METHOD FOR ENHANCING GLYCOPROTEIN STABILITY

Abstract: A method for modifiying eukaryotic and prokaryotic proteins to extend their in vivo circulatory lifetimes. In the preferred embodiment, enzymatic and/or chemical treatments are used to produce a modified protein carrying one or more covalently attached trisaccharide, sialic acid-->galactose-->N-acetylglucosamine--> (SA-->Gal-->GlcNAc-->), or tetrasaccharide (SA-->Gal-->GlcNAc-->GlcNAc-->)moieties. The method can be applied to any natural or recombinant protein possessing asparagine-linked oligosaccharides or to any non-glycosylated protein that can be chemically or enzymatically derivatized with the appropriate carbohydrate units. Following injection into an animal, the modified glycoproteins are protected from premature clearence by cells of the liver and reticulo-endothelial system which recognize and rapidly internalize circulating glycoproteins with carbohydrate containing terminal Gal, GlcNAc, fucose or mannose residues. The method can also be used to mask antigenic determinants on foreign proteins which would otherwise produce an immune response or to ''target'' a protein for recognition by sugar-specific cell surface receptors.

Description: METHOD FOR ENHANCING GIYCOPROTEIN STABILiπ BACKGROUND OF THE INVENTION The United States Government has certain rights in this invention by virtue of National Institutes of Health grants No. CA26712, GN31318, and CA14051. Glycoproteins, proteins with covalently bound sugars, are found in plants, animals, insects, and even many unicellular eukaryotes such as yeast. They occur within cells in both soluble and membrane-bound forms, in the intercellular matrix, and in extracellular fluids. The carbohydrate moieties of these glycoproteins can participate directly in the biological activity of the glycoproteins in a variety of ways: protection from proteolytic degradation, stabilization of protein conformation, and mediation of inter- and intracellular recognition. Examples of glycoproteins include enzymes, serum proteins such as immunoglobulins and blood clotting factors, cell surface receptors for growth factors and infectious agents, hormones, toxins, lectins and structural proteins. Natural and recombinant proteins are being used as tnerapeutic agents in humans and animals. In many cases a therapeutic protein will be most efficacious if it has an appreciable circulatory life¬ time. At least four general mechanisms can contribute to a shortened circulatory lifetime for an exogenous protein: proteolytic degrada¬ tion, clearance by the immune system if the protein is antigenie or i munogenic, clearance by cells of the liver or reticulo-endothelial system that recognize specific exposed sugar units on a giycoprotein, and clearance through the glomerular basement membrane of the kidney if the protein is of low molecular weight. The ol gosaccharides of a giycoprotein can exert a strong effect on the first three of these clearance mechanisms. The oligosaccharide chains of glycoproteins are attached to the polypeptide backbone by either N- or C-glycosidic linkages. In the case of N-l inked glycans, there is an amide bond connecting the anomeric carbon (C-1) of a reducing-terminal N-acetylglucosamine (GlcNAc) residue of the oligosaccharide and a nitrogen of an aspara- ' gine (Asn) residue of the polypeptide. In animal cells, 0-1 inked gl yeans are attached via a glycosidic bond between N-acetylgalactos- amine (GalNAc), galactose (Gal), or xylose and one of several hyαroxy- amino acids, most commonly serine (Ser) or threonine (Thr), but also hydroxyprol i ne or hydroxylysine in some cases. The 0-1 inked glycans in the yeast Sacc aromyces cerevisiae are also attached to serine or threonine residues, but, unlike the glycans of animals, they consist of one to several α-1 inked mannose (Man) residues. Mannose residues have not been found in the 0-1 inked oligosaccharides of animal cells. The biosynthetic pathways of N- and 0-1 inked oligosaccharides are quite different. O-Linked glycan synthesis is relatively simple, consisting of a step-by-step transfer of single sugar residues from nucleotide sugars by a series of specific glycosyltransferases. The nucleotide sugars which function as the onosaccharide donors are uridine-diphospho-Gal Ac (UDP-GalNAc), UDP-GlcNAc, UDP-Gal , guanidine- diphospho-fucose (GDP-Fuc), and cytidine-monophospho-sialic acid (CMP-SA). N-Linked oligosaccharide synthesis, which is much more complex, is described below. The initial steps in the biosynthesis of N-l inked glycans have been preserved with little change through evolution from the level of unicellular eukaryotes such as yeast to higher plants and man. For all of these organisms, initiation of N-l inked oligosaccharide assem¬ bly does not occur directly on the Asn residues of the protein, but rather involves preassembly of a lipid-1 inked precursor oligosaccha¬ ride which is then transferred to the protein during or very soon after its translation from mRNA. This precursor oligosaccharide, which has the composition Glc 3 Man Q GlcNAc 2 and the structure shown in Fig. 1A, is synthesized while attached via a pyrophosphate bridge to a polyisoprenoid carrier lipid, a dolichol. This assembly is a complex process involving at least six distinct membrane-bound glycosyltrans¬ ferases. Some of these enzymes transfer onosaccharides from nucleo¬ tide sugars, while others utilize dolichol -linked monosaccharides as sugar donors. After assembly of the lipid-1 inked precursor is complete, another membrane-bound enzyme transfers it to sterically accessible Asn residues which occur as part of the sequence -Asn-X- Ser/Thr-. The requirement for steric accessibility is presumably responsible for the observation that deπaturatioπ is usually required for in v tro transfer of precursor ol i gosaccharide to exogenous protei ns. Glycosylated Asn residues of newly-synthesized glycoproteins transiently carry only one type of oligosaccharide, Glc 3 Man g GlcNAc 2 . Modification, or "processing," of this structure generates the great diversity of structures found on mature glycoproteins, and it is the variation in the type or extent of this processing which accounts for the observation that different cell types often glycosylate even the same polypeptide differently. The processing of N-linked oligosaccharides is accomplished by the sequential action of a number of membrane-bound enzymes and begins immediately after transfer of the precursor oligosaccharide Glc,Man q - GlcNAc ? to the protein. In broad terms, N-linked oligosaccharide processing can be divided into three stages: removal of the three glucose residues, removal of a variable number of mannose residues, and addition of various sugar residues to the resulting trimmed "core," i.e., the Man 3 GlcNAc 2 portion of the original oligosaccharide closest to the polypeptide backbone. A simplified outline of tne processing pathway is shown in Fig. 2. Like the assembly of the precursor oligosaccharide, the removal of the glucose residues in the first stage of processing has been pre¬ served through evolution. In yeast and in vertebrates, all three glucose residues are trimmed to generate N-linked Man g GlcNAC - Pro¬ cessing sometimes stops with this structure, but usually it continues to the second stage with removal of mannose residues. Here the pathway for yeast diverges from that in vertebrate cells. As shown in Fig. IB, four of tne mannose residues of the Man g GlcNAc 2 moiety are bound by αl—>2 linkages. By convention the arrow points toward the reducing terminus of an oli osaccharide, or in this case, toward the protein-bound end of the gl can; α or β indicate the anomeric configuration of the glycosidic bond; and the two numbers indicate which carbon atoms on each monosaccharide are involved in the bond. The four αl—>2-linked mannose residues can be removed by Mannosidase I to generate N-linked Man ς 31cNAc 2 , all of which are commonly found on vertebrate glycoproteins. Ol gosaccharides with the composition Manj._gGlcNAc 2 are said to be of the "high-mannose" type. As shown in Fig. 2, protein-linked Man 5 GlcNAc 2 (Structure M-c) can serve as a substrate for GlcNAc transferase I, which transfers a 01—>2-linked GlcNAc residue from UDP-GlcNAc to the αl—>3-l inked mannose residue to form GlcNAcMan 5 GlcNAc 2 (Structure M-d) . Mannosi- dase II can then complete the trimming phase of the processing pathway by removing two mannose residues to generate a protein-linked oligo¬ saccharide with the composition GlcNAcMan.