JPS63502716A - How to enhance glycoprotein stability - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] How to enhance glycoprotein stability l Wanten 11 Glycoproteins are proteins that have conjugated bonds with sugars, but they are found in plants and animals. , even found in many unicellular eukaryotes such as insects and yeast. that They are present in both soluble and membrane-bound forms within cells and cells, such as intracellular matrices. Present in extravesicular secretions. The carbohydrate portion of these glycoproteins can be directly involved in the biological activity of glycoproteins: biodegradation by hydrolysis, It stabilizes protein structure and mediates inter- and intracellular recognition. sugar protein Examples of proteins include enzymes, serum proteins such as immunoglobulins or blood clotting factors, Cell surface receptors, hormones, toxins, lectins and structures of long factor or 5 chromosomes Contains protein.

Natural and recombinant proteins are used as human and animal therapeutics.

Therapeutic proteins are often the most effective if they have sufficient circulating life. At least four general mechanisms may shorten the circulating life of external proteins. Hydrolytic biodegradation, if the protein is antigenic or If it is immunogenic, it will be removed by the immune system, and certain exposed sugars on glycoproteins will be removed. removal by liver cells or reticuloendothelial cells that recognize the If the molecular weight is small, it is removed through the glomerular basement membrane of the kidney, and oligosaccharides of glycoproteins are removed. can have a strong influence on the first three of these removal mechanisms.

Oligosaccharide chains of glycoproteins! yo, either through N- or O-glycosidic bonds. attached to the polypeptide backbone. In the case of N-linked polysaccharides, the reducing end of the oligosaccharide N-acetylglucosamine (GlcNAe) residue anomeric carbon (C-1> and There is an amide bond that connects the nitrogen of asparagine (Asn) in the lipeptidone. .

In animal cells, 〇-linked polysaccharides are converted into N-acetylgalactosamine (Ga 　N　A　c　), galactose (G　al　) or xylose and number One of the amino acids with a hydroxyl group, most commonly serine (Ser) or threonine (Thr), or in some cases hydroxylated proline or water It is attached via a glycosidic bond with oxidized lysine. Yeast, Satsuka Roma 〇-linked polysaccharides of Ises cervisiae , attached to serine or threonine residues, but unlike animal polysaccharides, They consist of one to several α-linked mannose (Man) residues. Manno -ase residues have not been found in 0-linked oligosaccharides of animal cells.

The biosynthetic pathways of N- and O-linked oligosaccharides are quite different.

The synthesis of 〇-linked polysaccharides is relatively simple and involves a series of specific glycosyltransformations. It consists of transferring monosaccharide groups one by one from the sugars of nucleic acids by chelase. single The sugar of the nucleic acid that serves as a sugar donor is uridine-diphosphoric acid-GalNAc (UD P-GalNAC), UDP-GIcNAc, UDP-Gal, guanidine-ni Phosphoric acid-fucose (GDP-Fue) and cytidine-phosphoric acid-sialic acid be. N-linked oligosaccharide synthesis, which is much more complex, is described below. Ru.

The initiation step of N-linked polysaccharide biosynthesis begins in unicellular eukaryotes such as yeast. It has been conserved with little change throughout the evolution of higher plants and humans. these In all organisms, the initiation of N-linked oligosaccharide assembly occurs at the protein Asn. involves preassembly of lipid-linked oligosaccharide precursors, rather than occurring directly at the residues; The protein is isolated immediately or during translation from m-RNA. will be transferred to. This oligosaccharide precursor is G IcsMan, G IcN Ae2 Its structure is shown in Figure 1A. Synthesized while conjugated to a polyisoprenoid carrier lipid (dolichol) .

This assembly contains at least six different membrane-bound glycosyltransferases. It is a complex process that includes Some of these enzymes convert nucleotide sugars into monosaccharides. while other enzymes utilize dolichol-linked monosaccharides as sugar donors. . After assembly of the lipid-bound precursors is complete, they are processed by other membrane-bound enzymes. , the steric proximity that appears as part of the -Asn-X-3er/Thr- sequence It is easily transferred to the Asn residue. The need for 3D accessibility is important for in vitro Observation that transfer of oligosaccharide precursors to external proteins usually requires denaturation This is an inference based on the following.

The glycosylated Asn residues of newly synthesized glycoproteins temporarily become one type of glycoprotein. It has only oligosaccharides, G lc, MansG lcN Acz. this structure Modification or “processing” is a process that occurs in mature glycoproteins. It is this difference in the type and degree of processing that gives rise to the great structural diversity in , even the same polypeptide is often glycosylated differently in different cell types. This can explain some observations.

Processing of N-linked oligosaccharides is performed using oligosaccharide precursor G This is the sequence of many membrane-bound enzymes that starts immediately after the transfer of C2 to proteins. It is completed by the work done. Broadly speaking, N-linked oligosaccharide processing involves three steps. Removal of 23 glucose residues, some mannose residues removed and generated truncated “nuclei”, e.g. the original origin closest to the polypeptide backbone. Addition of various sugar residues to MansG IcN Ae2, which is a part of gosaccharide.

A simplified outline of this machining path is shown in Figure 2.

Assembly of oligosaccharide precursors as well as removal of glucose residues during the first step of processing has been conserved throughout evolution. In yeast and vertebrates, all three glucose is cleaved when creating N-linked MansG IeN Aez . Processing may stop at this structure, but a first step usually involves removal of the mannose residue. Two stages occur. Here the yeast pathway differs from that of vertebrate cells.

As shown in Figure 1B, the four mannos in the Man, G, IcN, and Ac2 parts The residues are linked by an α1-→22 linkage. As a promise, the arrow indicates the oligosaccharide It shows the direction of the reducing end or, in this case, the protein binding of the polysaccharide. The direction of the edge is indicated; α or β indicates the anomeric configuration of the glycosidic bond. The two numbers also indicate which carbon atoms in each monosaccharide are used for bonding. It shows that. The four α1-→22-linked mannose residues are removed, resulting in N-linked Mans-sGlcNAc2, all of which Commonly found in animal glycoproteins. Mans-@GIcNAcz composition These oligosaccharides are said to be of the “high mannose” type.

As shown in Figure 2, protein-bound MansG lcN Ac2 (structure M- c) acts as a substrate for GleNAe)transferase l, and the enzyme is β1-→22-linked G Ic N A c residue from UDP-GIcNAc α1 to form IcN AeMansG IcN Ac2 (structure M-d) --Transfer to 3-bound mannose residue. Mannosidase ■ produces two mannose residues. Complete the cutting step of the processing path by removing the base G IcN AcMa It produces a protein-bound oligosaccharide with the composition nsG IcN Acz (i-made M- e), this structure is a substrate for GlcNAc) transferase, which is β1 -→22-linked G1゜NAcW' group can be transferred to α1-→66-linked mannose residue ( (not shown).

At this stage, the true complexity of the machining path begins to explain itself. Simply put , monosaccharides are grown by a series of membrane-bound Golgi glycosyltransferases. each of them is added to the acceptor oligosaccharide, the donor oligosaccharide, the donor oligosaccharide, and It has high specificity regarding the type of bond formed between sugars and sugars.

Each type of cell has a large but distinct set of glycosyltransferases. have. These include at least four different GIeNAc) transfections. (makes β1-→3, β1-→4 or β1-→66 bonds); three galases tosyltransferase (β1-→4, β1-→3 and α1-→33) ); two sialyltransferases (one is α2-→3, the other is α 2--6 bonds); three fucosyltransferases (α1-→2, α1 −→3, α1−→4 or α1−→66); and various unusual Contains countless other enzymes for conjugation. These glycosyl transfer The synergistic action of enzymes allows the correct formation of various structures called “complex” oligosaccharides. I can make it. These are the 2( For example, M-f structure in FIG. 2), 3 (for example, M-g structure in FIG. 1C or FIG. structure), or contains four outer side chains. These structures depend on the number of outer side chains. Therefore, they are called as follows: 2 antennae (2 side M), 3 antennae (3 side chains) or four antennae (four side chains), the size of these complex polysaccharides is six monosaccharides (rhodo on pusin) to very large polylactose aminyl glycans; They are one or more repeating units (Galβ1-4GlcNAcβ1--3) (cells such as red blood cell glycoprotein band 3 and macrophage antigen Mac-2) on some surface glycoproteins).

Despite this diversity, the specificity of glycosyltransferases is For example, the outer side of many complex N-linked oligosaccharides The chain has the sequence SAα2-→3(6)Galβ1-→4 G lcN Acβ1-→ Consists of part or all of. Shown in M-f and M-gfl structures in Figure 2. As shown, one or two of these three monosaccharides link two α-linked manganese of the core five monosaccharides. It can also be attached to each of the north residues.

DNA transcription or w+RNA translation (these are highly reproducible phenomena) Unlike the conventional method, oligosaccharide biosynthesis is not performed by a template; As a result, considerable non-uniformity is commonly observed in the oligosaccharide structures of all glycoproteins. be noticed. The main reason for the difference is the variation in the degree of processing. There are at least 18 unique glycosylation sites on the egg white glycoprotein ovalbumin. It contains structurally related “families” of different oligosaccharides, the majority of which are High mannose or related “hybrid” types (e.g. structure Mh in Figure 2) , many glycoproteins have multiple glycosylated Asn residues, and Each of them can have a different oligosaccharide family. For example, one location is Can have mannose polysaccharides and other places are mainly fucosylated It has a composite chain of two antennae, and the third part is a fucose-free three or It can have a complex chain of four antennae. However, these glucan All have an invariant ManzG IcN Ac2 nucleus.

As discussed above, the yeast Saccharomyces cervicinii The initiation step of N-linked oligosaccharide synthesis in vertebrates This is very similar to what happens in cells.

In higher organisms, lipid-bound Glc3MansGle, NAe, is assembled The oligosaccharide chain is transferred to the Asn residue receptor of the protein, and the three Glucose residues are removed immediately after transfer. However, yeast cells are the smallest and most N-linked glycans due to small processing have the composition Mane-gG IcN Ac2. Only one mannose residue is removed in this way. Processing may stop at this stage. It is possible to add nearly 50 pieces to Man@G IeN ACz (structure Y-c in Figure 2). Remannan ( For example, one can proceed to yield structure Y-d). in mammalian cells Glycoproteins with a single glycosylated Asn residue are mainly high-mannan. - oligosaccharides and highly processed and complex glucans elsewhere. Similarly, yeast glycoproteins such as extracellular invertase are generally produced by Man @-IG　IcN　Has several sites glycosylated with Ac2, -Labor-management place I have mannan at the place.

Unlike eukaryotic cells, bacteria contain lipid-bound G lesMansG lcN Ac2. It lacks the enzymatic machinery to assemble or transfer it to proteins. that Therefore, the protein synthesized in Escherichia coli (emesis) contains many -A sn -X -Ser/Thr- sequences, but they are not glycosylated.

From the above discussion, the glycosylation status of glycoproteins depends on the cells in which they are produced. It is clear that there are. Protein glyca synthesized in cultured mammalian cells protein is similar to that of the same protein isolated from a natural animal, such as a tissue. proteins glycosylated by yeast, but not identical. has high mannose oligosaccharides and mannans, and has a high E. coli composition ratio. ), proteins synthesized in bacteria such as is not glycosylated.

