WO2023163040A1

WO2023163040A1 - SUGAR CHAIN-MODIFIED α1-MICROGLOBULIN AND PERFORMANCE EVALUATION REAGENT FOR BLOOD PURIFIER

Info

Publication number: WO2023163040A1
Application number: PCT/JP2023/006450
Authority: WO
Inventors: 隆木村; 泰史谷; 隆太戸部
Original assignee: ニプロ株式会社
Priority date: 2022-02-28
Filing date: 2023-02-22
Publication date: 2023-08-31

Abstract

This sugar chain-modified α₁-microglobulin is used for the performance evaluation of a blood purifier, contains a core glycan structure common in an N-type sugar chain, and has a modified N-type sugar chain in which an N-acetylneuraminic acid residue is not bonded to a non-reducing terminal. As a result, it is possible to produce a recombinant of α₁-microglobulin, which can be used for evaluating the performance of the blood purifier, by using a general method, and it is also possible to aspire to mass-production.

Description

Glycan variant α1-microglobulin and performance evaluation reagent for blood purifier

The present invention relates to sugar chain variant α ₁ -microglobulin and a reagent for evaluating the performance of a blood purifier using the same.

α ₁ -microglobulin is a glycoprotein having a molecular weight of about 30,000 and a sugar content of about 20% by mass, and is known to have an antioxidant action as a radical scavenger. α ₁ -microglobulin is produced mainly in the liver, passes through the renal glomerulus and is reabsorbed from the proximal renal tubules, so that it is normally hardly excreted in the urine. Therefore, the content of α ₁ -microglobulin in urine is known to serve as a marker for renal tubular and renal glomerular disorders.

α ₁ -microglobulin is also known as one of the low-molecular-weight proteins to be removed in blood purification. Therefore, performance evaluation of blood purifiers such as dialyzers or hemodiafilters may test the ability to remove α ₁ -microglobulin.

For example, Patent Document 1 discloses that, in a method for manufacturing a blood purifier, the clearance of α ₁ -microglobulin in the membrane area of a hollow fiber membrane provided in the blood purifier is set to a predetermined value or more. Specifically, in the examples of the document, a bovine plasma solution with an adjusted concentration of α ₁ -microglobulin is prepared, and the clearance of α ₁ -microglobulin in a blood purifier is measured (evaluated).

Japanese Patent No. 6030295

Here, commercially available α ₁ -microglobulin is not only very expensive but also difficult to obtain. Since a blood purifier is used to purify a large amount of blood, it is not realistic to use α ₁ -microglobulin for the purpose of evaluating the performance of the blood purifier. Therefore, for evaluating the performance of blood purifiers, other proteins, such as prolactin (molecular weight about 22 kDa, no glycosylation), which are considered to have similar removal behavior to α ₁ -microglobulin, have been substituted.

Needless to say, it is better to use α ₁ -microglobulin than other substitute proteins to evaluate the performance of blood purifiers. In general, as a method for producing proteins at low cost, it is assumed that a recombinant protein production system is constructed using genetic engineering techniques. However, a recombinant α ₁ -microglobulin that can be used for evaluating the performance of blood purifiers has not been known so far.

The present invention was made to solve such problems, and an object of the present invention is to provide a recombinant α ₁ -microglobulin that can be used for performance evaluation of blood purifiers.

In order to solve the above problems, the sugar chain variant α ₁ -microglobulin according to the present disclosure contains a core glycan structure common to N-type sugar chains and has an N-acetylneuraminic acid residue at the non-reducing end. It is a configuration having a mutant N-type sugar chain to which no groups are attached.

According to the above configuration, the sugar chain variant α ₁ -microglobulin has an N-type sugar chain structure that is not the same as the original N-type sugar chain structure, and has a non-reducing terminal N-acetylneuraminic acid It can be used to evaluate the performance of blood purifiers even though it does not have residues.

If it is not necessary to transglycosylate sialic acid such as N-acetylneuraminic acid to the non-reducing end of the sugar chain, genetic engineering techniques can be used using a common yeast that does not have a sialic acid biosynthetic pathway as a host. It becomes possible to construct a recombinant protein production system by using As a result, recombinant α ₁ -microglobulin, which can be used for performance evaluation of blood purifiers, can be produced using a general method, and mass production can be aimed at. The possibility of producing recombinant forms of ₁ -microglobulin at low cost can also be found.

In the sugar chain variant α ₁ -microglobulin having the above configuration, the mutant N-type sugar chain has two non-reducing ends in the core glycan structure represented as Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc. The configuration may include a sugar chain structure in which a mannose residue or an N-acetylglucosamine residue is bound to at least one of the mannose residues.

Further, in the sugar chain variant α ₁ -microglobulin having the above configuration, the mutant N-type sugar chain is expressed as Manα1-3(Manα1-3(Manα1-6)Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc. A configuration including a pentamannosyl structure may also be used.

Further, in the sugar chain variant α ₁ -microglobulin having the above configuration, the mutant N-type sugar chain is added to at least one of the three mannose residues at the non-reducing end of the pentamannosyl structure, A structure containing a sugar chain structure in which a group or an N-acetylglucosamine residue is glycoside-linked may also be used.

Further, in the sugar chain variant α ₁ -microglobulin having the above configuration, the mutant N-type sugar chain has N-acetyl It may have a structure including a sugar chain structure in which a glucosamine residue is bound and a galactose residue or a fucose residue is bound to the N-acetylglucosamine residue.

In addition, in the sugar chain variant α ₁ -microglobulin having the above configuration, even if it is produced by expressing the α ₁ -microglobulin gene in sugar chain variant yeast that does not cause hyperglycosylation. good.

Further, in the sugar chain variant α ₁ -microglobulin having the above configuration, the sugar chain variant yeast may be Pichia pastoris.

Further, in the sugar chain variant α ₁ -microglobulin having the above configuration, the sugar chain variant yeast may be SuperMan-5 strain (product name).

The present disclosure also includes a performance evaluation reagent for a blood purifier containing the sugar chain variant α ₁ -microglobulin having the above configuration. Furthermore, the present disclosure also includes a method for evaluating the performance of a blood purifier using the sugar chain variant α ₁ -microglobulin having the above configuration.

The above object, other objects, features, and advantages of the present invention will be made clear from the following detailed description of preferred embodiments with reference to the accompanying drawings.

The present invention has the effect of providing a recombinant α ₁ -microglobulin that can be used for performance evaluation of a blood purifier by the above configuration.

FIG. 1A shows a sugar chain structure of sugar chain variant α ₁ -microglobulin according to a representative embodiment of the present disclosure and a typical sugar chain structure of human-derived α ₁ -microglobulin. FIG. 1B is a schematic diagram for comparison, and FIG. 1B is a schematic diagram showing an example of a sugar chain structure when α ₁ -microglobulin is hyperglycosylated in common yeast. FIG. 2 is a schematic diagram showing representative variations of the mutant sugar chain shown in FIG. 1A. FIG. 3 is a schematic diagram showing representative variations of the mutant sugar chain shown in FIG. 1A. FIG. 4 shows electrophoresis results of mutant α ₁ -microglobulin in a representative example of the present disclosure.

Hereinafter, representative embodiments of the present invention will be described with reference to the drawings. In the following description, the same or corresponding elements are denoted by the same reference numerals throughout all the drawings, and duplicate descriptions thereof will be omitted.

[Sugar chain variant α ₁ -microglobulin]
α ₁ -Microglobulin and its family are known to exist in mammals including humans, birds, amphibians, fishes and the like. Human-derived α ₁ -microglobulin is generally a glycoprotein of about 33 kDa (molecular weight of about 30,000) containing two N-type sugar chains, the protein itself being about 21 kDa. α ₁ -microglobulin is mainly synthesized in the liver and secreted into blood, and is widely distributed in body fluids including blood. Since α ₁ -microglobulin has a low molecular weight, it easily passes through the renal glomerulus of the kidney from the blood, but is reabsorbed from the proximal renal tubules, so it is normally hardly excreted in the urine.

