Patent Application
20090275083
This application contains a sequence listing in computer readable form. The computer readable form is incorporated herein by reference.
The present invention relates to the field of lipases. In particular, it relates to isolated peptides having phospholipase inhibitory activity and isolated polynucleotides encoding the peptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the peptides. It furthermore relates to polypeptides having phospholipase inhibitory activity and lipases capable of being inhibited by such peptides and/or polypeptides.
Lipases (EC 3.1.1.3) hydrolyze ester bonds of triacylglycerols and catalyze esterification and transesterification (acidolysis, alcoholysis, and interesterification) in a non-aqueous system. Some lipases, such as phospholipases, hydrolyze ester bonds in phospholipids which are polar lipids that are of great importance for the structure and function of cell membranes and are the most abundant of membrane lipids. A 26 amino acid C-terminal peptide of Fusarium heterosporum phospholipase was described by Nagao et al., 1998, J. Biochem. 124:1124-29 to play an important role in increasing the thermostability of the lipase without the peptide having an effect on the enzymatic activity.
In light of the broad use of lipids such as e.g. as digestives, for the production of flavours, in dough and baked products of dough, as diagnostic reagents, ingredients of detergent, as catalysts of optical resolutions, etc. it would be desirable to have a mean of controlling the enzymatic activity of lipases. Furthermore, control of the lipolytic activity is desirable from a production point of view due to the option of making enzyme productions that are reproducible regarding their enzymatic activity.
It is an object of the present invention to provide peptides having lipase inhibitory activity and polynucleotides encoding the peptides, as well as nucleic acid constructs, vectors, and host cells comprising the polynucleotides and methods for producing and using the peptides. It is furthermore an object of the invention to provide polypeptides having lipase inhibitory activity and lipases capable of being inhibited by such peptides and/or polypeptides.
The present invention relates to an isolated peptide having phospholipase inhibitory activity, selected from: (a) an isolated peptide comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the residues 289-310 of SEQ ID NO: 1 or the residues 154-175 of SEQ ID. NO: 9; (b) an isolated peptide encoded by a polynucleotide that hybridizes under medium stringency conditions or high stringency conditions with a peptide coding sequence of SEQ ID NO: 1 or the complementary stand of said peptide coding sequence of SEQ ID NO: 1; (c) an isolated peptide encoded by a polynucleotide comprising a nucleotide sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the residues 289-310 of SEQ ID NO: 1; or (d) an isolated peptide comprising a motif with the following amino acid sequence: M1T2D3X4X5L6E7X8K9L10N11X12Y13V14X15X16D17X18E19Y20X21K22 where X4, X5, X8, X12, X15, X16, X18, and X21 independently may be any amino acid, wherein the size of the peptide is less than 60 amino acids (aa).
The present invention also relates to polypeptides comprising the peptide and a lipase, wherein said lipase has a phospholipase activity below 50 PHLU/mg, below 45 PHLU/mg, below 40 PHLU/mg, below 35 PHLU/mg, below 30 PHLU/mg, blow 25 PHLU/mg, below 20 PHLU/mg, below 15 PHLU/mg, below 10 PHLU/mg, below 5 PHLU/mg or below 1 PHLU/mg, and/or shows no phospholipase activity in a plate assay.
The present invention also relates to polypeptides having phospholipase inhibitory activity, wherein a parent protein with at least three solvent accessible residues of an alpha-helix localized at the surface of said protein has been amended in the alpha-helix at at least one of the solvent accessible residues corresponding to position D3, L6, L10, Y13, V14, D17, and X21 and/or the edge residues corresponding to position E7, K9, N11, X18, and Y20 of the motif.
The present invention also relates to isolated polynucleotide's, nucleic acid constructs, recombinant expression vectors, recombinant host cells comprising the peptides or the polypeptides and methods of producing the isolated peptides or polypeptides having phospholipase inhibitory activity.
The present invention also relates to use of the peptides or the polypeptides for inhibiting the enzymatic activity of a lipase upon binding of the peptides or the polypeptides to said lipase.
The present invention also relates to lipases having a phospholipase activity below 50 PHLU/mg, below 45 PHLU/mg, below 40 PHLU/mg, below 35 PHLU/mg, below 30 PHLU/mg, blow 25 PHLU/mg, below 20 PHLU/mg, below 15 PHLU/mg, below 10 PHLU/mg, below 5 PHLU/mg or below 1 PHLU/mg, and/or shows no phospholipase activity in a plate assay which is capable of being inhibited by the peptide, wherein said lipase comprises at least one alteration which independently is an insertion, a deletion or a substitution, whereby the activity of said lipase is inhibited upon binding of at least one of (a) the isolated peptide having phospholipase inhibitory activity; or (b) the peptide having phospholipase inhibitory activity comprised in the polypeptide.
