JP2007197434A - Peptide having reactivity as substrate of transglutaminase and utilization of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peptide having reactivity as a substrate of a transglutaminase. <P>SOLUTION: This peptide is provided by having the reactivity as the substrate of the transglutaminase consisting of a blood coagulation XIIIth factor and/or tissue type transglutaminase or having a specific amino acid sequence having transglutaminase inhibitor activity. The method for searching a compound having the transglutaminase inhibitory activity by utilizing the peptide presented by a phage-displaying method and a compound containing a primary amine is also provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a peptide having reactivity as a substrate for transglutaminase and use thereof.

Transglutaminase (TGase) is known as an enzyme that catalyzes an acyl transfer reaction between a γ-carboxyamide group on the side chain of a glutamine residue in a peptide or protein and a primary amine in a lysine residue or the like. Transglutaminase is responsible for cross-linking between proteins, which is a kind of post-translational modification of proteins. In higher animals such as humans, it is known that there are eight types of isozymes. Among them, the main isozymes are blood coagulation factor XIII responsible for blood coagulation (Factor XIII, or simply referred to as factor XIII). Tissue-type transglutaminase involved in dead cell processing and matrix enhancement.

Based on such physiological effects, transglutaminase, for example, forms an extracellular matrix in a supporting skeletal material used for repairing damaged tissue (Patent Document 1), and binds proteins such as enzymes to living tissues (Patent Document 2). Application to the medical field etc. is tried. In addition, attempts have been made to develop an inhibitor that inhibits the activity of transglutaminase as a therapeutic agent for blood coagulation-related diseases (Patent Document 3). Furthermore, it has been found that the bond between glutamine and lysine residues generated by transglutaminase is associated with cancer, apoptosis, aging, dementia, and the like (Non-patent Document 1).
Special table 2005-523733 Special table 2002-509160 Special table 2005-513141 Experimental Medicine 18, 1421-1425 (2000)

  Based on the above, in recent years, in the food and medical fields, rather than using transglutaminase itself, the reactivity of transglutaminase as a substrate has been increased in relation to the use of cross-protein cross-linking and the adhesion of proteins to other compounds. It is becoming more desirable to have peptides. Particularly in the medical field, an inhibitor or enhancer of the activity of transglutaminase is desired in connection with a prophylactic / therapeutic agent for transglutaminase-related diseases. However, to date, no transglutaminase substrate for cross-linking or adhesion use, effective inhibitors of transglutaminase, etc. have been found. In addition, no inhibitor has been found that has a substrate selectivity and a high selectivity for a specific isozyme among the isozymes of transglutaminase, or that inhibits a specific isozyme with a high selectivity. Furthermore, there has been no efficient method for searching for peptides having such substrate reactivity and inhibitory activity.

  Accordingly, an object of the present invention is to provide a peptide having reactivity as a substrate for transglutaminase, a method for searching the peptide, and a method for producing the peptide. Another object of the present invention is to provide a linker using a peptide having reactivity as a substrate for transglutaminase, and an artificial construct using the linker. Furthermore, another object of the present invention is to provide a peptide having high selectivity for substrate reactivity and inhibitory activity against transglutaminase isozymes and the like, a method for searching for it, and a method for producing the same. Furthermore, another object of the present invention is to provide a transglutaminase inhibitor and a composition for prevention / treatment of transglutaminase-related diseases.

  The present inventors search for a glutamine residue that is easily reacted with transglutaminase and its peripheral sequence using the “phage display method”, which allows a bacteriophage hosted in E. coli to display a random peptide sequence. And a search for two isozymes of tissue type transglutaminase (hereinafter also simply referred to as TGase2) and factor XIII. As a result, this system provides a reactive amino acid sequence as a substrate for transglutaminase. In addition to finding that it can be obtained, it has been found that a peptide having transglutaminase inhibitory activity can be obtained, and the present invention has been completed. Furthermore, the present inventors have found an inhibitor having high selectivity for a specific isozyme among transglutaminases, and completed the present invention. That is, according to the present invention, the following means are provided.

According to one aspect of the present invention, there is provided a peptide comprising one or more amino acid sequences selected from Table 7 and Table 8 below, or modified sequences thereof.

Here, in the present specification, the peptide is a concept including oligopeptides, polypeptides, and proteins. Accordingly, when simply referred to as a peptide in this specification, it is not intended to limit at least the number of amino acid residues.

  This peptide preferably has transglutaminase substrate activity, and is preferably a transglutaminase inhibitor. The transglutaminase is preferably Factor XIII and / or tissue type transglutaminase.

  The peptide is selected from T2, T5, T8, T12, T16, T20, T26, T29, T30, T32, F6, F17, F28 and F32 and their modified sequences described in Tables 7 and 8 above. It is preferable that it is a tissue type transglutaminase inhibitor containing 1 type or 2 types or more. More preferably, it contains one or more selected from T2, T5, T8, T12, T16, T20, T26, T29, T30, T32, F6, F17 and F28 and their modified sequences. In this embodiment, the amino acid sequence may include one or more amino acid sequences selected from T2, T5, T8, T16, T20, T26, T29, and T32 and modified sequences thereof. Moreover, in this aspect, it is preferable to include one or more amino acid sequences selected from the amino acid sequence set forth in SEQ ID NO: 22 and this modified sequence. Furthermore, it preferably consists of the amino acid sequence set forth in SEQ ID NO: 22.

  In addition, this peptide includes blood coagulation factor XIII containing one or more selected from T1, T30, F6, F11, F17, F28 and F32 and their modified sequences described in Tables 7 and 8 above. Inhibitors are preferred. More preferably, it includes one or more selected from T1, T30, F6, F11, F17 and F28 and their modified sequences. In this embodiment, the amino acid sequence preferably includes one or more amino acid sequences selected from T1 and F11 and modified sequences thereof, more preferably SEQ ID NO: 38 and SEQ ID NO: 59, and It is preferable to include one or two or more selected from these modified sequences, and it is more preferable to consist of the amino acid sequence described in SEQ ID NO: 38 or SEQ ID NO: 59.

According to another aspect of the present invention, there is provided a method for searching for a peptide having reactivity as a substrate for transglutaminase,
The following steps (a) to (c):
(A) reacting a peptide displayed using a phage display method with a compound containing a primary amine in the presence of transglutaminase,
And (b) a step of separating the phage retaining the transglutaminase reaction product in the step (a), and (c) a step of determining the amino acid sequence of the peptide displayed in the separated phage. The

According to another aspect of the present invention, there is provided a method for searching for a peptide having transglutaminase inhibitory activity,
About two or more isozymes of transglutaminase
The following steps (a) to (c):
(A) reacting a peptide displayed using a phage display method with a compound containing a primary amine in the presence of transglutaminase,
(B) a step of separating the phage retaining the transglutaminase reaction product in the step (a), and (c) a step of determining the amino acid sequence of the peptide displayed in the separated phage,
Carried out
(D) Provided is a search method for performing a step of evaluating reaction specificity of a peptide containing an amino acid sequence obtained as having substrate reactivity of the two or more isozymes to the two or more isozymes. .

According to another embodiment of the present invention, there is provided a method for producing a peptide having transglutaminase inhibitory activity,
About two or more isozymes of transglutaminase
The following steps (a) to (c):
(A) reacting a peptide displayed using a phage display method with a compound containing a primary amine in the presence of transglutaminase,
(B) a step of separating the phage retaining the transglutaminase reaction product in the step (a), and (c) a step of determining the amino acid sequence of the peptide displayed in the separated phage,
Carried out
(D) evaluating the reaction specificity of the peptide comprising an amino acid sequence obtained as having substrate reactivity of the two or more isozymes to the two or more isozymes; (e) in step (d) Producing a peptide evaluated to have reaction specificity for any of the two or more isozymes or a modified peptide thereof;
A manufacturing method is provided.

According to another embodiment of the present invention, an artificial structure,
An artificial construct comprising a peptide comprising one or more amino acid sequences selected from the amino acid sequences shown in Tables 7 and 8 and modified sequences thereof is provided.