The precise composition and structure of the carbohydrate chains on a glycoprotein has a direct impact on its serum longevity. It's echoing. This is because cells of the liver and reticuloendothelial system absorb circulating specific charcoal. This is because it can bind to or absorb hydrated glycoproteins. . Hepatocytes are attached to terminal (i.e., the outermost ends of glycans attached to polypeptides) ) has receptors on the cell surface that recognize oligosaccharide chains with Gal Phages have receptors for terminal Man or GlcNAC residues. Hepatocytes and leukocytes also have receptors for exposed fucose residues. . However, no specific receptor for sialic acid has been discovered; it may be due to the oligosaccharide space. The general rule is that cells in the liver and reticuloendothelial system The greater the number of exposed sugar residues recognized by surface receptors, the more Glycoproteins are quickly removed from the body. However, there are receptors specific for sialic acid. All side chains are terminated or “capped” with sialic acid. Oligosaccharides will not cause removal of proteins to which they are attached.

The nature and presence of oligosaccharide chains on glycoproteins are unique to sugars on liver and reticuloendothelial cells. In addition to its recognition by different receptors, it can also change important biochemical properties. . Removal of carbohydrates from a glycoprotein usually reduces its solubility, and it destabilizing the correct polypeptide fold and/or Increases susceptibility to biodegradation by exposing areas sensitive to protein-degrading enzymes. To increase. For similar reasons, the glycosylation status of proteins is determined by the immune system. can change the perception of

It is therefore an object of the present invention to provide yeasts, insects, plants isolated from natural sources or Oligosaccharide chains of glycoproteins produced from recombinant DNA of vertebrate cells The aim is to provide a method for modification and thereby extend lifespan in serum. or by targeting the protein to specific cell types.

Another object of the present invention is to provide m. seedlings with enhanced stability and effective biological activity. , protein glycosylation made from yeast, plant, virus, or animal DNA. The purpose of this invention is to provide an in vitro method for decoding.

Yet another object of the invention is to provide an efficient, reproducible and economical protein group. The object of the present invention is to provide a method for lycodylation or modification of oligosaccharide chains on glycoproteins.

and breasts Δi 扛 The present invention aims to prolong the circulatory life in the body or the location where it is absorbed into cells in the body. A method for protein modification of eukaryotic and prokaryotic cells to modulate Like In new embodiments, enzymatic and/or chemical treatments may be used to trimonosaccharide added by the above conjugate bond SAα2-6(3)Galβ1-4(3)GIcNAc-→or tetramonosaccharide 5Acr 2--6 (3) Galβ1--4 (3) G IcN Aeβ1- =4GlcNAc--” As an alternative example of producing a modified protein with moieties, or two GIcNAc residues attached to the appropriate glycosyl trans Used as a basis for building other oligosaccharides by elongation by ferases The method of the present invention can be applied to any natural or synthetic oligosaccharide containing an Asn-linked oligosaccharide. chemically or enzymatically by engineered proteins or appropriate carbohydrate residues. It can be applied to any non-glycosylated protein that can be induced to Wear.

Asn- case S A--G a l--G Ie N A c-- Protein@1min A preferred oligosaccharide modification method consists of the following steps, in which the N-linked oligosaccharides are All oligosaccharide chains except Asn-linked GIcNAc are enzymatically or chemically converted into proteins. and removed from trimonosaccharides made in situ.

U↓, GlcNAc-→Asn (protein) generation In the first step, E A suitable endo-β-N-acetylglucosaminida such as Endo H or Endo F. from all or some of the N-linked oligosaccharide chains of the glycoprotein, The glycosidic bond connecting the two most central GlcNAc residues is cleaved. let End H is Saccharomyces cervicinii mannan produced in yeast such as S. scerevisiae and eukaryotic cells. By cleaving the high mannose and hybrid oligosaccharide chains of glycoproteins produced in G　IcN　Ac attached to each glycosylated Asn residue in the peptide backbone Remove everything but residues. Endo F is a high-mannose and di-N-linked oligosaccharide. The present antennal complex can cleave both silver and each glycosylated site as well. Leave one GIcNAc attached, if a given glycoprotein has a known enzyme such as three- or four-antennary chains that cannot be efficiently cleaved by doglycosidases. If they have complex oligosaccharides, these chains can be processed by sialidases, β- or α- - such as galactosidase, α-fucosidase and β-hexosaminidase It can be excised by exoglycosidase. Next, the most of the generated nuclei The inner GIcNAe residue can be exposed by any of several techniques. Ru.

One approach is to use other endo-β-N-acetyl groups such as endo-F or endo-D. Degradation by lucosaminidase. The second method uses α-mannosidase and Subsequently either endoL or β-mannondase and β-hexo Decomposition by saminidase.

As an alternative, glycoproteins with complex Asn-linked oligosaccharides, swainsonine or deoxymannose In the presence of processing inhibitors such as deoxymannojirimycin can be produced in mammalian cell culture. The generated glycoprotein is It has a hybrid or high mannose chain that is susceptible to cleavage by Therefore, there is no need for initial treatment of the glycoprotein with exoglycosidases. . In a related variant, the glycoprotein is endo-H or endo-F. A mutant strain that lacks the ability to synthesize complex N-linked chains that are resistant to doglycosidases can be produced.

All sugars other than N-linked GIcNAc residues can be trifluorinated chemically instead of enzymatically. It can also be removed by treatment with dichloromethanesulfonic acid or hydrofluoric acid. can. Generally, chemical cleavage involves the use of relatively harsh conditions that result in denaturation. It is expected that this method will be less useful than the enzymatic method because of the effects of this method.

艮lλG Ic N A c --= G a to A s n < protein) The second step is through the action of galactosyltransferase. By enzymatically adding Gal residue to GlcNAc remaining on the protein, Ru. The preferred galactosyltransferase is UDP as the sugar donor. - Transfer GaI to GIcNAc in the presence of Gal to form β1-→4 bond It is a bovine milk enzyme. As a variant, galaxies from sources such as pig trachea can be Through the use of tosyltransferase, galactose is transferred through β1-→3 linkages. GlcNAc residues can also be added.

Modified llG a l-→G Ic N A c --S to A s n tamper A. The final stage of the number of mouths is Galβ1--=4 (3) GlcNAc--Asn (tan It is the enzymatic addition of sialic acid to (Pak). This reaction is similar to that in rats, for example. α1-→6-sialyltransferer isolated from liver or bovine colostrum The enzyme converts SA from CMP-3A into Galβ1--4 (3 ) G　lcN　Ac--Transferred to the terminal galactose residue of Asn (protein) , as an alternative method of forming α2-→6 bonds, α2-→3 sialyltrans Ferrase can be used to form α2-→3 bonds at each terminal G a l residue. Will. A preferred sialic acid is N-acetylneuramine Pi (NeuAe). However, sialyltransferase transfers galactose from CPM-3A derivatives. A chemically synthesized or naturally occurring irritant system that can be transferred to Alkalic acids can also be used, for example, N-glycolylneuraminic acid, 9-0-acetic acid, Tyl-N-acetylneuraminic acid and 4-0-acetyl-N-acetylneuraminic acid Minic acid is given.

Asn ASA--Gal-→GIcNAc--+GleNAc--*卆っ社 Eno! Jitsudo IA As a second specific example, oligosaccharide chains of glycoproteins (natural or processed either in the presence of an agent or produced by a mutated cell line ), the two innermost instead of one by the use of a suitable exoglycosidase For example, α- and β-mannosidases cut down to the nuclear GIcNAc residue of The product of this process, which could be used to cleave high mannose oligosaccharides, G leNAcβ1--4GIcNAC--Asn (protein), Next, tetramonosaccharide 5A (Z2--=6(3)Galβ1--"4(3)GlcN Acβ1-→4G! To convert cNAe-→Asn (protein), galacto Sequential treatment with syltransferase and sialyltransferase Now.

As a third specific example, as a third specific example, Monosaccharide SA-→G a l-→G lcN Ac-→ or dimonosaccharide SA-→Ga Expressing oligosaccharides such as 1−→ in either bacterial or eukaryotic systems For example, a trisaccharide SA- added to a non-glycosylated amino acid residue of a protein →Ga1-→To add GIcNAc, the protein is chemically reactive G　c　N　A　c　--*　　　G　a　l--+　G　l　c　N　A c--or SA-→G a l-→GIcNAc-→ glycoside induction processed by the body. In the first two cases, the monosaccharide or dimonosaccharide is then added as appropriate. It is converted into trimonosaccharides by glycosyltransferase. first carb The substance part is 1, a carbohydrate with an appropriate amino acid and an appropriately activated chemical group. can be added to proteins by a chemical reaction between glycoside derivatives of Ru. Due to the activated groups present in the glycosides, carbohydrates have free amino groups, Amino acids or aromatics with carboxyl, sulfhydryl or hydroxyl groups Added to amino acids.

Invention of oligosaccharides based on protein-rich ΔG Ic N A c-groups A modification of the method is to use glycoproteins with oligosaccharides other than the dicarcharoses or tetramonosaccharides mentioned above. It can also be used to make. for example, SAα2--6(3)Galβ1-4(GIcNAcβ1-→3G a lβ 1--4) n G I c N A c -- or SAα2-→6(3 )Galβ 1-→4 (GlcN Ac31-→3Galβ--4>n G1cNAcβ1--4GlcNAe-(in the formula, n is a number from 1 to 10) The extended oligosaccharide chain consisting of and β1-→3N-acetylglucosaminyltransferase. A series of mutual treatments are applied to glycoproteins with one or two nuclear GlcNAc residues. It can be constructed by The generated extended oligosaccharide chain is a polypeptide to hide antigenic or proteolytic enzyme-sensitive sites or to increase solubility. useful in doing.

Use of many other available oligosaccharide structures and appropriate glycosyltransferases It can be made by elongation of protein-bound monosaccharides or double saccharides.

- e.g. branched fucosylated bicarrier Ig Ga lβ1--”4CFu ca 1--3) GlcNAe-→. These and other structures have specific Targeting specific tissues known to have receptors for monosaccharides or oligosaccharides This would be effective in making the enemy selectively attack.

Brief description of the drawing Figure 1 shows (A) lipid-bound oligosaccharide precursor G Ic3MansG lcN A C2; (B) High mannose Asn-linked oligosaccharide MansGlcNAc2: and (C) shows the structure of a typical three-antennary Asn-linked oligosaccharide. of sugar residues The anomeric configuration and bond position are indicated and the invariant pentatom is enclosed by a dashed line. The glyconucleus is common in all known eukaryotic Asn-linked oligosaccharides .

Figure 2 shows a simplified example of Asn-linked oligomer biosynthesis in yeast and higher organisms. This is a biosynthetic pathway that has evolved into a biosynthetic pathway. Anomeric configuration and attachment position are not shown for simplicity. However, the arrangement of the side chains is the same as in Figure 1.

Figure 3 shows in vitro yeast invertase before and after treatment with glycosidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAG) E) Coma sheeple stained gel prepared by E). The concentration of acrylamide is 6 %.

(A>Untreated invertase, (B) Invertase after Endo H treatment under non-denaturing conditions. lutease; (C) Inver after Endo H treatment under denaturing conditions (0.7% 5DS) and (D) one of the samples initially treated with Endo H under non-denaturing conditions. Section where the portion was subsequently treated with jack bean α-mannosidase.

Figure 4 shows invertase treated with Endo H and denatured with SDS (Figure 3B). UDP-[3H]Gal and bovine milk β1-→4-galactosyl transfer time course (5 min, 1 h, 2 h, 3 h, 5 Samples of in vitro yeast invertase removed at 9 hours and 19 hours) Fluorescence analysis of a 6% 5DS-PAGE gel.