SEQ ID No. 1: Fusarium oxysporum, FoL
SEQ ID No. 2: Fusarium graminearium
SEQ ID No. 3: Nectria lipase 1
SEQ ID No. 4: Nectria lipase 2
SEQ ID No. 5: Fusarium heterosporum
SEQ ID No. 6: Fusarium semitectum.
SEQ ID No. 7: Fusarium solani LipC
SEQ ID No. 8: Fusarium solani LipD
SEQ ID No. 9: Thermomyces lanuginosus, TLL
SEQ ID No. 10: Fusarium venenatum PLA2, FVPLA2
SEQ ID No. 11: Plectasin
SEQ ID No. 12: Monellin
SEQ ID No. 13: Protegrin
SEQ ID No. 14: Barnase
SEQ ID No. 15: Cystatins
SEQ ID No. 16: Apolipoprotein E
Definitions
Phospholipase activity: The term “phospholipase activity” is defined herein as a phospholipolytic (EC number 3.1.1.4) activity that converts phospholipids into fatty acids and other lipophilic substances. For purposes of the present invention, phospholipase activity is determined according to the procedures described for PHLU and the plate assay described in the paragraph “Materials and Methods” below.
Phospholipase inhibitory activity: The term “phospholipase inhibitory activity” is defined herein as the activity that inhibits the phospholipase activity. The peptides of the present invention have at least 20%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the phospholipase inhibitory activity of the mature polypeptide of SEQ ID NO: 1.
Isolated peptide/polypeptide: The term “isolated peptide” or “isolated polypeptide” as used herein refers to a peptide or a polypeptide respectively that is isolated from a source. In certain aspects, the peptide/polypeptide is at least 1% pure, at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, or at least 90% pure, as determined by SDS-PAGE or HPLC. Peptide purity is determined by HPLC.
Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “identity”. For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment). For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNA-FULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment).
Homologous sequence: The term “homologous sequence” is defined herein as a predicted protein that gives an E value (or expectancy score) of less than 0.001 in a tfasty search (Pearson, W. R., 1999, in Bioinformatics Methods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219). The degree of homology may be suitably determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45). In the present invention, corresponding (or homologous) positions in the lipase sequences of Fusarium graminearium, Nectria lipase, Fusarium solani, Fusarium semitectum, Fusarium oxysporum, Fusarium heterosporum, and Thermomyces lanoginosus (synonym: Humicola lanuginose) are defined by the alignment shown in
Isolated polynucleotide: The term “isolated polynucleotide” as used herein refers to a polynucleotide that is isolated from a source. In certain aspects, the polynucleotide is at least 1% pure, at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, or at least 90% pure, as determined by agarose electrophoresis.
Coding sequence: When used herein the term “coding sequence” means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, synthetic or recombinant nucleotide sequence.
Nucleic acid construct: The term “nucleic acid construct” as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.
Control sequences: The term “control sequences” is defined herein to include all components necessary for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, pro-peptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.
Operably linked: The term “operably linked” denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.
Expression: The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: The term “expression vector” is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the present invention and is operably linked to additional nucleotides that provide for its expression.
Host cell: The term “host cell”, as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
Alteration: The term “alteration” means herein any chemical alteration of the polypeptide consisting of the mature polypeptide of SEQ ID NO: 1; or a homologous sequence thereof; as well as genetic manipulation of the DNA encoding such a polypeptide. The alteration can be a substitution, a deletion and/or an insertion of one or more (several) amino acids as well as replacements of one or more (several) amino acid side chains. In describing the amended amino acid sequences according to the invention, the following nomenclature is used for ease of reference: “Original amino acid:position:substituted amino acid(s)”. According to this nomenclature, for instance the substitution of glutamic acid (E) for glycine (G) in position 195 is shown as G195E. A deletion of glycine in the same position is shown as G195*, and insertion of an additional amino acid residue such as lysine (K) is shown as G195GK. Where a specific lipase contains a “deletion” in comparison with other lipases and an insertion is made in such a position this is indicated as *36D for insertion of an aspartic acid (D) in position 36. Multiple mutations are separated by pluses, i.e.: R170Y+G195E, representing mutations in positions 170 and 195 substituting tyrosine (Y) and glutamic acid (E) for arginine (R) and glycine (G), respectively. X231 indicates the amino acid in a parent polypeptide corresponding to position 231, when applying the described alignment procedure. X231R indicates that the amino acid is replaced with R. For SEQ ID NO: 2 X is T, and X231R thus indicates a substitution of T in position 231 with R. Where the amino acid in a position (e.g. 231) may be substituted by another amino acid selected from a group of amino acids, e.g. the group consisting of R and P and Y, this will be indicated by X231R/P/Y. In all cases, the accepted IUPAC single letter or triple letter amino acid abbreviation is employed.
