of covalent bonds between different polysaccharides, including between xyloglucans and cellulose, and between xyloglucans and (l,3;l,4)- ⁇ -D-glucans.
- the present invention provides an isolated or substantially purified polysaccharide transferase, wherein said polysaccharide transferase is capable of catalyzing the formation of a covalent bond between a donor polysaccharide and an acceptor polysaccharide; or a functionally active fragment or variant of said polysaccharide transferase.
- the polysaccharide transferase of the present invention is capable of forming a covalent bond wherein the donor polysaccharide and/or the acceptor polysaccharide is a polysaccharide other than a xyloglucan.
- the present invention also contemplates isolated or substantially purified polysaccharide transferases which are capable of catalyzing the formation of a covalent bond between plant cell w ⁇ all polysaccharides such as, for example, celluloses, hemicelluloses, xyloglucans, (l,3;l,4)- ⁇ -D-glucans, arabinoxylans, pectins, mannans and the like.
- polysaccharide transferases which are capable of catalyzing the formation of a covalent bond between plant cell w ⁇ all polysaccharides such as, for example, celluloses, hemicelluloses, xyloglucans, (l,3;l,4)- ⁇ -D-glucans, arabinoxylans, pectins, mannans and the like.
- polysaccharide transferases of the present invention may be produced in any suitable way or isolated from any suitable source.
- the polysaccharide transferase of the present invention is purified from a plant, or a part, organ, tissue or cell thereof.
- the polysaccharide transferase is purified from a plant cell w ⁇ all.
- the present invention provides a method for isolating or substantially purifying a polysaccharide transferase from a sample, the method comprising the steps of:
- the second aspect of the invention provides a method for isolating or substantially purifying a polysaccharide transferase to a homogenous form.
- the present invention provides a method for forming a covalent bond between a donor polysaccharide and an acceptor polysaccharide, the method comprising contacting said donor polysaccharide and said acceptor polysaccharide with either a polysaccharide transferase which is capable of catalyzing a covalent bond between said donor polysaccharide and said acceptor polysaccharide, or a functionally active fragment or variant of said polysaccharide transferase.
- the present invention provides an isolated polysaccharide of the structure:
- a - B wherein A and B are polysaccharides which are covalently linked to each other.
- Polysaccharide A and polysaccharide B may be any polysaccharides. However, in one embodiment, at least one of polysaccharide A and polysaccharide B is a polysaccharide other than a xyloglucan. Therefore, in this embodiment, the polysaccharide of the present invention comprises a xyloglucan covalently bonded to another polysaccharide of a different type and/or a polysaccharide comprising two non-xyloglucan polysaccharides (which may be the same or of a different type) covalently bonded to each other.
- the present invention also provides a method for modulating the extent of covalent bonding between a donor polysaccharide and an acceptor polysaccharide in a cell; the method comprising modulating the level or activity of either a polysaccharide transferase which is capable of catalyzing a covalent bond between said donor polysaccharide and said acceptor polysaccharide, or a functionally active fragment or variant of said polysaccharide transferase, in said cell.
- the method of the fifth aspect of the present invention is adapted to modulating the extent of covalent bonding between a donor polysaccharide and an acceptor polysaccharide in a plant cell. In another embodiment, the method of the fifth aspect of the present invention is adapted to modulating the extent of covalent bonding between a donor polysaccharide and an acceptor polysaccharide in a plant cell wall.
- sequence identifier number SEQ ID NO:
- a summary of the sequence identifiers is provided in Table 1.
- a sequence listing is provided at the end of the specification.
- the present invention is predicated, in part, on the isolation and functional characterisation of polysaccharide transferases which are capable of catalysing the formation of a covalent bond between a donor polysaccharide and an acceptor polysaccharide.
- polysaccharide transferase refers to a polysaccharide transferase that does not utilize nucleotide sugar substrates.
- polysaccharide transferase refers to a polysaccharide transferase which is a family GH16 glycoside hydrolase.
- GH16 glycoside hydrolases are described in Strohmeier et al. (Protein Science 13: 3200- 3213, 2004) and exemplary family GH16 glycoside hydrolases are described in "the family GH16 glycoside hydrolase database" (GHDB) at h
- GHDB family GH16 glycoside hydrolase database
- polysaccharide transferase refers to a xyloglucan:xyloglucosyl transferase/hydrolase (XTH).
- xyloglucan:xyloglucosyl transferase/hydrolase or an "XTH” should not be considered limiting to enzymes which act exclusively on xyloglucan substrates nor limiting to enzymes which exhibit both transferase and hydrolase activity.
- XTH should also be understood to include xyloglucan endotransglycosylases (XETs) which predominantly or exclusively exhibit transglycosylation activity.
- XETs xyloglucan endotransglycosylases
- the barley enzymes described herein in accordance with some embodiments of the invention may be classified as XETs.
- XTH enzymes in the XTH family act only as endohydrolases and these may also be designated XEHs.
- XTH should also be understood to include XEHs.
- Xyloglucans consist of a backbone of (l,4)-(3-D-glucan substituted with xylosyl, galactosyl and fucosyl residues.
- the molecular sizes of xyloglucans can be altered after their deposition into the cell wall and this is likely to be mediated by a class of enzymes known as xyloglucan endotransglycosylases/hydrolases (XTHs).
- the XTHs are abundant in the apoplastic space, they hydrolyse the (l,4)-[3-D-glucan backbone of xyloglucans and, in the case of XETs, transfer the aglycone product of hydrolysis directly onto the non-reducing terminus of another xyloglucan chain.
- Sequences encoding XTHs are surprisingly abundant in barley EST databases, given the relatively low levels of xyloglucans in walls of most barley tissues. There are at least 22 XTH genes in barley and about 30 in rice. In an attempt to reconcile the relatively low abundance of xyloglucans in barley cell walls against the large number of XTH genes and their high expression levels in many tissues of barley, it is postulated that some of the XTHs might be active on the more abundant matrix phase polysaccharides of barley cell walls, for example, the arabinoxylans and the (l,3;l,4)-[3-D-glucans.
- the plant XTHs and microbial (l,3;l,4)-[3-D-glucan endohydrolases are all classified in the family GH16 group of glycoside hydrolases, although a small number of microbial XTHs are classified within families GH12 and GH5 (http://afcnb.cnrs-mrs.fr/CAZY/).
- a role for XETs in the modification of highly abundant (l,3;l,4)- ⁇ -D-glucans and arabinoxylans in walls of the commelinoid monocots would be consistent with the abrupt increase in molecular size of heteroxylans that has been observed in suspension-cultured maize cells following the deposition of the polysaccharide into the walls.
- the present invention is predicated, in part, on polysaccharide transferases which are capable of catalysing the formation of a covalent bond between a donor polysaccharide and an acceptor polysaccharide.
- a "donor polysaccharide” refers to a polysaccharide which donates the energy to a transglycosylation reaction (catalysed by the polysaccharide transferase), while an “acceptor polysaccharide” is a polysaccharide which becomes covalently linked to the donor polysaccharide, through its non-reducing end, as a result of the transglycosylation reaction.
- the catalytic mechanism of the polysaccharide transferases of the present invention may be described as a "disproportionation reaction", as the degree of polymerization of both the donor and the acceptor polysaccharide are changed.
- the present invention provides an isolated or substantially purified polysaccharide transferase, wherein said polysaccharide transferase is capable of catalyzing the formation of a covalent bond between a donor polysaccharide and an acceptor polysaccharide; or a functionally active fragment or variant of said polysaccharide transferase.
- polysaccharide refers to polysaccharides which are differentiated on the basis of the backbone and/or side chains on the backbone of the polysaccharide molecule.
- polysaccharides may be classified as different "types" of polysaccharide on the basis of the monomeric composition (eg. glucose, mannose, xylose and the like) or the linkage types (eg. (3(1-4), ⁇ (l-3) and the like) in the molecular backbone of the polysaccharide molecule.
- polysaccharide may be differentiated on the basis of any side chains on the backbone of the polysaccharide.
- cellulose and xyloglucan should be considered different types of polysaccharide on the basis of differences in side chains, although both comprise a molecular backbone of
- polysaccharide should also be understood to include naturally occurring polysaccharides, synthetic polysaccharides and polysaccharide variants and derivatives. Examples of polysaccharide “derivatives” may be found in Liebert and Heinze (Biomacromolecules 6: 333-340, 2005) and Heinze (In S. Dimitriu (Ed.), Polysaccharides: Structural diversity and functional versatility, (2 nd ed.), pp. 551-590, New York: Marcel Dekker, 2004).
- polysaccharide should also be understood to specifically include the various different polysaccharide types found in plant cell walls, including, for example, celluloses, hemicelluloses, xyloglucans, arabinoxylans, glucomannans, galactomannans, (l,3)- ⁇ -D- glucans, mixed linkage [3-D-glucans (eg. (l,3;l,4)- ⁇ -D-glucans), and the like.
- the present invention contemplates a polysaccharide transferase which is capable of catalyzing the formation of a covalent bond between any suitable donor polysaccharide and any suitable acceptor polysaccharide.