Figure 5 shows UDP-[CH]Gal and bovine milk β1-→4galactosyltra Acid-precipitated radioactivity during treatment with spherase, treated with Endo H. This shows the rate of incorporation into yeast invertase denatured with SDS. .

Figure 6 shows CMP-[14C NeuAc and bovine colostrum (12--+6Cial). Various yeast in vitro inverters sialylated using transferase Fig. 2 is an autoradiograph of a 6% 5DS-PAGE gel of the enzyme derivative. (A) Produced from invertase denatured with SDS, treated with Endo H, and galactosylated. (B) Jack bean α-mannosidase and Endo H Sialyl generated from processed non-denatured invertase galactosylated samples (C) Sialyl compound produced from untreated invertase.

Figure 7 shows coma sheeple staining of a 6% 5DS-PAGE gel; (A) untreated Bovine serum albumin (BSA); (B) 0.25 at pH 8.5 for 24 hours at room temperature 2-Imino-2-methoxyethyl-1-thio-N-a in sodium morphoborate Approximately 48G per protein molecule by reacting with cetylglucosaminide. B converted to G lcN Ac -B S A with IcNAc residue S A ; (C) G lcN Ac -B S A is UDP-[3H]Gal or and bovine milk β1-→4 galactosyltransferase. and (D) Ga1−→G lcN Ae -B S　A to CMP-[”C]NeuAc and α2-→6Sia of bovine colostrum Sialylated BS formed by treatment with lyltransferase A.

Figure 8. Thioglycosylation as a function of the concentration of glycosylated BSA (μg/ml). Man/GlcNAc reception in murine peritoneal macrophages stimulated with cholate. G　a　l−−G　Ic　N　AC　−[+ts■]13SA ) and GIcNAc-[”’I]BSA (■) specific uptake (ny/2x This is a graph of the total uptake (mannan cell), and the specific uptake in the graph is the total uptake (mannan uptake in the absence) to nonspecific uptake (values obtained in the presence of mannan) is equal to minus .

Figure 9 shows the relationship between protein concentration (0.5 to 7.5 μg protein 7 ml). G a by G a l / G a I N A c receptor of HepG2 cells l--GlcNAc["J]BSA (intestine) and Ne u A c --G a l-→G l cNAc-[12J]BSA(・) specific uptake (n y/my cytoplasmic protein), and the specific uptake in the graph is the total uptake. (uptake in the absence of Asialo orosomucoid) and equal to the value obtained in the presence of dialoorosomucoid).

Figure 10 shows CMP-NeuAc and rat liver α2-→6 sialyl trans ['H]G before (O...O) and after (Δ-Δ) sialylation by ferase a1--” GIeNAc-RNase rapid protein liquid extraction with single-S column Analysis by romatograph (FPLC), where the column is as described below. Elute with a linear gradient.

Detailed description of the invention The present invention provides a method for increasing the stability of proteins in vivo or adding them to proteins. The protein activates cells that have specific receptors for the exposed sugars of the oligosaccharide chains. Modification of proteins, including attaching oligosaccharide chains to proteins for attack This is the way to do it. There are two basic aspects to this method. The first one is glycoprotein The Asn-linked oligosaccharide present on the protein is attached to the Asn site of the protein. cleavage leaving one or two GlcNAc residues and enzymatically converting G and extending the SA by attaching it to the terminal GlcNAc. The second method is Enzymatically or chemically convert GlcNAe or Gal residues into various proteins. Adds to any position of many amino acids and then forms oligosaccharides covered with sialic acid. is to enzymatically extend the terminal GlcNAe or Gal to achieve . Many enzymes and methods are used at each step, depending on the substrate and desired oligosaccharide structure. There is a change in

A, 5A--+Ga1--+GlcNAc-→A, sn protein for a long time 1st place allll, G Ic N A c −-+ A s n npa no Glico Glycoproteins with a single GIcNAc residue attached to a dylated asparagine residue There are several methods for preparing mulch. Six methods are shown below.

a, one or more oligosaccharides of high mannose or mannan type according to Endo H Enzymatically generates GlcNAc-→Asn (protein) on glycoprotein with In order to React with tilglucosaminidase. This enzyme produces glycosylated Asn leave a single GIcNAe residue attached to the sensitive N-linked oligo Hydrolyzes the bond between the two core GlcNAc residues of the sugar. A good choice for this purpose. The most desirable enzyme is Endo H, which is produced by Streptomyces plicatus (Danhong). It has been isolated from the plant. The enzyme is a naturally occurring protein or is Streptomyces levitasis (ratio c9LLL) or Streptomyces libitasis (Stre). Which of the recombinant DNA products expressed in tom ces 1ividans) It can also be used in the form of

Endo H contains all sensitive oligosaccharide structures of modified glycoproteins and natural sugar proteins. cleaves many oligosaccharides on the protein. However, in natural glycoproteins, the Due to steric factors such as folding of the lipeptidoid, it is protected from cleavage by EndoH. The GlcNAc2 core of high-mannose glycans may be protected. this problem often involves the use of one of several weak denaturants that partially widen the polypeptide. removed by. Examples of such weak modifiers include Triton X-100, NP-40, octyl glucoside, deoxycholate and dilute sodium dodecyl sulfate Surfactants such as thorium; dithiothreiotole and β-mercaptoethano Disulfide bond reducing agents such as urea, guanidine hydrochloride and isocyanogen chaotropic agents such as sodium chloride; alcohols (methanol, ethanol , propatool or butanol), low concentrations such as DMSO or acetone Examples include organic solvents. Endo H is a very stable enzyme, at pH 5 to 6. 5 in strong or weak ionic strength buffers and the denaturing agents mentioned above. or phenylmethanesulfonyl fluoride, EDTA, apro The presence of protease inhibitors such as tinine, leupeptide and pepstatin It is active even in the presence of Techniques for using End H are described below. Preserves biological activity The exact combination of reaction conditions that optimizes the cleavage of oligosaccharides by endoH will vary depending on the glycoprotein being modified, but common techniques in this field This can be determined by the surgeon using routine techniques.

Even after reaction under the most stringent endoH reaction conditions deemed safe for use. If one or more intact high mannose glycans continue to be present, they will not be commercially available. using α-mannosidase, such as that from jack bean, which is By doing so, the exposed mannose residue can be excised. Qualify this way The resulting high mannose glycans may not serve as substrates for further modification reactions described below. However, this treatment does not increase the mannose-specific receptor on macrophages or other cells. The high mannose glycans remaining on the body and glycoproteins bind, and the circulatory system should reduce the likelihood of causing premature removal from the

As mentioned earlier, yeast glycoproteins contain 1 to 4 α-linked mannose residues. sometimes have 〇-linked oligosaccharides consisting of

These bind to mannose-specific receptors and shorten the serum lifespan of glycoproteins. The protein found to contain such oligosaccharides is derived from jack bean. Treatment with the enzyme α-mannosidase is recommended. This is 〇 -Remove all but the innermost protein-bound mannose residues from the linked chain. α-Mannosidase treatment is followed by Endo H or Endo C. cleavage by these enzymes, it must be possible to do so after degradation by these enzymes. No.

The common 〇-linked oligosaccharide in animal cells is G a l- → G a lNAc- →Ser/Thr (protein), these glycans are endo-α-N-alpha It can be removed by the enzyme cetylgalactosaminidase, which Sold by GenzymCorp, Boston MA Ru. Many other mammalian 〇-linked oligosaccharides include sialidases, β-hexosaminidases, by treatment with exoglycosidases such as , converted to Ga1-→Ga1N Ac--+ Ser/Thr (protein) Ru. The resulting protein-bound double saccharide is endo-α-N-acetyl galacto It can be removed from the polypeptide by saminidase.

b, the enzyme--N cell control mini-ase and several other endo-β- N-acetylglucosaminidase also has two innermost cores G of various N-linked oligosaccharides. It can be cleaved between lNAc residues. The oligosaccharide characteristics of these enzymes The isomerism varies and is summarized in Table 1. Two of these enzymes, EndoC and E End F is used in place of End H to create a high manno-I Can be split open. However, unlike End H, End F has two The complex N-linked oligo-rings of the antennae are also active. The N-linked oligo ring of vertebrates is Although not a substrate for DoD, this enzyme is Husband by Robins (Robbins) et al. 5.82 (1984), in addition to high mannose oligosaccharides. Glycoproteins produced by insect cells that produce significant amounts of N-linked oligocycles It is active against chlorine. Endo-β-N-acetyl groups with different target glycoproteins If it has multiple oligo rings that are sensitive to lucosaminidase, Proteins are reacted with enzymes in order or in combination to maximize cleavage. .

c. 1 Go to end H of thin incubation in the presence of Loe ゛　Mammal cells contain all the endo-β-N-acetylglucosaminidas mentioned above. Oligo rings with structures that are resistant to It sometimes synthesizes glycoproteins that have complex oligo-rings (angular complex oligo rings).

If such oligocycles are synthesized in a cultured cell system, the oligocycles can be added to the culture medium. Later stages of oligosaccharide processing can be stopped by adding oligosaccharide processing inhibitors. It is possible. Two preferred processing inhibitors are deoxymannojirimycin and It's wine sonin. Cells treated with one of those inhibitors are endoH-sensitive This method preferentially synthesizes N-linked oligo rings with a neutral structure. Deoxymannojirimycin inhibits mannosidase I and therefore further inhibits high mannose N-linked oligosaccharides. Stop modifying. Swainsonine is an inhibitor of mannosidase■, Two α-linked mannoses on α1-→6-linked mannose residues of GlcNAc2 core Removal of residues (i.e., change from the M-d structure to the M-e structure shown in Figure 2) (exchange). As a result, under normal conditions it has endoH-resistant complex glycans. Instead, the glycosylated Asn residues containing endo H-sensitive “hybridized oligosaccharides” Swainsonine and deoxymannojirimycin are both commercially available. For example, Genzyme Corp., Boston MA. , or Boehringer Mannheim ), Indianapolis IN, etc. Often deoximer Modified glycoprotein produced in the presence of nojirimycin or swainsonine is nevertheless secreted in biologically active form. Similar to other oligosaccharide processing inhibitors, Properties and uses of insonine and deoxymannojirimycin are available from Siwarz (S. chwartz) and Datema, Adv, q door city υ, C he+s.

Biochem, 40.287-379 (1982) and Furman (Fu hrmann) et al. Biochim, 7. Aeta 8 2 5. 9 5- 110 (1985).