Whether a given amino acid residue is on the surface of the enzyme may be determined according to W. Kabsch and C. Sander (1983). Biopolymers 22, pp. 2577-2637. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features, for example if its solvent accessible surface as calculated with the program DSSP is over 30 Å2.
The inventors have found that a peptide, which may be isolated from the C-terminal of Fusarium oxysporum or Fusarium graminearium (Gibberella zeae) is able to bind to the lipase. They have furthermore shown that the peptide of Fusarium oxysporum has phospholipase inhibitory activity. It is thus suggested that a similar C-terminal peptide isolated from any suitable phospholipase such as e.g., Nectria lipase, Nectria haematococca (Fusarium eumartii), Fusarium solani, Fusarium culmorum, Fusarium semitectum and Fusarium heterosporum, may be used for inhibiting phospholipase activity.
In a first aspect, the invention relates to an isolated peptide having phospholipase inhibitory activity, selected from: (a) an isolated peptide comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the residues 289-310 of SEQ ID NO: 1 or the residues 154-175 of SEQ ID. NO: 9; (b) an isolated peptide encoded by a polynucleotide that hybridizes under medium stringency conditions or high stringency conditions with a peptide coding sequence of SEQ ID NO: 1 or the complementary stand of said peptide coding sequence of SEQ ID NO: 1; (c) an isolated peptide encoded by a polynucleotide comprising a nucleotide sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the residues 289-310 of SEQ ID NO: 1; or (d) an isolated peptide comprising a motif with the following amino acid sequence: M1T2D3X4X5L6E7X8K9L10N11X12Y13V14X15X16D17X18E19Y20X21K22 where X4, X5, X8, X12, X15, X16, X18, and X21 independently may be any amino acid, wherein the size of the peptide is less than 60 amino acids (aa).
In another aspect, the invention relates to the peptide which has a length of less than 55 aa, less than 50 aa, less than 45 aa, less than 40 aa, less than 35 aa, or less than 30 aa.
In another aspect, the invention relates to the peptide which has a length of at least 15 aa, at least 20 aa, or at least 25 aa.
In another aspect, the invention relates to the peptide wherein said peptide has a secondary structure of an alpha-helix.
In another aspect, the invention relates to the peptide wherein the amino acid at each of the positions X4, X5, X8, X12, X15, X16, X18, and X21 present in the motif independently are selected whereby X4 is A or E; X5 is E or Q; X8 is K or A; X12 is S or N; X15 is E, Q or A; X16 is L or M; X18 is K or Q; and X21 is I or V.
The peptide of Fusarium oxysporum lipase (FoL) may be altered to improve or reduce the capacity of said peptide of inhibiting the lipase. Examples of such alterations are shown in Table 1.
In another aspect, the invention relates to the peptide which compared to SEQ ID NO: 1 comprises at least one amino acid substitution at a position corresponding to FoL residues D291; L294; E295; K297; L298; N299; Y301; D305; K306; Y308; or V309.
In another aspect, the invention relates to the peptide which compared to SEQ ID NO: 1 comprises at least one amino acid substitution corresponding to FoL residues D291E; L294A; E295T,S; K297R; L298A; N299D,E; Y301W; D305A; K306R; Y308E,D; or V309I,N,Q.
It is contemplated that alterations in the peptide in some cases may result in a change and in other cases may result in no change in the binding between peptide and lipase. A change may lead to improved or reduced binding which accordingly may result in improved or reduced inhibition of the lipase by the peptide.
The interaction between the peptide and the lipase may in addition to the alterations in the peptide also be influenced by changes in the lipase. Such alterations may contribute to an improved inhibition or a reduced inhibition. Examples of alterations in the amino acid sequence of the Fusarium oxysporum lipase is shown in Table 2.
In another aspect, the invention relates to an isolated polynucleotide encoding the peptide.
In another aspect, the invention relates to a nucleic acid construct comprising the polynucleotide operationally linked to at least one control sequence that directs the production of the peptide in an expression host.
In another aspect, the invention relates to a recombinant expression vector comprising the nucleic acid construct.
In another aspect, the invention relates to a recombinant host cell comprising the nucleic acid construct or the recombinant expression vector.
In another aspect, the invention relates to a method of preparing the isolated peptide comprising the steps: (a) cultivating a host cell comprising the nucleic acid construct comprising the polynucleotide encoding at least one copy of the peptide under conditions conductive for production of the peptide; and (b) recovering the peptide.