-GlcNAc 2 (Structure M-e). This structure is a substrate for GlcNAc transferase II, which can transfer a SI—>2-linked GlcNAc residue to the αl—>6-l inked mannose residue (not shown) . It is at this stage that the true complexity of the processing pathway begins to unfold. Simply stated, monosaccharides are sequen¬ tially added to the growing oligosaccharide chain by a series of membrane-bound Golgi glycosyltransferases, each of which is highly specific with respect to the acceptor oligosaccharide, the donor sugar, and the type of linkage formed between the sugars. Each type of cell has an extensive but discrete set of these glycosyltrans- ferases. These can include at least four more distinct GlcNAc transferases (producing Bl—>3, βl—>4, or βl—>6 linkages); three galactosyltransferases (producing βl—>4, βl—>3, and αl—>3 link¬ ages); two sialyltransferases (one producing α2—>3 and another, α2—>6 linkages); three fucosyltransferases (producing αl—>2, αl—>3, αl—>4 or αl—>6 linkages); and a growing list of other enzymes responsible for a variety of unusual linkages. The cooperative action of these glycosyltransferases produces a diverse family of structures collectively referred to as "complex" oligosaccharides. These may contain two (for example, Structure M-f in Fig. 2), three (for example, Fig. 1C or Structure M-g in Fig. 2), or four outer branches attached to the invariant core pentasaccharide, Man,GlcNAc ? . These structures are referred to in terms of the number of their outer branches: biantenπary (two branches), triantennary (three branches) or tetraantennary (four branches). The size of these complex glycans varies from a hexasaccharide (on rhodopsin) to very large polylactos- a inylglycans, which contain one or more outer branches with repeating (Galβl—>4GlcNAcβl—>3) units (on several cell surface glycoproteins such as the erythrocyte giycoprotein Band 3 and the macrophage antigen Mac-2). Despite this diversity, the specificities of the glycosyl¬ transferases do produce some frequently recurring structures. For example, the outer branches of many complex N-linked oligosaccharides consist of all or part of the sequence SAα2— >3 ( 6 )Gal βl— >4G1 cNAcβl— > . One or two of these trisaccharide moieties may be attached to each of the two α-1 inked mannose residues of the core pentasaccharide, as in Structures M-f and M-g of Fig. 2. Unlike transcription of DNA or translation of mRNA, which are highly reproducible events, oligosaccharide biosynthesis does not take place on a template. As a consequence, considerable heterogeneity is usually observed in the oligosaccharide structures of every giycopro¬ tein. The differences are most commonly due to variations in the extent of processing. The single glycosylation site of the chicken egg giycoprotein oval umin, for example, contains a structurally related "family" of at least 18 different oligosaccharides, the great majority of which are of the high-mannose or related "hybrid" type (for example, Structure M-h in Fig. 2). Many glycoproteins contain multiple glycosylated Asn residues, and each of these may carry a distinct family of oligosaccnarides. For example, one site may carry predominantly high-mannose glycans, another may carry mostly fucosy- lated biantennary complex chains, and a third may carry fucose-free tri- and tetraantennary complex structures. Again, all of these glycans will contain the invariant Man,GlcNAc 2 core. As discussed above, the initial stages of N-linked ol gosaccharide synthesis in the yeast Saccharoyces cerev s ae closely resemble those occurring in vertebrate cells. As in higher organisms, lipid-1 inked Glc,Man g GlcNAc 2 is assembled, its oligosaccharide chain transferred to acceptor Asn residues of proteins, and its three glucose residues are removed soon after transfer. Yeast cells can remove only a single mannose residue, however, so that the smallest and least-processed N-linked glycans have the composition Man g _ Q GlcNAc 2 . Processing can stop at this stage or continue with the addition of as many as 50 or more α-linked mannose residues to Man g GlcNAc 2 (Fig. 2, Structure Y-c) to generate a annan (for example, Structure Y-d). Just as glycopro¬ teins in mammalian cells may have predominantly high-mannose oligosac¬ charides at one glycosylated Asn residue and highly processed complex glycans at another, yeast glycoproteins such as external invertase commonly have