Purchased from Genzyme Corp, Boston MA. Glycerols such as deoxymannojirimycin or castanosbermine, which can be Oligosaccharide processing inhibitors that inhibit cosidase I or ■ also inhibit endo H-sensitive structures. However, these inhibitors are less desirable as they sometimes inhibit secretion. , as well as many other oligosaccharide processing inhibitors mentioned in the two reviews cited in the previous paragraph. It can be used for the same purpose.

d. -N ~ acetylgluco Minidase splits N-linked oligo ring of glycoprotein Another way to deal with structure is to analyze cells with one or more mutations in the oligosaccharide processing pathway. The goal is to express it within the cells. Such mutations occur in the human MMA follicle. are easily separated. Many techniques are used to create engineered mutant strains. , resistance to one or more of various lectins as an indicator of the presence of processing mutations. Alternatively, selection based on sensitivity may be a useful method. DNA that codes for glycoproteins Conventional methods (e.g., transformation of an expression vector carrying the DNA) As an alternative method, processing can be introduced into mutant cell lines using The deficient supply line mutant strain is already capable of producing the desired glycoprotein. It can also be isolated from the stock. Various mutant cell lines depending on the desired phenotype 0 can be used for example CHO, ;[! I cell (mutation research and An established Chinese hamster strain that has long been used to express lactose glycoproteins. ovarian cell line) and BHK-21 cells (an established strain derived from newborn hamster kidney). Both highly active and fast-growing GlcNAc) transferase mutations There are stocks. Both CHO and BHK-218 cells are American type culture - Collection (American Type C1ture Co11ec tion) Bockville, MD. Due to loss of enzyme activity In addition, these mutant cells are unable to synthesize any complex or hybrid N-linked oligocycles. Glycosylated Asn residues that normally carry such glycans are I have MansG IcN Ac1. Therefore, glycosylated Asn residues is only for Mans-sG lcN Aez, which is the entire structure sensitive to Endo H. have. A number of other mutant cell lines have also been characterized, including Ga1N due to various deficiencies in fucosylation and deficiencies in galactosylation. Those that cannot extend the side chain outward beyond the Ae residue, three- or four-antenna complex ori Those that cannot add additional side chains to create Go rings and Ser/Thr-linked glycans. These include various strains deficient in protein synthesis. Regarding processing defects in animal cell mutants The plenum press was created by Stanley. u+sP　ress), New York, to L　ennarz. Biochemistry of Glycoproteins and Protein Sugars (1980) edited by TheB eochea+1str　of　Gd　and　Proteo　cans) is reviewed in.

Satucharomyces (edited by S. trachern et al. Molecular biology of yeast (S, cerevisiae) arBi9i9JILof the Yeast Saccharomces ) Cold Spring Harbor Laboratory Ballou (Lanoratory) 1982 states that A series of yeast mutant strains with various defects in mannan synthesis have been generated. Ru. Therefore, mutants that cannot elongate high mannose oligosaccharides into large mannans Glycoproteins in Tucharomyces cervicinii (Eervii spp.) strains It is possible to produce.

e, en゛L t-C en゛[): tf-gai is very difficult % 1 kosi As an alternative to -ze, however, G A less preferred method of producing proteins (proteins) is when the glycoprotein is high mannose or If it contains a nano-type oligosaccharide, it may be accompanied by the subsequent use of Endo L or It is the removal of monosaccharide units by exoglycosidase degradation. So The first step is α-mannosidase to remove all α-linked mannose residues. decomposition by enzymes. In the case of mannan from a certain yeast strain, the structure of mannan If other sugar or phosphate residues are present in the outer part of the It may be desirable to include cosidases or phosphatases. This decomposition In the second step, the last mannose residue is removed by β-mannosidase. Ru. Its product, GIcNAcnie→Asn (protein), is β-hexosamini processed by a third step carried out by Dase. This enzyme is GlcNAc -→Remove terminal G　a　1NAcli group to create Asn (protein) : The last G l c N A c is not a glycosidic bond with a protein but an amino acid bond. Because hexosaminidase is bound to proteins through double bonds, hexosaminidase is Its innermost GlcN'Ac cannot be removed.

As an alternative, α-mannosidase treatment of high mannose or mannan-type oligosaccharides Next to theory, Strebtomyces brecatus ) Any reaction can be carried out with purified Endo L. This enzyme is Manβ1-→4G lcNAcβ1-→4GIcNAc cleavage between GIcNAC residues Wear. For glycosaccharides containing complex or mixed oligosaccharides, sialidar ze, β and/or α-galactosidase, β-hegysosaminidase and sequential reaction with a suitable exoglycosidase such as α-fucosidase or , can be done simultaneously if enzyme requirements permit) to convert the oligosaccharide into M It will be cut up to anz G Ic N A e 2. This oligosaccharide can be cleaved by EndoD or EndoF. Alternatively, it is a tamper to generate Manβ1--4GIcNAcβ1--4GIcNAc. can be treated with α-mannosidase. This is the end L or α-ma Degradation by Mannosidase, β-mannosidase and β-hexosaminidase Can be cleaved by either

Sialidase is E. coli, Clostridium bellfrin Gens (C1 oatridiu…7, Vibrio cholera (V1brio ch olerae) and Arthrobacter bovine faciens (Arthrobae ter　urefaciens), and Calbiochem-Behring, S an Di+4o CA, or Sigma Chemical Coop (SiHma Che Many sponsors such as Michael Corp.), St. Louis M.O. Can be purchased from sources. β-galactosidase is Asparagillus niger (N-galactosidase). Katsura country direct picture work), Siepelfringens (C concave 2 u1 country and concave,), Tachinata mame Sigma Chemical Co., Ltd. or other suitable sources. From Ma Chemical Corp.), St. Louis Mo. I can do it. α-galacto from Ecoly (E, c9LL) or green coffee beans Sidase is from Böhringer Mannheim. , Indianapolis IN. β-hexaminida The enzyme may be purified from jack beans, bovine liver or testis or any other suitable source. Sigma Chemical Co., Ltd. p, ), SL, Louis Mo. β-mannosidase is , Enzymological Methods (Meth.

To Sugihara and Yamashita in 28,769-772<1972') The snake Aehatina fulica thus mentioned and enzymatic methods (Meth.

Danyu L11-. ) λ3yo777-782 (1972) ) was purified from the oviduct of a hen as described by et al. α from jack bean - Mannosidase is preferred and is available from Sigma Chemical Co. CalCorp, St., Louis Mo. en Do H, End D and End F are from New England. nd Nuclear), B oston MA; Miles Scientific ( Miles 5 scientific>, Naperville IL; also is Böhringer Mannheim, In Available from dianapdis IN. These and other endo-β-N -The conditions for using Endo CII or Endo L, which is acetylglucosaminidase, are mentioned in the publications cited in Table 1.

f, N AGIeNAcl: all the curves of the evening, chemically protein bound It is also possible to make GlcNAc. For example, Kalyan and By Bahl (Bahl), length and tando.

qα1.258.67-74 (as described in 198B> Hydrolysis with lomethanesulfonic acid (TFMS) leaves the protein backbone intact. is used to remove all sugars except N-linked GlcNAc residues in the . Similar results were obtained for Mort and Lamport. Anal, Biochem.

82.289-'309 (1977) using hydrofluoric acid. It is obtained by

Addition of a dose to the modified lλ GIcNAc-→Asn member. In the second step, the terminal GlcNAC residue generated in the first step is As sites for the addition of galactose, two galactosyltrans Either ferase can be used. UDP-G a l : G l c N A e-Rβ1-→4 galactosyltransferase or LIDP-Gal :GlcNAc-Rβ1-→3 galactosyltransferase. This stage The first change in the sequence is β1-→4 binding to GleNAc-→Asn (protein). Adds a combined galactose residue. UDP-Gal:GlcNAc-Rβ1-→4 Galactosyltransferases are obtained from a variety of sources, with the most common and economical An example of this is cow milk. Enzymes from this source are manufactured by Sigma Chemical Co. Sigma Che+s Corp), SL.

Available from Louis Mo. UDP-Gal to GlcNAc-→As Bovine milk galactosyltransfer for galactose transfer to n(protein) Reaction conditions for the use of chelase are as described by Trayer and H. 1ll) is J, Biol, Che+a, 246°6666-75 (197 The reaction conditions are similar to those described for natural substrates in 1). The preferred reaction pH is A 0M buffer having a concentration of 6.0 to 6.5 includes a phosphate buffer that inhibits enzyme activity. Other than that, most can be used and can be used in a wide range of salt concentrations.

The presence of 5 to 20 nM Mn'' or MgI2 is preferred.

Phenylmethanesulfonyl fluoride, TPCK, aprotinin, leupeptin and peptidase inhibitors such as pepstatin and galanthone-1,4-la Galactosyltransferase exoglycosidase inhibitors like Chton can be added without affecting the activity of

Removal of carbohydrates from proteins causes solubility problems, so proteins must be removed from solution. 2-3% nonionic surfactant, such as Triton X-100, to keep the DM Compare other suitable dissolved denaturants such as SO or 2 to 3M urea. Sometimes it is necessary to use high concentrations. We use bovine milk β1-→4 galactosyl The transferase maintains sufficient activity under these conditions, and this leads to galactosyl It was found that there was no effect on the transformation stage.

A second variant of this step is the G IcN A of the β1-→3-linked galactose residue. This is a transfer to e--Asn (protein). UDP-Gal:G IcN Ac -Rβ1-→3 galactosyltransferase is produced from pig trachea. There is. Transfer galactose of UDP-Gal to G l c N A c -H The conditions for use of this enzyme are as described by Sheares and Carlson. rlson) by 吏、dangoji、qgakirm、258.9893　98(198 3).

23, Sial to 0al l--”4 (3GlcNAc-→Asn protein) Name of acid■ “The term sialic acid J (SA) refers to naturally occurring or chemically synthesized sialic acids. Contains sialic acid or sialic acid derivatives. The preferred naturally occurring sialic acid is N-acetylneuraminic acid (NeuAc). Chau 7- (S chau Adv, Carb.

Chem, Biochem, 40.131 234 (1982). Other sialic acids are also transferred from CMP-3A to galactose, such as N-glycolylneuraminic acid, 9-0-acetylneuraminic acid and 4-0- Acetyl-N-acetylneuraminic acid. R, Schauer (R, S eha N-grouped sialic acid: Chemistry, Metabolism and Function (S1al icAcids: 7. Metabdisn and Function) ( Springer-Verlag, New Yor k.

(1982)), many sialic acids are potential substrates. Ru. Substrate made in steps 1 and 2, G a l β1-=4 (3) G IcN There are two modified methods of attaching sialic acid to Ac-→Asn (protein).

The first of the two is Galβ1-→4GIc in which sialic acid is α2-→6 linked. NAc-→Added to Asn (protein). CMP used at this stage -S A: Galβ1--4GlcNAc Rα2-→6 sialyltrans Ferrase can be obtained from a variety of sources, more commonly bovine milk and rat liver. Rat liver enzymes have recently been developed by Genzymecorp (G now available from enzymeCorp, Boston, MA .

CMP-3A to Ga lβ1--4 GIcNAc--+Asn (protein ) α2−→6 sialyltra in bovine colostrum and rat liver transferring hesialic acid. Reaction conditions for using spherase include the addition of additional enzyme to increase the reaction rate. According to Paulson et al., except that it is desirable to add Therefore, above.

Natural substrates are described in ``Tanse'', qbata rm, 252.2356 62 (1977). The conditions are the same as those described above. The preferred pH is 6.5 to 7.0. phosphoric acid Although most buffers except Buffer can be used, the preferred buffer is Lis-maleate or cacodylate. The enzyme is NP-40 and Weak surfactants such as TonX-100: phenylmethanesulfonyl fluoride , TPCK, aprotinin, leupeptin and peptidas such as pepstatin enzyme inhibitors; and exoglycosides such as galactono-1,4-lactone. active in the presence of enzyme inhibitors.