  In this artificial construct, a connecting part by an isopeptide bond formed between the γ-carboxyamide group of the glutamine residue side chain of the peptide chain and the primary amino group of the primary amino group-containing compound is provided. It is a preferable aspect to provide. In this artificial construct, the primary amino group-containing compound may be a compound containing two or more primary amino groups, and the primary amino group-containing compound may be cadaverine, spermidine, It is good also as 1 type, or 2 or more types selected from the group which consists of spermine and cardopentamine. Furthermore, in this artificial construct, one or more other peptide chains may be linked to the peptide chain via the linking part, and may have an artificial antibody part. Furthermore, a solid phase carrier may be provided via the connecting portion. Moreover, when a solid phase carrier is provided, it may be a protein-immobilized solid phase carrier. Moreover, this artificial construct may have a drug active part.

  According to another embodiment of the present invention, prevention of transglutaminase-related diseases comprising one or two or more amino acid sequences selected from Table 7 and Table 8 or peptides containing these modified sequences. A therapeutic composition is provided. In this composition, the peptide is preferably composed of any amino acid sequence selected from the amino acid sequences set forth in SEQ ID NO: 22, SEQ ID NO: 38 and SEQ ID NO: 59.

  The present invention is based on the finding that a novel amino acid sequence having reactivity as a substrate of transglutaminase (hereinafter also simply referred to as substrate reactivity) was found, and that a peptide having this amino acid sequence was found to have transglutaminase inhibitory activity. Is based. The present invention is also based on the finding that the phage display method could be used to search for peptides having transglutaminase substrate reactivity. The amino acid sequence of the present invention and the amino acid sequence specifying the peptide of the present invention are not derived from a sequence in a protein conventionally known as a substrate for transglutaminase, but are searched from a random peptide library. . Therefore, the peptide of the present invention can function as a transglutaminase inhibitor as well as function as a linker that mediates the cross-linking between peptides and the bond between the peptide and the primary amino group-containing compound. Furthermore, it can function as an inhibitor having high selectivity for TGase2 or factor XIII. No peptide sequence or peptide having such functions has been reported.

  Hereinafter, the peptide of the present invention and other embodiments, such as a linker, a transglutaminase inhibitor, an artificial construct, a method for searching for a transglutaminase substrate-reactive peptide, a method for searching for a peptide having transglutaminase inhibitory activity, and the like will be described in detail. .

(peptide)
The peptide of the present invention includes one or more amino acid sequences selected from Table 7 and Table 8, or modified sequences thereof. That is, this peptide may contain only one amino acid sequence described in Table 7 and Table 8 and its modified sequence, or two or more of these sequences (same type or different type). May be included in tandem. Moreover, it may be composed only of the displayed amino acid sequence and modified sequence, or may contain a separate amino acid sequence in addition to these sequences. In particular, when two or more of the displayed amino acid sequences and these modified sequences are included, a linker or spacer for linking these amino acid sequences is preferably included.

  Therefore, this peptide may be constituted as a hybrid peptide containing a physiologically active protein molecule such as an enzyme or a fragment or peptide thereof as another amino acid sequence, a protein for labeling or separation, its It may be a hybrid peptide containing a fragment or peptide.

Examples of the modified sequence include sequences in which one, two or more amino acid residues are inserted, substituted, deleted and added to the amino acid sequences shown in Tables 7 and 8. The modification of amino acid residues in such a modified sequence is not particularly limited as long as it is possible as long as the intended transglutaminase substrate reactivity is maintained in the present peptide. Preferably, the number of amino acid residues to be modified depends on the amino acid modification form (position and type of modification), but is preferably 5 or less, more preferably 3 or less, except for addition. is there. In the modification, the amino acid residues shaded in Tables 7 and 8 are maintained as motifs (the positions of the amino acid residues in the peptide may be changed, but the relationship between amino acid residues (sequence order and It is preferred that the (interval) is maintained). However, the amino acid residue at position 6 in Table 7 is a hydrophobic amino acid (G (glycine), W (tryptophan), M (methionine), P (proline), F (phenylalanine), A (alanine), V ( Valine), L (leucine) and I (isoleucine), etc.) can also be substituted. In addition, the amino acid residue at the fifth position in Table 8 can be substituted if it is a hydrophobic amino acid. The amino acid residue at the first position in Table 8 is preferably D (aspartic acid) or E (glutamic acid), and the sixth position is preferably P (proline) or A (alanine).

In these modified sequences, it is preferable to maintain the following motif. As a substrate-reactive sequence or inhibitory activity sequence (Table 7) of tissue-type transglutaminase,
Motif 1: QxPf
Motif 2: QxPffD- (P)
Motif 3: QxxxxDP
(Wherein f represents a hydrophobic amino acid residue, x represents an arbitrary amino acid, and the amino acid residue in () represents an amino acid residue that may be provided).

In addition, as a substrate reactive sequence or inhibitory activity sequence (Table 8) of Factor XIII,
Motif 4: D / EQxxxfP / AWP
Is mentioned.

  Among them, the TGase2 inhibitor includes one or more selected from T2, T5, T8, T12, T16, T20, T26, T29, T30, T32, F6, F17, F28 and F32 and their modified sequences. Peptides containing can be preferably used. Among these, T16, T26, T29, T30, T32, and F6 and their modified sequences have good TGase2 inhibitory activity. Also preferred are T2, T5, T8, T16, T20, T26, T29 and T32 and their modified sequences. This is because these sequences can particularly inhibit TGase2 with high selectivity. Further, an amino acid sequence of T26 (described in SEQ ID NO: 22) and a peptide containing this modified sequence are preferable, and a peptide consisting of the amino acid sequence described in SEQ ID NO: 22 is more preferable.

  Moreover, as a factor XIII inhibitor, the peptide containing 1 type, or 2 or more types of T1, T30, F6, F11, F17, F28, and F32 and these modified sequences can be used preferably. Among these, T1, T30 and F11 and these modified sequences have good factor XIII inhibitory activity. T1 and F11 and these modified sequences are particularly preferable from the viewpoint of inhibiting factor XIII with high selectivity. More preferably, F11 (SEQ ID NO: 38) and SEQ ID NO: 59 and peptides containing these modified sequences are preferred, and peptides consisting of the amino acid sequences set forth in SEQ ID NO: 38 and SEQ ID NO: 59 are more preferred.

  T30 has good inhibitory activity against both TGase2 and factor XIII transglutaminase.

Peptides consisting of the various sequences described in Tables 7 and 8 and the above-mentioned preferred sequences are all short sequences of 11 to 12 amino acid residues. Such peptides can be synthesized easily and in large quantities, and more effective inhibitors can be developed by amino acid substitution. In addition, many of the inhibitors so far were obtained from the natural world using only inhibition of activity as an index, and the mechanism of inhibition was not clear. On the other hand, the inhibitory activity of the present peptide and its modified peptide is superior in actual applications because it is caused by the enzyme incorporating the present peptide instead of the substrate to inhibit the reaction. That is, since the inhibition mechanism and the product of the inhibition reaction are clear, the burden of study on the influence on the living body is reduced.

  The peptide having transglutaminase substrate reactivity is not limited to Tables 7 and 8, and may be composed of other amino acid sequences. Tables 7 and 8 are amino acid sequences that are substrate-reactive with TGase2 and / or Factor XIII, respectively, as shown in the Examples, but for example against other isozymes in higher animals such as humans. A substrate-reactive sequence and a peptide can be easily obtained by searching for a peptide-reactive amino acid sequence using a peptide search method described later by the phage display method. By using the amino acid sequence thus obtained in the same manner as described above, the peptide of the present invention can be obtained.

(Linker)
Since this peptide has transglutaminase substrate reactivity, it can be used as a linker capable of binding to a compound having a primary amino group by transglutaminase. Although it does not specifically limit as a form of a linker, it may have only one said amino acid sequence, and may have two or more. If it has two or more, it may be arranged so that the glutamine part of the amino acid sequence comes to the part where it wants to bind to the primary amino group-containing compound, and binds to the primary amino group-containing compound at both ends of the linker If desired, the amino acid sequence is arranged on at least one end, and glutamine is arranged appropriately or the amino acid sequence is arranged on the other end. Using this linker, a primary amino group-containing compound can be introduced at a desired position where a glutamine residue is arranged in the linker sequence.