The amino acid sequence of human-derived α ₁ -microglobulin (hereinafter simply referred to as α ₁ -microglobulin) is known as shown in SEQ ID NO: 1 in the sequence listing, and consists of 183 amino acid residues. N-glycans are attached to the 17th and 96th asparagine (Asn, N) residues. The amino acid sequence shown in SEQ ID NO: 1 is truncated.

FIG. 1A schematically shows the sugar chain structure of the N-type sugar chain possessed by α ₁ -microglobulin. Of the three sugar chain structures shown in FIG. 22 is an example of a representative sugar chain structure of a human-derived N-type sugar chain. These

glycans

21 or 22 are referred to as "human-derived glycans 21" or "human-derived glycans 22" for convenience of explanation.

In addition, as will be described later, the glycan 20 shown on the left side of the paper surface of the sugar chain structure shown in FIG. 1A is a typical example of the N-type sugar chain possessed by the sugar chain variant α ₁ -microglobulin according to the present disclosure. . This glycan 20 is referred to as mutated glycan 20 for convenience of explanation.

In the sugar chain structures schematically shown in FIGS. 1A to 3, among monosaccharide residues, N-acetylglucosamine (GlcNAc) is indicated by a rectangular symbol 11, and mannose (Man) is indicated by a circular symbol 12. Galactose (Gal) is indicated by the double circle symbol 13, N-acetylneuraminic acid (NeuNAc) is indicated by the diamond symbol 14 and fucose (Fuc) is indicated by the triangular symbol 15. In addition, in FIGS. 1A to 3, the sugar chain structure surrounded by dotted lines indicates the core glycan structure 10 of the N-type sugar chain. In FIGS. 1A-3, asparagine residues (Asn) or peptides are not shown as specific symbols, but simply shown as thick curves.

The core glycan structure 10, also sometimes referred to as the tri-mannosyl core structure, is shown in FIG. An N-acetylglucosamine residue 11 and a mannose residue 12 are bonded in this order, and two mannose residues 12 are bonded to this mannose residue 12 in a branched manner.

Although not specifically illustrated in FIG. 1A (and FIGS. 1B to 3), the first N-acetylglucosamine residue 11 is glycosidic-bonded to the asparagine residue in the β-form, and the first The second N-acetylglucosamine residue 11 to the N-acetylglucosamine residue 11 is glycosidically bonded in the β1-4 type, and the first to the second N-acetylglucosamine residue 11 The mannose residue 12 is β1-4 type and is glycosidic bonded, and the mannose residue 12 is glycosidic bonded so that the two mannose residues 12 are each branched, but one mannose residue 12 is bound in the α1-3 form and the other mannose residue 12 is bound in the α1-6 form.

This core glycan structure 10 can be described as Manα1-6 (Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAcβAsn, in order from the non-reducing end to the reducing end, and the monosaccharide residues as general abbreviations. In this method of describing the sugar chain structure, the type of glycosidic bond is indicated by the carbon position of the 6-membered ring to which it is bound together with the bond type of α or β, and the branched chain is indicated in parentheses. .

Various methods have been proposed for notation of sugar chain structures, that is, sugar chain nomenclature, including the nomenclature recommended by the IUPAC (International Union of Pure and Applied Chemistry). However, at present, no internationally standardized sugar chain nomenclature can be confirmed, so in the present specification (and the scope of claims), especially from the viewpoint of facilitating recognition of monosaccharide residues, it is commonly used The sugar chain structure is described based on the description method described above (hereinafter simply referred to as "this sugar chain description method"). When describing only the sugar chain structure, the description of Asn β-bonded to the sugar chain residue at the reducing end is omitted.

In the future, when an internationally standardized sugar chain nomenclature is established, in the present specification (and the scope of claims), the sugar chain structure described based on the above-described present sugar chain description system will be Descriptions in standardized carbohydrate nomenclature can be substituted.

Human-derived glycan 21 shown in the center of FIG. Galactose residue 13 is bound to -acetylglucosamine residue 11, and N-acetylneuraminic acid residue 14 is bound to the galactose residue 13. In addition, N-acetylglucosamine residue 11 is branched and bound to mannose residue 12 serving as a branching source of coagulan structure 10, and N-acetylglucosamine residue 11 (which binds to asparagine) is a reducing end. , the fucose residue 15 is branched and bound.

Therefore, human-derived glycan 21 has four branched chains on the non-reducing end side, and has a sugar chain structure containing two branched chains each consisting of one monosaccharide residue in core glycan structure 10 . This type of sugar chain structure is called a “complex type” that does not contain mannose residues 12 other than the core glycan structure 10 .

In the human-derived glycan 22 shown on the right side of FIG. 1A, one N-acetylglucosamine residue 11 is bound to each of two mannose residues 12 that are the non-reducing ends of the core glycan structure 10 . Galactose residue 13 is bound to each of these N-acetylglucosamine residues 11, and N-acetylneuraminic acid residue 14 is bound to the galactose residue 13, human-derived glycan 21 is similar to In addition, human-derived glycan 22 is similar to human-derived glycan 21 in that fucose residue 15 is branch-bonded to N-acetylglucosamine residue 11, which is the reducing end. The N-acetylglucosamine residue 11 is not bound to the mannose residue 12 that becomes .

Therefore, the human-derived glycan 22 has two branched chains on the non-reducing end side, and has a sugar chain structure in which the core glycan structure 10 includes only one branched chain consisting of one monosaccharide residue. This human-derived glycan 22 is also “complex” because it does not contain mannose residues 12 other than core glycan structure 10 .

Here, for the convenience of describing the sugar chain structure, the mannose residue 12 that serves as the starting point for branching of the core glycan structure 10 is referred to as the "branch point mannose residue 12", and α1-3 type is used for the branch point mannose residue 12. The binding mannose residue 12 shall be referred to as the "α3 mannose residue 12" and the mannose residue 12 which binds in the α1-6 form to the branch point mannose residue 12 shall be referred to as the "α6 mannose residue 12". .

In human-derived glycan 21, for α3 mannose residue 12, two N-acetylglucosamine residues 11 are bound in the β1-2 or β1-4 form, respectively, and for α6 mannose residue 12, Thus, two N-acetylglucosamine residues 11 are linked in the β1-2 or β1-6 form, respectively. In human-derived glycan 22, one N-acetylglucosamine residue 11 is linked to both α3 mannose residue 12 and α6 mannose residue 12 in the β1-2 form.

In both human-derived glycan 21 and human-derived glycan 22, galactose residue 13 is linked to N-acetylglucosamine residue 11 in the β1-3 form, and N-acetylneuraminic acid residue 14 is , bound to galactose residue 13 in the α2-3 form. In addition, the branched fucose residue 15 is bound to the N-acetylglucosamine residue 11 in the α1-6 type, and the branched N-acetylglucosamine residue 11 in the human-derived glycan 21 is the branch point It binds to mannose residue 12 in the β1-4 form.

In addition, in FIG. 1A, for convenience of illustration, the types of glycosidic bonds are basically not described. However, with respect to the mutant glycan 20 shown on the left side in FIG. 1A, the type of glycosidic bond is added in the abbreviated form of “α3” or “α6” for the branched portion of the mannose residue 12 in the core glycan structure 10 . “α3” indicates an α1-3 type glycosidic bond, and “α6” indicates an α1-6 type glycosidic bond.

Human-derived glycan 21 or human-derived glycan 22 shown in FIG. 1A is a representative example of the N-type sugar chain possessed by human-derived α ₁ -microglobulin, and wild-type α ₁ -microglobulin. does not necessarily have the sugar chain structure shown in human-derived glycan 21 or human-derived glycan 22. Similarly, the bond type between monosaccharide residues in human-derived glycan 21 or human-derived glycan 22 is also a representative example. does not necessarily match the bond types described above.