Alternatively the peptides of the invention may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example automated synthesizers by Applied Biosystems Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids, particularly D-isomers (or D-forms) e.g. D-alanine and D-isoleucine, diastereoisomers, side chains having different lengths or functionalities, and the like. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.
The peptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.
In another aspect, the invention relates to use of the peptide for inhibiting the enzymatic activity of a lipase upon binding of the peptide to said lipase.
Some lipases which are not classified as phospholipases and it is desirable to derive a variant with phospholipase activity from a parent lipolytic enzyme having no or very little phospholipase activity, e.g. corresponding to a ratio of phospholipase activity to lipase activity below or below 50 PHLU/mg, below 45 PHLU/mg, below 40 PHLU/mg, below 35 PHLU/mg, below 30 PHLU/mg, blow 25 PHLU/mg, below 20 PHLU/mg, below 15 PHLU/mg, below 10 PHLU/mg, below 5 PHLU/mg or below 1 PHLU/mg, and/or shows no phospholipase activity in a plate assay.
In a further aspect, the invention relates to a polypeptide comprising the peptide and a lipase, wherein said lipase has a phospholipase activity below 50 PHLU/mg, below 45 PHLU/mg, below 40 PHLU/mg, below 35 PHLU/mg, below 30 PHLU/mg, blow 25 PHLU/mg, below 20 PHLU/mg, below 15 PHLU/mg, below 10 PHLU/mg, below 5 PHLU/mg or below 1 PHLU/mg, and/or shows no phospholipase activity in a plate assay.
In another aspect, the invention relates to the polypeptide comprising a lipase which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to Thermomyces lanuginosus (SEQ ID NO: 9).
Lipases which do not naturally comprise a peptide having inhibitory activity may be altered for the purpose of obtaining lipase variants that are inhibited by the isolated peptide of the invention. These lipase variants may be inhibited by either of (a) the isolated peptide, (b) the peptide which is attached to such lipase variants or (c) a polypeptide comprising peptides having inhibitory activity. The binding interaction between the peptide and the lipase variants may be further modified by amending the amino acid sequence of the peptide.
Thermomyces lanuginosus lipase (TLL) has essentially no phospholipase activity and there is currently no information indicating that this lipase comprises any C- or N-terminal peptides to control and/or inhibit its activity. This lipase has a structure that makes it suitable for introducing amino acid alterations that will render TLL susceptible for binding of the Fusarium oxysporum lipase (FoL) peptide and thereby inhibiting its lipolytic activity. Examples of such alterations are disclosed in Table 3 (A). The interaction of TLL and isolated peptides of the invention may further be optimized by introduction of alterations in the peptide. Examples of alterations in the C-terminal peptide of the FoL are shown in Table 3 (B).
It is contemplated that where TLL residues is substituted with FoL residues all the residues may be substituted, or alternatively at least one residue selected from the TLL sequence may be substituted with the corresponding residue selected from the FoL sequence according to the following alignments:
In another aspect, the invention relates to the polypeptide wherein the lipase comprises at least one alteration which independently is an insertion, a deletion or a substitution.
In another aspect, the invention relates to the polypeptide wherein the at least one alteration of the lipase comprises one or more amino acid substitutions corresponding to the residues E87R, I90L, G91T, I202P, R209L, E210I, or T244L of Thermomyces lanuginosus (SEQ ID NO: 9).
In another aspect, the invention relates to the polypeptide wherein the lipase comprises a substitution of the residues corresponding to 248-255 of Thermomyces lanuginosus (SEQ ID NO: 9) with the residues 246-254 of Fusarium oxysporum (SEQ ID NO: 1), or 248-253 of Thermomyces lanuginosus (SEQ ID NO: 9) with the residues 247-252 of Fusarium oxysporum (SEQ ID NO: 1).
In a further aspect, the invention relates to a polypeptide having phospholipase inhibitory activity, wherein a protein with at least three solvent accessible residues of an alpha-helix localized at the surface of said protein has been amended in the alpha-helix at at least one of the solvent accessible residues corresponding to position D3, L6, L10, Y13, V14, D17, and X21 and/or the edge residues corresponding to position E7, K9, N11, X18, and Y20 of the motif of the invention as described.
In another aspect, the invention relates to the polypeptide wherein the protein has an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the Plectasin (SEQ ID NO: 11); Monellin (SEQ ID NO: 12); Protegrin (SEQ ID NO: 13); Barnase (SEQ ID NO: 14); Cytostatins (SEQ ID NO: 15); or Apolipoprotein E (SEQ ID NO: 16).