- the polysaccharide transferase of the present invention is capable of forming a covalent bond wherein the donor polysaccharide and/or the acceptor polysaccharide is a polysaccharide other than a xyloglucan. Therefore, in this embodiment, the polysaccharide transferase of the present invention is capable of catalyzing the formation of a covalent bond between a xyloglucan and another polysaccharide of a different type and/or capable of catalyzing the formation of a covalent bond between two non- xyloglucan polysaccharides (which may be the same or of a different type).
- the polysaccharide transferase of the present invention may catalyse the formation of a covalent bond between the same or different types of polysaccharide.
- the polysaccharide transferase of the present invention is capable of catalyzing the formation of a covalent bond between a donor polysaccharide and an acceptor polysaccharide which are of a different type.
- the polysaccharide transferase of the present invention may catalyse the formation of a covalent bond between any appropriately sized donor and acceptor polysaccharides.
- the donor polysaccharide comprises a backbone of at least 10 monomer units.
- the acceptor polysaccharide comprises a backbone of at least 4 monomer units.
- the present invention provides an isolated or substantially purified polysaccharide transferase which is capable of forming a covalent bond between a donor polysaccharide comprising:
- a glucose polymer other than a xyloglucan which comprises at least one [3(1-4) linkage between two glucose monomers; and an acceptor polysaccharide comprising:
- a xyloglucan (i) a xyloglucan; or (ii) a glucose polymer (other than a xyloglucan) which comprises at least one [3(1-4) linkage between two glucose monomers.
- glucose polymer should be understood to include any polysaccharide which includes a glucose monomer backbone.
- the glucose polymers contemplated herein may also comprise glucose or non-glucose containing side chains.
- one or more of the glucose monomers comprising the backbone of the glucose polymer may be derivatised (eg. see below regarding cellulose derivatives).
- the donor polysaccharide comprises a glucose polymer (other than a xyloglucan) which comprises at least one (3(1-4) linkage between two glucose monomers and the acceptor polysaccharide comprises a xyloglucan.
- the donor polysaccharide comprises a xyloglucan and the acceptor polysaccharide comprises a glucose polymer (other than a xyloglucan) which comprises at least one [3(1-4) linkage between two glucose monomers.
- the glucose polymer comprises [3(1-4) linkages between all of the glucose monomers in the backbone of the polysaccharide, and in one embodiment, the glucose polymer comprises cellulose, or an oligomer or derivative thereof.
- Cellulose comprises a backbone of [3-glucose monomers linked together through (1 ⁇ 4) glycosidic bonds.
- the hydroxyl groups of cellulose can also be partially or fully reacted with various chemicals to generate cellulose derivates.
- Exemplary cellulose derivatives include: cellulose esters, such as cellulose acetate and triacetate and nitrocellulose; cellulose ethers including ethylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose; acid-swollen celluloses such as sulfuric acid swollen cellulose and phosphoric acid swollen cellulose.
- the glucose polymer may comprises at least one (3(1-3) linkage between two glucose monomers in addition to at least one (3(1-4) linkage between two glucose monomers, and in one embodiment, the glucose polymer comprises a l,3;l,4-(3-D- glucan or an oligomer or derivative thereof.
- (l,3;l,4)- ⁇ -D-Glucans should be understood to include linear, unbranched polysaccharides in which (3-D-glucopyranosyl monomers are polymerized through a mixture of both (1-4)- and (l-3)-linkages.
- the present invention also contemplates polysaccharide transferases which are capable of catalyzing the formation of a covalent bond between other polysaccharides.
- the present invention also contemplates isolated or substantially purified polysaccharide transferases which are capable of catalyzing the formation of a covalent bond between other plant cell wall polysaccharides such as, for example, cellulosic and non-cellulosic polysaccharides (eg.
- xyloglycans mannoglycans, xyloglucans, glucans
- arabinoxylans comprise a backbone of 1,4-beta- linked xylosyl residues, which is similar in overall structure to cellulose (which comprises a backbone of 1,4-beta-linked glucosyl residues).
- the backbone of the arabinoxylan further comprises mostly single substituents of arabinose that interfere with alignment of the backbones and hence solubilise the polysaccharide.
- arabinoxylan and xyloglucan may be functionally equivalent with respect to their structures and properties in a plant cell wall and, accordingly, arabinoxylans may also serve as donor and/or acceptor polysaccharides for the polysaccharide transferases of the present invention.
- the present invention provides an isolated or substantially purified polysaccharide transferase which is capable of forming a covalent bond between a donor polysaccharide and an acceptor polysaccharide wherein at least one of the donor and/or acceptor polysaccharide comprises an arabinoxylan.
- the present invention contemplates any polysaccharide transferase which is capable of forming a covalent bond between a donor polysaccharide and an acceptor polysaccharide.
- the isolated or substantially purified polysaccharide transferases contemplated by the present invention may comprise any suitable amino acid sequence.
- the polysaccharide transferase comprises: (i) the amino acid sequence set forth in SEQ ID NO: 1; or
- polysaccharide transferase comprises:
- polysaccharide transferase comprises: (i) the amino acid sequence set forth in SEQ ID NO: 3; or
- the present invention also contemplates "functionally active fragments or variants" of polysaccharide transferases comprising the amino acid sequence set forth in any of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
- “Functionally active fragments”, as contemplated herein, may be of any length wherein the fragment retains the capability to catalyse a covalent bond between a donor and acceptor polysaccharide.
- the fragment may comprise at least 100 amino acid residues, at least 150 amino acid residues, at least 200 amino acid residues or at least 250 amino acid residues.
- a fragment at least 100 amino acid residues in length comprises fragments which include 100 or more contiguous amino acids from, for example, the amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
- “Functionally active variants” of the polysaccharide transferases of the invention include orthologs, mutants, synthetic variants, analogs and the like which retain the capability to catalyse the formation of a covalent bond between a donor polysaccharide and an acceptor polysaccharide.
- variant should be considered to specifically include, for example, orthologous polysaccharide transferases derived from different organisms; naturally occurring or synthetic mutants of the polysaccharide transferase; variants of the polysaccharide transferase wherein one or more amino acids within the sequence has been substituted, added or deleted; analogs that contain one or more modified amino acids modified for stability or for other reasons; and chemically synthesised forms of the polysaccharide transferase.
- the functionally active fragment or variant comprises at least 30% sequence identity, at least 45% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity, at least 94% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence set forth in any of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
- the compared amino acid sequences should be compared over a comparison window of at least 20 amino acid residues, at least 50 amino acid residues, at least 100 amino acid residues, at least 200 amino acid residues, at least 250 amino acid residues, or over the full length of any of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
- the comparison window may comprise additions or deletions (ie. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
- Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms such the BLAST family of programs as, for example, disclosed by Altschul et al.
- polysaccharide transferase of the present invention is glycosylated, or otherwise modified by post-translational processes.
- glycosylated would be readily determined by one of skill in the art and should be understood to specifically include, among other things, the post- translational attachment of N-linked and O-linked oligosaccharides to the polysaccharide transferase protein.
- other post-translational modifications should be understood to refer to post-translational modifications such as, for example, phosphorylation, proteolytic cleavage, acylation, methylation, sulphation, prenylation, selenocysteine incorporation and the like.
- the polysaccharide transferase of the present invention comprises a plant-type glycosylation or other post-translational modification pattern, or a functionally equivalent glycosylation or other post-translational modification pattern.
- a polysaccharide transferase comprising a "plant-type glycosylation or other post-translational modification pattern” refers to the polysaccharide transferase having a glycosylation pattern or other post-translational modification pattern that is the same as, or functionally equivalent to, the glycosylation or other post-translational modification pattern that is used by plants.
- a polysaccharide transferase comprising a "plant- type glycosylation or other post-translational modification pattern” refers to the polysaccharide transferase having the same glycosylation pattern or other post-translational modification pattern, or a functional equivalent thereof, of a polysaccharide transferase isolated from a plant.
- the polysaccharide transferases of the present invention may be produced in any suitable way or isolated from any suitable source.
- the polysaccharide transferase of the present invention is purified from a plant, or a part, organ, tissue or cell thereof.
- the polysaccharide transferase is purified from a plant cell wall.
- the present invention contemplates the purification of polysaccharide transferases from any plant, plant cell or plant cell wall. As such, in some embodiments, the present invention contemplates the purification of polysaccharide transferases from monocotyledonous angiosperm plants, dicotyledonous angiosperm plants and gymnosperm plants.
- the present invention provides a polysaccharide transferase isolated from a monocotyledonous plant. In another embodiment, the present invention provides a polysaccharide transferase purified from a cereal crop plant or other member of the Poaceae.
- the term "cereal crop plant” includes members of the order Poales and/or the family Poaceae, which produce edible grain for human or animal food.
- Examples of cereal crop plants that in no way limit the present invention include barley, wheat, rice, maize, millets, sorghum, rye, triticale, oats, teff, rice, spelt and the like.
- the term “cereal crop plant” should also be understood to include a number of non-Poales species that also produce edible grain, which are known as pseudocereals, and include, for example, amaranth, buckwheat and quinoa.
- the polysaccharide transferase of the present invention is purified from a barley (Hordeum vulgare) plant or a cell or cell wall thereof.
- the present invention also contemplates an isolated or substantially purified polysaccharide transferase produced in a recombinant expression system.