  Moreover, it does not specifically limit as a usage form as a linker. As a form of the linker, for example, as shown in FIG. 1, a transglutaminase is allowed to act on a primary amino group-containing compound immobilized on a solid phase carrier and a desired peptide having this linker, thereby causing a desired The peptide can be immobilized on a solid phase carrier via a primary amino group-containing compound and the present linker. By doing so, peptides such as enzymes and antibodies can be selectively applied to the solid phase carrier at a glutamine site in this linker without considering non-selective ligation as in the past and under mild conditions. It can be easily fixed. For this reason, it is possible to immobilize a peptide or the like in a steric configuration effective for expression of its activity with respect to the solid phase carrier, and to obtain a solid phase carrier that effectively retains the function of the peptide.

  Further, for example, as shown in FIG. 2, it contains a desired peptide (here, a single-chain antibody in which a light chain and a heavy chain of an antibody are linked via a spacer) and two or more primary amino groups. When transglutaminase is allowed to act on a compound, an artificial antibody construct that mimics an antibody in which two antibody-like peptide chains are linked via a primary amino group-containing compound can be obtained. By doing so, the number of antigen-binding sites and intermolecular distance of the single-chain antibody can be appropriately designed according to the distance and number of primary amino groups in the primary amino group-containing compound. Furthermore, modification of an antibody molecule can be easily performed by adding a modification reaction or a label to a primary amino group-containing compound.

In addition, as a primary amino group containing compound, it should just contain at least 1 primary amino group, and may contain 2 or more primary amino groups. Examples of such a primary amino group-containing compound include a monoamine having one primary amino group, a diamine having two primary amino groups, and a polyamine having two or more same, in addition to a peptide having a lysine residue. Such monoamines, diamines, and polyamines can be used without any particular limitation. Examples of monoamines include R-NH ₂ (wherein R is an optionally substituted alkyl group). Examples include histamine. Examples of the diamine include R (NH ₂ ) ₂ (R is a divalent carbonization such as an optionally substituted alkylene chain, an optionally substituted alkenylene chain, and an optionally substituted alkynylene chain. Hydrogen group), specifically, alkylenediamines such as ethylenediamine, trimethylenediamine, hexanediamine, octanediamine, dodecanediamine, tetramethylenediamine (putrescine), pentamethylenediamine (cataverine), spermidine, spermine and Examples include polyalkylene polyamines such as cardopentamine. Putrescine, catavelin, spermidine, spermine and cardopentamine are biological polyamines. Examples of diamines are shown in FIG.

  Thus, according to the present linker, various forms of artificial constructs can be obtained depending on the number and position of primary amino groups in the primary amino group-containing compound used. In particular, when a peptide consisting only of the amino acid sequence set forth in SEQ ID NOs: 1 to 59 or having a length similar to that is used as a linker, an artificial construct having a desired structure is obtained because the length of the linker is appropriate. be able to.

(Transglutaminase inhibitor)
The peptide also has transglutaminase substrate reactivity and transglutaminase inhibitory activity. The transglutaminase inhibitory activity may be for any transglutaminase, but is preferably TGase2 inhibitory activity and / or factor XIII inhibitory activity. The amino acid sequences shown in Table 7 and these modified sequences are preferred for inhibitors having inhibitory activity against TGase2, and the amino acid sequences shown in Table 8 and these modified sequences are preferred for inhibitors against Factor XIII. The preferred amino acid sequences and peptides for use in such a transglutaminase inhibitor are as described above.

  Since the inhibitory activity of the transglutaminase inhibitor of the present invention is based on the substrate reactivity of transglutaminase, it can function as a linker as well as a transglutaminase inhibitor.

  An inhibitor having an inhibitory activity with a high selectivity for TGase2 is useful, for example, as a research use or a prophylactic / therapeutic agent for diseases requiring inhibition of the activity of TGase2. For example, an inhibitor that has a high inhibitory activity against TGase2 in higher animals but only a lower inhibitory activity against factor XIII is a cell death or cell that does not affect the blood coagulation system. The matrix can be applied to research reagents and related disease therapeutic agents. On the other hand, an inhibitor having a high inhibitory activity against factor XIII but only a lower inhibitory activity against TGase2 is used for research on a blood coagulation system that does not affect cell death or the like. It can be applied to reagents and therapeutic agents for related diseases.

(Method for searching for peptides having transglutaminase substrate reactivity)
The search method of the present invention includes the following steps (a) to (c):
(A) reacting a peptide displayed using a phage display method with a compound containing a primary amine in the presence of transglutaminase,
(B) separating the phage retaining the transglutaminase reaction product in the step (a), and (c) determining the amino acid sequence of the peptide displayed in the separated phage.

  The phage display method is a system in which a foreign gene is expressed as a fusion protein on the N-terminal side of a coat protein (such as g3p) of a filamentous phage such as M13 which is a kind of E. coli virus so as not to lose the infectivity of phage particles. In order to display the peptide on the phage surface layer, for example, a display kit containing a bacteriophage displaying a peptide having a random amino acid sequence of about 12 predetermined lengths can be used. The peptide thus displayed is reacted with a primary amino group-containing compound in the presence of transglutaminase. In this way, when the presented peptide has transglutaminase substrate reactivity, it binds to the primary amino group-containing compound. As a result, the phage is in a state of presenting the peptide to which the primary amino group-containing compound is bound.

  As the primary amino group-containing compound, it is preferable to use a compound provided with various tags in addition to a labeling substance such as biotin so as to facilitate separation and detection in the next step.

  Next, the phage particles displaying the peptide bound to the primary amino group-containing compound are separated. Separation of phage particles can be performed according to the type of tag or labeling substance attached to the primary amino group-containing compound.

  Next, the amino acid sequence of the peptide on the surface layer of the phage particles thus separated is analyzed. In the analysis, it is preferable that the separated phage particles are propagated by infecting a host such as Escherichia coli. The amino acid sequence of the peptide bound to the primary amino group-containing compound can be obtained by obtaining a predetermined region of DNA from such a large amount of phage particles and analyzing the base sequence.

  By using such a search method, it is possible to search for a peptide having a highly selective inhibitory activity for the transglutaminase isozyme. That is, the above steps (a) to (c) are performed on two or more types of isozymes, and (d) the peptide containing the amino acid sequence obtained as having the substrate reactivity of these two or more types of isozymes. What is necessary is just to implement the process of evaluating the reaction specificity with respect to 2 or more types of isozymes. Further, if necessary, it is preferable to evaluate the inhibition specificity for a peptide evaluated to have a high reaction specificity.

  In obtaining amino acid sequences and peptides having transglutaminase substrate reactivity and amino acid sequences and peptides having transglutaminase inhibitory activity, the phage display method is advantageous in comparison with conventional methods in the following points. That is, conventionally, in order to search for an amino acid sequence or peptide that is a substrate for an enzyme, a substrate-reactive site such as a protein having substrate reactivity is specified by a site-specific displacement introduction method or the like. This was done by modifying the surrounding sequence. However, according to the phage display method, amino acid sequences having high selectivity and high reactivity can be efficiently searched from random amino acid sequences based on substrate reactivity. Conventionally, the phage display method has been used exclusively to search for displayed peptides that interact with specific compounds, but it has been reported as a method for screening peptides having substrate reactivity of enzymes such as transglutaminase. There were no examples and it was not considered suitable for such screening methods. For example, in the present invention, when a substrate peptide is bound to a primary amino group-containing compound such as transglutaminase, the enzyme reaction caused by the substrate peptide being displayed on the phage is suppressed. It was considered that the reduction of the infectious ability of phages by binding to the display peptide, and the cross-linking between the display peptides by transglutaminase were obstacles. However, as a result of adopting the phage display method, the present inventors have been able to identify a good or highly selective substrate-reactive amino acid sequence despite the anticipated obstacles. Although not restricting the present invention, the phage display method selects amino acid sequences and peptides that are substrate-reactive in the presence of elements that interfere with the reactivity between the enzyme and the substrate. Even so, it is presumed that an amino acid sequence having transglutaminase substrate reactivity having good and high selectivity that can exhibit highly selective inhibitory activity was obtained.