A second variant of this step converts sialic acid into Galβ1−− by an α2−→3 linkage. 4 (3) GlcNAc--It is attached to Asn (protein). This bond Two sialyltransferases have been described that produce . First, CMP- 3A: Galβ1-→4GlcNAcα2-→3 sialyltransferase , van den E 1jnden and Siifo J, Biol, C Heng by Schiphorst! I11. 256.3159-3162 (1981) in the human placenta. Identified. This enzyme has not yet been purified, but can be purified by conventional methods. can be done. The second enzyme, CMP-8A:Galβ1--3 (4) G　lcN　Acα2--3 sialyltransferase is :m, 257.13835 44 (1982) Purified from rat liver by Weinstein et al. It is. The rat liver enzyme has a somewhat loose specificity, and CMP- From sialic acid to C-3 site of galactose, G a lβ1-→4GIeNAc and Galβ1-→3GlcNAe sequences can transfer sialic acid. α2 The conditions for use of −→3 sialyltransferase are described in the two publications cited in full. It is being

B, S A-→GJLI-→G IcN Ac --G lcN Ac -- A protein containing Asn protein; 1 S A-→Ga1--G IcN Ac-4G IcN Ac-→Asn (Tan The method used to make SA-→Ga l-→GlcNAc-→A To create a modified glycoprotein that has a sn (protein) dicarboxylic sugar sequence, This is similar to the method described above. Preferred embodiments include both parent N-linked oligosaccharides. A nuclear GlcNAc residue of is left attached to the protein, and a sequence of tetramonosaccharides, SA-→G a l--” G l c N A c --G I c N A c −→ is enzymatically constructed.

I GlcNAc 1-4GIcNAc--Asn A normal N-linked oligosaccharide chain consists of two outermost GlcNAC residues. processed by a selected exoglycosidase to remove all of the side carbohydrates. be managed. In the case of high mannose or mannan type oligosaccharides, α- and β- Mannosidase is used. In the case of complex or hybrid oligosaccharides, additional Soglycosidases are required, but the specific enzymes are Depends on structure.

Often sialidase, β- and/or α-galactosidase, β- Treatment with hexosaminidase and, if necessary, α-fucosidase and β-mannosidase treatment. The β-hexosaminider enzyme treatment removes GleNAc residues only from the outer side chains of oligosaccharides, not from the nucleus. and β-hexosaminidase is present during and after β-mannosidase treatment. Care must be taken to ensure that there are no Sources of exoglycosidases and The reaction conditions are S A--* G a I-- + G l c in the first step described above. This is the same as in the production of N A c-- + A s n (protein).

G lc N A cβ1--”4 G lcN Ac--Asn<Protein ) and addition of galactose to G a Iβ1--4 (3) G IcN A The method for adding sialic acid to cβ1-→4GlcNAc-→Asn (protein) is , N-bond S A a 2-→3 (6) Galβ1-4 (3) GleNAc- →Same as previously described for the preparation of modified glycoproteins with Asn(protein). It is the same.

Addition of oligosaccharides to toglycosylated amino acids of C1 protein SA-→Ga I-→GlcNAc-→ to non-glycosylated amino acid residues The basic method for addition of oligosaccharides is to attach the sugar that will be the innermost sugar residue (in this case activated glycoside derivatives of GIcNAc) and their proteins. glycosyltransferase to react with the oligosaccharide chain and elongate the oligosaccharide chain. The solution is to use chelase. Chemical and/or enzymatic protein and glycan The binding of cosides is linked to various activation groups, such as Aplin and Liston (W　r　　　　　　s　　on　n　) by CRC mm±, ev, As stated in Bioche+n, pp. 259-306 (1981). This can be done using . The advantages of chemical bonding technology are its relative simplicity and natural It does not require the complex enzyme systems required for N-linked glycosylation. use Depending on the mode of attachment, the sugar can be attached to arginine, histidine or the amino acid of the polypeptide. (b) free calcium such as glutamic acid or aspartic acid; Bonyl group or carboxy-terminal amino acid of a polypeptide; (c) cysteine free sulfhydryl groups such as; (d) serine, threonine or hydroxypropyl groups; Free hydroxyl groups such as phosphorus; (e) phenylalanine, tyrosine or tryptophenyl; or (f) added to the amide group of glutamine.

As shown below, the aglycone, R, is combined with a sugar to form a glycoside. The chemical part that reacts with amino acids to bond sugars and proteins. Rz□-5(C)12)nNH2; R-=-0(CH2)nN) Iz; GIcNAc residue was determined by Stowell and Lee ee) in Meth, Tanji 15 Yama, 83.278 288 (1982) As stated, protein and 2-imino-2-methoxyethyl-1-thousand – Treatment with β-N-acetylglucosaminide inhibits non-glycosylated proteins. It can be added to the ε-amino group of a lysine residue. with activated carboxylic acid For example, thioglycosylation of GlcNAc containing R3 or R2 Other bonding methods, such as by processing glycoside derivatives or glycoside derivatives with proteins, etc. method can also be used.

The GlcNAc residue can be an aglycone containing a free amino group, such as R3 or is a thioglycoside derivative or glycoside of GIcNAc having R1. treating the protein in the presence of a coupling agent such as a carbodiimide Calculation of aspartic acid and glutamic acid in non-glycosylated proteins by Can be added to a boxyl group.

Compounds with free amino groups (e.g. with aglycones R1 or R4) GlcNAe derivatives) were also described by Yan and Woid. This is stated in Dankisho, Yamayama Yu, λ3.3759-3465 (1984). Derivatizes the amide group of glutamine through the action of transglutaminase It can be used for.

GIcNA to the thiol group of cysteine residue of non-glycosylated protein Attaching the e residue makes the protein contain an electronic moiety, such as an acrylic acid unit. G1eNAc glycocontaining aglycones, such as R1 or R6 This can be done by treatment with sedatives or thioglycosides.

Protein aroma due to monosaccharides such as GleNAc, glycosyl amino acid residues The aglycone having a diazo group, such as aglycon R1 or R6, is This can be done by treatment with glycosides or thioglycosides.

Numerous other coupling methods and aglycone structures with GlcNAe derivatives Used for protein synthesis.

After chemical induction of the protein with GIeNAc residues, the sequence SAa 2- -3(6) GJIIβ1--4 (3) G lcN Ac-= is Asn-bonded G Sequential fermentation of galactose and sialic acid residues as described for IcNAc residues Constructed by prime addition.

In other variations, the protein is induced by: Galβ1--4(3)GleNAc-X, Galβ1--4(3)GIcNA cal −→4 G IeN Ae −X, SAα2−→3(8) Galβ1 -4(3)GleNAc-X, or 5Aa2-→3(6')Galβ1-→4(3)GIcNAcal-→4GIe NAc-X, where X is a free amino group, an active ester of a carboxylic acid, a diazo group, or It is an aglycone that has other groups.

The same technique chemically adds galactose directly to amino acids rather than GlcNAc. As previously mentioned, the galactose can be used to synthesize the sialic acid. It can be enzymatically extended or capped as such.

D, GIcNAc member GleNAe protein or Gle NAe--GIcNAc protein is a protein similar to the outer side chain of a complex oligosaccharide. Others found on the GlcNAc residue attached to the terminal mannose unit of the linked oligosaccharide can be used to construct the carbohydrate structure of

Fire 1 Salmon 1 < ai c N A cβ1--3Galβ1--4) unit repetition Generation of proteins with repeats, using the method described above, GICNA After preparation of e protein or GIcNAcal-→4GIcNAc protein , U D P -Gal:G IcN Ac -Rβ1-→4 galactosyl Transferase and U D P -G lcN Ac:Galβ1-→4G IcNAc-Rβ1--3N-acetylglucosaminyltransferase Long carbohydrate chains can be created by several consecutive reactions with each other. This is the starting material GIcNAc protein or GIcNAcal-→4GIcNAe protein (GIcNAcal--3Galβ1--4) n-type polylactosamide attached to It is possible to create a nyl-type structure. Kaur, Turco and Laine are UDP-Gal:GIcNAcal- of bovine milk. →4 galactosyltransferase converts polylactosaminyl oligosaccharide into β1-→ 44-linked galactosyl residues can be transferred. rnatiobol 4, 345-351 (1982), and β1-→3N-acetylglucosaminyltransferase was determined by J. Biol. , Chem.

258.3435-37 (1983) van den Eijden It has been identified in Novikotsu ascites tumor cells by E. Ijnden et al.

The number of repeats of GIcNAc-→Gal units in the structure depends on the desired length. can be varied: 1 to 10 units is sufficient for most applications. . The necessary requirement is that after the addition of the dimonosaccharide unit, the carbohydrate chain changes to α2-→3- or The exposed The presence of galactose residues.

Therefore, the final structure is S A a 2-6 (3) Galβ1--”4[GIcNAcal-→3G a lβ1-→4]nG lcN Ac protein or SAα2--6 ( 3) Galβ1--4 [G IcN Acβ1-3Galβ1--4]nG leNAcβ1--4GlcNAc protein (wherein n is 1 to 10) It is.

The advantage of introducing such a polylactosaminyl structure is that it increases solubility or protein-based to protect it from degradation by rotease or recognition by the immune system. The idea is to hide the chain better.

ILLL Galβ1-4(3) [FLl(!(Z1-3(4)GIcN Ac or SAα2-3Galβ1-3 (Fucα1 = 4) GlcNA Production of glycoproteins with eiill structures at their ends.

Galβ1 = 4 (3) GIeNAe-protein, G a lβ1--3 (4 ) [G IcN Acβ1-3Galβ1--4]nGlcNAe-protein , Galβ1 = 4 (3) GIcNAcal--4GIcNAc-protein, G a lβ1-→3 (4) [GleN Acβ1-3Galβ1--4]n GleNAcβ1--4GlcNAc-protein, 5Aa2-3Galβ1- →3　G　lcN　Ac-protein, SAα2　-30alβ1--3 [GIc NAcal--3Galβ1-4]nGlcNAc-protein, 5A (22-30alβ1--3GlcNAcβ1--4 GlcNActa Npaku or SAa'2 = 3Galβ1--3 [GIcNAcal-3Galβ1=4] nGleNAcβ1--4GlcNAc-protein (where n is a number from 1 to 10 After the preparation of the compound of the formula uc and GDP-Fuc: GIcNAcal-→3(4) fucosyl transf. GIcNAe residues can be attached to any of the receptors by treatment with chelase. Wear. Purification of this fucosyltransferase, its substrate specificity and preference The reaction conditions were according to Prieels et al. 256.104456-63 (1981). sialylated The activity of this enzyme on substrates was determined by Johnson and Watki. E.A. Davidson (W.B.Jkins) on), J.C. Williams and and N, M, Diferente (N, M.

Proc edited by D i F errante, Vlllth Int.

Danr1. Published in GI Ocon'ates (1985). if Attach fucose to appropriate G Ic N A e residue receptors only through α1--3 bonds. GDP-Fuc:GIcNAcal--+3fucosilt Transferase can be used. This enzyme was developed by Johnson ) and Watkins E, A.

Davidson (E, A, Davidson), J, C, Williams (J.

C, Williams) and N, N, DiFerrente (N, M, DiFerra) Proc edited by nte>, Vlllth, th, 1.

Published in Gl coconjugates (1985).

Eo, has a cell surface receptor specific for sucrose of 1 cosyl lymphoma to 4 The cells in the body are able to recognize and absorb glycoproteins that have the appropriate carbohydrate structure. can.

The best-characterized glycosylated cell surface receptors are the hepatocyte Gal receptors; Man/GlcNAc receptors on reticuloendothelial cells and hepatocytes, lymphocytes and It is a fucose receptor found in tetracarcinoma cells. sugar-special The issue of heterosexual cell surface receptors is a sugar edited by Lennarz. Biochemistry of proteins and protein polysaccharides co roteins and proteo l cans) prembreth (P lenum P ress), New York (1980), pp. Neufeld and Aswell, 241-266. Hwell).

Proteins are produced by creating glycoproteins that have appropriate sugars at the non-reducing end positions. Thus, it can be directed to cells that have sugar-specific cell surface receptors.