α ₁ -microglobulin is one of the indicators of removal ability in evaluating the performance of blood purifiers such as dialyzers and hemodiafilters. However, commercially available α ₁ -microglobulin is very expensive and difficult to obtain. Therefore, with the aim of producing α ₁ -microglobulin at low cost, it is envisioned to construct a recombinant protein production system using genetic engineering techniques.

However, α ₁ -microglobulin is a glycoprotein as described above. Normally, proteins are not glycosylated in prokaryotic cells such as E. coli even if they are expressed. In order to produce a recombinant α ₁ -microglobulin, it is considered preferable to select prokaryotic cells as a host from the viewpoint of productivity. is not suitable as a host. Therefore, eukaryotic cells that allow post-translational modifications, including sugar chain modifications, to the expressed proteins should be selected as hosts.

Yeast can be mass-cultured together with eukaryotic cells like prokaryotic cells such as E. coli. Other eukaryotic cells include, for example, cultured cells of animals and plants, but such cultured cells of animals and plants require an expensive culture solution, are slow to grow, and are more difficult to grow than unicellular organisms. Concerns such as relatively strict legal regulations are known. Therefore, it is considered that yeast is suitable as a host for producing a recombinant α ₁ -microglobulin.

However, as a result of investigations by the present inventors, when trying to produce a recombinant α ₁ -microglobulin using yeast as a host, α ₁ -microglobulin expressed in yeast is hyperglycosylated (or hyperglycosylated). It was found that excessive sugar chain modification called mannosylation occurs.

Glycan 23 shown in FIG. 1B is an example of a sugar chain structure when hyperglycosylation occurs. In glycan 23, two mannose residues 12 bind to α6 mannose residue 12 in the core glycan structure 10, and one of these mannose residues 12 has mannose residue 12 and mannose phosphate residue. 16 are bound.

On the other hand, the α3 mannose residue 12 has the same binding structure as the α6 mannose residue 12 in that two mannose residues 12 are directly bound. On the other hand, a large number of mannose residues 12 are bonded in series, branched chains in which a plurality of (for example, three) mannose residues 12 are bonded in series branch from each mannose residue 12, and partially mannose residues 12 Acid residue 16 is attached.

The sugar chain structure that binds to this α3 mannose residue 12 is referred to as a “hypermannose structure 120” for convenience of explanation, and is shown surrounded by a dashed line in FIG. 1B. Some hypermannose structures 120 have 100 or more mannose residues 12, and the number of mannose residues 12 is not constant. Therefore, the molecular weight of hyperglycosylated α ₁ -microglobulin varies. As shown in the examples below, when hyperglycosylated α ₁ -microglobulin is electrophoresed, it is not identified as a relatively distinct single band, but is continuously elongated along the direction of electrophoresis. A blurred band (a so-called smear state) results.

Thus, hyperglycosylation of α ₁ -microglobulin not only increases the molecular weight of α ₁ -microglobulin compared to standard human-derived α 1 -microglobulin, but also causes variation in the molecular weight itself. become. This makes it difficult to use the blood purifier to evaluate the ability to remove α ₁ -microglobulin.

Another concern is that sialic acid is not bound to the non-reducing end of the sugar chain structure when attempting to produce a recombinant α ₁ -microglobulin using yeast as a host. Among sialic acids, N-acetylneuraminic acid is an acidic monosaccharide residue that is confirmed in many biological species, including humans. Furthermore, human-derived α ₁ -microglobulin has an N-acetylneuraminic acid residue 14 at the non-reducing end. In humans, it is generally localized at the non-reducing ends of not only α ₁ -microglobulin but also many glycoproteins or glycolipids, and is thought to be involved in cell surface signaling. On the other hand, it is known that many yeast species do not have a sialic acid synthesis pathway.

Suppose that when expressing α ₁ -microglobulin in yeast as a host, hyperglycosylation could be suppressed and a sugar chain structure similar to human-derived glycan 21 or human-derived glycan 22 could be reproduced. Since yeast does not have a sialic acid synthesis pathway, for example, in the example of human-derived glycan 21, a sugar chain structure in which sialic acid is not bound to the tips of the four branched chains at the non-reducing end, or It is assumed that a sugar chain structure in which other sugar chain residues are bound to is generated. In this case, compared with the sugar chain structure of wild-type α ₁ -microglobulin, the sugar chain structure of recombinant α ₁ -microglobulin lacks as many as four sugar chain residues at the non-reducing end. , it is thought that a large deviation occurs in the molecular weight of the glycoprotein.

In addition, since sialic acid is an acidic sugar, it is assumed that it may have some influence on the behavior of α ₁ -microglobulin released into the blood. Therefore, in evaluating the removal ability of α ₁ -microglobulin in a blood purifier, not only the molecular weight problem but also the removal ability of α ₁ -microglobulin should be considered if sialic acid is not present at the tip of the sugar chain structure. may not be properly evaluated.

On the other hand, as a result of intensive studies by the present inventors, as demonstrated in Examples described later, human-derived α ₁ -microglobulin has It has become clear that it is not necessary to reproduce a sugar chain structure similar to that of the original sugar chain structure, such as human-derived glycan 21 or human-derived glycan 22. It was also found that no residue was required.

The N-type sugar chain possessed by the α ₁ -microglobulin recombinant obtained in the example described later is the mutant glycan 20 shown on the left side of FIG. 1A, and the human-derived glycan 21 or human Compared to derived glycan 22, it is significantly different in that it does not have N-acetylneuraminic acid residue 14 at the non-reducing end, which is essentially essential for human-derived glycan structures. On the other hand, what is common between the mutant glycan 20 and the human-derived glycan 21 or the human-derived glycan 22 is that they have a core glycan structure 10 .

What is derived from these points is that in evaluating the performance of blood purifiers, the N-type sugar chain of α ₁ -microglobulin is the original N-type sugar chain derived from humans (wild-type N-type sugar chain). It is derived that it does not need to have the same structure as , and that it suffices to have at least the coagulcan structure 10, and that the N-acetylneuraminic acid residue at the non-reducing end is not necessary.

Therefore, the sugar chain variant α ₁ -microglobulin according to the present disclosure can be used for evaluating the performance of blood purifiers, and has a mutated N-type sugar chain. N-acetylneuraminic acid is not bound to at least one non-reducing end of the N-type sugar chain of human-derived α ₁ -microglobulin, in other words, a core glycan structure common to N-type sugar chains Any sugar chain structure may be used as long as it contains N-acetylneuraminic acid residue 14 and the monosaccharide residue at the non-reducing end is not N-acetylneuraminic acid residue 14 .

If the mutant glycan 20 shown in FIG. 1A is described based on the present sugar chain description method described above, it becomes Manα1-6 (Manα1-3) Manα1-6 (Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc (reducing end Asn β-bonded to is omitted). In addition, this mutant glycan 20 has a structure in which two mannose residues 12 are branched and bound to the α6 mannose residue 12 in the core glycan structure 10 . Because the core glycan structure 10 has three mannose residues 12, it is sometimes referred to as the trimannosyl core structure, as described above. Based on this, in the present embodiment, since the mutant glycan 20 has five mannose residues 12, it may be referred to as a pentamannosyl structure for convenience of explanation.

[Examples of Variations of Mutant N-Glycans]
As described above, the mutant N-type sugar chain possessed by the sugar chain variant α ₁ -microglobulin according to the present disclosure contains a core glycan structure common to N-type sugar chains and has N-acetylneuramin at the non-reducing end. It is not particularly limited as long as it has a sugar chain structure to which no acid is bound. Representative mutant N-glycans can include, for example, hybrid-type glycans 20a-20e or high-mannose-type glycans 20f-20h shown in FIG. 2, or complex-type glycans 30a-30l shown in FIG. .

