Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
  • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
  • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a novel method for enzymatic modification of proteins, especially to a method for coupling a peptide or protein bound glutamine residue by acyl transfer, catalysed by a transglutaminase (TGase) activity.
• peptide or protein bound glutamine refers to an amino acid sequence (i.e. a peptide or a protein) , wherein the glutamine residue is at least two amino acids spaced away from the N-terminus of the said amino acid sequence .
• transglutaminase is known to catalyse an acyl transfer reaction, wherein the ⁇ -carboxamine group of glutaminyl residues is the acyl donor.
• Substrates that can act as the acyl acceptor are less defined and can roughly be divided into three groups, wich will be discussed below.
• the ⁇ -aminogroup of protein bound lysine residues can serve as substrate, leading to the generation of intra- and inter olecular ⁇ - ( ⁇ -glutamyl) -lysine isopeptide bonds.
• transglutaminase is used to couple a glutamine comprising peptide or protein to lysine residues of a protein, in order to change certain properties of the said protein, such as improvement of the texture of a protein food product, for improvement of the solubility of a protein etc. (for a review, see Nielsen, Food Biotechnology, 9(3), 119-156 (1995) ) .
• the said proteins should have sufficient suitable lysine residues as acyl acceptors. Whereas the peptide or protein must comprise at least a suitable glutamine residue as acyl donor.
• acyl donors are peptide or protein bound glutaminyl residues.
• Ikura et al. (Agric. Bio. Chem, 45 (11), 2587 - 2592, 1981) describes the use of transglutaminase for the incorporation of methionine ethyl ester into food proteins, wherein the amine group of the amino acid ethyl ester is coupled to the ⁇ carbon of protein bound glutamine residues.
• the food proteins could be enriched with limiting essential amino acids by covalent attachment thereof to the said protein. In this way, food proteins of altered amino acid composition could be produced.
• TGase reactivity could therefore only be shown using citraconylated legumin and in the presence of DTT; both aspects imply that modification is unsuited for food applications.
• TGase reaction on unmodified legumin has not been shown nor suggested, as the goal of the article is to study the reactivity of the ⁇ -amino groups without considering the implications for protein functionality.
• EP-A-0 725 145 describes the coupling of a number of molecules to a physiologically active protein with the purpose to improve the adaptability of this protein to a medicine.
• the molecules contain an alkylamine, which was introduced into the molecule via chemical synthesis .
• transglutaminase can catalyse the acyl transfer reaction between peptide or protein bound glutamine and a number of novel substrates.
• the invention provides a method for coupling a peptide or protein bound glutamine residue to a substrate by acyl transfer, catalysed by a transglutaminase activity, charactarized in that the substrate is chosen from the group, consisting of lysine-free peptides of at least 2 amino acids, lysine-free proteins, aminosaccharides, aminopolysaccharides, saccharides and alcohols.
• the peptide or protein of the peptide or protein bound glutamine is preferably a peptide or protein, derived from its natural environment, without being subjected to further modification in vitro.
• said peptide may be originated from a part of a naturally occurring protein, and be obtained from the protein by e.g. enzymatic hydrolysis or any other method known in the art. However, it is preferred that such a peptide or protein is not subjected to any further in vitro modification, including e.g. modification of amino acid residues in the peptide or protein.
• the above defined substrates are therefore preferably free of any alkylamine group, that would function as an acyl acceptor in a transglutaminase reaction.
• Transglutaminase activity is defined as the activity of a compound to catalyse an acyl transfer reaction, wherein the ⁇ - carboxamide group of glutamine residues is the acyl donor. Said glutaminase activity therefore comprises the transglutaminases that are presently known in the art, which are widely distributed in nature and have been isolated from e.g. mammals, plants, fish and micro-organisms.
• the substrate is chosen from peptides and proteins, the N-terminal thereof being preferably glycine, alanine, serine or valine, more preferably glycine.
• the N-terminal thereof being preferably glycine, alanine, serine or valine, more preferably glycine.
• such peptides could successfully be used by several transgluta inases and could be coupled to the glutamyl residues of a protein.
• the N- terminal amino acid of the peptide is glycine, a very versatile peptide is obtained that can be coupled to a peptide or protein bound glutamine by several tested transglutaminases.