  A peptide having substrate reactivity or inhibitory activity can be easily obtained by producing a peptide obtained by a method for searching for peptides having such transglutaminase substrate reactivity or inhibitory activity or a modified peptide thereof.

(Artificial structure)
The artificial construct of the present invention comprises a peptide chain comprising one or more amino acid sequences selected from the amino acid sequences shown in Tables 7 and 8 and modified sequences thereof. The artificial construct of the present invention utilizes the function of this peptide as a linker. This artificial construct further comprises a connecting portion by a peptide bond formed between the γ-carboxyamide group of the glutamine residue side chain of the peptide chain and the primary amino group of the primary amino group-containing compound. I have. That is, the artificial construct can include a linking portion formed by binding the peptide and a primary amino group-containing compound with transglutaminase. As already explained, the artificial construct can take a wide variety of structures by using the third material such as the form and combination of the peptide and the primary amino group-containing compound, and also a solid phase carrier. .

  That is, as shown in FIGS. 1 (a) and (b), the artificial construct may have a form including a solid phase carrier having another peptide chain or the like via a connecting portion, as shown in FIG. As described above, an artificial antibody portion such as one or two or more single-chain antibodies may be provided. Further, a part having a drug activity may be provided in part. The drug activity here is not limited to those intended for prevention or treatment of diseases, but includes those intended for diagnosis or research. Further, the drug-active moiety may be a peptide such as an antibody or an enzyme linked to the present peptide or a primary amino group-containing compound, or the present peptide itself. This is because this peptide is also a transglutaminase inhibitor. In addition, the artificial construct having such a pharmaceutically active moiety can be used as a reagent composition for research, and includes, for example, a pharmaceutically acceptable solid phase carrier or the like, or a pharmaceutically acceptable primary amino acid. When it has a coupling | bond part with a group containing compound etc., it can also be used as a pharmaceutical composition itself.

  For example, as shown in FIG. 1B, a peptide chain that functions as a linker is a peptide chain that has substrate reactivity with factor XIII, and the other peptide chains are peptide chains that have TGase2 inhibitory activity. A solid phase composed of a pharmaceutically acceptable material that synthesizes a peptide of a type (which may be provided with an appropriate spacer) by a genetic engineering technique and retains a primary amino group using this linker moiety By binding to the surface of the carrier, a drug containing a TGase2 inhibitor as an active ingredient can be obtained. For the production of the connecting part, by using Factor XIII, even a tandem type peptide (or another peptide containing glutamine) can be selectively connected to the solid phase carrier in the linker part.

  Moreover, in this artificial construct, by providing this peptide as a linker part by transglutaminase, it is possible to control the orientation when the artificial construct is bound to a solid phase carrier. This is because the linker moiety having this peptide is selectively bound to the surface of the solid phase carrier having a primary amino group. As a result, for example, in the example shown in FIG. 1B, the orientation of the portion (drug portion) having TGase2 inhibitory activity is controlled so as to be presented on the surface of the solid phase carrier. Further, according to an artificial construct having a single amino acid sequence as a linker in the vicinity of the C-terminal of the heavy chain constant region of a double chain antibody, the linker moiety is selectively bound to a solid support. As a result, the orientation is controlled so that the variable region is presented on the surface of the solid support. Such orientation control can also be applied to other peptides such as enzymes and receptors. According to this artificial construct, a solid-phase carrier (protein chip / bead or antibody chip) having an antibody or enzyme capable of exhibiting high reactivity. / Bead etc.) and solid phase carriers (surface layered preparations, capsules, films, patches, etc.) provided with peptide drugs.

  In addition, the kind of material which comprises a solid-phase carrier is not ask | required in particular, Glass, ceramics, a plastics, paper, a metal, a gel etc. can be used as needed. Further, it may be dense or porous, and may be liquid permeable like a filter. Furthermore, the form is not particularly limited, such as a particulate form such as a bead form, a plate form, a sheet form, a film form, and a fiber form.

  In addition, the artificial construct can be a construct including a scaffold used for cell proliferation and cell retention in, for example, transplantation and diagnostic applications. In this case, it is preferable that the scaffold has a biological affinity that allows cells to grow and be retained, and has a biodegradability particularly in the case of transplantation. Such scaffold materials are well known to those skilled in the art, such as collagen, gelatin, and hyaluronic acid. By causing transglutaminase to act on the primary amino group-containing compound and the present peptide bound to such a scaffold, a predetermined shape, strength, and the like can be easily imparted to the scaffold. Further, when the scaffold itself is a primary amino group-containing composition having an amino group such as a lysine residue, the same effect can be obtained by causing transglutaminase to act on the scaffold and the present peptide. . Such a scaffold-type artificial construct may be seeded with cells, or may have already been grown.

  The artificial construct described above can be obtained, for example, by reacting a desired present peptide with a primary amino group-containing compound in the presence of transglutaminase as appropriate.

(Pharmaceutical composition for prevention / treatment of transglutaminase related diseases)
The pharmaceutical composition of the present invention contains the present peptide. The present pharmaceutical composition is used for prevention / treatment of transglutaminase-related diseases, and in particular, for prevention / treatment of diseases that are improved by inhibiting the activity of transglutaminase. As such a disease, for example, when it has plasma transglutaminase inhibitory activity, it is useful as an antithrombotic agent, a thrombolytic agent, an antiarteriosclerotic agent, and an agent for preventing restenosis after percutaneous coronary angioplasty. In addition, when it has TGase2 inhibitory activity, etc., the thickening of the blood vessel wall in the processes of Crohn's disease, tumor transplantation, atherosclerosis, for example, thrombotic microangiopathy in the kidney, fibrous growth of skin such as scleroderma , Membranous glomerulonephritis, repair of retinal injury, cataracts, acne, scar tissue formation, and infection by various filaria nematodes. Furthermore, when it has TGase2 inhibitory activity, Alzheimer's disease, hemophilia, apoptosis, celiac disease, Huntington's disease, skin disease, cataract, spinal cord medulla atrophy (Kennedy disease), (spinal cord) cerebellar ataxia, parietal nucleus Inflammatory diseases of the central nervous system, including rheumatoid atrophy of the brain stem, multiple sclerosis, rheumatoid arthritis, diabetes such as insulin-dependent diabetes, tetanus and other Clostridium related symptoms, Let's syndrome, human immunity Deficiency virus infections and inflammatory diseases.

  The pharmaceutical composition can be combined with a transglutaminase inhibitor, optionally in a pharmaceutically acceptable form, optionally with a pharmaceutically acceptable carrier. In such a composition, an appropriate administration route is selected depending on the dosage form, etc., and the amount and management of administration of the transglutaminase inhibitor in the pharmaceutical composition are easily determined by those skilled in the art treating these diseases. Is.

  The composition containing the transglutaminase inhibitor of the present invention can be used as a pharmaceutical composition, prevents diseases associated with transglutaminase, and is used in individuals who are at risk for transglutaminase-related diseases. It can also be used as a preferred food composition for improving risky conditions. The form of the food composition is not particularly limited. In addition to beverages, processed foods, seasonings and tube feeding agents, pharmaceutical preparation forms such as tablets, capsules, powders and granules may be employed.

  Such a composition can also be used as a food processing composition for meat and fish. Such a composition for food processing is useful for adjusting the texture of meat or fish meat or processed products thereof, or molding and processing meat or fish meat.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to a following example.