First, the mutant N-type sugar chain according to the present disclosure may be the sugar chain structure (pentamannosyl structure) of the mutant glycan 20 shown in FIG. 1A. As shown, the three non-reducing terminal mannose residues 12 in the mutated glycan 20 (i.e., α3 mannose residue 12, mannose residue 12 glycosyl-linked to α6 mannose residue 12 in the α1-6 form, and α6 mannose residue 12) Examples include those containing a sugar chain structure in which mannose residue 12 and N-acetylglucosamine residue 11 are linked to at least one of mannose residues 12) glycosyl-linked to residue 12 in the α1-3 type.

At this time, one or two mannose residues 12 may be bound to one mannose residue 12 at the non-reducing end of the pentamannosyl structure, or one mannose residue 12 at the non-reducing end Alternatively, one or two N-acetylglucosamine residues 11 may be attached. If only the mannose residue 12 is bound to the mannose residue 12 at the non-reducing end, it is a "high mannose type" sugar chain structure, and if at least one N-acetylglucosamine residue 11 is bound. For example, it is a “hybrid” sugar chain structure.

Further, as shown in FIG. 2, when the N-acetylglucosamine residue 11 is bound to at least one non-reducing terminal mannose residue 12 of the pentamannosyl structure, the N-acetylglucosamine residue 11 Furthermore, it may contain a sugar chain structure in which galactose residue 13 is bound, or a sugar chain structure in which N-acetylglucosamine residue 11 is bound to branch point mannose residue 12 .

Furthermore, the fucose residue 15 may be branched and bound to the N-acetylglucosamine residue 11 (directly bound to Asn) at the reducing end, or the N-acetylglucosamine residue 11 located at the non-reducing end side. The fucose residue 15 may be branched and bound to .

For convenience of explanation, for example, the α1-3 type glycosyl bond is abbreviated simply as "α1-3 bond", and the β1-2 type glycosyl bond is simply abbreviated as "β1-2 bond". In addition, the mannose residue 12 that is α1-3 bonded to the α6 mannose residue 12 is referred to as α6α3 mannose residue 12, and the α6α6 mannose residue that is α1-6 bonded to the α6 mannose residue 12. 12. Therefore, in FIG. 2, for α6 mannose residue 12 in the pentamannosyl structure (variant glycan 20), as well as for branch point mannose residue 12, the type of glycosidic bond is indicated in the abbreviated form “α3” or “α6”. is appended.

For example, the hybrid glycan 20a shown on the left side of the page (first row) in FIG. It has a glycosyl-linked sugar chain structure.

Based on the present sugar chain description method described above, the hybrid glycan 20a is Manα1-6 (Manα1-3) Manα1-6 (GlcNAcβ1-2Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc.

The hybrid-type glycan 20b shown in the upper center of FIG. 2 has a sugar chain structure in which galactose residues 13 are β1-4-linked to the N-acetylglucosamine residue 11 at the non-reducing end of the hybrid-type glycan 20a. Based on mutant glycan 20, N-acetylglucosamine residue 11 is β1-2 bound to α3 mannose residue 12, and galactose residue 13 is β1 to the N-acetylglucosamine residue 11. -4 has a linked sugar chain structure.

Based on the present sugar chain description method described above, the hybrid glycan 20b is Manα1-6 (Manα1-3) Manα1-6 (Galβ1-4GlcNAcβ1-2Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc.

The hybrid glycan 20c shown on the right side of the upper page of FIG. 2 has a sugar chain structure in which the fucose residue 15 is α1-3 linked to the N-acetylglucosamine residue 11 of the hybrid glycan 20b. Based on mutant glycan 20, N-acetylglucosamine residue 11 is β1-2 bound to α3 mannose residue 12, and galactose residue 13 is β1 to the N-acetylglucosamine residue 11. -4 linkage and α1-3 linkage of fucose residue 15.

Based on the present sugar chain description system described above, the hybrid glycan 20c is Manα1-6 (Manα1-3) Manα1-6 (Galβ1-4 (Fucα1-3) GlcNAcβ1-2Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc becomes.

The hybrid glycan 20d shown on the left in the middle (second row) of FIG. 2 has a sugar chain structure in which the N-acetylglucosamine residue 11 is β1-4 linked to the branch point mannose residue 12 in the hybrid glycan 20a. are doing. Based on mutant glycan 20, N-acetylglucosamine residue 11 is β1-2 linked to α3 mannose residue 12, and N-acetylglucosamine residue 11 is linked to branch point mannose residue 12. It has a β1-4 linked sugar chain structure.

Based on the present sugar chain description method described above, the hybrid glycan 20d is Manα1-6 (Manα1-3) Manα1-6 (GlcNAcβ1-2Manα1-3) (GlcNAcβ1-4) Manβ1-4GlcNAcβ1-4GlcNAc.

The hybrid-type glycan 20e shown in the center of the middle row of FIG. Group 11 is branched and β1-4 linked, N-acetylglucosamine residue 11 is β1-4 linked to branch point mannose residue 12, and N-acetylglucosamine at the reducing end (directly linked to Asn) It has a sugar chain structure in which fucose residue 15 is α1-6 linked to residue 11. Based on the mutant glycan 20, the first N-acetylglucosamine residue 11 is β1-2 linked to the α3 mannose residue 12, and the second N-acetylglucosamine residue 11 is β1-4 linked. Then, the N-acetylglucosamine residue 11 is β1-4 bonded to the branch point mannose residue 12, and the galactose residue 13 is β1-4 bonded to the first N-acetylglucosamine residue 11. , has a sugar chain structure in which fucose residue 15 is α1-6 linked to N-acetylglucosamine residue 11 at the reducing end.

Based on the present sugar chain description system described above, the hybrid glycan 20e is Manα1-6 (Manα1-3) Manα1-6 (Galβ1-4GlcNAcβ1-2 (GlcNAcβ1-4) Manα1-3) (GlcNAcβ1-4) Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc.

The high-mannose glycan 20f shown in the center right of FIG. 2 has a sugar chain structure in which the mannose residue 12 is α1-2 linked to the α3 mannose residue 12 of the mutant glycan 20.

Based on the present sugar chain description method described above, the high-mannose glycan 20f is Manα1-6 (Manα1-3) Manα1-6 (Manα1-2Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc.

The high-mannose glycan 20g shown in the lower (third row) left of FIG. In addition, it has a sugar chain structure in which α6α6 mannose residue 12 and mannose residue 12 are linked α1-2. Based on the mutant glycan 20, mannose residue 12 is α1-2 bonded to α3 mannose residue 12, and mannose residue 12 is further α1-2 bonded to this mannose residue 12, It has a sugar chain structure in which 12 α6α6 mannose residues are further α1-2 linked to 12 mannose residues.

Based on the present sugar chain description method described above, 20 g of high-mannose glycans are Manα1-2Manα1-6 (Manα1-3) Manα1-6 (Manα1-2Manα1-2Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc.

The high-mannose glycan 20h shown in the lower right of FIG. 2 has a sugar chain structure in which the mannose residue 12 is further α1-2 linked to the α6α3 mannose residue 12 in the high-mannose glycan 20g. Based on the mutant glycan 20, mannose residue 12 is α1-2 bonded to α3 mannose residue 12, and mannose residue 12 is further α1-2 bonded to this mannose residue 12, It has a sugar chain structure in which α6α6 mannose residue 12 is further α1-2 bonded to mannose residue 12, and α6α3 mannose residue 12 is further α1-2 bonded to mannose residue 12.

Based on the present sugar chain description method described above, the high-mannose glycans 20 g are Manα1-2Manα1-6 (Manα1-2Manα1-3) Manα1-6 (Manα1-2Manα1-2Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc. Become.

In the example of the sugar chain structure shown in FIG. 2, when the mannose residue 12 is branched, it binds to other mannose residues 12 via the α1-3 bond and the α1-6 bond, but when it is not branched, is α1-2 linked to another mannose residue 12. The sugar chain binding type is not limited to this, and various binding types may be used within a known range. Moreover, in the high-mannose glycans 20f to 20h, a galactose residue 13 may be bound to at least one of the plurality of mannose residues 12 at the non-reducing end. Alternatively, two mannose residues 12 may be branched and linked to the α3 mannose residue 12 in high-mannose glycans 20f-20h.