- a vast array of recombinant expression systems that may be used to express a polysaccharide transferase-encoding a nucleic acid are known in the art.
- Exemplary "recombinant expression systems” include: bacterial expression systems such as E. coli expression systems (reviewed in Baneyx, Curr. Opin. Biotechnol. 10: 411-421, 1999; eg. see also Gene expression in recombinant microorganisms, Smith (Ed.), Marcel Dekker, Inc.
- the present invention provides an “isolated” or “substantially purified” polysaccharide transferase.
- isolated refers to the polysaccharide transferase being removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
- substantially purified polysaccharide transferases may be either pure or part of a mixture. When part of a mixture, the polysaccharide transferase activity is generally higher in the mixture than any other enzymatic activity in the mixture.
- the isolated or substantially purified polysaccharide transferase of the present invention is purified to a substantially homogenous form.
- polysaccharide transferases of the present invention may be isolated or purified from any source using any suitable method.
- the present invention provides a method for isolating or substantially purifying a polysaccharide transferase from a sample, the method comprising the steps of:
- sample contemplated for use in the method of the second aspect of the invention may be any sample comprising a polysaccharide transferase protein.
- sample is of biological origin, and in another embodiment, the sample comprises one or more cells, or a homogenate thereof. In yet another embodiment, the sample comprises any of one or more plant cells, a homogenate thereof, or one or more plant cell walls.
- the salt fractionation step comprises an ammonium sulphate fractionation step.
- the ammonium sulphate fractionation step comprises fractionation using ammonium sulphate at a concentration of at least 50%, a concentration of at least 65%, a concentration of at least 80% or a concentration of about 90%.
- the ion-exchange chromatography step comprises an anion exchange chromatography step.
- the anion exchange chromatography step comprises anion exchange using a stationary phase comprising Sepharose Q anion exchange resin.
- the anion exchange step is performed at a mildly acidic pH, at a pH of between 6.0 and 7.0, at a pH of between 6.5 and 7.0 or at a pH of about 6.8.
- the hydrophobic interaction chromatography step comprises hydrophobic interaction chromatography using a stationary phase comprising non-polar groups, such as phenyl non-polar groups.
- the stationary phase comprises phenyl Sepharose.
- the hydrophobic interaction chromatography step is performed at a mildly acidic pH, at a pH of between 5.0 and 7.0, at a pH of between 5.5 and 6.5 or at a pH of about 6.0.
- the chromatofocussing step comprises using a PBE-94 ion exchange resin. In another embodiment, the chromatofocussing step comprises elution in a pH range of about 5.0 to about 8.3.
- the size exclusion chromatography step comprises using a porous polyacrylamide resin. In another embodiment, the size exclusion chromatography step comprises a fractionation range of about 3000 to about 60000 Daltons. In yet another embodiment, the size exclusion chromatography step comprises using a Bio-Gel P-60 stationary phase. In a further embodiment, the size exclusion chromatography step is performed at substantially neutral pH.
- the second aspect of the invention provides a method for isolating or substantially purifying a polysaccharide transferase to a homogenous form.
- the present invention provides a method for forming a covalent bond between a donor polysaccharide and an acceptor polysaccharide, the method comprising contacting said donor polysaccharide and said acceptor polysaccharide with either a polysaccharide transferase which is capable of catalyzing a covalent bond between said donor polysaccharide and said acceptor polysaccharide, or a functionally active fragment or variant of said polysaccharide transferase.
- the donor and/or acceptor polysaccharides used in accordance with the method of the third aspect of the invention are as described above in connection with the first aspect of the invention.
- polysaccharide transferase used in accordance with the method of the third aspect of the invention is as described above in connection with the first aspect of the invention.
- the method of the third aspect of the invention may be applied to the formation of a covalent bond between a donor polysaccharide and an acceptor polysaccharide under any suitable conditions.
- the method of the third aspect of the invention is adapted to forming the covalent bond in vitro.
- polysaccharide transferases of the present invention have broad-ranging utility.
- labelled polysaccharides either radioactively labelled or labelled by introducing fluorescent tags onto their reducing termini
- fluorescent tags could be introduced onto different types of polysaccharides by various types of polysaccharide transferases described herein.
- the present invention provides an isolated polysaccharide of the structure:
- a - B wherein A and B are polysaccharides which are covalently linked to each other.
- Polysaccharide A and polysaccharide B may be any polysaccharides. However, in one embodiment, at least one of polysaccharide A and polysaccharide B is a polysaccharide other than a xyloglucan. Therefore, in this embodiment, the polysaccharide of the present invention comprises a xyloglucan covalently bonded to another polysaccharide of a different type and/or a polysaccharide comprising two non-xyloglucan polysaccharides (which may be the same or of a different type) covalently bonded to each other.
- Polysaccharides A and B in the polysaccharide of the present invention may be of any size. However, in one embodiment polysaccharide A comprises a backbone of at least 10 monomer units and polysaccharide B comprises a backbone of at least 4 monomer units. In another embodiment, polysaccharide A comprises a backbone of at least 4 monomer units and polysaccharide B comprises a backbone of at least 10 monomer units.
- polysaccharide A comprises: (i) a xyloglucan; or (ii) a glucose polymer (other than a xyloglucan) which comprises at least one [3(1-4) linkage between two glucose monomers and polysaccharide B comprises: (i) a xyloglucan; or
- a glucose polymer (other than a xyloglucan) which comprises at least one [3(1-4) linkage between two glucose monomers
- polysaccharide A comprises a glucose polymer (other than a xyloglucan) which comprises at least one (3(1-4) linkage betw ⁇ een two glucose monomers and polysaccharide B comprises a xyloglucan, or polysaccharide A comprises a xyloglucan and polysaccharide B comprises a glucose polymer (other than a xyloglucan) which comprises at least one (3(1-4) linkage between two glucose monomers.
- the glucose polymer comprises (3(1-4) linkages between all of the glucose monomers in the backbone of the polysaccharide and in one embodiment, the glucose polymer comprises cellulose, or an oligomer or derivative thereof.
- the glucose polymer may comprises at least one (3(1-3) linkage between two glucose monomers in addition to at least one (3(1-4) linkage between two glucose monomers and in another embodiment, the glucose polymer comprises a l,3;l,4-(3-D- glucan or an oligomer or derivative thereof.
- polysaccharides A and/or B being other polysaccharides, particularly plant cell wall polysaccharides such as, for example, arabinoxylans, pectins, mannans and the like.
- the polysaccharide of the fourth aspect of the invention is produced according to the method of the third aspect of the invention.
- the present invention also provides a method for modulating the extent of covalent bonding between a donor polysaccharide and an acceptor polysaccharide in a cell; the method comprising modulating the level or activity of either a polysaccharide transferase which is capable of catalyzing a covalent bond between said donor polysaccharide and said acceptor polysaccharide, or a functionally active fragment or variant of said polysaccharide transferase, in said cell.
- the donor and/or acceptor polysaccharides used in accordance with the method of the firth aspect of the invention are as described above in connection with the first aspect of the invention.
- polysaccharide transferase used in accordance with the method of the fifth aspect of the present invention is as described above with regard to the first aspect of the invention.
- the method of the fifth aspect of the present invention is adapted to modulating the extent of covalent bonding between a donor polysaccharide and an acceptor polysaccharide in a plant cell. In another embodiment, the method of the fifth aspect of the present invention is adapted to modulating the extent of covalent bonding between a donor polysaccharide and an acceptor polysaccharide in a plant cell wall.
- the chemistry and enzymology of interactions between polysaccharides and other components in the wall are crucial determinants of overall plant strength, wall porosity, and susceptibility of the wall to enzymatic degradation.
- decreased polysaccharide cross-linking in plant cell walls could be induced late in the development or during senescence of cereal straws, corn stover, sugarcane bagasse, etc. to enhance the digestibility of these residues and grasses more generally in animals, and to facilitate industrial processes such as pulp and paper manufacture.
- increased polysaccharide cross-linking in plant cell walls could be used to increase stem strength and hence to reduce crop losses due to lodging.
- the method of the fifth aspect of the present invention may be applied to modulating the extent of covalent bonding between a donor polysaccharide and an acceptor polysaccharide in any plant cell or cell wall thereof, including, for example, monocotyledonous angiosperm plant cells, dicotyledonous angiosperm plant cells and gymnosperm plant cells.
- the plant cell is a monocotyledonous plant cell and in another embodiment the plant is a cereal crop plant cell or other member of the Poaceae family.
- the "modulating the extent of covalent bonding between a donor polysaccharide and an acceptor polysaccharide in a cell” should be understood to include an increase or decrease in the extent of covalent bonding between a donor polysaccharide and an acceptor polysaccharide in the cell or cell wall thereof, relative to the wild type of the cell.
- modulation of the extent of covalent bonding between a donor polysaccharide and an acceptor polysaccharide in a cell is effected by modulating the level or activity of either a polysaccharide transferase which is capable of catalyzing a covalent bond between said donor polysaccharide and said acceptor polysaccharide, or a functionally active fragment or variant of said polysaccharide transferase, in said cell.