Example 1
[Search for transglutaminase highly reactive substrate sequences by phage display]
First, the transglutaminase preparation used in all experiments was as follows. TGase2 was provided by Mr. Koji Ikura, Professor, Faculty of Textiles, Kyoto Institute of Technology, a purified preparation derived from guinea pig liver. FactorXIII in Germany Behring Inc., was used Fibrogammin ^R P (derived from human plasma). Factor XIII was subjected to an activation reaction by limited decomposition using thrombin (Sigma, derived from bovine plasma) (reaction buffer: 10 mM Tris-HCl, pH 7.5, 150 mM).
NaCl, 20 mM CaCl ₂ ). This is because Factor XIII is synthesized as an inactive precursor and activated by decomposition. After the cleavage, thrombin was inactivated by adding thrombin inhibitor PMSF (phenylmethylsulfonyl fluoride) at a final concentration of 1 mM.

(1) Binding reaction of peptide and primary amine by transglutaminase A display kit (New England Biolabs, No. 1) containing a bacteriophage (hereinafter referred to as phage) to which a random 12-mer (consisting of 12 amino acids) peptide is bound (displayed). The search was performed as follows using a single chemistry). For a liquid containing phage (1.5 × 10 ¹¹ ), the total reaction volume is 0.1 ml, and the enzyme (TGase 2 is 0.5 ng / μl, or Factor XIII is 5 ng / μl: both have the same enzyme activity. And biotin-labeled primary amine (biotinylated pentylamine, Pierce). These were added so that {20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM DTT (Dithiothreitol, reducing agent) was present as a reaction buffer. Since the enzyme requires calcium ions, the reaction was started by adding a CaCl ₂ solution to a final concentration of 5 mM, and the reaction was carried out in a warm bath at 37 ° C. for a reaction time of 15 minutes. The situation of the reaction start is the same). The divalent ion chelator EDTA (ethylene diamine tetraacetate) was added to a final concentration of 10 mM to stop the reaction.

(2) Separation of amine-bound phage Next, phages were precipitated and separated by adding polyethylene glycol (final concentration 3.3%) and NaCl (final concentration 0.4 M) to the reaction. The phage was again suspended in TBS buffer {20 mM Tris-HCl (pH 8.0), 150 mM NaCl} and bound to avidin gel (SoftLink ™ SoftRelease Avidin Resin, Promega). This is generally used in a method of specific purification utilizing a strong non-covalent bond between avidin and biotin. In these processes, unreacted biotin-labeled primary amine and phage that did not incorporate biotin-labeled primary amine were removed. Phage bound to the avidin gel was competitively eluted by adding TBS buffer containing high concentration of biotin (5 mM).

(3) Peptide analysis When phages are mixed with the growing Escherichia coli ER2738, they are infected and propagated in E. coli to be lysed. For this reason, infected phages are propagated and then recovered in the culture solution of E. coli. The culture solution was centrifuged to remove E. coli, and the phage-containing solution was precipitated with polyethylene glycol and NaCl, and then resuspended in enzyme reaction buffer {20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM DTT}. The steps from the enzymatic reaction were repeated. The enzyme reaction time was 15 minutes for the first time, 10 minutes for the second time, and 2 minutes for the third time and thereafter. As for the amount of phage, 1 × 10 ^12-13 was used for the second and subsequent times. The base sequence encoding the peptide region of the phage was determined using PRIM310 from ABI. The analysis result of the amino acid sequence of the peptide displayed by the phage conjugated with amine is shown in FIG.

(Example 2)
[Evaluation of sequence obtained by production of fusion protein]
In this example, a fusion protein shown in FIG. 5 was prepared using a certain amino acid sequence and evaluated as a substrate for transglutaminase.

As for the peptide sequence in the phage obtained as an excellent substrate in Example 1 (added with an asterisk in FIG. 4), the gene sequence encoding it exists in a specific region in the DNA of the phage. The phage DNA selected by displaying the peptide was purified and used as a template, and the gene sequence corresponding to the peptide sequence was amplified by the PCR method. For amplification by PCR, a primer DNA that synthesizes an NcoI restriction enzyme recognition site is synthesized on the 5 ′ side for incorporation into an expression vector, and an endogenous EagI site in the phage DNA on the 3 ′ side. Primers that can be used were prepared for PCR reaction.

On the other hand, GST (glutathione S-transferase) as a fused protein is derived from the pGEX4T-1 vector (Novagen) with a gene region encoding GST, restriction enzyme recognition sites on both ends (EagI, 3 on the 5 ′ side). A primer DNA was synthesized so that XhoI) was synthesized on the “side” and amplified by PCR. This was cloned into a cloning vector (pCRTopo2.1 Invitrogen), and all five glutamine residues in GST were replaced with asparagine residues. As the replacement method, QuikChange ^R mutation genes kit (Stratagene), which applied the PCR method, was used.

Thereafter, the gene part encoding the modified GST was inserted into E. coli expression vector pET24d (Novagen) (EagI and XhoI), and then the gene part encoding the optimal substrate sequence was inserted (NcoI and EagI). In the expression vector (pET24d), a histidine tag useful for purification is added to the carboxy terminal side of the protein to be expressed. A vector plasmid constructed to express a protein fused with peptide and GST was transformed into E. coli BL21 (DE3) LysS strain. E. coli was cultured, and expressed in E. coli as a recombinant fusion protein by adding an expression inducer (final concentration 0.1 mM IPTG: isopropyl 1-thio β-D-galactoside). The expressed recombinant protein was purified using the affinity of the histidine tag {TALON Metal Affinity Resin (BD Bioscience)}. As a result, GST fusion proteins having several types of optimal substrate sequences were obtained.

Next, the purified GST fusion protein was used for the enzyme reaction of TGase2 and FactorXIII to evaluate whether it was an effective substrate. The fusion protein with the sequence obtained as an optimal substrate with TGase2 was reacted with TGase2, and the fusion protein with the sequence obtained with FactorXIII was reacted with FactorXIII. TGase2 reaction conditions were as follows: GST fusion protein (0.2 mg / ml) and monodansyl cadaverine (fluorescently labeled pentylamine, Sigma) were added as a substrate at 0.5 mM, and a reaction buffer (10 mM Tris-HCl (pH 8.0)) was added. ), 150 mM NaCl, 5 mM CaCl ₂ , 1 mM DTT). The concentrations of TGase2 and FactorXIII were 0.5 ng / μl and 5 ng / μl, respectively. The enzyme reaction was performed at 37 ° C. at different times, and SDS (lauryl sulfate, final concentration 2%) and a reducing agent (mercaptoethanol, 5%) were added to the reaction product, followed by heat denaturation. Polyacrylamide electrophoresis with a gel concentration of 12.5% was performed. After electrophoresis, the gel was irradiated with ultraviolet rays (280 nm) and photographed. Similar experiments were performed for TGase2 and FactorXIII for all GST fusion proteins with a fixed reaction time (10 minutes) and analyzed for specificity to each isozyme. The reaction conditions are exactly the same as described above. The results are shown in FIGS.

Further, among the GST fusion proteins, the fusion with the most reactive and specific sequences (T26, F11 in FIG. 4) was analyzed in more detail. That is, the uptake amount of the radiolabeled primary amine ( ³ H-labeled putrescine, Perkinelmer) was measured for these two types of GST fusion proteins and the GST fusion protein containing no glutamine residue as a control. The buffer composition of the reaction solution was 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM as before.
It was made to be CaCl ₂ and 1 mM DTT. In the reaction solution, 1.6 mg / ml of the fusion protein, 1 mM of the labeled primary amine, 4 ng / μl of TGase2 and 40 ng / μl of FactorXIII were added, respectively. The reaction was started by adding a calcium solution. After 10 minutes of reaction time, 100 μl of the reaction product was soaked in filter paper (2.4 cm diameter, Whatman). The filter paper was previously treated by being dipped in trichloroacetic acid (TCA), which is a strong acid, and dried, and the enzymatic reaction was stopped when the solution after the reaction was soaked. The filter paper was washed 3 times in 10% TCA solution and once with acetone to remove unreacted labeled putrescine. The filter paper was dried and placed in a vial containing scintillation solvent to measure radioactivity and measured with a scintillation counter. The results are shown in FIG.