Thus, in the sugar chain variant α ₁ -microglobulin according to the present disclosure, the mutant N-type sugar chain is expressed as Manα1-3(Manα1-3(Manα1-6)Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc For example, as shown in FIG. 2, for at least one of the three mannose residues 12 at the non-reducing end of the pentamannosyl structure, further mannose residue 12 Alternatively, those containing a sugar chain structure in which N-acetylglucosamine residue 11 is glycoside-linked can be mentioned.

Alternatively, the mutant N-type sugar chain according to the present disclosure, as shown in FIG. 3, is a complex type sugar chain that includes core glycan structure 10 (trimannosyl core structure) but does not include the sugar chain structure of mutant glycan 20 (pentamannosyl structure). may be a sugar chain structure of That is, a sugar chain in which N-acetylglucosamine residue 11 is bound to at least one of two non-reducing terminal mannose residues 12 (that is, α3 mannose residue 12 and α6 mannose residue 12) of coagulan structure 10. Those containing structures can be mentioned.

At this time, only one mannose residue 12 binds to one mannose residue 12 at the non-reducing end of the coagulan structure 10 . Further, when the N-acetylglucosamine residue 11 is bound to at least one non-reducing terminal mannose residue 12 of the coagulan structure 10, the galactose residue 13 is further attached to the N-acetylglucosamine residue 11. It may contain a binding sugar chain structure, the fucose residue 15 may be branched and bound, or the fucose residue 15 may be branched and bound to the N-acetylglucosamine residue 11 at the reducing end. .

For example, the leftmost complex glycan 30a in the upper (first) row of FIG. have.

Based on the present sugar chain description method described above, the complex glycan 30a is Manα1-6 (GlcNAcβ1-2Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc.

The complex-type glycan 30b, shown second from the left in the upper part of FIG. .

Based on the present sugar chain description system described above, complex glycan 30b is GlcNAcβ1-2Manα1-6 (Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc.

Complex-type glycan 30c, shown third from the left in the upper part of FIG. It has a sugar chain structure in which N-acetylglucosamine residue 11 is β1-2 linked.

Based on the present sugar chain description system described above, the complex glycan 30c is GlcNAcβ1-2Manα1-6 (GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc.

Complex-type glycan 30d shown on the right in the upper part of FIG. 11 has a sugar chain structure in which fucose residue 15 is α1-6 linked.

Based on the present sugar chain description method described above, complex glycan 30d is Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc.

Complex-type glycan 30e shown on the far left in the middle (second) of FIG. It has a sugar chain structure in which galactose residue 13 is β1-4 linked to glucosamine residue 11.

Based on the present sugar chain description method described above, complex glycan 30e is Manα1-6(Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc.

Complex-type glycan 30f, shown second from the left in the middle of FIG. 11, it has a sugar chain structure in which galactose residue 13 is β1-4 linked.

Based on the present sugar chain description method described above, complex glycan 30f is Galβ1-4GlcNAcβ1-2Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc.

Complex-type glycan 30g, shown third from the left in the middle of FIG. 11, it has a sugar chain structure in which galactose residue 13 is β1-4 linked and fucose residue 15 is α1-3 linked.

Based on the present sugar chain description method described above, the complex glycan 30 g is Manα1-6(Galβ1-4(Fucα1-3)GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc.

Complex-type glycan 30h shown on the far right in the middle of FIG. On the other hand, it has a sugar chain structure in which galactose residue 13 is β1-4 linked and fucose residue 15 is α1-3 linked.

Based on the present sugar chain description method described above, complex glycan 30h is Galβ1-4(Fucα1-3)GlcNAcβ1-2Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc.

Complex-type glycan 30i shown on the far left in FIG. Galactose residue 13 is further β1-4 bonded to glucosamine residue 11, and N-acetylglucosamine residue 11 is β1-2 bonded to α6 mannose residue 12, and the N-acetylglucosamine residue It has a sugar chain structure in which galactose residue 13 is β1-4 linked to group 11.

Based on the present sugar chain description method described above, complex glycan 30i is Galβ1-4GlcNAcβ1-2Manα1-6(Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc.

Complex-type glycan 30j shown second from the left in the lower part of FIG. Galactose residue 13 is β1-4 linked to 11, and N-acetylglucosamine residue 11 is β1-2 linked to α6 mannose residue 12.

Based on the present sugar chain description method described above, the complex glycan 30j is GlcNAcβ1-2Manα1-6(Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc.

The complex-type glycan 30k shown third from the left in the lower part of FIG. Furthermore, galactose residue 13 is β1-4 linked to 11, fucose residue 15 is β1-3 linked, and N-acetylglucosamine residue 11 is β1-2 linked to α6 mannose residue 12. , has a sugar chain structure in which galactose residue 13 is β1-4 linked to the N-acetylglucosamine residue 11.

Based on the present sugar chain description method described above, complex glycan 30k is Galβ1-4GlcNAcβ1-2Manα1-6(Galβ1-4(Fucα1-3)GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc.

Complex-type glycan 30l shown on the right in the lower part of FIG. Furthermore, galactose residue 13 is β1-4 bonded, and N-acetylglucosamine residue 11 is β1-2 bonded to α6 mannose residue 12, and to the N-acetylglucosamine residue 11, Furthermore, galactose residue 13 has a β1-4 bond, and fucose residue 15 has a sugar chain structure in which fucose residue 15 is α1-6 linked to N-acetylglucosamine residue 11 at the reducing end of coagulan structure 10 .

Based on the present sugar chain description method described above, complex glycan 30l is Galβ1-4GlcNAcβ1-2Manα1-6(Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc.

Thus, in the sugar chain variant α ₁ -microglobulin according to the present disclosure, the variant N-type sugar chain contains the core glycan structure 10 denoted as Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc. When the mannose residue 12 binds to at least one of the two mannose residues 12 at the non-reducing end in the core glycan structure 10, the hybrid glycans 20a to 20e or the high mannose glycans 20f to 20f shown in FIG. 20h, but binding of N-acetylglucosamine residue 11 to at least one of the two mannose residues 12 at the non-reducing end can result in complex glycans 30a-30l shown in FIG.

When the mutant N-type sugar chain binds to at least one of the two mannose residues at the non-reducing end in the coagulan structure 10, the N-acetylglucosamine residue 11 binds to the N-acetylglucosamine residue 11. In contrast, galactose residue 13 or fucose residue 15 may be bound.

[Method for production and use of sugar chain variant α ₁ -microglobulin]
The method for producing the sugar chain variant α ₁ -microglobulin according to the present disclosure is not particularly limited. A recombinant protein production system can be constructed using a known genetic engineering technique using a common yeast that does not have a sialic acid synthesis pathway as a host.

In the present disclosure, yeast that can be used as a host is not particularly limited, but for example, Saccharomyces species such as Saccharomyces cerevisiae; Schizzosaccharomyces pombe; Pichia pastoris, Pichia finlandica (Pichia Finlandica), Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta, Pichia lindneri, Pichia opuntiae , Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, etc. Kluyveromyces species such as Kluyveromyces lactis; Candida species such as Candida albicans and Candida utilis; In the examples described later, P. pastoris is used as a host.

In these yeasts, proteins expressed in cells undergo post-translational modifications such as glycosylation, and the glycosylation does not cause hyperglycosylation. When hyperglycosylation occurs in wild-type yeast, a sugar chain mutant strain introduced with a mutation that prevents hyperglycosylation may be used. In the examples described later, a commercially available sugar chain mutant strain of P. pastoris (manufactured by BioGrammatics, product name: SuperMan-5 strain) is used.