- Modulation of the "level” of the polysaccharide transferase or functionally active fragment or variant thereof should be understood to include modulation of the level of polysaccharide transferase or functionally active fragment or variant thereof transcripts and/or polypeptides in the cell.
- Modulation of the "activity" of the polysaccharide transferase or functionally active fragment or variant thereof should be understood to include modulation of the total activity, specific activity, half-life and/or stability of the polysaccharide transferase or functionally active fragment or variant thereof in the cell.
- moduleating with regard to the level and/or activity of the polysaccharide transferase or functionally active fragment or variant thereof includes decreasing or increasing the level and/or activity of polysaccharide transferase or functionally active fragment or variant thereof in the cell.
- decreasing is intended, for example, at least a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% reduction in the level or activity of polysaccharide transferase or functionally active fragment or variant thereof in the cell.
- increasing is intended, for example, at least a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20 fold, 50-fold, 100- fold increase in the level of activity of polysaccharide transferase or functionally active fragment or variant thereof in the cell.
- Modulating also includes introducing a polysaccharide transferase or functionally active fragment or variant thereof into a cell which does not normally express the introduced enzyme, or the substantially complete inhibition of polysaccharide transferase or functionally active fragment or variant thereof activity in a cell that normally has such activity.
- the extent of covalent bonding between a donor polysaccharide and an acceptor polysaccharide in a cell is increased by increasing the level and/or activity of a polysaccharide transferase or functionally active fragment or variant thereof in the cell. In another embodiment, the extent of covalent bonding between a donor polysaccharide and an acceptor polysaccharide in a cell is decreased by decreasing the level and/or activity of a polysaccharide transferase or functionally active fragment or variant thereof in the cell.
- the method of the fifth aspect of the invention contemplates any means known in the art by which the level and/or activity of a polysaccharide transferase or functionally active fragment or variant thereof may be modulated in a cell. This includes, for example:
- the level and/or activity of a polysaccharide transferase or functionally active fragment or variant thereof is modulated by modulating the expression of a nucleic acid which encodes a polysaccharide transferase or functionally active fragment or variant thereof (referred to hereafter as 'a polysaccharide transferase-encoding nucleic acid') in the cell.
- a nucleic acid which encodes a polysaccharide transferase or functionally active fragment or variant thereof referred to hereafter as 'a polysaccharide transferase-encoding nucleic acid'
- modulating with regard to the expression of a polysaccharide transferase- encoding nucleic acid is intended decreasing or increasing the transcription and/or translation of the nucleic acid.
- decreasing is intended, for example, a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% reduction in transcription and/or translation.
- Modulating is intended, for example a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50- fold, 100-fold or greater increase in transcription and/or translation.
- Modulating also comprises introducing expression of a nucleic acid not normally found in a particular cell; or the substantially complete inhibition (eg. knockout) of expression in a cell that normally has such activity.
- the expression of a polysaccharide transferase-encoding nucleic acid is modulated by genetic modification of the cell.
- exemplary types of genetic modification include, for example:
- insertional mutagenesis of a polysaccharide transferase-encoding nucleic acid in a cell including knockout or knockdown of a polysaccharide transferase-encoding nucleic acid in a cell by homologous recombination with a knockout construct (for an example of targeted gene disruption in plants see Terada et al., Nat. Biotechnol. 20: 1030-1034, 2002);
- the present invention also contemplates the downregulation of a polysaccharide transferase- encoding nucleic acid in a cell via the use of synthetic oligonucleotides, for example, siRNAs or microRNAs directed against a polysaccharide transferase-encoding nucleic acid (for examples of synthetic siRNA mediated silencing see Caplen et al, Proc. Natl. Acad. Sci. USA 98: 9742-9747, 2001; Elbashir et al, Genes Dev. 15: 188-200, 2001; Elbashir et al, Nature 411: 494- 498, 2001; Elbashir et al, EMBO J. 20: 6877-6888, 2001; and Elbashir et al, Methods 26: 199-213, 2002).
- synthetic siRNA mediated silencing see Caplen et al, Proc. Natl. Acad. Sci. USA 98: 9742-9747, 2001; Elbashir
- an introduced nucleic acid may also comprise a nucleotide sequence which is not directly related to a polysaccharide transferase-encoding nucleic acid but, nonetheless, may directly or indirectly modulate the expression of a polysaccharide transferase-encoding nucleic acid in a cell.
- examples include nucleic acid molecules that encode transcription factors or other proteins which promote or suppress the expression of an endogenous polysaccharide transferase-encoding nucleic acid molecule in a cell; and other non-translated RNAs which directly or indirectly promote or suppress endogenous polysaccharide transferase expression and the like.
- the method of the fifth aspect of the invention may involve transformation of a plant.
- Plants may be transformed using any method known in the art that is appropriate for the particular plant species. Common methods include Agrobacterium-mediated transformation, microprojectile bombardment based transformation methods and direct DNA uptake based methods. Roa-Rodriguez et ⁇ l. ⁇ Agrobacte ⁇ um-medi ⁇ ted transformation of plants, 3 rd Ed. CAMBIA Intellectual Property Resource, Canberra, Australia, 2003) review a wide array of suitable Agrobacterium-mediated plant transformation methods for a wide range of plant species. Other bacterial-mediated plant transformation methods may also be utilized, for example, see Broothaerts et ⁇ l.
- Microprojectile bombardment may also be used to transform plant tissue and methods for the transformation of plants, particularly cereal plants, and such methods are reviewed by Casas et al. ⁇ Plant Breeding Rev. 13: 235-264, 1995).
- Direct DNA uptake transformation protocols such as protoplast transformation and electroporation are described in detail in Galbraith et al. (eds.), Methods in Cell Biology Vol. 50, Academic Press, San Diego, 1995).
- a range of other transformation protocols may also be used. These include infiltration, electroporation of cells and tissues, electroporation of embryos, microinjection, pollen-tube pathway, silicon carbide- and liposome mediated transformation.
- the introduced nucleic acid may be operably connected to one or more transcriptional control sequences, such as a promoter.
- a promoter may regulate the expression of an operably connected nucleotide sequence constitutively, or differentially, with respect to the cell, tissue, organ or developmental stage at which expression occurs, in response to external stimuli such as physiological stresses, pathogens, or metal ions, amongst others, or in response to one or more transcriptional activators.
- the promoter used in accordance with the methods of the present invention may include, for example, a constitutive promoter, an inducible promoter, a tissue- specific promoter or an activatable promoter.
- a promoter or other transcriptional control sequence derived from a native polysaccharide transferase-encoding nucleic acid may be used.
- the present invention also contemplates the use of any promoter which is active in a plant. Accordingly, plant-active constitutive, inducible, tissue-specific or activatable promoters may be used.
- Plant constitutive promoters typically direct expression in nearly all tissues of a plant and are largely independent of environmental and developmental factors.
- Examples of constitutive promoters that may be used in accordance with the present invention include plant viral derived promoters such as the Cauliflower Mosaic Virus 35S and 195 (CaMV 35S and CaMV 19S) promoters; bacterial plant pathogen derived promoters such as opine promoters derived from Agrobacterium spp., eg.
- rbcS rubisco small subunit gene
- Pubi plant ubiquitin promoter
- Fact rice actin promoter
- “Inducible” promoters include, but are not limited to, chemically inducible promoters and physically inducible promoters.
- Chemically inducible promoters include promoters which have activity that is regulated by chemical compounds such as alcohols, antibiotics, steroids, metal ions or other compounds. Examples of chemically inducible promoters include: alcohol regulated promoters (eg. see European Patent 637 339); tetracycline regulated promoters (eg. see US Patent 5,851,796 and US Patent 5,464,758); steroid responsive promoters such as glucocorticoid receptor promoters (eg. see US Patent 5,512,483), estrogen receptor promoters (eg.
- the inducible promoter may also be a physically regulated promoter which is regulated by non-chemical environmental factors such as temperature (both heat and cold), light and the like.
- physically regulated promoters include heat shock promoters (eg. see US Patent 5,447858, Australian Patent 732872, Canadian Patent Application 1324097); cold inducible promoters (eg. see US Patent 6,479,260, US Patent 6,184,443 and US Patent 5,847,102); light inducible promoters (eg. see US Patent 5,750,385 and Canadian Patent 132 1563); light repressible promoters (eg. see New Zealand Patent 508103 and US Patent 5,639,952).
- heat shock promoters eg. see US Patent 5,447858, Australian Patent 732872, Canadian Patent Application 1324097
- cold inducible promoters eg. see US Patent 6,479,260, US Patent 6,184,443 and US Patent 5,847,102
- light inducible promoters eg. see US Patent
- tissue specific promoters include promoters which are preferentially or specifically expressed in one or more specific cells, tissues or organs in an organism and/or one or more developmental stages of the organism. It should be understood that a tissue specific promoter may be either constitutive or inducible.
- plant tissue specific promoters include: root specific promoters such as those described in US Patent Application 2001047525; fruit specific promoters including ovary specific and receptacle tissue specific promoters such as those described in European Patent 316 441, US Patent 5,753,475 and European Patent Application 973 922; and seed specific promoters such as those described in Australian Patent 612326 and European Patent application 0 781 849 and Australian Patent 746032.
- the promoter may also be a promoter that is activatable by one or more transcriptional activators, referred to herein as an "activatable promoter".