Proteins suitable for generating a polypeptide having phospholipase inhibitory activity must comprise at least one alpha-helix which is exposed to the surface of the protein. It is necessary that the some of the amino acids comprised in the alpha helix are solvent accessible to be able to bind to a lipase and exert its inhibitory effect. The size of the alpha helix may be 22 amino acids or 20, 18, 16, 14, 12, or 10 amino acids. In addition to the at least one alpha-helix other secondary structure elements such as e.g. beta-strands may be present. Based on their secondary structures three main groups of proteins may be defined: an All-alpha group, an all beta-group and an Alpha-beta group.
The alpha-helix of the phospholipase inhibitory peptide may be superimposed and aligned with the alpha-helix of the selected protein (underlined) in a two-step procedure as illustrated below for monellin:
Select the direction, either parallel or anti-parallel of the sequences to be aligned,
Once the direction is selected there are several possibilities of aligning the sequences of the alpha-helices. The most optimal alignment is the one comprising the maximum length in common and the minimum number of troubles (clashes).
After identifying the most optimal alignment, modifications in the protein alpha-helix may be made to change the amino acid residues to the alpha-helix of the phospholipase inhibitory peptide. An example is shown in
Finally, parts outside the alpha-helix in the protein are analyzed to identify if there are other and/or new clashes that may affect binding of the alpha-helix of the scaffold protein to the lipase or affect the accommodation of the alpha-helix within the protein. Such residues are then optionally changed to maximize the phospholipase inhibitory effect. These are the mutations outside the helix.
In another aspect, the invention relates to a method of preparing the polypeptide of the invention comprising a step of attaching the peptide to a lipase, wherein said lipase has a phospholipase activity below 50 PHLU/mg, below 45 PHLU/mg, below 40 PHLU/mg, below 35 PHLU/mg, below 30 PHLU/mg, blow 25 PHLU/mg, below 20 PHLU/mg, below 15 PHLU/mg, below 10 PHLU/mg, below 5 PHLU/mg or below 1 PHLU/mg, and/or shows no phospholipase activity in a plate assay.
In another aspect, the invention relates to a method of preparing a polypeptide comprising the steps: (a) selecting a protein having an alpha-helix localized at the surface of the protein whereby at least three, at least four, at least five, or at least six residues of said alpha-helix is solvent accessible; (b) aligning the alpha-helix of the protein with the peptide; (c) identifying the residues in the alpha-helix of the protein that are solvent accessible corresponding to position D3, L6, L10, Y13, V14, D17, and X21 of the motif; (d) altering at least one, at least two, at least three, at least four, at least five, at least six or at least seven of the amino acids in the protein identified in step (c); (e) testing the polypeptide for phospholipase inhibitory activity; (f) selecting the polypeptide having phospholipase inhibitory activity; and (g) producing the polypeptide selected in (f).
In another aspect, the invention relates to the method further identifying in step (c) the residues in the alpha-helix of the protein that are potentially solvent accessible corresponding to position E7, K9, N11, X18, and Y20 of the motif.
In another aspect, the invention relates to the method further comprising a step of making at least one alteration at one or more positions in the protein.
In another aspect, the invention relates to use of the polypeptide, for inhibiting the enzymatic activity of a lipase upon binding of the peptide comprised in the polypeptide to said lipase.
In a further aspect, the invention relates to a lipase having a phospholipase activity below 50 PHLU/mg, below 45 PHLU/mg, below 40 PHLU/mg, below 35 PHLU/mg, below 30 PHLU/mg, blow 25 PHLU/mg, below 20 PHLU/mg, below 15 PHLU/mg, below 10 PHLU/mg, below 5 PHLU/mg or below 1 PHLU/mg, and/or shows no phospholipase activity in a plate assay which is capable of being inhibited by the peptide, wherein said lipase comprises at least one alteration which independently is an insertion, a deletion or a substitution, whereby the activity of said lipase is inhibited upon binding of at least one of (a) the isolated peptide having phospholipase inhibitory activity; or (b) the peptide having phospholipase inhibitory activity comprised in the polypeptide.
In another aspect, the invention relates to the lipase wherein said lipase is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to Thermomyces lanuginosus (SEQ ID. NO: 9).
In another aspect, the invention relates to the lipase wherein the at least one alteration is a substitution corresponding to the residues L92; R96; L203; I207; R211; L243; L250; or L252 of Thermomyces lanuginosus (SEQ ID NO: 9).
In another aspect, the invention relates to the lipase wherein the at least one alteration is a substitution corresponding to the residues L92D,E,W; R96E,D,A; L203W,K,M; I207D,E; R211 H; L243W,K L250D,E,R; and L252S,T of Thermomyces lanuginosus (SEQ ID NO: 9).