In the present disclosure, the α ₁ -microglobulin gene used in the sugar chain variant α ₁ -microglobulin production system is, for example, a DNA having the base sequence shown in SEQ ID NO:2. The base sequence shown in SEQ ID NO: 2 is an optimized sequence that also includes a restriction enzyme sequence. Therefore, DNA encoding α ₁ -microglobulin used in the present disclosure is not limited to DNA having the nucleotide sequence shown in SEQ ID NO:2. For example, it may be a DNA having a nucleotide sequence homologous to the nucleotide sequence shown in SEQ ID NO: 2, or a DNA having another nucleotide sequence encoding the α ₁ -microglobulin amino acid sequence shown in SEQ ID NO: 1. There may be.

In addition, the α ₁ -microglobulin gene used in the present disclosure may be one in which the transcriptionally translated α ₁ -microglobulin has reactivity with a commercially available or known anti-α ₁ -microglobulin antibody. Mutant DNA in which at least one mutation has been introduced in the base sequence shown in SEQ ID NO: 2 may also be used. This antibody may have at least a portion of the human-derived α ₁ -microglobulin protein itself as an epitope.

In this mutant DNA, a base sequence in which one or more bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 2 to the extent that the antigenicity of the encoded α ₁ -microglobulin is maintained. Those having The number of bases to be deleted, substituted or added may generally be in the range of 1 to 120, may be in the range of 1 to 60, or may be in the range of 1 to 30. good too.

Alternatively, the α ₁ -microglobulin shown in SEQ ID NO: 1 has at least 95% homology (or sequence identity) with the amino acid sequence shown in SEQ ID NO: 1 as long as it retains its antigenicity. , may have 97% or more homology, or may have 98% or more homology.

The sugar chain variant α ₁ -microglobulin according to the present disclosure has the above-described variant N-type sugar chain and retains the antigenicity of α ₁ -microglobulin. The amino acid sequence shown may contain amino acid substitutions, and the nucleotide sequence shown in SEQ ID NO: 2 may contain mutations. Such amino acid substitutions or base sequence mutations are advantageous from the viewpoint of low-cost production of recombinant α ₁ -microglobulin, and the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. It can be positively introduced into the indicated nucleotide sequence.

A DNA encoding the amino acid sequence shown in SEQ ID NO: 1 or a homologous sequence thereof, a DNA having the base sequence shown in SEQ ID NO: 2, or a DNA obtained by introducing mutations into these DNAs, that is, a sugar according to the present disclosure A DNA encoding the chain variant α ₁ -microglobulin is constructed as a recombinant DNA by inserting it into a known vector. A method for constructing such a recombinant DNA is not particularly limited, and a known genetic engineering technique may be used.

In general, for example, a DNA encoding a sugar chain variant α ₁ -microglobulin and a vector are digested (cleavage) with a known type II restriction enzyme, and these DNA fragments and vector fragments are cut, if necessary. A method of ligation using DNA ligase or the like after annealing treatment may be mentioned, but is not particularly limited.

The specific vector used in the present disclosure is not particularly limited as long as it is capable of autonomous replication in host cells. Representative phases include, for example, pHV14, TRp7, YEp series plasmids, pBS7, YAC, pBR series plasmids, shuttle vector plasmids such as pAUR112, or commercial yeast expression vector plasmids. In the examples described later, a vector commercially available as a Pichia yeast expression vector (manufactured by ATUM, product name pD912 vector) is used.

The promoter used for gene expression is not particularly limited as long as it functions in the host yeast cell. Representative examples include promoters such as GAPDH, PGK, GAL1, CUP1, AOX and FLD. This promoter sequence may be included in the vector in advance. The vector may also contain regulatory sequences other than promoters, such as terminators, and may also contain restriction enzyme recognition sequences and the like.

In addition, the recombinant DNA having the structure described above may contain a DNA encoding the sugar chain variant α ₁ -microglobulin and a DNA other than the vector. For example, the vector may contain a separate DNA encoding a control sequence (promoter, terminator, etc.) not contained in the vector, or may contain a DNA (or gene, etc.) encoding another peptide. For example, in Examples to be described later, a base sequence encoding α-factor, which is a secretory signal, is included in order to secrete sugar chain variant α ₁ -microglobulin outside the host cell.

The method of introducing the recombinant DNA having the above structure into the host cell, that is, the transformation method, is not particularly limited, and known methods can be used depending on the type of host cell or the type of autonomously replicable vector. A representative transformation method is not particularly limited, but if the host cell is yeast, for example, a method of partially removing the cell wall of the yeast cell to form a spheroplast, a lithium acetate method, or electroporation. method, particle gun method, transfection method, etc. can be used. In the examples described later, the electroporation method is used.

The transformant or DNA-integrated cell thus obtained may be cultured using a known nutrient medium depending on various conditions such as the type of host cell and the purpose of culture. For example, when replicating recombinant DNA using Escherichia coli as a host, LB medium (LB culture solution) or the like may be used. In addition, when α ₁ -microglobulin is produced by culturing the transformant having the above constitution, a known medium (culture solution) suitable for the type of transformant or cell may be used. In addition, various known additives may be added to the medium depending on various conditions. In the examples described later, a commercially available P. pastoris sugar chain mutant strain is used as a host, and transformants are cultured according to the specifications of the commercially available mutant strain.

The sugar chain variant α ₁ -microglobulin according to the present disclosure can be obtained by introducing (or integrating) DNA encoding α ₁ -microglobulin into a host cell capable of sugar chain modification by various methods to obtain a transformant. (or DNA-integrating cell) and culturing this transformant (or DNA-integrating cell). Therefore, the present disclosure also includes a method for culturing such cells and obtaining sugar chain variant α ₁ -microglobulin from the resulting culture, that is, a method for producing sugar chain variant α ₁ -microglobulin. can contain.

When producing the sugar chain variant α ₁ -microglobulin according to the present disclosure, the culture scale of the transformed cells is not particularly limited. Alternatively, it may be a small-scale culture using a flask, a large-scale culture using a jar fermenter, or a large-scale culture using a tank at an industrial level.

The method for obtaining sugar chain variant α ₁ -microglobulin from cultured cells is not particularly limited, and known methods can be used. When the expressed sugar chain variant α ₁ -microglobulin accumulates intracellularly, the cultured cells are harvested and disrupted by a known method to obtain a crude enzyme solution. may be purified or concentrated by a known method. If purification or concentration is not required, the crude enzyme solution may be used as the sugar chain variant α ₁ -microglobulin according to the present disclosure.

Further, when the expressed sugar chain variant α ₁ -microglobulin is significantly secreted extracellularly, the sugar chain variant α ₁ -microglobulin may be obtained from the culture medium (recovery and purification), Sugar chain variant α ₁ -microglobulin may be obtained (collected and purified) from the entire culture including the cultured cells and the culture medium. In the examples described later, the sugar chain variant α ₁ -microglobulin is secreted (released) outside the host cell using the α factor, so the sugar chain variant α ₁ -microglobulin is collected and purified from the culture medium. are doing.

In sugar chain variant α ₁ -microglobulin collected and purified from host cells, if necessary, the state of sugar chain modification may be confirmed by a known technique. When a commercially available yeast strain is used as a host, there is no particular need to confirm the glycosylation structure of the expressed protein, since the specifications of the yeast strain, research reports, etc. reveal the structure of the glycosylation. For example, in the examples described later, a commercially available sugar chain mutant strain of P. pastoris, SuperMan- ₅ strain (product name), was used. is revealed to be the mutated glycan 20 shown on the left in FIG. 1A, the pentamannosyl structure.

On the other hand, for example, when sugar chain mutant yeast was newly constructed instead of using commercially available host cells, it was collected using an electrophoresis method such as SDS-PAGE as in the examples described later. Hyperglycosylation may be confirmed in the sugar chain variant α ₁ -microglobulin. Moreover, when the structure of the N-type sugar chain is unknown or when confirmation of the structure of the N-type sugar chain is required, the structure of the N-type sugar chain may be analyzed by a known technique. For example, a sugar chain is excised from sugar chain variant α ₁ -microglobulin by a known method, subjected to necessary or suitable known treatment, mass spectrometry, method using lectin, antibody The sugar chain structure may be analyzed by a method using

The sugar chain variant α ₁ -microglobulin according to the present disclosure can be suitably used for performance evaluation of blood purifiers. Specific types of blood purifiers are not particularly limited, and devices using dialysis membranes for various blood purification methods such as dialysis, filtration, plasma exchange, and adsorption can be mentioned. The specific structure of the blood purifier is not particularly limited, either, and a known structure can be suitably adopted.