- the activatable promoter may comprise a minimal promoter operably connected to an Upstream Activating Sequence (UAS), which comprises, inter alia, a DNA binding site for one or more transcriptional activators.
- UAS Upstream Activating Sequence
- the term "minimal promoter” should be understood to include any promoter that incorporates at least an RNA polymerase binding site and, optionally a TATA box and transcription initiation site and/or one or more CAAT boxes.
- the minimal promoter may be derived from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter.
- the CaMV 35S derived minimal promoter may comprise, for example, a sequence that substantially corresponds to positions -90 to +1 (the transcription initiation site) of the CaMV 35S promoter (also referred to as a -90 CaMV 35S minimal promoter), -60 to +1 of the CaMV 35S promoter (also referred to as a -60 CaMV 35S minimal promoter) or -45 to +1 of the CaMV 35S promoter (also referred to as a -45 CaMV 35S minimal promoter).
- the activatable promoter may comprise a minimal promoter fused to an Upstream Activating Sequence (UAS).
- UAS Upstream Activating Sequence
- the UAS may be any sequence that can bind a transcriptional activator to activate the minimal promoter.
- Exemplary transcriptional activators include, for example: yeast derived transcription activators such as Gal4, Pdrl, Gcn4 and Acel; the viral derived transcription activator, VP16; Hapl (Hach et al, J Biol Chem 278: 248-254, 2000); Gafl (Hoe et al, Gene 215(2): 319-328, 1998); E2F (Albani et al, J Biol Chem 275: 19258-19267, 2000); HAND2 (Dai and Cserjesi, / Biol Chem 277: 12604-12612, 2002); NRF- 1 and EWG (Herzig et al, ] Cell Sci 113: 4263-4273, 2000); P/C
- the UAS comprises a nucleotide sequence that is able to bind to at least the DNA-binding domain of the GAL4 transcriptional activator.
- UAS sequences which can bind transcriptional activators that comprise at least the GAL4 DNA binding domain, are referred to herein as UASG.
- the UASG comprises the sequence 5'- CGGAGTACTGTCCTCCGAG-3' or a functional homolog thereof.
- a "functional homolog" of the UASG sequence should be understood to refer to any nucleotide sequence which can bind at least the GAL4 DNA binding domain and which may comprise a nucleotide sequence having at least 50% identity, at least 65% identity, at least 80% identity or at least 90% identity with the UASG nucleotide sequence.
- the UAS sequence in the activatable promoter may comprise a plurality of tandem repeats of a DNA binding domain target sequence.
- UASG comprises four tandem repeats of the DNA binding domain target sequence.
- the term "plurality" as used herein with regard to the number of tandem repeats of a DNA binding domain target sequence should be understood to include, for example, at least 2 tandem repeats, at least 3 tandem repeats or at least 4 tandem repeats.
- nucleic acid which encodes a polysaccharide transferase or functionally active fragment or variant thereof also referred to as "a polysaccharide transferase-encoding nucleic acid”.
- Nucleic acid sequences which encode a particular polysaccharide transferase will be readily determined by one of skill in the art.
- the amino acid sequence of a particular polysaccharide transferase or functionally active fragment or variant thereof may be used to identify a suitable encoding nucleic acid from nucleotide sequence data such as, for example, genomic nucleotide sequence data and/or Expressed Sequence Tag (EST) data.
- EST Expressed Sequence Tag
- nucleic acid which encodes a polysaccharide transferase or functionally active fragment or variant thereof may be identified and/or isolated from an organism.
- an amino acid sequence from a polysaccharide transferase or functionally active fragment or variant thereof may be used to determine a corresponding nucleotide sequence which may then be chemically synthesized.
- the codon usage in the synthetic nucleic acid may be adapted to the particular cell type into which the nucleic acid is to be introduced.
- the fifth aspect of the invention contemplates modulating the extent of covalent bonding between a donor polysaccharide and an acceptor polysaccharide in a cell by modulating the expression of a nucleic acid which encodes a polysaccharide transferase comprising any of:
- the nucleic acid which encodes a polysaccharide transferase comprising the amino acid sequence set forth in SEQ ID NO: 1 comprises the nucleotide sequence set forth in SEQ ID NO: 4.
- the nucleic acid which encodes a polysaccharide transferase comprising the amino acid sequence set forth in SEQ ID NO: 2 comprises the nucleotide sequence set forth in SEQ ID NO: 5.
- nucleic acid which encodes a polysaccharide transferase comprising the amino acid sequence set forth in SEQ ID NO: 3, comprises the nucleotide sequence set forth in SEQ ID NO: 6.
- the isolated or substantially purified polysaccharide transferases of the present invention may also be useful, for example, in the generation of antibodies that bind to the polysaccharide transferase polypeptides.
- the present invention provides an antibody or an epitope binding fragment thereof, raised against a polysaccharide transferase polypeptide as defined in the first aspect of the invention.
- antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds an antigen.
- the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
- the antibodies of the present invention may include, for example: polyclonal, monoclonal, multispecific, chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library and epitope-binding fragments of any of the above.
- antibody should also be understood to encompass derivatives that are modified, eg. by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to a polysaccharide transferase polypeptide or an epitope thereof.
- the antibody derivatives include antibodies that have been modified, eg., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
- any of numerous chemical modifications may also be made using known techniques. These include specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.
- the derivative may contain one or more non-classical amino acids.
- the antibodies of the present invention may be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.
- a heterologous epitope such as a heterologous polypeptide or solid support material.
- the antibodies of the present invention may act as agonists or antagonists of a polysaccharide transferase.
- the antibodies of the present invention may be used, for example, to purify, detect, and target the polysaccharide transferases of the present invention, including both in vitro and in vivo diagnostic methods.
- the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of polysaccharide transferase polypeptide in biological samples. See, e.g., Harlow et ah, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
- Antibodies may be generated using methods known in the art, such as in vivo immunization, in vitro immunization, and phage display methods. For example, see Bittle et al. (J. Gen. Virol. 66: 2347-2354, 1985).
- animals may be immunized with free peptide; however, anti- peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
- KLH keyhole limpet hemacyanin
- peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.
- polyclonal antibodies to a polysaccharide transferase polypeptide can be produced using methods known in the art.
- animals such as rabbits, rats or mice may be immunized with either free or carrier-coupled peptides.
- intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein may be used to induce the production of sera containing polyclonal antibodies specific for the antigen.
- adjuvants may also be used to increase the immunological response, depending on the host species, for example, Freund's (complete and incomplete), mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
- BCG Bacille Calmette-Guerin
- Corynebacterium parvum Such adjuvants are also well known in the art.
- booster injections may be needed, for example, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
- the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods known in the art.
- monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
- monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et ah, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988) and Hammerling et ah, in: Monoclonal Antibodies and T-CeIl Hybridomas (Elsevier, NY, 1981).
- the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
- the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
- Antibody fragments which bind to a polysaccharide transferase of the present invention may also be generated by known techniques.
- Fab and F(ab')2 fragments may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
- F(ab')2 fragments contain the variable region, the light chain constant region and the CHl domain of the heavy chain.
- the antibodies of the present invention can also be generated using various phage display methods known in the art.
- phage display methods that can be used to make the antibodies of the present invention include those disclosed by Brinkman et a (J. Immunol. Methods 182: 41-50, 1995), Ames et al. (J. Immunol. Methods 184: 177-186, 1995), Kettleborough et al. (Eur. J. Immunol. 24: 952-958, 1994), Persic et al. (Gene 187: 9-18, 1997), Burton et al.
- antibody coding regions from the phage can be isolated and used to generate whole antibodies or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria.
- techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al. (BioTechniques 12(6): 864-869, 1992); and Sawai et al. (AJRI 34:26-34, 1995); and Better et al. (Science 240: 1041-1043, 1988).
- the present invention also contemplates an aptamer that binds to the polysaccharide transferase of the first aspect of the invention.
- Nucleic acid aptamers that bind to a particular protein may be produced using methods known in the art. For example, in-vitro selection methods (eg. see Ellington and Szostak, Nature 346(6287): 818-22, 1990) and SELEX methods (eg. see Tuerk and Gold, Science 249(4968): 505-510, 1990) may be used. Further details relating to the production and selection of aptamers may also be found in the review of Osborne and Ellington (Chem Rev 97(2): 349-370, 1997).
- FIGURE 1 SDS-PAGE, IEF and a primary sequence of the purified HvXET5.
- A A monodisperse HvXET5 (2.4 ⁇ g of protein) after 5 purification steps, detected with a colloidal Coomassie Brilliant Blue G-250.
- B IEF of the purified HvXET5; the activity of the enzyme was detected by zymogram detection with TXG and XGO-SR. The molecular mass standards, pH boundaries of the IEF gel, and the pi point of HvXET5 are indicated.
- C A primary sequence of HvXET5 (TrEMBL accession number P93668). The first 30 amino acid residues (underlined) were determined by Edman degradation.
- FIGURE 2 pH and Temperature dependences of relative activities of HvXET5.
- A), pH and (B), temperature dependences were determined radiometrically after 15 min incubation at the indicated pH or temperatures values.