• the said coupling reaction has been found to be improved when the second amino acid from the N-terminus of the substrate is chosen from glycine, alanine and valine, but the best coupling was obtained when the second amino acid from the N-terminal of the peptide was glycine. More preferably, the substrate comprises at least 3 amino acids.
• the substrate is a peptide of 3-20 amino acids, although longer amino acid chains can be used as well.
• the said substrates can be coupled to one or more glutamines of the protein or peptide, therewith altering the properties of the peptide/protein in a desired manner.
• the person, skilled in the art will be aware of the possibilities for improving certain qualities or activities of proteins and peptides by coupling to one or more of the glutamine residues thereof a substrate according to the present invention.
• the substrate is an aminosaccharide.
• aminosaccharides are defined as any monosaccharide, oligosaccharide or polysaccharide containing a primary amino group and any monosaccharide, oligosaccharide or polysaccharide prepared by means of reductive amination of a monosaccharide, oligosaccharide or polysaccharide.
• the nitrogen of the aminogroup of the aminosugar is directly linked to a carbon atom of the sugar backbone, i.e. without the presence ofany spacer moieties there between, such as e.g. an alkyl group.
• an advantageous aminosaccharide is aminosorbitol.
• saccharides are defined as any reducing monosaccharide, oligosaccharide or polysaccharide, such as starch hydrolysates, enzymatic prepared oligosaccharide syrops, cellulose and b-glucans .
• the substrate is a saccharide having a terminal hydroxy group. It has surprisingly been shown that the terminal hydroxy group of such saccharides can act as effective acyl acceptor in the transglutaminase katalysed acyl transfer reaction. The terminal hydroxy group can in that case be covalently linked to the ⁇ -carbon of the glutamine residue.
• Such a saccharide therefore is preferably void of amino groups that would act as acyl acceptors .
• the peptide or protein bound glutamyl residue is part of a biologically functional peptide or protein or functional fragment thereof.
• the biologically functional peptide or protein or functional fragment thereof may be a natural peptide, protein or fragment, but may also be a manipulated peptide, protein or fragment, e.g. by genetic manipulation techniques.
• the peptide or protein bound glutamine residue is part of an epitope.
• "Epitope" is defined as an amino acid sequence that forms a specific binding site for an immunoglobuline molecule.
• the said epitope may be masked, by which the protein can loose its antigenic activity.
• the glutamyl residue is in close vicinity of an epitope, e.g. 1 to 10 amino acids spaced therefrom; when a sufficient long or bulky substrate is coupled to the said glutamine residue, the said epitope can be successfully masked.
• the epitope, or the surrounding amino acid sequence may also be engineered in such a way, that it comprises one or more glutamines on a position where it is not present in the natural counterpart of the said epitope, in order to couple, at the said glutamines, the substrates according to the invention.
• the transglutaminase activity comprises a transglutaminase of microbial origin or a functional derivative thereof.
• transglutaminase of microbial origin has a versatile utility with respect of the method according to the present invention.
• Microbial transglutaminase is e.g. capable of successfully couple peptides of two or three amino acids to a peptide or protein bound glutamine residue.
• Transglutaminase derived from guinea pig liver is however also suitable, although the versatility thereof appears to be somewhat less than that of microbial origin.
• the calcium independent transglutaminase of Streptovertic ⁇ ll ⁇ um sp. is used, more preferably from Streptovert ⁇ c ⁇ llium mobaraense (Ando et al. Agric. Biol. Chem., 53(10), 2613-17, 1989).
• the transglutaminase of the said micro-organism has been shown very versatile for the method according to the present invention and has the additional advantage that no calcium is necessary for enzymatic activity thereof.
• the invention further relates to a method as described above, for conferring to the peptide/protein comprising the glutamine residue, the capacity to substantially keep its activity upon passing the stomach of a mammal, which mammal preferably is human. Many functional peptides and proteins are degraded upon passing the stomach and therewith loosing their activity. In order to maintain the activity, one or more of the glutamine residues of the said peptide or protein is coupled to a substrate according to the present invention.
• the N-terminus of peptides or proteins is often susceptible to degradation in the stomach.
• the N-terminus thereof can be coupled to a glutamine comprising carrier peptide/protein, using transglutaminase according to the invention.