Several techniques are used to expose the desired sugar. One method is generally Adv by Ashwell and Morell, 　Hio Mito.

Exoglycosides, as described in It involves the processing of natural glycoproteins by enzymes.

Another approach is described by Stahl et al. in Roe, Natl.

Described in M.A. Ashizumi, USA 1399-1403 (1978) As such, it is the addition of monosaccharides to proteins.

The third method is one button φ” mountain by Yan and Wald. As reported in 23, 3759-3765 (1984), The addition of oligosaccharide derivatives isolated from natural sources such as rubumin The glycosylated proteins that are the subject of this invention depend on the specific sugars added. can be directed to specific cells.

Gal--”G lcN Ac-protein, Ga1--”GIcNAc-→G IcNAc-protein, (Gal-→GIeNAc)n--G, at--GIc NAc-protein, and (G a I-→G I c N A c) n-→ G a I--+ G Ie N A c -- G l c N A e - protein, (where n is a number from 1 to 10). These target liver cells.

G Ic N A c-protein, G Ic N A c - → G I c N A c-protein, (G Ic N Ac--+Ga1)n--+G IcN Ac-protein, and (Gl c N A c −-+ G at) n −- + G lcN Ac-→G lc N Ac-protein, where n is a number from 1 to 10. These are macros Target phages. lastly Ga1--(Fue-→)GIcNAc-protein, Ga1--(Fuc--) GIeNAc-→GlcNAc- protein, Gal--+ (Fuc-- +) G IcN A c −-+ [G al −-+ (F uc −-+) + G 1cNAc]n-protein, and Gal　--=(F　uc--)G　lcN　Ac　--[Ga1--(F　u c --)mG lcN Ac]n --G lcN Ac-protein, (in the formula , n is a number from 1 to 10, and 语 is 0 or 1).

These target liver cells, lymphocytes and cancer cells. One example of directivity application is It is an enzyme replacement treatment. For example, glucocerebrosidase has been used to treat Gauche disease. can be directed to macrophages. The second application example is to treat teratocarcinoma cells. Attack with drugs or toxins.

The following non-limiting examples have multiple high mannose and mannan oligosaccharides. Figure 3 illustrates the method of the present invention on yeast glycoproteins.

Stage ILL Endo H treatment of yeast extracellular invertase Yeast extracellular invertase is a glycoprotein that has approximately two high mannose and seven mannan oligosaccharides. It is Satsuri Romyces cerevisi A commercial product of extracellular invertase prepared from Ae) is available from Sigma Chemical Co., Ltd. (Sigma, Chem, Corp,) SL, Louis Mo, This commercially available product is available from Trimble and Maley. left by Dan io1. chem, 252.4409 12 (1977 ) and Tri+5ble et al. Therefore, it is basically based on Danyu of Chew, 258.2562-67 (1983). Processed in End H as described in . Purified invertase is The glycoprotein was denatured by placing a 1% SDS solution in a boiling water bath for 5 minutes. I made her have sex. Then, the denatured invertase (250 μ9) was added to Endo H (0 , 3 μg Miles Scientific (Miles 5 scientific) N aperville IL) and 0.1M sodium citrate for 20 hours at 37°C. Mu buffer pH 5,5,175μ! It reacted inside. After End H treatment, the reaction mixture The compound was applied to a Bio-Gel (Bio-Get) P-4 column (IX 10c m) equilibrate and elute with ammonium acetate at 5C)+M pH 6,5 Desalination was carried out by The desalting method is not limited to this. dialysis or Protein precipitation can also be used. The raw material eluted into the void volume of the column is pooled. and lyophilized.

Analysis of Endo H-treated preparations of 5DS-denatured invertase by 5DS-PAGE As shown in Figure 3, the analysis shows that the glycoprotein has a single glycosylation site at each glycosylation site. It was confirmed that the invertase was converted to a form consistent with invertase having only GlcNAc residues. Indicated.

In a parallel experiment, native invertase was combined with 5DS-modified invertase. Treated with Endo H in the same manner. Analysis of desalting reaction products by 5DS-PAGE As shown in Figure 3b, the 2-3 oligosaccharide chain of natural invertase is It was shown to be resistant to cleavage by H. Exposed manno on the resistant strand To remove S residues, 250 μg of Endo H-treated invertase was desalted and frozen. Dry and add 50mM Na(J, 4mM ZnC1z and 20 wheel units). R of New York State University Albany NY for Chinese bean α-mannosidase, 5 of pH 5.0 containing (gift from Dr. Rimple (R, Tri+*ble)) 0mM sodium acetate 100μ! The reaction was carried out for 17 hours at 37°C. 5DS-P Analysis of the reaction mixture by AGE shows a shift to lower molecular weights, as shown in Figure 3d. The attached mannose residues are removed by α-mannosidase treatment during the reaction. It showed that it was done.

A sample of Endo H-treated samples of invertase in vitro, both native and denatured yeast. ctosylation Endo H-treated sample of modified yeast extracorporeal invertase [85 μs, approximately 15 nm 0.8 % Triton X-100, 25TaM MnC1z, 1.25mM UDP-[Co H]Ga1 (specific activity, 8Ci/mol) and bovine milk UDP-Gal: G leN Acβ1-→4 galactosyltransferase (100 mU, Sigma Chemical Coop (Sigma Chew.

Corp, ) SL, Louis Mo) at pH 6.3. React in M2-(N-morpholino)ethanesulfonic acid (MES>180 μl) I let it happen. As shown in Figure 4, a portion was taken out over time and 5DS-PAGE Analyzed by. A slow increase in molecular weight was clearly visible up to 1 hour reaction time. It was. This result shows that precipitation with 0.5M HC1/1% phosphotungstic acid This was confirmed by measuring tritium incorporation into the material.

The results are shown in FIG.

Non-radiolabeled galactosylated samples of native and modified yeast invertase in vitro was prepared as a substrate for the sialylation reaction. Endo H-treated modified invertase and endoH and α-mannosidase-treated native invertase using the method described above. and galactosylated with non-radioactive UDP-Gal.

Step 1: Simulation of galactosylated samples of natural and modified yeast invertase in vitro. Allylation Natural and denatured samples of non-radioactive galactosylated yeast invertase ( 50μg of protein) was added to 0.7% Triton X-100, 2mM CMP-[”C NeuAc (specific activity 1, ICi/mmol) and bovine colostrum CM P- S A: Galβ1-4GIcNAc-Ra2-→6 sialyltransfer [1,1 mU, J, Dan Yuri 9q Ishibe, 252.2356-2362 (1 977) purified according to the method of Paulson et al. React at 37°C for 17 hours in 70 μp of Tris-maleate at pH 6, 7, 0, 1M. The 0.0 reaction mixture was subjected to 5DS-PAGE and automated 5DS-PAGE as shown in Figure 6. Analyzed by radiograph. The radioactivity associated with the invertase band is Invertase is converted into galactic acid by α2-→6 sialyltransferase. It was shown that it was added to a tos residue.

The following non-limiting example shows the conversion of proteins to proteins that are not glycosylated in their native form. 1 illustrates the method of the invention using chemical and enzymatic techniques.

Step 1 - Thioglycoside induction of GIcNAe to bovine serum albumin (BSA) body chemical additions BSA (Lee) et al.

3956-63 (1976), John Hopkins University. (Johns Hopkins) and Dr. R. S. Chnaar (Johns Hopkins). ) by 2-imino-2-methoxyethyl-1-thio-N-acetylglucosa induced by treatment with minido. Glycosylated BSA has an average of 1 molecule per molecule. It contains 48 lysine-linked GIcNAe residues.

Stage R12GIcNAc4s BSA Tsugalactosylation G IcN Ac4BB S A (0.9my), 0.6% Triton X-100, 20mM MnCf 2.5MM UDP [koHkoGal　(specific activity, ICi/5ol), 1m Mm preload tonneau 1.4-lactone, 1mM phenylmethanesulfonyl fluoride TPCK (21 μg), aprotinin (12 μTIU), leupeptin (0 , 6 μg), pepstatin (0.6 μg) and bovine milk UDP-Gal : G leN Ac - p containing Rβ1-→4 galactosyltransferase The reaction was carried out in H6,3,0,12MMES600AL1 for 17 hours at 37°C. So The glycosylated BSA was filtered by Bio-Gal P4 gel filtration. The reaction components were separated from each other. By measuring the amount of radioactivity incorporated into BSA, It is calculated that 46% of the available GIcNAc residues are galactosylated. 6 Under the same conditions, the second reaction of galactosylated BSA increased from 46% to 51%. Increased response rate. Galactosylated BSA is available from Cooper Biomedical (Coop). Anti-BS available from Biomedical) Malvern PA, Purified by A antibody column.

[Sialylation of galactosylated BSA Galactosylated BSA (240μf) was added to 3n+MCM P-[”CcoN euAc (specific activity 0.55 Ci/mol) and bovine colostrum CMP-3A:Ga lβ1-4GIcNAc-Ra2-→6 sialyltransferase (2,1 mU) for 16 hours in 120 μl of pH 6, 7, 0, 1 M trismaleate. The reaction was carried out at 37°C. Glycosylated BSA was separated from other components by gel filtration.

By measuring the ratio of I40 and 3H radioactivity incorporated into the sample, Ga It is calculated that 42% of the 1-→GlcNAc-→ protein residues are sialylated. It was. 25 mLJ of sialyltransferase and sialylated BSA for the second time. The reaction increased the rate of sialylation to 51%. The glycoprotein is anti-BSA isolated by immunoaffinity chromatography in a body column.

As shown in Figure 7, three types of glycosylated BSA were analyzed by 5DS-PAGE. Analysis showed a significant increase in molecular weight with each step of the process. This evidence is SA- This proves that the →Ga1-→GlcNAc-→ portion was built horizontally on the protein. Ru.

The following non-limiting example shows that GIcNAc/Man-specific receptors on macrophages G lcN Ac -B S A and G a lβ1-→4G lcN A It shows the differential uptake of e-BSA.

Mice have cell surface receptors that recognize terminal GIcNAc and Man residues. Peritoneal macrophages were cultured in thioglycollate medium (1.5 + Il per mouse). obtained from mice 4-5 days after intraperitoneal injection of. The peritoneal cells were 10% bovine. washed with Dulbecco's modified minimal medium (DME) containing fetal serum (Fe2), and A 96-well tissue culture plate was plated at a concentration of 2×10'4111 cells/6. After 4 hours, remove the hole. Washed twice with phosphate buffered saline (PBS) to remove adherent cells. that hole The remaining adherent cells were radiolabeled with +25I using the chloramine T method. G leN Ac - ['25I] BS8 and G a lβ1 -4G Used for uptake experiments with IcNAc-[”’I]BSA. Radiolabeled The protein preparations were prepared in 10% FCS and 10mM HEPES [4(2-hydrogen). DM containing (loxyethyl)-1-piperazineethanesulfone fi] pH 7,4 E was added to 100 μl at a concentration of 0.1-1.2 μm 17×1. Glycodyl Yeast mannan (ltng /11) Parallel experiments were conducted in the presence of Cells were incubated with the sample for 30 minutes at 37°C. 5 minutes with PBS to remove residual protein not taken up by the cells. Washed twice.

Washed cells were dispersed in 200 μl of 1% 5DS and their radioactivity was measured.

Non-specific uptake (CPM in the presence of yeast mannan) and total uptake (CPM in the presence of yeast mannan) Man/Gl by mouse peritoneal macrophages (in the absence of CPM) cNAc receptor-specific uptake was calculated.