Materials and Methods
Phospholipase Activity (PHLU)
Phospholipase activity (PHLU) is measured as the release of free fatty acids from lecithin. 50 μl 4% L-alpha-phosphatidylcholine (plant lecithin from Avanti), 5 mM CaCl2 in 50 mM HEPES, pH 7 is added 50 μl enzyme solution diluted to an appropriate concentration in 50 mM HEPES, pH 7. The samples are incubated for 10 min at 30° C. and the reaction stopped at 95° C. for 5 min prior to centrifugation (5 min at 7000 rpm). Free fatty acids are determined using the NEFA C kit from Wako Chemicals GmbH; 25 μl reaction mixture is added 250 μl Reagent A and incubated 10 min at 37° C. Then 500 μl Reagent B is added and the sample is incubated again, 10 min at 37° C. The absorption at 550 nm is measured using an HP 8452A diode array spectrophotometer. Samples are run in at least in duplicates. Substrate and enzyme blinds (preheated enzyme samples (10 min at 95° C.)+substrate) are included. Oleic acid is used as a fatty acid standard. 1 PHLU equals the amount of enzyme capable of releasing 1 μmol of free fatty acid/min at these conditions. Specific phospholipase activity (PHLU/mg) is calculated at the phospholipase activity (PHLU) per amount protein (mg).
Plate assay: 50 ml 2% agarose is dissolved by heating in purified water for 5 minutes and subsequently cooled to 60-63° C. 50 ml 2% plant L-alpha-Phosphatidylcholine 95% in 0.2M NaOAc, 10 mM CaCl2, pH 5.5 at 60° C. in 30 min. is blended for 15 sec. with ultrathorax. Equal volumes of 2% agarose and 2% Lecithin are mixed. 250 μl 4 mg/ml crystal violet in purified water is added as indicator. The mixture is poured into appropriate petri dishes (e.g. 30 ml in 14 cm Ø dish), and appropriate holes (3-5 mm) are made in the agar for application of enzyme solution. The enzyme sample is diluted to a concentration corresponding to OD280=0.5 and 10 microliter and applied into holes in the agarose/lecithin-matrix. Plates are incubated at 30° C. and reaction zones in the plates are identified after approximately 4-5 hours and/or after approximately 20 hours incubation. The Humicola lanuginosa lipase is used as a control, and the presence of a larger clearing zone than the control is taken as a positive result for phospholipase activity.
The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
Protein expression and purification: The Gibberella zeae/Fusarium graminearum lipase (GZEL) gene was amplified by PCR and further confirmed by sequencing. Then the GZEL gene was expressed in yeast with a significant protein band shown on SDS-PAGE after Comassie staining. And the activity was detected against the olive oil/Bright Green plate at pH 7. The positive candidate clones showed dark green zones around the holes.
The culture supernatant separated from cells by centrifugation and the pH of supernatant was adjusted to pH 7.0. The supernatant was then filtrated and applied into Ni-Sepharose FF equilibrated with 25 mM Tris-HCl at pH 7.0 with 0.3 M NaCl. The target protein was eluted by imidazole at a gradient from 0M to 1M. Fractions from the column were analyzed for activity. Fractions with enzyme activity were pooled and concentrated. Then the samples were loaded into a gel filtration column Superdex 75 equilibrated by 25 mM Tris-HCl at pH 8.0 with 0.15 M NaCl. The eluted active lipase was concentrated and dialyzed with 25 mM Tris-HCl at pH 8.0. The lipase was checked by SDS-PAGE and the pure fractions were prepared for crystallization trails.
Crystallization: The freshly prepared protein was concentrated to 10 mg/ml and crystallized by the hanging drop vapor diffusion method at 291K. The initial crystallization conditions were screened using several Crystal Screen Kits (Hampton Research screen kit 1 and 2, Index screen kit, MembFac screen kit). One microliter of protein solution was mixed with 1 microliter of reservoir solution and equilibrated against 200 microliter of reservoir solution. Small crystals could be found in many different conditions within three days. Many of the crystals are hollow sticks and have poor diffraction quality. Fine shaped and good quality crystals were selected from the condition with 0.2 M Ammonium Sulfate, 0.1 M Bis-Tris (pH 5.5), 25% w/v PEG3350 within 2-4 days (see
Data collection and processing: A 2.8 Å resolution set of diffraction data sets were collected at 100K from a single GZEL derivative crystal using an in-house Rigaku MM-007 generator and a Mar345dtb detector. The beam was focused by osmic mirrors. For a more detailed analysis, flash-cooled crystals were used. Crystals were immersed in cryoprotectant for 5-10 sec., picked up with a loop and flash-cooled in a stream of nitrogen gas cooled to 100K. The cryoprotectant was prepared by adding 25% glycerol to the mother liquor reservoir. The crystal form belongs to space group P212121 (a=78.4, b=91.0, c=195.8, α=β=γ=90°) with four GZEL molecules per asymmetric unit and a VM of 2.6 Å3 Da-1 (Matthews 1968), corresponding to a solvent content of 48%. Processing of diffraction images and scaling of the integrated intensities were performed using the HKL2000 software package (Otwinowski et al. 1997).