- FIGURE 3 Rate of synthesis of HECXGO-SR conjugate and transfer of XXXGoI onto
- A The synthesis of high molecular mass HEQXGO-SR from unlabelled HEC and low molecular mass, fluorescent XGO-SR was monitored by HPLC for up to 24 h. Progressive formation of high molecular mass HEQXGO-SR indicates that HEC and XGO-SR molecules are linked together by the enzymic action of HvXET5. The grey shaded area indicates the material that was pooled for the reverse reaction shown in panel B.
- B In the reverse reaction, the progressive transfer of fluorescent label from the HEQXGO- SR conjugate (the grey shaded area in panel A) to a non-fluorescent XXXGoI acceptor was followed by HPLC up to 16 h.
- FIGURE 4 Characterization of HEQXXXG-SR conjugate synthesized by HvXET5.
- B Purification by HPLC of three major oligosaccharide fractions from the degradation products of HEOXXXG-SR conjugate.
- FIGURE 5 Characterization of (l,3;l,4)-[3-D-glucan:XXXG-SR conjugate synthesized by
- HvXET5. HPLC chromatogram of high molecular mass, fluorescent (l,3;l,4)-(3-D- glucan:XXXG-SR synthesized after 144 h from unlabelled (l,3;l,4)-[3-D-glucan and XXXG-SR by HvXET5. The material eluting between 8-12 min was pooled for hydrolysis by B. subtilis (l,3;l,4)-[3-D-glucan endohydrolase. Fluorescence and ELSD profiles have non-linear detection responses.
- (B) Purification by HPLC of five major oligosaccharides released from (l,3;l,4)-[3-D-glucan:XXXG-SR by (l,3;l,4)-[3-D-glucan endohydrolase.
- (C) MALDI-TOF mass spectra of (l,3;l,4)-[3-D-glucan-derived oligosaccharide conjugates 1-5 from panel B showed the presence of XXXG-SR with 2-6 covalently attached glucosyl residues at its non-reducing termini. The position of XXXG-SR is marked by an arrow. The positions of molecular mass standards of (l,3;l,4)-[3-D-glucan (40 kDa), polyethylene glycol 1450 and glucose (180) are indicated.
- FIGURE 6 A barley xyloglucan xyloglucosyl transferase HvXET5 covalently links xyloglucan, cellulose and (l,3;l,4)-[3-D-glucan.
- the polymers xyloglucan, cellulose and (l,3;l,4)-[3-D-glucan are shown in blue, brown, and green.
- the distance between individual cellulosic microfibrils (d) could be altered after HvXET5 links xyloglucan and cellulosic microfibrils.
- AmpholineTM isoelectric focussing (IEF) polyacrylamide gels (pH range 3.5-9.5), molecular mass marker proteins (20-94 kDa) and dextran 500 were from GE Healthcare Biosciences (NSW, Australia), Bio-Gel P-60, Phenyl-Sepharose and pi marker proteins 4.45-9.6 were from Bio-Rad Laboratories (Hercules, CA, USA), Microcon microconcentrators were from Amicon (Beverly, MA, USA), Whatman 3MM paper was from Whatman (Brentford, UK), Miracloth (22-25 ⁇ m pore size) was from Calbiochem (San Diego, CA, USA) and ampholines were from Serva (Heidelberg, Germany).
- Lissamine rhodamine B sulfonyl chloride (sulforhodamine, SR) w ⁇ as from Acros Organics (Morris Planes, NJ, USA), the Coomassie Protein Assay Reagent was from Pierce (Rockford, Illinois, USA), EcoLume scintillation fluid was from MP Biomedicals (Irvine, CA, USA), chromatography 3MM paper was from Whatman (Brentford, Middlesex, UK) and acetonitrile was from BDH Laboratory Supplies (Poole, England).
- Barley (l,3;l,4)-(3-D-glucans (average molecular masses of 450 and 40 kDa), [3-D-galactans (from lupin and potato), lichenin, ivory nut mannan, konjac glucomannan, barley arabinoxylan, tamarind xyloglucan (TXG), rhamnogalactouronan (soybean pectic fibre), xyloglucan-derived heptasaccharide (XXXG), its reduced form XXXGoI, and (l,3;l,4)-(3-D-glucanase from Bacillus subtilis were from Megazyme (Bray, Ireland).
- Carboxymethyl cellulose (CMC) of degree of substitution 0.54 was from Imperial Chemical Industries (Dingley, Australia), arabinogalactan protein (from gum arabic) was from Aldrich Chemical Corporation (Milwaukee, WI, USA), and cello-oligosaccharides (CEO) of degree of polymerization (DP) of DP 2-6 and laminari-oligosaccharides (LAO) of DP 2-6, were from Seikagaku Corporation (Tokyo, Japan).
- Cello-heptaose and cello-octaose were prepared by acid hydrolysis from Antigum CS6 (System Bio-Industries, Paris, France).
- HEC Hydroxyethylcellulose
- Beechwood 4-O-methyl-(l,4)- ⁇ -D-glucuronoxylan was from Institute of Chemistry (Slovak Republic), a low viscosity locust-bean gum galactomannan was donated by Dr Peter Biely (Institute of Chemistry), sulfuric acid swollen cellulose of average molecular mass of 12-15 kDa and a degree of substitution approximately 0.25, was provided by Professor Bruce Stone (La Trobe University, Australia), and 1,4-[3-D- glucan endohydrolase EGII from Trichoderma reesei was kindly donated by the late Dr Marianne Hayn (University of Graz, Austria).
- the germinated grain and young seedlings were homogenised at 4 0 C in 2.0 volumes of homogenization buffer, pH 6, containing 0.1 M imidazole-HCl buffer, IM NaCl, 2 mM EDTA, 1 mM 2-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride (buffer A), in a Waring Blender for 6x1 min intervals with intermittent cooling (2 min) on ice.
- the homogenate was held for 1 h at 4 0 C to extract proteins, insoluble material was removed by centrifugation (4000 g, 60 min, 4 0 C), and the extract filtered through Miracloth.
- the extract was precipitated to 90% with solid (NH ⁇ SCk, the precipitate collected (8000 g, 45 min, 4 Q C) and resuspended in 4 litres of buffer A (without NaCl).
- the extract was stored in 0.5 litre aliquots at -20 0 C.
- the HvXET5 enzyme was purified from extracts of seven-day-old barley seedlings using Sepharose Q, Phenyl-Sepharose, chromatofocussing on PBE-94 and size-exclusion chromatography on Bio-Gel P-60, as shown in Table 2.
- Sepharose Q pH 6.8 1,254 7,771 6 100 5
- Bio-Gel P-60, pH 7.0 0.1 363 3,630 5 3,025 a As a total enzyme activity in selectively pooled fractions assayed radiometrically.
- b Recoveries are expressed as % of a total enzyme activity in a crude homogenate.
- c Purification factors are based on specific activities.
- HvXET5 The activity of HvXET5 during enzyme purification was determined radiometrically at 3O 0 C in 100 mM succinate or ammonium acetate buffers, pH 6.0, containing 5 mM calcium chloride, 0.3% (w/v) TXG and [ 3 H]-labelled xyloglucan-derived saccharide heptaitol (XXXGoI, specific radioactivity 83 MBq- ⁇ mol 1 ) (Fry, et al., Biochem. J. 282, 821-828, 1992) with approximately 30,000 dpm per reaction mixture.
- HvXET5 Protein concentration determinations during purification and characterization of HvXET5 and SDS-PAGE, were performed as described by Hrmova et al. (Biochem. J. 399, 77-90, 2006). During HvXET5 purification, protein was detected with colloidal Coomassie Brilliant Blue G- 250 in methanol for 30 h at ambient temperature (Neuhoff et ah, Electrophoresis 9, 225-262, 1988). This staining technique detects approximately 6-10 ng protein per band or 0.7 ng per mm 2 . Automated amino acid sequence analysis of HvXET5 was performed by Edman degradation as described by Hrmova et al (J. Biol. Chem. 271, 5277-5286, 1996). A single primary sequence was detected, with no secondary sequence. Clear signals for each phenylthiohydantoin amino acid residue derivative were detected.
- the crude protein extracts and purified preparations were separated on a flatbed IEF apparatus (GE Healthcare Biosciences) in 1 mm polyacrylamide gels using a pH gradient of 3.5-9.5. Pre-focused gels were run at 600 V for 30 min, followed by 800V for a further 20 min. Proteins were detected with a Coomassie Brilliant Blue dye after the gels were fixed in 20% (w/v) trichloroacetic acid. Apparent pi values were estimated by reference to marker proteins with pi values of 4.45-9.6.
- Enzyme activity in gels was detected by overlaying the separation gels with a 1.5-mm 1.3% (w/v) agarose detection gel containing 0.2 % (w/v) TXG and 5-10 ⁇ M of SR-labeled xyloglucan-derived oligosaccharides XGO-SR (XXXG-SR, XXLG- SR, XLLG-SR molar ratios were 1:1.6:1.8) (Fry, Plant J. 11, 1141-1150, 1997; Farkas et al. Plant Physiol. Biochem. 43, 431-435, 2005) in 0.1 M succinate buffer, pH 6, containing 5 mM calcium chloride.
