JP4251399B2 - Screening method for peptides to which O-linked sugar chains are added - Google Patents

Description

  The present invention relates to a method for identifying and analyzing a peptide to which an O-linked sugar chain is added.

  It is known that post-translational modification of proteins, especially addition of sugar chains, plays an important role in protein functions. Sugar chains are (1) for controlling protein activity by determining the higher-order structure of the protein, (2) as a tag to identify the protein, for quality control of the protein and for intracellular transport, and (3) as a direct information molecule for the cell. It is becoming clear that it plays an important function in intercommunication. In addition, cases in which congenital diseases, cancer, immunodeficiency, etc. are caused by abnormal sugar chain modification have been known. Therefore, in order to elucidate the function and control of glycoproteins, it is necessary to analyze the structure of glycosylation.

  Protein sugar chain modification is broadly divided into N-type sugar chains starting with glucosamine (GlcNAc) bound to asparagine residues and O-linked sugar chains starting with galactosamine (GalNAc) bound to serine / threonine residues. Is done. For the N-type sugar chain, the enzyme group involved in the synthesis of the basic structure is almost determined, and the consensus sequence at the additional position is also known. On the other hand, for O-linked sugar chains, an enzyme that specifically releases GalNAc O-linked to serine or threonine residues is not known, and O-linked sugar chains are removed from glycoproteins under non-denaturing conditions. It was extremely difficult. For this reason, the structure and function of sugar chains have not yet been elucidated, and no consensus sequence has been found. The enzyme that first adds GalNAc to a protein is called ppGalNAcT (UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferase). Based on the achievements of the genome project, the gene's Analysis is ongoing. As a result, it has been clarified that there are 20 types of ppGalNAcT and their expression patterns vary depending on tissues.

  To determine where in the protein an O-linked sugar chain is added, digest the glycoprotein with protease and separate the different fragments by HPLC, then determine the amino terminal sequence of each fragment, or HPLC and A method of determining the sequence of a peptide by mass spectrometry has been used. However, since this method requires a lot of labor and time, it is not suitable for analyzing many types of glycoproteins. Therefore, in this technical field, development of a method for comprehensively analyzing O-linked glycoprotein expressed in a specific organism or a specific tissue is required.

Prior art document information related to the present invention includes the following.
Van denSteen, et al., Trends in Glycosicence and Glycotechnology, Vol. 12 (63), 35-49,2000

  An object of the present invention is to provide a method capable of comprehensively analyzing a peptide to which an O-linked sugar chain is added.

The present invention is a method for identifying a peptide to which an O-linked sugar chain is added,
(A) producing a peptide that can serve as a substrate for glycosyltransferase in a protein expression system in which a sugar chain cannot be added;
(B) adding an O-linked sugar chain by allowing a glycosyltransferase to act on the peptide;
(C) selecting a peptide to which an O-linked sugar chain has been added;
(D) analyzing the structure of the glycosylated peptide;
The method including each of these steps is provided.

  In a preferred embodiment, the production of the peptide in step (a) of the method of the present invention is performed by displaying the peptide on the phage surface. Also preferably, the expression of the peptide in the step (a) is performed by displaying the peptide on the surface of the phage, and the analysis of the structure of the peptide in the step (d) is performed by the sequence of the DNA inserted into the phage. This is done by analyzing Preferably, the method of the present invention further includes a step of analyzing the structure of the O-linked sugar chain added to the glycosylated peptide.

  In the method of the present invention, first, a peptide that can serve as a substrate for glycosyltransferase is produced in a protein expression system in which a sugar chain cannot be added. As used herein, the “peptide” may be a short peptide consisting of several amino acid residues, a protein, or a protein fragment. As the glycosyltransferase, any enzyme may be used as long as it has an activity of adding GalNAc, GlcNAc, Man, Fuc, Glu or Xyl to the serine residue or threonine residue of the peptide. For example, ppGalNAcT, O-GlcNAcT (OGT), O-FucT, XylT (XT-1), or the like can be used. The glycosyltransferase may be a natural enzyme extracted from animals, plants, microorganisms, etc., may be produced using genetic recombination technology, or may be an artificially created enzyme mimic. Good.