(Example 3)
In this example, two peptide sequences (SEQ ID NO: 22 (pepT26) and SEQ ID NO: 59 (pepF11KA)) that were assumed to be highly reactive as substrates for transglutaminase in Example 2 were synthesized (consignment: finite). (Peptide Support Co., Ltd. Kitakyushu City) Note that pepF11KA is a peptide obtained by substituting the 11th K in the amino acid sequence of F11 with A. In addition, biotinylation modification was applied to the N-terminal side of the peptide. Were 94% (pepT26) and 93% (pepF11KA), both of which were dissolved in dimethyl sulfoxide (DMSO) to prepare a 100 mM solution and added to the reaction system.

[Binding mode of pepT26 and pepF11KA to milk protein casein]
The biotinylated peptide, transglutaminase (enzyme), and casein (as lysine-providing substrate) were mixed in a reaction buffer {10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM CaCl ₂ , 1 mM DTT}. The peptide in the reaction solution is 0.5 mM, and casein is 200 ng / μl. The enzyme was reacted by mixing TGase 2 at 0.5 ng / μl and Factor XIII at 5 ng / μl so that the enzyme had almost the same activity. 50 μl of the product reacted at different times is placed in a well of a 96-well (Maxisorp, Nalgenunc) plate. In the well, 50 μl of ice-cooled 0.5 M EDTA is placed in advance, and the reaction stops when mixed.

  Since the reaction product is coated (adsorbed) to the well, casein having more biotin molecules is adsorbed when a large amount of biotinylated peptide is introduced into casein by transglutaminase. Adsorption is completed by heating at 37 degrees for 1 hour. Thereafter, the whole well was washed with a washing solution (0.01% triton-X, 10 mM phosphate buffer (pH 8.0), 150 mM NaCl) three times. The amount of biotinylated can be determined by color development by adding avidin-binding peroxidase (Rockland) and a substrate for peroxidase (o-phenylenediamine). This is due to the affinity between avidin and biotin, and the more biotin that is adsorbed, the more peroxidase reaction occurs. Avidin-bound peroxidase was diluted 2000 times with a commercially available solution with a washing solution, and heated at 37 degrees for 1 hour. Further, a washing operation was performed to prepare a coloring solution in which o-phenylenediamine was dissolved in citrate buffer (pH 5.0) at 0.4 mg / ml, and hydrogen peroxide was added at a final concentration of 0.003%. 150 μl was added to the wells. After standing for several minutes at room temperature for color development, an equal amount of 2.5N sulfuric acid is added to stop the color development reaction, and this color development amount is measured using a spectrophotometer (absorbance 450 nm). The results are shown in FIG.

As shown in FIG. 10, it was found that PepT26 was incorporated into casein significantly more than pepF11KA in the TGase2 reaction. pepF11KA was incorporated into casein more than pepT26 in the enzyme reaction of FactorXIII, although the reactivity was somewhat low.

[Inhibitory effect on the reaction of TGase2 and FactorXIII]
Next, whether or not these peptides exhibited an inhibitory action on the reaction of TGase2 and FactorXIII was analyzed using the cross-linking of the substrate protein as an index.

As a substrate for TGase2, casein, a milk protein, is polymerized by cross-linking between molecules by an enzymatic reaction. Therefore, when analyzed by electrophoresis, a cross-linked high molecular weight protein band appears with a decrease in the original molecule (molecular weight 32,000 band). On the other hand, in Factor XIII, fibrin which is a physiological substrate (generated by fibrinogen cleaved by thrombin) was used as an index. Fibrin before cross-linking is composed of three kinds of molecules, α, β, and γ. Fibrin molecules newly generated by cross-linking are α polymer (multi-molecule bond), γ dimer (2 molecules). (In the figure, indicated as α-polymer, γ-γ).

The buffer used for the enzyme reaction is the same as the experimental conditions in FIG. 10 (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM CaCl ₂ , 1 mM DTT). In both cases, casein (Hanai Chemical Co., Ltd.) and fibrin (Calbiochem) were both added to a 200 μl reaction system at a final concentration of 200 ng / μl and reacted at 37 ° C. Factor XIII and fibrin are added as precursors, and bovine plasma-derived thrombin is simultaneously added to the reaction system for activation. The final concentration of the enzyme added is 0.5 ng / μl for TGase 2 and 5 ng / μl for Factor XIII. Here, pepT26 or pepF11 was added at different concentrations from 0.01 mM to 0.5 mM. At this time, DMSO as a solvent is added so that the final concentration is the same. In each reaction time, casein crosslinking with TGase2 was performed for 30 minutes, and fibrin crosslinking with FactorXIII was performed for 10 minutes. SDS (lauryl sulfate, final concentration 2%) and reducing agent (mercaptoethanol, final concentration 5%) are added to the reacted product, followed by heat denaturation. For casein, the gel concentration is 12.5%. For fibrin, 7.5% polyacrylamide electrophoresis was performed. After electrophoresis, the gel was stained by CBB (Coomassie Brilliant Blue) dye staining, decolorized with an acetic acid solution, and the degree of crosslinking was observed. The results are shown in FIG. In FIG. 11A, casein crosslinking reaction by TGase2, and FIG. 11B shows fibrin crosslinking reaction by Factor XIII.

As shown in FIG. 11, pepT26 and pepF11KA were added at different concentrations, and it was confirmed that their crosslinking (crosslinking polymerization) was inhibited. In these figures, the reaction labeled EDTA has a chelating agent (EDTA: sodium ethylenediaminetetraacetate) that captures calcium ions necessary for the cross-linking enzyme reaction, and the enzyme reaction is completely inhibited. Products appear in the situation. The right side shows the reaction product in which cross-linking occurs when no peptide is added. Thus, in FIG. 11 (a), pepT26 was significantly inhibited by addition of 0.1 mM final concentration, while pepF11KA was not inhibited at all. On the other hand, in FIG.11 (b), pepF11KA shows clear inhibition from 0.1 mM. Although the tendency to inhibit also to pepT26 is seen, when the reaction pattern in 0.1-0.5 mM is compared, pepF11KA clearly inhibits predominately. As described above, it was found that the peptide prepared based on the substrate recognition sequence exhibited an inhibitory effect specific to each isozyme in the crosslinking reaction with TGase2 and FactorXIII.

FIGS. 12 and 13 display numerical results of the results obtained in FIG. Since the intensity of the band detected by electrophoresis is proportional to the amount of the protein, the amount of protein present can be compared by quantifying the intensity of the band. FIG. 12 and FIG. 13 show the band obtained by scanning a specific protein band, converting it into an area using software “SCION” based on the quantified darkness, and digitizing it.

  As shown in FIG. 12, the inhibition efficiency of pepT26 to the crosslinking reaction by TGase2 is 30% at 0.01 mM and 80-100% at 0.05 mM to 0.5 mM. On the other hand, pepF11KA is hardly inhibited at 0-10%. In FIG. 13, the band (crosslinked γ-γ dimer) was numerically converted. From the viewpoint of the crosslinking rate (dimer formation rate), the inhibition rate is 15% for pepF11KA at 0.01 mM, about 40% at 0.1 mM, and 75% at 0.5 mM. PepT26 was not inhibited at 0.1 mM or less, but some inhibition was observed at 0.5 mM. Thus, it was shown that the optimal substrate sequence peptide has a competitive inhibitory activity with significant specificity against the original physiological reaction of protein cross-linking.

Example 4
In this example, a fusion protein of pepT26 peptide and GST and a fusion protein of pepT26 peptide and a single chain antibody against BSA (serum albumin) were prepared, and the activity of these fusion proteins was evaluated.

(Preparation of pepT26 peptide fusion protein)
The method for producing a fusion protein of a predetermined peptide and GST is as already described. However, in this example, since it is necessary to measure the GST enzyme activity, the glutamine residue in GST is not converted, and a pepT26 peptide is genetically engineered on the N-terminal side of the same GST protein as in nature. Fused.