- HvXET5 The effect of pH on the activity of HvXET5 was determined by incubating 1 nM HvXET5 at 30 0 C for 60 min in 50 mM citric acid/100 mM sodium dihydrophosphate (Mcllvaine) buffers, pH 4.0-8.5 in the presence of 0.02 % (w/v) BSA. A comparison of succinate, ammonium acetate or sodium phosphate buffers, each at 50-200 mM, indicated that HvXET5 activity was unaffected by the ionic strength of these buffers. The thermal stability of HvXET5 was determined after 15 min incubation at 0-70 0 C.
- the freeze/thaw stability of HvXET5 at 1 nM concentration was determined after three cycles of freezing (-80 0 C) and thawing (4 0 C), each of 3 min duration. Activity was subsequently measured at 30 0 C in 100 mM ammonium acetate buffer, pH 6, containing 5 mM calcium chloride, without and with the addition of 10% (v/v) glycerol. Enzyme activity was determined radiometrically as specified above, and expressed as % activity relative to maximal activity. Assays were performed in triplicate and standard errors of 8-14% were observed.
- EXAMPLE 8 Methods - Substrate Specificities
- the incubation mixtures contained 1.2% (w/v) soluble polysaccharides as donor substrates, and as acceptor substrates either 23-27 ⁇ M XGO-SR, SR-labelled cello-oligosaccharides (CEO-SR) (DP 2-8) or SR-labelled laminari-oligosaccharides (LAO-SR) (DP 2-6) (Farkas et al. Plant Physiol. Biochem. 43, 431-435, 2005) in 100 mM ammonium acetate buffer, pH 6, containing 5 mM calcium chloride and 0.5-1 nM purified HvXET5.
- the molar ratios of individual oligosaccharides in the two oligosaccharide-SR mixtures were 1:0.78:0.62:0.41:0.16:0.03:0.013 for C2-SR to C8-SR, and 1.0:1.7:0.87:0.41:0.2 for L2-SR to L6-SR. All incubations proceeded for 18h at 30 0 C. Enzymes inactivated by boiling for 3 min served as controls. The efficiency of transfer of selected polysaccharides onto fluorescent acceptors was determined by size exclusion HPLC.
- SR-labelled oligosaccharides and polysaccharides were detected following HPLC by fluorescence detection (excitation 568 nm and emission 584 nm) or by evaporative light scattering detection (ELSD) at 568 nm, and by MALDI TOF mass spectrometry analyses. Enzyme activities were determined by integrating peak areas, after subtracting background level obtained from boiled enzyme control reactions. Relative activities of HvXET5 are expressed as % of activity observed with TXG as a donor substrate and XGO-SR as an acceptor. In all instances assays were performed in duplicate with standard errors of 8-12%.
- Detection limits of the fluorescence assays and ELSD were better than 0.1 pmol XGO-SR or CEO-SR, or MO 5 % of the amount of XGO-SR or CEO-SR acceptors used in standard enzyme reactions, with a standard error of 6%.
- the efficiency of transfer of selected polysaccharides onto [ 3 H]-XXXGoI was further evaluated by ascending chromatography in 60% (v/v) ethanol on Whatman chromatography 3MM paper strips, and radioactivity in paper strips was determined by liquid scintillation counting as specified above.
- XXXG-SR was prepared as described by Fry (Plant J. 11, 1141-1150, 1997) and Farkas et al. (Plant Physiol Biochem. 43, 431-435, 2005) and purified on a reversed phase column with a water/acetonitrile gradient.
- HEC and (l,3;l,4)-[3-D-glucan fractions collected after separations by size-exclusion chromatography) at 0.8% (w/v) concentrations were dissolved in 100 mM ammonium acetate buffer, pH 6, containing 5 mM calcium chloride, and incubated with 8 ⁇ M XXXG-SR, in the presence of 4 nM purified HvXET5 at 3O 0 C with shaking. Every 16 h (HEC) or 24 h [(l,3;l,4)-[3-D-glucan] a fresh HvXET5 enzyme (1/4 of the original amount) was added. The reactions w ⁇ ere terminated by boiling after 48 h and 144 h, for HEC and (l,3;l,4)-[3-D-glucan, respectively.
- 3-D-glucan:XXXG-SR was dissolved in 50 mM ammonium acetate buffer, pH 5 and incubated with 10 nM of the purified 1,4-[3-D- glucan endohydrolase from Trichoderma reesei (family GH5 glycoside hydrolase; Suurnakki, et al, Cellulose 7, 189-209, 2000) or 1 nM of the purified (on Bio-Gel P-60) (l,3;l,4)-[3-D- glucanase (a family GH16 glycoside hydrolase; Hahn et al, Eur. ]. Biochem.
- Native polysaccharides were fractionated by size-exclusion chromatography on either a P3000 or P4000 PolySep GFC columns (particle size not specified, 300 x 7.8mm) (Phenomenex, Torrance, CA, USA) with water or 100 mM ammonium acetate as eluant at a flow rate of 0.8 ml/min.
- a model 1090 liquid chromatograph with diode-array detector controlled by ChemStation software (Agilent Technologies, Palo Alto, CA, USA), and fluorescence (model RF-IOAXL, Shimadzu, Kyoto, Japan) and ELSD (model 800, Alltech Associates Inc., Deerfield, IL, USA) connected in series to the 1090 DAD, w ⁇ ere used for analyses of enzymatic reactions.
- the eluant flow from the fluorescence detector to the ELSD was split in the ratio 5 (to collect) to 1 (ELSD).
- the ELSD was operated at 4O 0 C and a nitrogen pressure of 1.5 bar and the column temperature was 21 0 C.
- the barley HvXET5 enzyme was purified approximately 3,000-fold from extracts of 7-day- old barley seedlings, using ammonium sulphate precipitation, ion exchange and hydrophobic chromatography, chromatofocussing and size exclusion chromatography (see Table 1).
- the numbering of the barley XET isoenzymes is based upon the gene nomenclature proposed by Strohmeier et al. (Protein Sci. 13, 3200-3213, 2004).
- the specific activity of the purified HvXET5 was 3,630 pkat-mg "1 .
- the final purified enzyme preparation showed a single band of 34 kDa on SDS gels at high protein loadings ( Figure IA), with no other protein species present. These data indicated that contaminating proteins accounted for less than 6 ng, or 0.25% of the protein used for SDS-PAGE analysis. Furthermore, a single protein and activity band of pi 7.6 was detected on an isoelectric focusing gel, and on a zymogram detection gel containing TXG and SR- labelled xyloglucan oligosaccharides XGO-SR ( Figure IB).
- SEQ ID NO: 1 A single sequence (SEQ ID NO: 1) was detected during NH2-terminal amino acid sequence analysis of the purified HvXET5 enzyme, where a 97% yield of a phenylthiohydantoin-alanine in the 1 st cycle, normalised per mole of the total protein of HvXET5, was obtained.
- HvXET ⁇ SEQ ID NO: 2
- the pH optimum of the purified HvXET5 was 6.0 and the temperature optimum varied between 28 0 C and 30°C ( Figure 2).
- the enzyme also operated efficiently at O 0 C, where it showed approximately 40% of the activity observed at 3O 0 C.
- the purified enzyme retained its activity for at least a year when stored at -20 0 C, and did not lose activity after several freeze-thaw cycles.
- the addition of 10% (v/v) glycerol had no affect on HvXET activity after several freeze- thawing cycles.
- Xyloglucan oligosaccharides XGO-SR
- CEO-SR cello-oligosaccharides fluorescently labelled with sulforhodamine (SR) were used as acceptor substrates for the purified HvXET5.
- Transferase activity was observed when tamarind xyloglucan (TXG), hydroxyethylcellulose (HEC), sulfuric acid-swollen cellulose and barley (l,3;l,4)-[3-D-glucan were used as donor polysaccharides, as shown in Table 3.
- FIG 3B the transfer reaction by HvXET5 is shown with the high molecular mass product of the reaction presented in Figure 3A, whereby the fluorescent component XGO-SR of the high molecular mass HEQXGO-SR material from Figure 3A (shaded fractions) was removed from the reducing end of the polysaccharide and replaced with the non-fluorescent oligosaccharide XXXGoI. As this occurred, low molecular mass fluorescent oligosaccharides XGO-SR were progressively released.
- a schematic representation of the transfer reaction shown in Figures 3A and 3B is summarised in Figure 3C.
- the HvXET5 enzyme also catalyzes the transfer of TXG, celluloses and (l,3;l,4)-[3-D-glucan onto fluorescently labelled CEO-SR, albeit at low levels (see Table 2).
- the fluorescence assay technique used in this study was sufficiently sensitive to confidently measure activities that were better than 0.1 pmol XGO-SR or 1-10 5 % of the amount of XGO-SR acceptor used in standard enzyme reactions.
- HvXET5 The capacity of HvXET5 to form covalent linkages between xyloglucan fragments and either cellulose or (l,3;l,4)-[3-D-glucan was first show ⁇ n with the oligosaccharide mixture XGO-SR and later confirmed with the single xyloglucan-derived heptasaccharide XXXG-SR ( Figures 4-5). Similarly, formation of covalent linkages by HvXET5 between xyloglucan fragments and cellulose or (l,3;l,4)-(3-D-glucan was confirmed independently using radiometric analysis with [ 3 H]-labelled xyloglucan-derived heptaitol and paper-chromatography.