  In particularly preferred embodiments, the methods of the invention are performed using a phage display system. First, DNA encoding a peptide to be analyzed is incorporated into the phage genome, and the peptide is displayed on the surface of the phage by infecting the phage with a host according to a conventional method and allowing it to propagate. The DNA may be a cDNA library derived from a specific organism or a specific organ, or may be a library of synthetic oligonucleotides. Phage display is a technique for displaying a peptide on the surface of a phage as a fusion protein with a capsid protein of a phage, and is generally used for screening a peptide that binds to a receptor. Various phage vectors for peptide display are commercially available, and can be appropriately selected and used depending on the size of the cDNA library and the size of the protein to be displayed. An example of such a phage vector is T7Select® phage (Novagen). It should be noted that at this time, the phage capsid protein displayed on the surface of the phage must not contain an amino acid sequence which is a target to which a glycosyltransferase first adds a sugar.

  Particularly preferably, a phage displaying a multi-copy peptide on the surface of the phage can be used according to the method described in JP-A-2004-89069. Briefly, a recombinant vector for phage display in which a stop codon is inserted between a DNA encoding a phage coat protein and a DNA encoding a target peptide, and a suppressor tRNA suitable for the stop codon is encoded. A recombinant vector containing DNA is prepared, a suppressor tRNA is introduced and expressed in the host body to be infected, and the suppressor activity of the suppressor tRNA to be used is controlled by an appropriate method. By using this method, phages that display multiple copies of larger size peptides can be obtained.

Next, an O-linked sugar chain is added by allowing glycosyltransferase to act on the peptide. As the conditions for the action of glycosyltransferase, conditions suitable for each glycosyltransferase can be used. For example, TBST containing 5 mM MnCl ₂ and 250 μM UDP-GalNAc (10 mM TB (pH 7.4), 0.15 M) It can be carried out by incubating in a buffer such as NaCl, 0.1% Tween 20) at 37 ° C. for 2 to 24 hours. When a phage display system is used, an O-linked sugar chain can be added to the peptide displayed on the phage surface by allowing a glycosyltransferase to act on the phage.

  Next, a peptide to which an O-linked sugar chain is added is selected. Selection can be made by using a lectin, for example, by adsorbing a peptide having glycosyltransferase acting on a column on which the lectin is immobilized, washing it appropriately, and adding an added sugar or a sugar having a similar lectin binding activity, for example, , Elution using GalNAc, galactose or the like. The lectin can be appropriately selected according to the type of O-linked sugar chain to be added. For example, when a peptide to which GalNAc is added is selected, a preferred lectin is jacalin. Moreover, after modifying the sugar chain couple | bonded with the peptide with biotin, you may carry out by collect | recovering biotin modification sugar chain binding peptides using avidin, streptavidin, etc.

  For example, as described in the Examples below, Muc5AC peptides and IgA hinge peptides are used as a model system, oligonucleotides encoding these peptides are incorporated into phages and the peptides are expressed on the phage surface, and the phages are ppGalNAcT It is possible to recover the GalNAc-containing glycopeptide by treating with the above and purifying with a jacarin column.

  Next, the structure of the glycosylated peptide thus selected is analyzed. HPLC, amino acid sequencer, mass spectrometry (MS) and the like are used to identify amino acid residues to which sugar chains on peptides are added. In the case of using a phage display system, the phage obtained according to the above-mentioned method is collected, and then the sequence of the DNA inserted into the genome is analyzed, whereby the peptide to which the O-linked sugar chain is added is analyzed. Determine the amino acid sequence. The sequence of the inserted DNA can be easily determined using, for example, a primer designed based on the sequence near the DNA insertion site in the phage genome.