Therefore, the sugar chain variant α ₁ -microglobulin according to the present disclosure can be used as a performance evaluation reagent for blood purifiers. The form of such a performance evaluation reagent is not particularly limited, and any form may be used as long as it is stable and can be maintained for a certain period of time without denaturing or aggregating the sugar chain variant α ₁ -microglobulin. . Generally, a protein solution in which sugar chain variant α ₁ -microglobulin is dissolved or dispersed in a known buffer solution can be mentioned. The specific composition of the buffer solution is not particularly limited, and known compositions can be suitably used.

In addition, the sugar chain variant α ₁ -microglobulin according to the present disclosure can be used in a method for evaluating the performance of blood purifiers. A specific configuration of the performance evaluation method of the blood purifier is not particularly limited, and a known method can be suitably used. As a representative performance evaluation method, for example, the performance evaluation method for a blood purifier described in Reference 1: Japanese Patent No. 4973194 can be cited. The contents of reference 1 are incorporated herein by reference.

Even when the sugar chain variant α ₁ -microglobulin according to the present disclosure is used in the performance evaluation method described in Reference Document 1, the specific configuration of the performance evaluation method is the content described in Reference Document 1. Needless to say, it is not limited to In other words, the method for evaluating the performance of the blood purifier according to the present disclosure may use the sugar chain variant α ₁ -microglobulin according to the present disclosure. , the content described in Reference Document 1 can be appropriately changed or modified within the scope of common general technical knowledge.

In addition, the form in which the sugar chain variant α1-microglobulin according to the present disclosure is used in the performance evaluation method of a blood purifier is not particularly limited, and it may be used as the performance evaluation reagent described above, or a known performance evaluation. The sugar chain variant α ₁ -microglobulin according to the present disclosure may be contained in known reagents used in the method. Therefore, the present disclosure also includes a method for evaluating the performance of a blood purifier using the aforementioned performance evaluation reagent (reagent containing sugar chain variant α ₁ -microglobulin).

Thus, the sugar chain variant α ₁ -microglobulin according to the present disclosure is α ₁ -microglobulin used for performance evaluation of blood purifiers, contains a core glycan structure common to N-type sugar chains, and , a configuration having a mutated N-type sugar chain in which an N-acetylneuraminic acid residue is not bound to the non-reducing end.

According to such a configuration, the sugar chain variant α ₁ -microglobulin has an N-glycan structure that is not identical to the original N-glycan structure, and has a non-reducing terminal N-acetylneu Although it has no laminic acid residue, it can be used for performance evaluation of blood purifiers.

The present disclosure will be described more specifically based on Examples and Comparative Examples, but the present disclosure is not limited thereto. Various changes, modifications, and alterations can be made by those skilled in the art without departing from the scope of this disclosure.

(Construction of Expression System for α ₁ -Microglobulin Recombinant)
As a host, the methanol-utilizing yeast Pichia pastoris Δaox1 wild strain or P. pastoris sugar chain mutant strain SuperMan-5 (product name: BioGrammatics) was used. As a vector, pD912 vector (product name) (manufactured by ATUM) containing the nucleotide sequence of α-factor, which is a secretory signal, was used.

For the pD912 vector, the α ₁ -microglobulin gene having the nucleotide sequence shown in SEQ ID NO: 2 (optimized sequence including restriction enzyme sequence), or the α ₁ -microglobulin gene and 10 consecutive histidine residues An expression plasmid was constructed by inserting a nucleotide sequence encoding a group (histidine tag). The expression plasmid was introduced into a host by electroporation to obtain a transformant.

By culturing the transformant according to the SuperMan-5 specifications, the α ₁ -microglobulin recombinant was expressed in host cells (transformant cells). Since the expressed α ₁ -microglobulin recombinant is released outside the host cell by α factor, the α ₁ -microglobulin recombinant can be obtained from the culture supernatant. The α ₁ -microglobulin recombinant was obtained by purifying the culture supernatant by column chromatography (purifying by affinity chromatography when having a histidine tag).

In this expression system, EAEA or EA (E: glutamic acid (Glu), A: alanine (Ala)), which is a fragmented structure of α factor, is the N of the expressed protein (α ₁ -microglobulin recombinant). The possibility of appending to the ends is known. However, in this example, whether or not EAEA or EA is added to the N-terminal side is considered to have no effect on the evaluation in the present disclosure _. The terminal side is not particularly analyzed.

(Evaluation of α ₁ -microglobulin recombinants)
The obtained α ₁ -microglobulin recombinant was reacted with a commercially available anti-α ₁ -microglobulin antibody reagent (manufactured by Eiken Chemical Co., Ltd., LZ test 'Eiken' α ₁ -M), and its reactivity ( The quality of the α ₁ -microglobulin recombinant was evaluated by confirming whether it has antigenicity or not. If reactivity could be confirmed, it was evaluated as ○, and if reactivity could not be confirmed, it was evaluated as ×.

In addition, the obtained α ₁ -microglobulin recombinant was subjected to SDS-PAGE and stained with CBB to confirm and evaluate the molecular weight and band state. The state of the band was evaluated as "absent" when no smear was observed, and as "presence" when smear was observed.

(Evaluation of permeability of dialysis membrane)
A polyethersulfone membrane dialyzer PES-15Eαeco (product name, manufactured by Nipro Corporation) having a membrane area of 1.5 m ² was used to measure α ₁ -micro Sieving coefficients of globulin recombinants were determined.

(Comparative example 1)
A P. pastoris Δaox1 wild strain was used as a host, the expression plasmid was introduced into the host, and the α ₁ -microglobulin recombinant according to Comparative Example 1 was obtained by the above-described expression system. The α ₁ -microglobulin recombinants were evaluated by body reactivity and SDS-PAGE and sieving coefficients were determined as described above. The results are shown in Table 1, and the results of SDS-PAGE are shown in FIG. In addition, in the results of SDS-PAGE shown in FIG. 4, the leftmost lane is the molecular weight marker. In Table 1, the host P. pastoris Δaox1 wild strain is abbreviated as "WT".

(Comparative example 2)
An α ₁ -microglobulin recombinant according to Comparative Example 2 was obtained in the same manner as in Comparative Example 1, except that the expression plasmid contained a base sequence encoding an N-terminal histidine tag. The α ₁ -microglobulin recombinant was evaluated by antibody reactivity and SDS-PAGE as described above. The results are shown in Table 1, and the results of SDS-PAGE are shown in FIG.

(Example 1)
The α1-microglobulin recombinant according to Example 1 was obtained in the same manner as in Comparative Example 1, except that the SuperMan-5 strain (manufactured by BioGrammatics) was used as the host. The α ₁ -microglobulin recombinants were evaluated by antibody reactivity and SDS-PAGE and sieving coefficients were measured as described above. The results are shown in Table 1, and the results of SDS-PAGE are shown in FIG. In Table 1, the host SuperMan-5 strain is abbreviated as "SM5".

(Example 2)
The α ₁ -microglobulin recombinant according to Example 2 was obtained in the same manner as in Example 1, except that the expression plasmid contained a base sequence encoding an N-terminal histidine tag. The α ₁ -microglobulin recombinant was evaluated by antibody reactivity and SDS-PAGE as described above. The results are shown in Table 1, and the results of SDS-PAGE are shown in FIG.

(Reference example)
The sieving coefficient was measured as described above using commercially available α ₁ -microglobulin derived from human urine (manufactured by MyBioSource) as a comparison target for the permeability of the dialysis membrane. The sieving coefficient results are shown in Table 1.