• the N-terminus of the peptide/protein to be stabilised is coupled to a glutamine residue of the carrier, resulting in protection of the N-terminus of the stabilised peptide/protein.
• substrates or carriers are to be coupled to the protein in order to obtain the desired resistance against degradation.
• proteins that can serve as carrier are gluten proteins, soy proteins and milk proteins.
• peptides to be stabilised are bio-active peptides, in particular anti-microbial peptides which are effective in the gastrointestinal tract.
• the invention further relates to a method as described above for masking an epitope of a peptide/protein.
• Transglutaminase is used to covalently link one or more of the abovementioned substrates to a glutamine that is part of the epitope or located in close vicinity thereof, but according to the invention it may also be used to covalently link a lysine within the epitope or in close vicinity thereof to a glutamine of a masking peptide or protein.
• the said peptide/protein is rendered less antigenic.
• the epitope comprises a glutamine, or a glutamine is in close vicinity of the said epitope.
• the invention also provides a method for improving the emulsion properties of a glutamin containing peptide or protein.
• a transglutaminase katalysed reaction By the transglutaminase katalysed reaction according to the invention, the emulsion properties of a protein/peptide can be altered.
• substrates that are positively or negatively charged at the emulsion conditions are preferred, such as short glycine chains, in particular gly-gly-gly.
• the invention in particular provides a method for improving the functionality of a glutamin containing protein by coupling a food peptide or food protein bound glutamine residue to a substrate by acyl transfer, catalysed by a transglutaminase activity according to the invention as described above.
• the functionality of food grade peptides or proteins can effectively be improved using the TGase reaction according to the invention.
• the invention also relates to a peptide or protein obtainable by the method according to the invention, i.e. to proteins and peptides, one or more glutamine residues of which are coupled to one or more of the substrates that are discussed above.
• the invention relates to a peptide or protein comprising at least a glutamine residue, the ⁇ -carbon thereof being covalently linked to a • substrate, the substrate being chosen from: a second lysine-free peptide or lysine-free protein, at least comprising 2 amino acids, the free ⁇ -aminogroup thereof being covalently linked to the ⁇ -carbon of the glutamine residue, an aminosaccharide or aminopolysaccharide, the aminogroup thereof being covalently linked to the ⁇ -carbon of the glutamine residue.
• the substrate is chosen from peptides and proteins, the N terminal thereof preferably being as described above.
• Figure 1 is a schematic diagram of the acyl transfer reaction from a ⁇ -carboxamide group of a peptide or protein bond glutaminyl residue, catalysed by transglutaminase, with different substrates as acyl acceptor.
• A the tripeptide gly-gly-gly as substrate
• B the aminosaccharide aminosorbitol as substrate
• C sorbitol as substrate.
• Fig. 2 MA DI-TOF analysis of dBi before (A) and after modification with TG and gly-gly-gly (B) .
• Fig. 3 SDS-PAGE analysis of dBi incubated with TG. Samples were taken after different time points as indicated at the top of each lane. Lane 2 shows unmodified dBi .
• Fig. 4. SDS-PAGE analysis of ⁇ -lactalbumin incubated with TG and with or without gly-gly-gly. Samples were taken at time points indicated at the top of each lane. Due to cross-linking by TG, protein polymers in lanes 5 and 6 at the interface of the stacking and running gel are too large to enter the running gel.
• Fig. 6 Inhibition ELISA of casein and casein modified with CBZ-gln- gly (CBZ-Q-G) peptides using microbial TG.
• Fig. 7A and B show graphs of turbidity of emulsions.
• Fig. 8 shows emulsions, being incubated for three days at room temperature, the samples being numbered according to table 3.
• the dBi protein is a model protein that corresponds with a part of the central domain of high molecular weight (HMW) wheat gluten and contains a high number of glutamines but no lysines. This makes the protein a suitable substrate to study incorporation of primary amines and deamidation catalysed by transglutaminase, since no glutamine- lysine cross-linking can occur.
• HMW high molecular weight
• Fig. 1 shows incorporation of glycine-tripeptide into mdBl. Peaks correspond to mdBl with varying numbers of glycine-tripeptide incorporated, the number being indicated.