- HEQXXXG-SR conjugate generated by incubation of the HvXET5 with XXXG-SR and non- fluorescent HEC ( Figure 4A) was partially hydrolysed with a highly purified (l,4)-(3-D- glucan endohydrolase from Trichoderma reesei that contained no [3-D-glucosidase or other contaminating activities.
- Three major fluorescent oligosaccharide fractions were released from the HEC:XXXG-SR ( Figures 4A and 4B) during the partial hydrolysis with the (l,4)-(3-D- glucan endohydrolase.
- Xyloglucans consist of a backbone of (l,4)-(3-D-glucan substituted with xylosyl, galactosyl and fucosyl residues.
- the molecular sizes of xyloglucans can be altered after their deposition into the cell wall and this process is likely to be mediated by a class of enzymes broadly known as xyloglucan endotransglycosylases/hydrolases (XTHs).
- enzymes within this group can have xyloglucan endotransglycosylase (XET) activity or both xyloglucan endotransglycosylase and xyloglucan endohydrolase (XEH) activities.
- the XETs are abundant in the apoplastic space, they cleave the (l,4)-(3-D-glucan backbone of xyloglucans and, in the case of xyloglucan endotransglycosylases (XETs), transfer the non-reducing fragment of the original substrate that remains bound to the enzyme directly onto the non- reducing terminus of another xyloglucan chain.
- the xyloglucan molecule that is cleaved by the enzyme initially is referred to as the donor substrate, while the xyloglucan chain to which the product of hydrolysis is transferred is known as the acceptor substrate.
- the transglycosylation activity of XETs can theoretically result in the disproportionation of xyloglucan molecules, such that some will increase in molecular mass while others will decrease in molecular mass.
- Sequences encoding XTHs are surprisingly abundant in barley EST databases, given the relatively low levels of xyloglucans in walls of most barley tissues. There are at least 22 XTH genes in barley, about 30 in rice, about 40 in Populus trichocarpa and about 33 in Arabidopsis. In an attempt to reconcile the relatively low abundance of xyloglucans in cell walls of barley against the large number of XTH genes and their high expression levels in many tissues of barley, it was contemplated that some of the XTHs might be active on the more abundant matrix phase polysaccharides of cell walls in barley, namely the arabinoxylans and the (l,3;l,4)-[3-D-glucans.
- molecular modelling established a potential structural connection between XTHs and (l,3;l,4)-[3-D-glucan endohydrolases, and with certain (l,4)-(3-D-xylan endohydrolases.
- the models were subsequently supported by the published 3D structure of the Populus tremula x tremuloides XET.
- the plant XTHs and microbial (l,3;l,4)-(3-D-glucan endohydrolases are all classified in the family GH16 group of glycoside hydrolases, although a small number of microbial XEHs are also classified within families GH5, GH12, GH44 and GH74.
- the polysaccharides are linked from reducing to non-reducing ends of donor and acceptor substrates, respectively, rather than by cross-linking of the type observed between arabinoxylan chains through esterified hydroxycinnamic acids or between pectic polysaccharides through borate.
- the HvXET5 activity represents a non-Leloir type of biosynthetic reaction, insofar as the energy required for the formation of the new glycosidic linkage is provided from an existing glycosidic linkage rather than from a sugar nucleotide activated donor.
- the data shown in Figure 3A is particularly important with respect to the action pattern of the barley XET.
- the presence of fluorescent material of intermediate molecular mass, that is with a molecular mass between that of the starting HEC and the fluorescent acceptor substrate XGO-SR indicates that the enzyme acts in an essentially stochastic manner.
- the substrate specificity of XET enzymes which involves cleaving a (l,4)-[3-D-glucosyl linkage in the donor substrate before transfer to the non-reducing end of the acceptor substrate, would suggest that the HvXET5 re-forms a (l,4)-[3-linkage between the reducing end glucosyl residue of the donor polysaccharide, whether that be the HEC or the barley (l,3;l,4)-[3-D-glucan, and the non-reducing end of the XXXG-SR acceptor substrate. It is considered unlikely that the polymeric donor molecules would be attached to the xylosyl residues of the XXXG-SR acceptor substrate.
- the rate of the reaction catalyzed by the HvXET5 enzyme described here with HEC is comparable with that on TXG (see Table 2).
- Values for the K m and kcat constants with TXG were 3 mg-rnl 1 and 1-1O 7 s 1 , respectively, and for the acceptor substrate XXXGoI the values of Km and kcat were 69-10 6 M and 1.5-10 7 s 1 , respectively.
- the rate of the reaction with (l,3;l,4)-[3-D-glucan is relatively slow (see Table 2), but it would be anticipated that contact between a large molecular mass donor-enzyme complex and the non-reducing terminus of the acceptor substrate might not occur quickly.
- 3-D-glucans and xyloglucans in plant cell walls could be linked by XTH enzymes (such as HvXET5) to create a very large, continuous molecular network within the wall and would significantly alter the strength of walls, their porosity and flexibility (see Figure 6).
- XTH enzymes such as HvXET5
- Potential accessibility and diffusion limitations in the cell wall environment could greatly reduce the catalytic rates in muro.
- the cell might compensate for this through the synthesis of relatively large amounts of stable enzyme and this would be consistent not only with the high levels of XET mRNA transcripts that are found in plant cells, but also with the long term stability of the HvXET5 observed here.
- GPI-anchored transferase enzymes some of which are members of family GH16, might also be involved in linking different polysaccharides such as [3-D- glucans and chitin in the wall.
- pectic polysaccharides might be covalently linked with xyloglucans in plant cell walls.
- the purified HvXET5 enzyme did not link polygalacturonan or [3-D-galactans to xyloglucan, nor did the HvXET5 enzyme link arabinoxylans to xyloglucans, despite suggestions based on molecular modeling that this was a possibility.
- a polysaccharide transferase includes a single polysaccharide transferase as well as two or more polysaccharide transferase;
- a donor (or acceptor) polysaccharide includes a polysaccharide cell as well as two or more polysaccharides; and so forth.
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US12/375,237 US20100009414A1 (en) | 2006-07-27 | 2007-07-27 | Polysaccharide transferase |
EP07784690A EP2082034A4 (en) | 2006-07-27 | 2007-07-27 | Polysaccharide transferase |
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WO1999062343A1 (en) * | 1998-05-29 | 1999-12-09 | Novo Nordisk A/S | Methods for using xyloglucan endotransglycosylase in baking |
GB0307294D0 (en) * | 2003-03-29 | 2003-05-07 | Univ Southampton | A novel genetic approach to improve the processability of fresh produce |
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Non-Patent Citations (13)
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DATABASE GENPEPT [online] OKAZAWA K. ET AL.: "Molecular cloning and cDNA sequencing of endoxyloglucan transferase, a novel class of glycosyl transferase that mediates molecular grafting between matrix polysaccharides in plant cell walls", Database accession no. (Q41542) * |
DATABASE GENPEPT [online] SMITH C.R. ET AL.: "The regulation of leaf elongation and xyloglucan endotransglycosylase by gibberellin in 'Himalaya' barley (Hordeum vulgare L.)", XP002597155, Database accession no. (CAA62847) * |
FEMINA A. ET AL.: "Investigation of the occurrence of pectic-xylan-xyloglucan complexes in the cell walls of cauliflower stem tissues", CARBOHYDRATE POLYMERS, vol. 39, 1999, pages 151 - 164, XP004162713 * |
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HRMOVA M. ET AL.: "A barley xyloglucan xyloglucosyl transferase covalently links xyloglucan, cellulosic substrates, and (1,3:1,4)-beta-D-glucans", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 282, no. 17, 2007, pages 12951 - 12962, XP008109611 * |
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NISHITANI K. ET AL.: "Endo-xyloglucan transferase, a novel class of glycosyltransferase that catalyses transfer of a segment of xyloglucan molecule to another xyloglucan molecule", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, no. 29, 1992, pages 21058 - 21064, XP002158858 * |
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Cited By (7)
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WO2015044209A1 (en) * | 2013-09-24 | 2015-04-02 | Bayer Cropscience Nv | Hetero-transglycosylase and uses thereof |
CN105612251A (en) * | 2013-09-24 | 2016-05-25 | 拜尔作物科学公司 | Hetero-transglycosylase and uses thereof |
US10093907B2 (en) | 2013-09-24 | 2018-10-09 | Basf Se | Hetero-transglycosylase and uses thereof |
AU2014327258B2 (en) * | 2013-09-24 | 2020-05-07 | Basf Se | Hetero-transglycosylase and uses thereof |
US10647965B2 (en) | 2013-09-24 | 2020-05-12 | Basf Se | Hetero-transglycosylase and uses thereof |
EA036403B1 (en) * | 2013-09-24 | 2020-11-06 | Басф Се | Protein having cellulose:xyloglucan endotransglucosylase (cxe) activity and use thereof |
CN105612251B (en) * | 2013-09-24 | 2021-01-26 | 拜尔作物科学公司 | Isotransglycosylase and use thereof |
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