(Comparison of Examples and Comparative Examples)
In both Comparative Examples 1 and 2 and Examples 1 and 2, as shown in Table 1, α ₁ -microglobulin recombinants showed antibody reactivity, and these recombinant proteins were regarded as antigens as proteins themselves. , ie suitable as α ₁ -microglobulin.

As shown in Table 1 and FIG. 4, the α ₁ -microglobulin recombinant according to Comparative Example 1 has a molecular weight of 37 kDa. It was confirmed (staining was confirmed from around 30 kDa to over 40 kDa, centering on a darkly stained portion around 37 kDa).

The α ₁ -microglobulin recombinant according to Comparative Example 2 (having a histidine tag on the N-terminal side) has a molecular weight of 39 kDa. Significant smearing was confirmed in the same manner as in Example 1 (staining was confirmed from around 31 kDa to nearly 50 kDa, centering on the darkly stained portion around 39 kDa).

In contrast, the α ₁ -microglobulin recombinant according to Example 1 had a molecular weight of 31 kDa, and the α ₁ -microglobulin recombinant according to Example 2 had a molecular weight of 33 kDa. No smear was observed in the band (see Figure 4).

In Comparative Example 1 or Comparative Example 2, wild-type P. pastoris was used as the host, and the results of these Comparative Examples confirmed a broad smear in the SDS-PAGE band. As is clear from this, it can be seen that α ₁ -microglobulin is hyperglycosylated when wild-type yeast is used. On the other hand, in Example 1 or Example 2, sugar chain mutant P. pastoris was used as the host, but the results of these Examples show clear SDS-PAGE bands and no smearing. Therefore, even if the host is yeast, hyperglycosylation of α ₁ -microglobulin can be avoided by using the sugar chain mutant strain.

In the α ₁ -microglobulin recombinant according to Comparative Example 2 or Example 2, as shown in FIG. 4, the effect of the N-terminal histidine tag did not appear significantly, so the performance evaluation of the blood purifier was also performed. , the sieving coefficient was not measured because it was thought that the results would be nearly equivalent to those of Comparative Example 1 or Example 1, respectively.

As shown in Table 1, the α ₁ -microglobulin recombinant according to Example 1 had a sieving coefficient comparable to that of the reference example, that is, human-derived α ₁ -microglobulin. Therefore, it is judged that the α ₁ -microglobulin recombinant according to Example 1 has sufficient practical utility in evaluating the performance of blood purifiers.

On the other hand, the α ₁ -microglobulin recombinant according to Comparative Example 1 had smaller sieving coefficient values than those of Example 1 or Reference Example. As is clear from the results shown in FIG. 4 (results of SDS-PAGE), when the P. pastoris Δaox1 wild strain was used as the host, the α ₁ -microglobulin recombinant was hyperglycosylated. This is thought to be due to the occurrence of

On the other hand, as shown in FIG. 4, no significant difference can be confirmed between the band of Comparative Example 2 and the band of Comparative Example 1, or between the band of Example 2 and the band of Example 1. Therefore, the α ₁ -microglobulin recombinant according to Example 2, which has a histidine tag on the N-terminal side, is also considered to have sufficient practicality for performance evaluation of blood purifiers.

In addition, in the recombinant protein expressed using the SuperMan-5 strain, which is a sugar chain mutant strain, as a host, the N-type sugar chain does not undergo hyperglycosylation as shown in FIG. The structure of the mutant glycan 20 shown, that is, the sugar chain structure (pentamannosyl structure) in which two mannose residues 12 are bound to the α6 mannose residue 12 in the core glycan structure 10, has been clarified. Therefore, the sugar chain structures of the α ₁ -microglobulin recombinants according to Examples 1 and 2 also have a pentamannosyl structure.

That is, the sugar chain structure (variant glycan 20) of the recombinant α ₁ -microglobulin according to Example 1 (or Example 2) and the original sugar chain structure of α ₁ -microglobulin derived from human Comparing human-derived glycan 21 or human-derived glycan 22, α ₁ -microglobulin does not need to reproduce the entire carbohydrate chain structure for the permeation behavior of the dialysis membrane. It turned out that the (N-acetylneuraminic acid) residue was also not necessary.

Therefore, in the present disclosure, N-glycans such as hybrid-type glycans 20a-20e or high-mannose-type glycans 20f-20h illustrated in FIG. 2, or complex-type glycans 30a-30l illustrated in FIG. If it is a sugar chain variant α ₁ -microglobulin having a mutant N-type sugar chain that contains a common core glycan structure 10 and has a non-reducing end bound to an N-acetylneuraminic acid residue 14 , like the original human-derived α ₁ -microglobulin, can be used to evaluate the performance of blood purifiers.

It should be noted that the present invention is not limited to the description of the above embodiments, and various modifications are possible within the scope of the claims, and different embodiments and multiple modifications are disclosed respectively. Embodiments obtained by appropriately combining the above technical means are also included in the technical scope of the present invention.

From the above description, many modifications and other embodiments of the invention will be apparent to those skilled in the art. Accordingly, the above description is to be construed as illustrative only and is provided for the purpose of teaching those skilled in the art the best mode of carrying out the invention. Substantial details of construction and/or function may be changed without departing from the spirit of the invention.

The present invention can be widely and suitably used in the field of performance evaluation of blood purifiers.

10: Coaglycan structure (trimannosyl core structure)
11: N-acetylglucosamine residue (GlcNAc)
12: Mannose residue (Man)
13: Galactose residue (Gal)
14: N-acetyl neuraminic acid residue (NeuNAc)
15: Fucose residue (Man)
16: mannose phosphate residue 20: mutated glycan (mutated N-type sugar chain)
20a-20e: Hybrid glycans (mutated N-type sugar chains)
20f-20i: high mannose type glycans (mutated N-type sugar chains)
30a-30l: Complex-type glycans (mutated N-type sugar chains)
21: Human-derived glycan (human-derived original N-type sugar chain)
22: Human-derived glycan (human-derived original N-type sugar chain)
23: glycans (hyperglycosylated sugar chains)
120: Hypermannose structure

Claims

A mutant N-type sugar chain comprising a core glycan structure common to N-type sugar chains and having a non-reducing end to which an N-acetylneuraminic acid residue is not bound,
Sugar chain variant α 1 -microglobulin.
The mutant N-type sugar chain has a mannose residue or an N- Containing a sugar chain structure to which an acetylglucosamine residue is bound,
The sugar chain variant α 1 -microglobulin according to claim 1.
The mutant N-type sugar chain comprises a pentamannosyl structure denoted as Manα1-3(Manα1-3(Manα1-6)Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc,
The sugar chain variant α 1 -microglobulin according to claim 2.
The mutant N-type sugar chain comprises a sugar chain structure in which a mannose residue or an N-acetylglucosamine residue is glycoside-bonded to at least one of the three mannose residues at the non-reducing end of the pentamannosyl structure. ,
The sugar chain variant α 1 -microglobulin according to claim 3.
The mutant N-type sugar chain binds an N-acetylglucosamine residue to at least one of the two mannose residues at the non-reducing end in the coagulan structure, and binds to the N-acetylglucosamine residue , containing a sugar chain structure to which a galactose residue or a fucose residue is bound,
The sugar chain variant α 1 -microglobulin according to claim 2.
Produced by expressing the α 1 -microglobulin gene in sugar chain mutant strain yeast in which hyperglycosylation does not occur,
The sugar chain variant α 1 -microglobulin according to any one of claims 1 to 5.
The sugar chain mutant yeast is Pichia pastoris,
The sugar chain variant α 1 -microglobulin according to claim 6.
The sugar chain mutant strain yeast is SuperMan-5 strain (product name),
The sugar chain variant α 1 -microglobulin according to claim 7.
containing the sugar chain variant α 1 -microglobulin according to any one of claims 1 to 8,
Performance evaluation reagent for blood purifiers.
A method for evaluating the performance of a blood purifier, using the sugar chain variant α 1 -microglobulin according to any one of claims 1 to 8.