• incorporation of this peptide via coupling of the ⁇ -amino group occurs to a high extent, as roughly half of the 64 glutaminyl residues present in mdBl can be coupled. (Increased TG concentration and extended incubation time increased the number to a maximum of 31 peptides incorporated.)
• the second amino acid gives similar results as for the third amino acid. Even when the amino acid at the N-terminus is changed, incorporation is observed, although to a relatively low extent.
• the ⁇ -amino group of peptides can serve as a substrate for transglutaminase and the presence of a glycine residue at the N- terminus has been shown to be important for a high rate of incorporation into proteins.
• Amyloid peptide GAIIGLMVGGW Induce fusion of liposomes in vitro
• Dynorphin GGFLRRI Potentiates the dorsal immobility response in rats
• Enkephalinase GGFM Inhibitor of enkephalinase inhibitor Neuromedin B GNLWATGHFM-NH A potent stimulant effect on the smooth muscle preparation of rat uterus
• ⁇ -lactalbumin was incubated with TG and with or without glycine-tripeptide (Fig. 3) .
• Fig. 3A protein crosslinking is largely inhibited by the presence of glycine-tripeptide, strongly suggesting that the rate of the incorporation reaction is significantly higher than that of the crosslinking reaction.
• Fig. 3B shows the NH 3 production of the two reactions in time. Since NH 3 is a product in all types of reactions catalysed by TG, production of NH 3 represents a summation of protein crosslinking, incorporation and deamidation.
• Fig. 4 The structure of 1-amino-l-deoxy-D-sorbitol (aminosorbitol) is depicted in Fig. 4. This compound was incubated with dBl and TG similar to the experiments described in example 1. Surprisingly, aminosorbitol could be incorporated into dBl as could be shown by MALDI-TOF and NH 3 production (data not shown) . This incorporation is relatively efficient, since up to 10 molecules of aminosorbitol per dBl could be coupled.
• Factor XIII did not show any incorporation of the glycine-tripeptide into mdBl, showing that the ⁇ -amino group is not a substrate for this enzyme.
• glycine- tripeptide was incorporated to about the same extend as when used microbial TG.
• the level of incorporation rapidly decreased (data not shown).
• casein As a source of IgE directed against casein, serum form 6 patients with a casein allergy was used. Affinities of casein for IgE were measured using IgE binding experiments as described earlier for another allergen (Koppelman et al . J. Biol. Chem. 274, 4770-7, 1999). Dilutions of casein (final concentrations 1 to 100000 ng/ml) , either or not modified with mTG and CBZ-gln-gly under conditions described in Example 1, were incubated with a 1 to 30 dilution of patient serum pool in phosphate buffered saline (PBS) containing 1% BSA and 0.1% Tween 20.
• PBS phosphate buffered saline
• casein was allowed to bind to IgE for one hour at room temperature under gently shaking conditions .
• the incubation mixtures were transferred to the 96-well plates pre-treated as follows. 96-well plates were coated with 10 ⁇ g/ml native casein in PBS and subsequently blocked with BSA (1% in PBS containing 0.1% Tween 20) to diminish the non-specific binding. IgE bound to the casein coated wells was detected using an anti-human IgE antibody conjugated to horse radish peroxidase. Between each step, plates were washed five times with PBS containing 0.1% Tween 20.
• the inhibition of IgE binding as a function of the amount of casein present in the pre-incubation sample reflects the affinity of casein for IgE.
• concentrations needed for half-maximal inhibition were calculated using a semi-logarithmic equation and were used to compare the affinities of the different forms of casein for IgE.
• Fig. 5 shows that the affinity of mTG/CBZ-gln-gly treated casein for IgE was decreased 5 times compared to native casein.
• Emulsions were made by adding 2 ml of soy bean oil to 4 ml of 1% (modified) Solpro in 20 mM phosphate buffer, pH 7.0. The mixture was sonicated four times 15 seconds (at 18 microns peak to peak) . Turbidity of the emulsions, defined as the absorbance at 500 nm multiplied by 2.303, was followed in time (see Fig. 7). This shows that incorporation of gly-gly-gly into Solpro results in a strong increase of the turbidity of the corresponding emulsion and also in an increased stability in time.

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