  In this embodiment, various DNA other than the cDNA library can be used as the DNA to be incorporated into the phage. For example, in order to map the glycosylation position of a specific protein, a fragment obtained by cleaving DNA encoding the protein, a library of fragments produced with random primers using DNA as a template, and the like can be used. . In addition, oligonucleotides encoding peptides into which site-specific mutations or random mutations have been introduced may be used in order to determine the characteristics of each glycosyltransferase and to study recognition sequences.

  According to this method, a peptide to which an O-linked sugar chain has been added by a specific glycosyltransferase is purified using a lectin column or the like, and an O-linked sugar chain is analyzed by analyzing the gene inserted into this phage. Can be identified. Furthermore, by using a cDNA library derived from a specific cell or tissue or a specific organism, it is possible to comprehensively identify proteins modified by glycosyltransferases. That is, the present invention is useful for analyzing the characteristics and action mechanisms of glycosyltransferases, analyzing the structure, function and activity of glycoproteins, and analyzing intracellular and intercellular signal transduction via glycoproteins.

  Furthermore, the structure of the added sugar chain can be analyzed from the glycosylated peptide obtained according to the method of the present invention. The analysis of the sugar chain structure can be carried out using methods well known in the art, such as assays using various lectins and sugar chain-specific antibodies, and mass spectrometry.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Preparation of Peptide Displaying Phage Clones Oligonucleotides having the following sequences were synthesized based on the sequences of Muc5AC peptide and IgA hinge peptide.

  This oligonucleotide was incorporated in-frame into the EcoRI-HindIII site of T7Select (registered trademark) 10-7 phage (Novagene). T7Select (registered trademark) is a phage display system based on bacteriophage T7, and a peptide encoded by DNA incorporated into a multicloning site can be expressed on the phage surface as a fusion protein with a phage capsid protein.

  Phages were grown according to a conventional method, recovered by plate lysate, and concentrated by polyethylene glycol precipitation to purify the phages. The phage was dialyzed against TBST (10 mM TrisHCl (pH 7.4), 0.15 M NaCl, 0.1% Tween-20).

  Secreted human ppGalNAcT3 was synthesized in a baculovirus system and purified according to the method described in the literature (FEBS Letters, 531, 115-121 (2002)). In order to confirm the activity of ppGalNAcT, a synthetic mucin peptide with a fluorescent label was used as a substrate, and analysis was performed based on the shift of the elution position by sugar chain modification using HPLC.

The reaction was carried out under the conditions shown in the following table.

  After reacting at 37 ° C. for 12.5 hours with stirring, the reaction was stopped by adding 10 μl of 0.5 M EDTA for positive control and 450 μl of TBST for others.

  Phage reacted with ppGalNAcT and jacalin agarose (Vector Laboratories, UK) were treated in TBST at 4 ° C. overnight, packed in a minicolumn, washed with a large excess of TBST, and eluted with 10 mM GalNAc. The number of phages contained in each fraction was counted. The results are shown in FIG.

  For both the Muc5AC peptide and the IgA hinge peptide, the GalNAc-containing glycopeptide was eluted from the jacalin column. From this, it was shown that the glycosylated peptide displayed on the phage surface can be isolated using the method of the present invention.

FIG. 1 shows the purification of GalNAc-added peptide using a jacalin column.

Claims (2)

A peptide library prepared using a phage display system is allowed to act on an O - linked glycosyltransferase, and a peptide to which an O - linked sugar chain has been added is converted using a lectin that recognizes the corresponding sugar chain. selected, and wherein the analyzing child nucleotide sequence of the DNA in the phage that display a sugar chain added peptide, O - a method of identifying and / or analyzing a peptide-linked sugar chain is added. A kit for identifying and / or analyzing a peptide to which an O-linked sugar chain has been added, comprising the following (a) to (c);
(A) a peptide library prepared using a phage display system;
(B) an O-linked glycosyltransferase,
(C) A lectin that recognizes a sugar chain added by the enzyme of (b).