Results: Initially, although we have obtained dozens of GZEL crystals in many conditions of the screening kits, they are unsuitable for X-ray diffraction. Many of the crystals are hollow fibre. Therefore, further crystallization optimization was performed and better crystals were obtained in the following condition: 0.2 M Ammonium Sulfate, 0.1 M Bis-Tris, pH 5.5, 25% w/v PEG3350. Drops containing 2 μl protein solution and 2 μl of reservoir solution were equilibrated against 200 microliter of reservoir solution. Crystals grown from the optimized reservoir solution (0.2 M Ammonium Sulfate, 0.1 M Bis-Tris, pH 5.5, 25% w/v PEG3350) were more suitable for X-ray diffraction and diffracted to 2.8 Å. A set of data was subsequently collected from one single crystal (see
References: Matthews, B. W., Solvent content of protein crystals. J. Mol. Biol., 1968. 33: p. 491-497. Otwinowski, Z. and W. Minor, Processing of X-ray diffraction data collected in oscillation mode, in Macromolecular Crystallography, part A, C. W. Carter Jr. and R. M. Sweet, Editors. 1997, Academic Press. p. 307-326.
There are four GZEL-peptide complexes in the asymmetric unit of the crystal structure. The four complexes were refined independently from each other. They constitute four different entities. In this four different complexes the peptide sits in exactly the same way with respect to the lipase core domain, see Table 5 below. This provides another set of evidence for a specific binding of the peptide to the lipase core.
FoL was purified from a Pilot fermentation LVF 57 UF concentrate PPW6523. PPW6523 was 0.22 μm filtered and loaded onto a butyl-sepharose fast flow column, washed with 1.8M NH4acetate and eluted with MilliQ H2O. Datasheet on purified FoL: 2003-04317-01. On an isoelectric focusing gel IEF pH 3-10 (Novex precast) 20070628 the sample gave a single band. N-terminal sequencing of the band showed only one sequence—the expected FoL N-terminus showing that C-terminal peptide was not bound.
The substrate used for these experiments was para-nitropenyl (p-NP) butyrate purchased from Sigma Aldrich. A substrate stock solution was prepared by adding 18 μL of p-NP Butyrate to 1 ml of 2-propanol giving a 100 mM stock solution. The working solution was prepared by diluting 10 μL stock solution in final volume of 1 ml 50 mM Tris pH 7 buffer. 45 μL of the FoL 1 mg/ml in 50 mM Tris buffer pH 7 was preincubated with two concentrations of the peptide and control was carried out in absence of peptide using the same buffer. After preincubation for 5 minutes at room temperature the enzyme solution was diluted in 50 mM Tris pH 7 buffer and 100 μL diluted enzyme solution was mixed with the 100 μL substrate solution and reaction was followed in microtiter plate spectrophotometer under constant shaking at room temperature. Table 6 below shows the color development over time monitored at A405 (every 30 sec. for 10 minutes) of p-nitrophenol, one of the degradation products of p-NP butyrate, liberated by the enzyme which was preincubated with the peptide in two different concentrations and a control which was treated exactly the same way in the absence of peptide.
The samples prepared in Example 4 were loaded to a Nowex gel IEF 3-10. Lane 1: Marker. Lane 2: Sample 1 (10 μL). Lane 3: Sample 1 (20 μL). Lane 4: Sample 2 (10 μL). Lane 5: Sample 2 (20 μL). Lane 6: Sample 3 (10 μL). Lane 7: Sample 3 (20 μL). A photo of the gel is shown in
A lecithin plate without Triton-X 100 was prepared as described in the Materials and Methods. The same amounts based on ODA280 of purified TLL and FoL was added to the holes: TLL to the two top holes, FoL to the two bottom holes; left holes (lower concentration, ODA280 equal to 0.2); right holes (higher concentration, ODA280 equal to 0.5). After 20 hours of incubation FoL exhibited a 7/10 mm clearing zone at the lower/higher concentration. TLL did not show a clearing zone.
The recently solved structure of the Fusarium graminearum, also known as Gibberella zeae, phospholipase has shown that the C-terminal peptide normally cleaved for maturation of the enzyme was present in the structure. This peptide adopts an alpha-helical structure and packs against the catalytic domain of the phospholipase. The peptide has been shown to inhibit the phospholipase activity against small esters.