(Preparation of pepT26-anti-BSA single chain antibody)
In MRC (UK Medical Research Council), a vector capable of expressing an antibody-like variable region protein against BSA (bovine serum albumin) (hereinafter, single-chain antibody) as a secreted recombinant protein in Escherichia coli was constructed by the phage display method. (Professor Hiroshi Ueda, professor of the University of Tokyo, modified the grant provided by MRC; plasmid name pIT2-29IJ6). This plasmid was modified to insert the corresponding synthetic oligonucleotide 3 'to the antibody gene so that a spacer sequence (GGGSGGGSGGGS), pepT26 and a histidine tag for purification could be added. Escherichia coli serving as a host was HB2151 strain, and expression induction as a recombinant protein was performed with 1 mM IPTG. However, expression in Escherichia coli was carried out at 30 ° C. for 16 hours, and the culture supernatant was concentrated by ammonium sulfate precipitation and purified almost uniformly using a TALON gel (BD Bioscience).

(3) Introduction of fluorescently labeled primary amine TGase can introduce an amine into a glutamine residue in a target amino acid sequence. T26-GST fusion protein (T26-GST) and T26 fusion BSA single chain antibody protein (antiBSA-T26) were reacted with fluorescently labeled primary amine for 30 minutes with TGase2. Thereafter, the fusion protein was recovered and electrophoresed, and fluorescence was observed. As a control, the enzyme reaction was similarly performed on the protein before fusion. Furthermore, in order to show that the uptake of the fluorescence-labeled amine was due to the TGase reaction, a system containing a calcium chelating agent (final concentration 5 mM EDTA) was also performed. The enzyme reaction conditions were as follows. These results are shown in FIG.
(1) Composition of reaction solution 10 mM HEPES · NaOH (pH 8.0), 150 mM NaCl, 1 mM DTT, 5 mM CaCl ₂ , 0.5 mM MDC (Monodansyl cadaverine, Sigma)
(2) Substrate protein final concentration 200 ng / μl each
(3) TGase concentration 5 ng / μl
(4) 37 ° C, 30 minutes

As shown in FIG. 14, fluorescence was observed in all fusion proteins comprising the pepT26 peptide, whereas no fluorescence was observed in the pre-fusion protein without the pepT26 peptide. From this, it was found that TGase introduced a fluorescently labeled primary amine to the pepT26 peptide portion of these fusion proteins. In addition, since no fluorescence was observed in the absence of calcium ions, it was supported that the primary amine was introduced into the pepT26 peptide moiety by TGase.

(Example 5)
In this example, GST mediated by the substrate peptide sequence, that is, the two fusion proteins prepared in Example 4 were immobilized on a solid phase carrier (gel) according to the scheme shown in FIG. As the gel for immobilization, NHS activated Sepharose manufactured by Pharmacia (GE Health Science) was used. This is one in which an amino group is covalently bonded to an N-hydroxysuccinimide group on the gel surface only by mixing with a molecule having a primary amino group, and is widely used for immobilizing proteins and peptides. In general, there are many primary amino groups such as lysine residues on the surface of proteins, and how they are bound cannot be predicted at all.

As shown in FIG. 15, octanediamine was immobilized on this gel by the following method. That is, 950 mM Tris-HCl (pH 8.0) buffer containing 50 mM octanediamine (1,8-octanediamine; Aldrich) was added per 1 ml of gel, and reacted at room temperature for 1 hour. After completion of the reaction, TBS (10 mM Tris-HCl, 150 mM NaCl) was added to wash and block the activated N-hydroxysuccinimide group.

Next, the pepT26-GST fusion recombinant protein produced in Example 4 was added to the gel, and TGase reaction was performed. The reaction solution was 10 mM HEPES · NaOH (pH 8.0), 150 mM NaCl, 1 mM DTT, 5 mM CaCl ₂ and TGase ₂ (derived from guinea pig liver, provided by Prof. Ikura, Kyoto Institute of Technology) was added at a concentration of 5 ng / μl. The enzyme reaction was carried out for 30 minutes at 37 ° C. with stirring. Thereafter, the enzyme reaction was terminated by adding EDTA to a final concentration of 50 mM EDTA. As a control, GST not having pepT26 peptide was similarly immobilized with TGase. The supernatant after completion of the enzyme reaction was collected and electrophoresed to detect the fusion protein and GST remaining without being immobilized on the gel. The results are shown in FIG. In addition, it preserve | saved at 4 degreeC until the enzyme activity as GST was measured.

FIG. 16 shows an electrophoretogram of the fusion protein and GST that remain without being immobilized in the supernatant after the immobilization reaction, and a graph diagram in which the band concentration is quantified. As shown in FIG. 16, the fusion protein was immobilized on the gel more efficiently than GST.

Further, the other fusion protein pepT26-anti-BSA single chain antibody prepared in Example 4 was immobilized on the gel in the same manner. The enzyme reaction conditions were the same as the enzyme reaction conditions for the pepT26-GST fusion recombinant protein in this example. This fusion protein was also immobilized on the anti-BSA single chain antibody as a control in the same manner as described above. Moreover, electrophoresis was similarly performed on the supernatant after completion of the enzyme reaction. The results are shown in FIG. After completion of the enzyme reaction, the enzyme was stored at 4 ° C. until the activity was measured.

As shown in FIG. 17, the pepT26-anti-BSA single chain antibody fusion protein was immobilized on the gel more efficiently than the anti-BSA single chain antibody.

(Example 6)
In this example, the enzyme activity of GST in a GST fusion protein immobilized on a gel was measured. A commercially available measurement kit (GST • Tag assay kit; Novagen) was used for the enzyme activity of GST. 10 μl of the gel was mixed with 200 μl of a substrate solution (1 mM glutathione, 1 mM chloro-2,4, -dinitrobenzene), and allowed to react at room temperature for 1 minute while stirring. Trichloroacetic acid (final concentration 2%) was added to terminate the enzyme reaction, and the absorbance (340 nm) was measured. As a comparative control, the pepT26-fused GST protein was directly immobilized on an NHS gel without forming a peptide bond with TGase. 18 and 19 show these results.

FIG. 18 shows the GST enzyme activity per gel, and FIG. 19 shows the GST activity (specific activity) per protein bound to the gel. From the results shown in FIG. 18, the enzyme activity was not significantly different for a certain amount of gel. When bound directly to the gel, it was equivalent to 2.7 μg of GST, and it was found that the gel bound by TGase via the pepT26 sequence has an activity equivalent to 3.2 μg of GST. Furthermore, when converted based on the amount of recombinant GST fusion protein binding per ml of gel, as shown in FIG. 19, the one immobilized with TGase and the substrate sequence is slightly more than the GST in a considerable amount of solution state. It was rising. On the other hand, when directly immobilized, the specific activity was lower than that in the solution state. The fusion protein immobilized by TGase was found to have a specific activity approximately 1.8 times higher than the directly immobilized fusion protein.

(Example 7)
In this example, the antigen-binding ability of pepT26-anti-BSA single chain antibody fusion protein immobilized on a gel was measured. A solution (HBS: 10 mM Hepes-NaOH (pH 8.0), 150 mM NaCl, 0) containing 12 μg each of BSA solution (100 μg / ml) as an antigen of this single-chain antibody and OVA (ovalbumin; 100 μg / ml) as a control. 1% tween 20) and a single-chain antibody-immobilized gel were mixed in a buffer and reacted at 37 ° C. for 1 hour. The gel was washed by repeating centrifugation and suspension with a buffer, and then the gel was heated and centrifuged in the presence of SDS buffer to obtain a supernatant. Since BSA bound to the gel is present in the supernatant, its abundance was analyzed by 12.5% SDS-PAGE and CBB staining. As a control, the pepT26-anti-BSA single chain antibody fusion protein was directly immobilized on an NHS gel without forming a peptide bond with TGase. The results are shown in FIG. The electrophoretic BSA pattern was analyzed by quantifying the abundance (amount bound to the gel) with Scion image software. The results are shown in FIG. 21 and FIG.