GIcNAc-[12’I]BSA and Galβ1--4GIcNAc-[” J] The specific uptake of BSA is shown in Figure 8 as a function of BSA concentration. Ru. The result was Gαlβ1-→4GIcNAc-BSA (GIc NAc-BSA is recognized and taken up by mouse peritoneal macrophages. This indicates that the

The following non-limiting example demonstrates how galactose-specific receptors in the hepatoma cell line HepG2 G a lβ1-→4 GleNAc BSA and 5A (22-→6 Gal Differential uptake of β1-4G IcN Ac-BSA is shown.

G IcN Ac -B S A and Ga1-→GlcNAe-BSA The samples were radiolabeled using the chloramine T method for 125 days. HepG2 Cells were cultured in DME containing 10% fetal calf serum. Intake experiment is 35mm set This cannot be done if the cells planted in a fiber culture dish are approximately 70% confluent. . The cells were washed with protein-free medium and 2On+M HEPES, pH 7. ,3, cytochrome C (0.2my/xl) and Ga lβ1-→4GIc NAc-[12J]BSA or 5A(Z2--6Galβ1-→4GlcNA c-[+2'I] DME1zz containing 0.5-7.5 μg of BSA! It was cultured in

Parallel experiments were carried out using non-radioactive Asialo orosomes to measure non-specific uptake. The experiment was carried out in the presence of id (0,2m++/x1). Cells were radiolabeled with protein solution Incubate at 37°C for 2.5 h in a 5% CO2 atmosphere, and at 1.7+*M Ca” Washed 5 times with cold PBS containing +. Wash cells with IMNaOH/10% 5DS Dispersed in 1zN. Another part measures radioactivity and protein content per culture dish. was used to do. Assume that the amount of protein in each dish is proportional to its number of cells. Ta. Complete removal of non-specific uptake (CPM in the presence of asialoorosomucoid) HepG2 (CPM in the absence of asialoorosomucoid) Galactose receptor-specific uptake by cells was calculated.

Figure 9 shows galactose receptor-specific uptake as a function of glycosylated BSA. It shows. The result is SAα2-→6G a lβ1--4 G IcN Ac　-B　S　A (Galβ1-→4G　IcN　Ac　-B　S　A It is shown that is recognized and taken up by HepG2 cells.

The following non-limiting example is a high mannose type oligo I! Mammals that have one chain 1 shows the method of the invention on glycoproteins.

111L A glycoprotein with 11 high-mannose oligosaccharides, ribonuclea. Deglycosylation of enzyme B Sigma Chemical Corp (Sigma Chem, Corp,) St, Lo Natural seaweed bonuclease B (490 μfI) available from uisMo was added to the Setsuji, Tanbebe, 251.6016-24 (1976) The affinity of concanavalin A announced by The product further purified by take chromatography was purified using Endo H (50 clear U, Gen. Team Corp (Genzy Haku eCorp, ) Obtained from B oston M A ) and 100μ of sodium acetate at pH 5,5,510mM! 24 hours inside37 The reaction was carried out at ℃. 5DS-PAGE shows that the glycoprotein is a single G The form containing the Ac residue is biogel (Bio-Get) P6DG The column desalted and the ribonuclease fraction was lyophilized.

[Treatment with endo H or galactosylation of bonuclease B] Treated with Endo H or bonuclease B (400 μg), 0.1% Triton X-100,0,OIM MnC4! z, 100mU bovine milk UDP-G al　:G　IcN　Ac　-Rβ1-→4 galactosyltransferase and 300nmolUD　P　-[koHkoG　al　(specific activity 17.3Ci/ +on+ol) in pH 6,3,0,1M ME3250μN for 3 hours 37 The reaction was carried out at ℃. Galactosylated ribonuclease to FPLC using Mono S column Therefore, I analyzed it. 2011 IM sodium phosphate to LM Na at pH 7,95 Elution was with a linear gradient of 20mM sodium phosphate containing C1. Galactosylated The phosphoric acid eluted at a NAC1 concentration of 0.13M. Tan by UV absorption (A28°) The Pak peak coincides with the radioactivity peak as shown in Figure 10 (0...0) did. Collect the protein peaks eluted with 0.13M NaCl' and apply them to 5DS-P. Analyzed by AGE. The only protein band detected after staining with Comaseaple was , co-migrated with Endo H-treated ribonuclease B (not shown).

艮ll Antiobtained from sialylation step 2 of galactosylating ribonuclease B Add 40 μl of reaction mixture to 0 μl of 8.5 mM CMP-NeuAcl and rat Liver CM P -NeuAc:Gal -Rα2-→6 sialyltransfera ese (1,6+ntl, G enzyme Corp, ) and reacted at 37°C for 18 hours. I let it happen. The sialylated ribonuclease was used under the conditions described in step 2 to Analyzed by FPLC on a NoS column. Sialyl ribonuclease is A2 ．． . Eluted at a NaCl concentration of 0.188, judging from the radioactivity graph. It was. A graph of radioactivity is shown in Figure 10 (Δ-Δ). Ga1--” Conversion from GleNAc-RNAse to SA--Ga1-→RNA5e was observed to be quantitative.

Although the invention has been described with respect to specific embodiments, it is possible to modify or glycosylate proteins. glycosylated proteins and modifications and variations of glycosylated proteins. It is understood that the contractor can consider it. All such modifications and changes are included within the scope of the claims. I think it is included.

, and... Feeling μN-mu1go-4 X Cyto ■ Yeast mannan, all high macinoses (Streptomyces bricata Oligosaccharides and hybrid oligosaccharides, (1, 2) The enzyme is a C1 attached to the C1-→67-nose residue of the MANzCIcNAc2 nucleus. -→3-linked 1-ninose, and we found that the enzyme is the innermost GlcNAc discovered that it was not inhibited by the C1-→6-linked fucose attached to the fi group. . ) Endo CII Certain high mannose oligosaccharide-(3)(cross)lysium lueringens (C1-→3 bond to β-bonded mannose (Clostrid ium 7)) When the base sugar is replaced with another sugar at the C-4 position or β -bond When the base residue is replaced with a β1-→4-linked GlcN^C residue, the group except that the quality cannot be cleaved It is the same as End ■. ) Gindo D Innermost 1cNAc1pi base (4) (Jib U Cocas New Moni A　It has 7 courses that are connected by C1-→6 (Kan drμm 徂 Want ■ White group)) Mans-sGIcNAc2 with or without x) Do L ManGIcNAc2 (5) (S, Bricatas (Turtle 1 color I 1 cat gyos>) Endo F High Mannose and Dual Antennary Complex (6) (Flavobacterium menishigosepticum oligosaccharide References: 1. Cresotin (Torentino) et al., Meth, 7.50.

574-580 (1978>.

2.Tai and Kobata, Biochem, Dan 4° Rcs, Commun, 78.434 441 (1977).

3. Kobata, Meth, Kunwang Annotation, 50, 567-574 (1978).

4. Trimble et al., J. Biol, Chem, 254 ゜9708-13 (1979).

5, Plummer et al., L. io1. Chem, 259 .

10700-4 (1984).

200-; Fig, 3 71 people, 4 Figure 5 Oiten-roi (crow) Fig, 6 Fig, 7 Figure 8 5 Natsume (pq/ml) Figure 10 international search report 1ms+n°″0°alAopHc+lle″)Iap “T,■l586.o, , 49゜lmemmanml^””””OR” PCT/US86100495

Claims (45)