Monellin is a sugar tasting protein from the African serendipity berry. The structure of Monellin consists of a long partially exposed alpha-helix packed perpendicularly against a 5-stranded beta-sheet. The presence of the solvent accessible alpha-helix makes the protein well suited for the purpose of modifying the alpha-helix in the attempt of transferring the inhibitory properties of the C-terminal peptide of the Fusarium oxysporum lipase (FoL), which is a very close homologue of Fusarium graminearum lipase (FGL). Two different Monellin variants were designed as FoL inhibitors.
Variant summary: Two variants were found after analyzing all the possible ways of superimposing the C-terminal peptide alpha-helix of FoL onto the alpha-helix of Monellin. In selection of the variants the visual inspection focused on; a) maximizing the alignment of the alpha-helix of Monellin with the C-terminal peptide residues that interact with the catalytic domain of the lipase; and b) minimizing the conflicts of other parts of Monellin with the catalytic domain. Two different possibilities of superimposing the helices either matching the N to C-terminal direction (parallel) or inverting it (anti-parallel) were identified.
Sequence/structure: The sequence of Monellin used on this study is shown in
Variants: Two Monellin variants were the result of this study. They are described in what follows.
MON1 variant: When the superposition where of the lipase peptide alpha-helix is aligned parallel to the Monellin alpha-helix the amino acid substitutions F11E+Q13K+N14L+L15V+K17Y+F18V+N24Y+K25V+I74A+E77R+R82G+R83G are to be made. These substitutions will mimic the interaction of the peptide with the lipase catalytic domain and eliminate possible conflicts of the remaining parts of the molecules. The complete sequence of MON1 is shown in
MON2 variant: When the superposition of the lipase peptide alpha-helix is aligned antiparallel to the Monellin alpha-helix the amino acid substitutions F11Y+Q13N+N14L+K17A+F18L+K25H+F34V+R81G+R84G are to be made. These substitutions will mimic the interaction of the lipase peptide with the lipase catalytic domain and eliminate possible conflicts of the remaining parts of the molecule. The complete sequence of MON2 is shown in
Enzyme assay: The substrate used for these experiments was para-nitropenyl (p-NP) butyrate (N9876 Sigma Aldrich). A substrate stock solution was prepared by adding 18 μL of p-NP butyrate to 1 ml of 2-propanol giving a 100 mM stock solution. The working solution was prepared by diluting 500 μL stock solution in final volume of 50 mL 500 mM phosphate pH 7, 0.4% triton X-100. 40 μL of the enzyme 38 ng/mL in 500 mM phosphate pH 7, 0.4% triton X-100, was pre-incubated with 20 μL of peptide having various concentrations giving peptide/lipase ratios from 0.5 to 1000 increasing with a factor of 2 through 12 steps. An enzyme substrate blank containing 60 μL was included. After pre-incubation in minimum 15 minutes, 100 μL of substrate was added and the reaction was followed in microtiter plate spectrophotometer (SPECTRAmax PLUS 384 from Molecular Devices) after an initial shaking (5 sec.) at room temperature. Colour development over time was monitored at A405 (every 20 sec. for 30 minutes), measuring p-nitrophenol, one of the degradation products of p-NP butyrate, liberated by the hydrolysis.
Initial reaction rates (determined as the linear initial slope of A405 plotted against time) were plotted against the logarithm to the inhibitor/lipase ratio. From the following model IC50 values were determined:
v0 is the initial reaction rate, vmax is the maximum rate, vmin is the minimum reaction rate and IC50 is the half maximal inhibitory concentration. GraphPad Prism (v.5.00, Mar. 12, 2007) from GraphPad Software, Inc. was used for regression.
% Inhibition is the percentage reduction in initial reaction rate where 0% inhibition is defined as the initial reaction rate without peptide.
2871
(208-397)2
1IC50 value (half maximal inhibitory concentration).
295% Confidence interval.
Results:
Enzyme assay: As described above. Peptides P11 and P13 were tested in concentrations giving peptide/lipase ratios from 5 to 5000 increasing with a factor of 2 through 10 steps. Peptide P12 was tested in concentrations giving peptide/lipase ratios from 6 to 6000 increasing with a factor of 2 through 10 steps.
1IC50 value (half maximal inhibitory concentration).
295% Confidence interval.
Results:
| Number | Date | Country | Kind |
|---|---|---|---|
| 08155438.8 | Apr 2008 | EP | regional |
This application claims priority or the benefit under 35 U.S.C. 119 of European application no. 08155438.8 filed Apr. 30, 2008 and U.S. provisional application No. 61/050,317 filed May 5, 2008, the contents of which are fully incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61050317 | May 2008 | US |