As shown in FIG. 17, the amount of BSA bound to the antibody was higher than that of the single-chain antibody fusion protein bound via pepT26. According to FIG. 21, in which this result was quantified as the amount of antigen bound per fixed amount of gel, the superiority of the antigen-binding ability of the single-chain antibody gel bound through pepT26 was apparent. Further, when quantified as the amount of bound antigen per amount of immobilized single-chain antibody, the superiority of the antigen-binding ability of the single-chain antibody immobilized via pepT26 was further evident.

It is a figure which shows the example of the artificial structure of this invention. It is a figure which shows another example of the artificial structure of this invention. It is a figure which shows the example of diamine. It is a list of sequences that are likely to be substrates for transglutaminase. It is a figure which shows the structure of a fusion protein. It is a figure (electrophoretic diagram) which shows evaluation as a substrate in the crosslinking reaction by TGase2 of a fusion protein and factor XIII. It is a figure (figure by a time course) which shows the evaluation as a substrate in the crosslinking reaction by TGase2 of a fusion protein and a factor XIII. It is a figure which shows the evaluation (reaction time 10 minutes) as a substrate in the crosslinking reaction by TGase2 and factor XIII of fusion protein. It is a graph which shows the uptake | capture result of the labeled primary amine (putrescine) to the fusion protein containing two types of amino acid sequences. It is a figure which shows the uptake | capture amount to the casein of 2 types of peptides. It is a figure (a) and (b) which shows inhibition of a crosslinking reaction by two kinds of peptides. It is a figure which shows the inhibitory effect with respect to TGase2 which made cross-linking of casein (amount of monomer) an index. It is a figure which shows the inhibitory effect (crosslinked product) with respect to the factor XIII which used the bridge | crosslinking of fibrin as a parameter | index. It is an electrophoretic diagram which shows the result of the presence or absence of the introduction | transduction of the fluorescence labeled amine by TGase with respect to the fusion protein in Example 4. It is a figure which shows the scheme which adds pepT26 fusion protein to the solid-phase carrier in Example 5. It is the electrophoretic diagram and graph which show the coupling | bonding result to the solid phase of pepT26GST fusion protein. It is the electrophoretic diagram and graph which show the coupling | bonding result to the solid phase of the pepT26 single chain antibody fusion protein. It is a graph which shows the GST activity per gel. It is a graph which shows the GST activity per GST protein fix | immobilized in the gel. It is an electrophoretic diagram which shows the amount of antigen (BSA) couple | bonded with the gel. The graph which shows the quantity of the antigen (BSA) couple | bonded with respect to 10 microliters gel. The graph which shows the quantity of the antigen (BSA) couple | bonded with respect to 1 microgram of antibodies fix | immobilized on the gel.

Sequence number: 1-60: Synthetic peptide

Claims (28)

A peptide comprising one or more amino acid sequences selected from the amino acid sequences shown in Tables 1 and 2 below and modified sequences thereof.
  The peptide according to claim 1, which has a substrate activity of transglutaminase.   The peptide according to claim 1 or 2, which is a transglutaminase inhibitor.   The peptide according to claim 2 or 3, wherein the transglutaminase is blood coagulation factor XIII and / or tissue type transglutaminase.   Tissue type trans, comprising one or more amino acid sequences selected from T2, T5, T8, T12, T16, T20, T26, T29, T30, T32, F6, F17, F28 and F32 and their modified sequences The peptide according to any one of claims 1 to 4, which is a glutaminase inhibitor.   Tissue type transglutaminase inhibition comprising one or more amino acid sequences selected from T2, T5, T8, T12, T16, T20, T26, T29, T30, T32, F6, F17 and F28 and their modified sequences The peptide according to any one of claims 1 to 4, which is an agent.   The peptide according to claim 5 or 6, comprising one or more amino acid sequences selected from T2, T5, T8, T16, T20, T26, T29, and T32 and modified sequences thereof.   The peptide according to claim 7, comprising the amino acid sequence set forth in SEQ ID NO: 22 and a modified sequence thereof.   The peptide of Claim 8 which consists of an amino acid sequence of sequence number: 22.   The amino acid sequence is a blood coagulation factor XIII inhibitor comprising one, two or more amino acid sequences selected from T1, T30, F6, F11, F17, F28 and F32 and modified sequences thereof. The peptide in any one of 1-4.   The amino acid sequence is a blood coagulation factor XIII inhibitor comprising one or more amino acid sequences selected from T1, T30, F6, F11, F17 and F28 and modified sequences thereof. 5. The peptide according to any one of 4.   The peptide according to claim 10 or 11, wherein the amino acid sequence includes one or more amino acid sequences selected from T1 and F11 and modified sequences thereof.   The peptide according to claim 12, comprising one or more amino acid sequences selected from the amino acid sequences set forth in SEQ ID NO: 38 and SEQ ID NO: 59 and modified sequences thereof.   The peptide of Claim 13 which consists of an amino acid sequence of sequence number: 38 or sequence number: 59. A method for searching for a peptide having reactivity as a substrate of transglutaminase, comprising:
The following steps (a) to (c):
(A) reacting a peptide displayed using a phage display method with a compound containing a primary amine in the presence of transglutaminase,
(B) separating the phage retaining the transglutaminase reaction product in the step (a), and (c) determining the amino acid sequence of the peptide displayed in the separated phage. A method for searching for a peptide having transglutaminase inhibitory activity,
About two or more isozymes of transglutaminase
The following steps (a) to (c):
(A) reacting a peptide displayed using a phage display method with a compound containing a primary amine in the presence of transglutaminase,
(B) a step of separating the phage retaining the transglutaminase reaction product in the step (a), and (c) a step of determining the amino acid sequence of the peptide displayed in the separated phage,
Carried out
(D) A search method for carrying out a step of evaluating reaction specificity of the peptide comprising an amino acid sequence obtained as having the substrate reactivity of the two or more isozymes with respect to the two or more isozymes. A method for producing a peptide having transglutaminase inhibitory activity, comprising:
About two or more isozymes of transglutaminase
The following steps (a) to (c):
(A) reacting a peptide displayed using a phage display method with a compound containing a primary amine in the presence of transglutaminase,
(B) a step of separating the phage retaining the transglutaminase reaction product in the step (a), and (c) a step of determining the amino acid sequence of the peptide displayed in the separated phage,
Carried out
(D) evaluating the reaction specificity of the peptide comprising an amino acid sequence obtained as having substrate reactivity of the two or more isozymes to the two or more isozymes; (e) in step (d) Producing a peptide evaluated to have reaction specificity for any of the two or more isozymes or a modified peptide thereof;
A manufacturing method comprising: An artificial construction,
An artificial construct comprising a peptide comprising one or more amino acid sequences selected from the amino acid sequences shown in Tables 3 and 4 below and modified sequences thereof.
  The apparatus further comprises a linking portion formed by an isopeptide bond formed between a γ-carboxyamide group of a glutamine residue side chain of the peptide chain and the primary amino group of a primary amino group-containing compound. The described artificial construction.   The artificial structure according to claim 19, wherein the primary amino group-containing compound is a compound containing two or more primary amino groups.   21. The artificial construct according to claim 20, wherein the primary amino group-containing compound is one or more selected from the group consisting of cataverine, spermidine, spermine and cardopentamine.   The artificial construct according to any one of claims 18 to 21, wherein one or more other peptide chains are linked to the peptide chain via the linking part.   The artificial construct according to any one of claims 18 to 22, which has an artificial antibody portion.   The artificial structure according to any one of claims 16 to 21, comprising a solid phase carrier via the connecting portion.   The artificial construct according to claim 24, which is a protein-immobilized solid phase carrier.   26. The artificial construct according to any one of claims 18 to 25 having a pharmaceutically active moiety. A composition for prevention / treatment of transglutaminase-related diseases, comprising a peptide comprising one or more amino acid sequences selected from Table 5 and Table 6 below or a modified sequence thereof.
  The composition for prevention and treatment according to claim 27, wherein the peptide consists of any amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 38 and SEQ ID NO: 59.