[Claims] (1) Gal-G by adding galactose residues to nuclear N-acetylglucosamine forming an lcNAc sequence; and attaching a sialic acid residue to the galactose. In addition, glycoprotein repair consists of forming the SA-Ga1-GlcNAc sequence. Decoration method. (2) First develop asparagine-linked oligosaccharide chains of glycoproteins, and Removes all sugars except for the nuclear N-acetylglucosamine residues that are bound to the protein. 2. The method of claim 1, further comprising: (3) Claim 2, wherein the oligosaccharide chain is cleaved by endoglycosidase. Method described. (4) Endoglycosidase is endo-β-N-acetylglucosaminidase H, endo-β-N-acetylglucosaminidase F, endo-β-N-acetylglucosaminidase luglucosaminidase CII, endo-β-N-acetylglucosaminidase D , endo-β-N-acetylglucosaminidase L, and combinations thereof. 4. The method of claim 3, wherein the method is selected from the group consisting of: (5) α-mannosidase, endo-α-N-acetylgalactosaminidase, and combinations thereof. 5. The method according to claim 4, which also includes cleavage of sugar chains. (6) α-mannosidase, endo-α-N-acetylgalactosaminidase, and combinations thereof. The method according to claim 1, which also includes cleavage of sugar chains. (7) Claims in which the oligosaccharide chain is cleaved by exoglycosidase degradation The method described in Scope No. 2. (8) Exoglycosidase is sialidase, α-mannosidase, β-mannosidase nosidase, α-galactosidase, β-galactosidase, α-fucosidase , β-hexosaminidase, and combinations thereof. 8. The method according to claim 7. (9) The method according to claim 8, wherein the oligosaccharide chain is cleaved in the following order: . 1) Decompose glycoproteins with α-mannosidase to remove α-mannose residues that; and 2) Decompose the product of 1) above with β-mannosidase to remove β-mannose residues. to leave (10) Selected from the group consisting of exoglycosidase and phosphatase. Claim 9 also includes degrading the glycoprotein by selected additional enzymes. How to put it on. (11) Decomposing the product of step 2) with β-hexosaminidase Claim 1 The method described in item 0. (12) Glycoproteins are first processed by exoglycosidases and then by endoglycosidases. Claim 2, in which oligosaccharide chains are opened by sequential decomposition with enzymes. The method described in section. (13) The method according to claim 12, wherein the oligosaccharide chain is cleaved by the following method. 1) Degrading glycoproteins using α-mannosidase to remove α-mannose residues and 2) converting the product of 1) to endo-β-N-acetylglucosa Glucosaminidase D and endo-β-N-acetylglucosaminidase D. Degradation with an endoglycosidase selected from the group. (14) Sialidase, α-galactosidase, β-galactosidase, β-galactosidase A group consisting of xaminidase, α-fucosidase, and combinations thereof The glycoprotein is degraded by a selected enzyme, followed by endo-β-N-acetate. Tylglucosaminidase D and endo-β-N-acetylglucosaminidase The oligosaccharide chain is cleaved by decomposition by an enzyme selected from the group consisting of 13. The method according to claim 12 for modifying glycoproteins. (15) Cleave high mannose oligosaccharide chains with α-mannosidase to leave mannose residue. 3. The method of claim 2, which also includes removing the group. (16) The method according to claim 2, wherein the oligosaccharide chain is cleaved by chemical treatment. (17) Group consisting of trifluoromethanesulfonic acid and hydrofluoric acid The method according to claim 16, wherein the oligosaccharide chain is cleaved by a compound selected from Law. (18) Initial production of glycoproteins in cells in the presence of glycosidase inhibitors The method according to claim 1, which also includes: (19) Glycosidase inhibitors include deoxymannojirimycin, swainsoni selected from the group consisting of sterine, castanospermine and deoxynojirimycin. 19. The method of claim 18, wherein the method is selected. (20) Cells with one or more mutations in the oligosaccharide processing pathway initially produce sugar proteins. 2. The method according to claim 1, which also includes producing parka. (21) Glycoside or thioglycoside S-X (wherein S is N-acetyl group is the first sugar selected from the group consisting of lucosamine and galactose , X is an aglycone), and galactose, N-acetylglucosamine, fucose, and sialic acid. a protein consisting of the enzymatic addition of a second sugar selected from the group consisting of How to modify Park. (22) The aglycone is an activated carboxylic acid; a free amino group; an electrophilic site; and Claim 21 consisting of an active group selected from the group consisting of diazo group How to put it on. (23) Glycosides or glycans with aglycones containing activated carboxylic acids Oglycosides and lysine, arginine, histidine, amino terminal amino acids of proteins acids, and other amino acids containing free amino groups. 22. The method according to claim 21, which comprises reacting the protein with amino acids of protein. (24) Glycosides or thioglycosides with aglycones containing free amino groups acid, glutamic acid, aspartic acid, amino acids at the carboxy-terminus of proteins, and other amino acids containing a free carbonyl group. 22. The method according to claim 21, which comprises reacting with an amino acid of. (25) Glycosides or thioglycosides with aglycones containing electrophilic sites and cysteine and other amino acids containing free sulfhydryl groups. Claim 21, which comprises reacting the selected protein with an amino acid. the method of. (26) Glycosides or thioglycosides with aglycones containing free amino groups and hydroxyproline, serine, threonine, and other amines with free hydroxyl groups. comprising reacting with an amino acid of a protein selected from the group consisting of amino acids. The method according to claim 21. (27) Glycoside or thioglycoside with aglycone containing diazo group From phenylalanine, tyrosine, tryptophan, and other aromatic amino acids Claims comprising reacting with an amino acid of a protein selected from the group consisting of: The method according to paragraph 21. (28) Glycosides or thioglycosides with aglycones containing free amino groups The method involves reacting the compound with glutamine using transglutaminase. Scope of Claim 22. The method according to item 22. (29) The first sugar is GlcNAc, and the enzyme addition described above is: Gal-GlcNAc is created by adding a galactose residue to N-acetylglucosamine. −→ formation of the sequence and addition of sialic acid residues to galactose to form SA-G forming a1-GlcNAc-→ sequence according to claim 21. Method. (30) The first sugar is galactose, and the enzyme addition described above is: Adding a sialic acid residue to galactose to form an SA-Gal sequence. 22. The method according to claim 21. (31) Galactose residues are converted into N-acetyltransferases by galactosyltransferase. 30. The method according to claim 1 or 29, wherein the method is added to tilglucosamine. (32) Galactosyltransferase is UDP-Gal:GlcNAc- Rβ1-→4 galactosyltransferase and UDP-Gal:GlcN selected from the group consisting of Ac-Rβ1-→3 galactosyltransferases 32. The method of claim 31. (33) Galactose is added to N-acetylglucosamine by the following process. The method according to claim 1 or 29: 1) UDP-Gal:GlcN Ac-Rβ1-→4 galactosyltransferase, the terminal galactose is 2) Step 1: incubate with protein derivative to add to lcNAc; ) to add terminal GlcNAc to terminal galactose using UDP- GlcNAc: Galβ1-→4GlcNAc-Rβ1-→3N-acetylglu incubating with cosaminyltransferase; 3) To add terminal galactose to terminal GlcNAc from the product of step 2) UDP-Gal:GlcNAc-R1-→4 galactosyltransferase incubate with; and 4) Oligosaccharide chain (Galβ1-→4GlcNAcβ1-→3) n units (in the formula, n is a number between 1 and 10) is produced. Repeat steps 2) and 3). (34) A claim that also includes the addition of fucose to the Gal-→GlcNAc-→ sequence The method according to item 1, item 29 or item 33. (35) GDP-Fuo: GlcNAcα1-→3 fucosyltransferase Claim 3 in which fucose is added to the Gal-GlcNAc-→ sequence by The method described in Section 4. (36) Nonionic surfactant, chaotropic agent, organic solvent, urea, protea -ase recombinants, exoglycosidase inhibitors, disulfide bond reducing agents, or Adding galactose residues to N-glucosamine in solutions containing these combinations 30. The method according to claim 1 or 29, further comprising: (37) Sialyltransferase converts sialic acid residues into Gal- 2. The method according to claim 1, wherein the GlcNAc sequence is added to the GlcNAc sequence. (38) Sialyltransferase is CMP-SA:Galβ1-→4Gl eNAc-Rβ2-→6 sialyltransferase, CMP-SA:Galβ 1-→3(4)GlcNAcα2-→3 sialitransferase, and CM P-SA: Galβ1-→4GlcNAcα2-→3 sialyltransferer 38. The method of claim 37, wherein the method is selected from the group consisting of: (39) Oligosaccharides with exposed sugars and Gal-→GlcNAc sequences were l-→GlcNAc sequence is added to the protein, and the exposed sugar is added to the sugar. The protein is recognized by a cell surface having a specific surface receptor for the protein. A method of directing the molecule to the cell. (40) Claim that the oligosaccharide chain is a dimonosaccharide consisting of Galβ1-→4GlcNAc The method according to item 39. (41) The oligosaccharide chain is Galβ1-→3(4) [Fucα1-→4(3)]Gl cNAc, SAα2-→3Galβ1-→3(Fucα1-→4)GlcNAc , Galβ1-→3(4) [Fucα1-→4(3)] GlcNAcβ1-→4 GlcNAc, and SAα2-→3Galβ1-→3(Fucα1-→4)G A branched oligosaccharide selected from the group consisting of lcNAcβ1-→4GlcNAc. 40. The method according to claim 39. (42) Glycosylated proteins containing any of the following oligosaccharide sequences: Galβ1-→4GlcNAc-→; Galβ1-→3GlcNAc-→; SAα2-→6Galβ1-→4GlcNAc-→;SAα2-→3Galβ1 -→4GlcNAc-→;SAα2-→3Ga1β1-→3GlcNAc-→; Galβ1-→4(Fucα1-→3)GlcNAc-→;Galβ1-→3( Fucα1-→4)GlcNAc-→;SAα2-→3Galβ1-→3(Fu cα1-→4) GlcNAc-→;Galβ1-→4GlcNAcβ1-→4G lcNAc-→;Galβ1-→3GlcNAcβ1-→4GlcNAc-→; SAα2-→6Galβ1-→4GlcNAcβ1-→4GlcNAc-→;S Aα2-→3Galβ1-→4GlcNAcβ1-→4GlcNAc-→;SA α2-→3Galβ1-→3GlcNAcβ1-→4GlcNAc-→;Gal β1-→4(Fucα1-→3)GlcNAcβ1-→4GlcNAc-→;G alβ1-→3(Fucα1-→4)GlcNAcβ1-→4GlcNAc-→ ;SAα2-→3Galβ1-→3(Fucα1-→4)GlcNAcβ1-→ 4GlcNAc-→; [GlcNAcβ1-→3Galβ1-→4]nGlcNAc-→, where n is 1 is a number between and 10; [GlcNAcβ1-→3Galβ1-→4]nGlcNAcβ1-→4Glc NAc-→, where n is a number between 1 and 10; Galβ1-→4[GlcN Acβ1-→3Galβ1-→4]nGlcNAc-→, where n is from 1 to 10 The number is between; Galβ1-→3 [GlcNAcβ1-→3Galβ1-→4] nGlcNAc-→, where n is a number between 1 and 10; SAα2-→6Ga lβ1-→4[GlcNAcβ1-→3Galβ1-→4]nGlcNAc-→ , where n is a number between 1 and 10; SAα2-→3Galβ1-→4 [GlcNAcβ1-→3Galβ1-→4] nGlcNAc-→, where n is a number between 1 and 10; SAα2-→3Galβ1-→3 [GlcNAcβ1-→3Galβ1-→4] nGlcNAc-→, where n is a number between 1 and 10; Ga1β1-→4 (Fucα1-→3) GlcNAcβ1-→3 [Galβ1- →4(Fucα1-→3)mGlcNAcβ1-→3]n-Galβ1-→4G lcNAc-→, where m is zero or 1 and n is a number between 1 and 10 Galβ1-→3(Fucα1-→4)GlcNAcβ1-→3[Ga lβ1-→4(Fucα1-→3)mGlcNAcβ1-→3]n-Galβ1 −→4GlcNAc−→, where m is zero or 1, and n is between 1 and 10. SAα2-→3Galβ1-→3(Fucα1-→4)GlcNAc β1-→3[Galβ1-→4GlcNAcβ-→3]n-Galβ1-→4G lcNAc-→, where n is a number between 1 and 10; Galβ1-→4[GlcNAcβ1-→3Galβ1-→4]nGlcNAc β1−→4GlcNAc−→, where n is a number between 1 and 10; Galβ1-→3[GlcNAcβ1-→3Galβ1-→4]nGlcNAc β1−→4GlcNAc−→, where n is a number between 1 and 10; SAα2-→6Galβ1-→4 [GlcNAcβ1-→3Galβ1-→4] nGlcNAcβ1−→4GlcNAc−→, where n is a number between 1 and 10. Ru; SAα2-→3Galβ1-→4 [GlcNAcβ1-→3Galβ1-→4] nGlcNAcβ1−→4GlcNAc−→, where n is a number between 1 and 10. Ru; SAα2-→3Galβ1-→3 [GlcNAcβ1-→3Galβ1-→4] nGlcNAcβ1−→4GlcNAc−→, where n is a number between 1 and 10. Ru; Galβ1-→4 (Fucα1-→3) GlcNAcβ1-→3 [Galβ1- →4(Fucα1-→3)mGlcNAcβ1-→3]n-Galβ1-→4G lcNAcβ1-→4GlcNAc-→, where m is zero or 1, and n is 1 is a number between and 10; Galβ1-→3 (Fucα1-→4) GlcNAcβ1-→3 [Galβ1- →4(Fucα1-→3]mGlcNAcβ1-→3]n-Ga1β1-→4G lcNAcβ1-→4GlcNAc-→, where m is zero or 1, and n is 1 is a number between and 10; and SAα2-→3Galβ1-→3 (Fucα1-→4) GleNAcβ1-→3 [Galβ1-→4GlcNAc-→3]n-Galβ1-→4GlcNAcβ 1-→4GlcNAc-→, where n is a number between 1 and 10; (43) Protein containing any of the following oligosaccharide sequences; (GlcNAcβ1-→ 3Galβ1-→4)nGlcNAcβ1-→3Gal-→, where n is 1 to 1 is a number between 0; SAα2−→6Gal−→; SAα2-→6Galβ1-→4 (GlcNAcβ1-→3Galβ1-→4) nGlcNAcβ1-→3Gal, where n is a number between 1 and 10; SAα2-→3Gal-→; and SAα2-→3Galβ1-→4(GlcNAcβ1-→3Galβ1-→4n GlcNAcβ1-→3Gal-→, where n is a number between 1 and 10. (44) consists of Gal-GlcNAc-protein, and the Gal is UDP-Gal :GlcNAc-Rβ1-→4 galactosyltransferase and UDP- Gal: Consists of GlcNAc-Rβ1-→3 galactosyltransferase a glycosylation tag added to the GlcNAc by an enzyme selected from the group Npaku. (45) SA-Gal-GlcNAc-protein, and the SA is CMP- SA: Galβ1-→4GlcNAc-Rα2-→6 sialyltransferer ze; CMP-SA: Galβ1-→3(4)GlcNAc-Rα2-→3 siali and CMP-SA: Galβ1-→4GlcNAc- Enzymes selected from the group consisting of Rα2-→3 sialyltransferases A glycosylated protein added to the Gal by.