some glycosylation sites with Man 8 _ 0 GlcNAc 2 chains, while other sites carry mannans. Unlike eukaryotic cells, bacteria lack the enzymatic machinery to assemble lipid-1inked Glc 3 Man g GlcNAc 2 or transfer it to proteins. Thus, although proteins synthesized in E_. col i contain many -Asn-X- Ser/Thr- sequences, they are not glycosylated. From the foregoing discussion, it is apparent that the glycosy¬ lation status of a giycoprotein will depend on the cell in which it is produced. The glycans of a protein synthesized in cultured mammalian cells will resemble those of the same protein isolated from a natural animal source such as a tissue but are unlikely to be identical. Pro¬ teins glycosylated by yeast contain high-mannose oligosaccharides and mannans, and proteins synthesized in a bacterium such as F.. coli will not be glycosylated because the necessary enzymes are absent. The precise composition and structure of the carbohydrate chain(s) on a giycoprotein can directly influence its serum lifetime, since cells in the liver and reticulo-endothelial system can bind and inter¬ nalize circulating glycoproteins with specific carbohydrates. Hepato- cytes have receptors on their surfaces that recognize oligosaccharide chains with terminal (i.e., at the outermost end(s) of glycans rela¬ tive to the polypeptide) Gal residues, macrophages contain receptors for terminal Man or GlcNAc residues, and hepatocytes and lymphocytes have receptors for exposed fucose residues. No sialic acid-specific receptors have been found, however. Although somewhat dependent on the spatial arrangement of the oligosaccharides, as a general rule, the greater the number of exposed sugar residues recognized by cell surface receptors in the liver and reticulo-endothelial system, the more rapidly a giycoprotein will be cleared from the serum. Because of the absence of sialic acid-specific receDtors, however, oligosac¬ charides with all branches terminated, or "capped," with sialic acid will not promote the clearance of the protein to which they are attached. The presence and nature of the oligosaccharide chain(s) on a giycoprotein can also affect important biochemical properties in addition to its recognition by sugar-specific receptors on liver and reticulo-endothelial cells. Removal of the carbohydrate from a giycoprotein will usually decrease its solubility, and it may also increase its susceptibility to proteolytic degradation by destabi- lizing the correct polypeptide folding pattern and/or unmasking protease-sensitive sites. For similar reasons, the glycosylation status of a protein can affect its recognition by the immune system. It is therefore an objective of the present invention to provide a method for modifying ol gosaccharide chains of glycoproteins isolated from natural sources or produced from recombinant DNA in yeast, insect, plant or vertebrate cells in a manner that increases serum lifetime or targets the protein to specific cell types. It is another objective of the invention to provide an in vitro method for glycosylating proteins produced from bacterial, yeast, plant, viral or animal DNA in a manner that enhances stability and effective biological activity. It is a further objective of the invention to provide a method for glycosylation of proteins or modification of oligosaccharide chains on glycoproteins which is efficient, reproducible and cost-effective. SUWARY OF THE INVENTION A method for modifying eukaryotic and prokaryotic proteins to extend their in vivo circulatory lifetimes or to control their site of cellular uptake in the body. In preferred embodiments, enzymatic and/or chemical treatments are used to produce a modified protein carrying one or more covalently attached trisaccharide SAα2—>6(3)Galβl—>4(3)GlcNAc—> or tetrasaccharide SAα2—>6(3)Galβl—>4(3)G1cNAcBl—>4G1cNAc—> moieties. In alternative embodiments, one or two GlcNAc residues bound to the protein are used as a basis for construction of other oligosaccharides by elongation with the appropriate glycosyltransfer¬ ases. The method can be applied to any natural or reco binant protein possessing Asn-1inked oligosaccharides or to any non-glycosylated pro¬ tein that can be chemically or enzy atically derivatized with the appropriate carbohydrate residues. Generation of glycoproteins containing Asn-1 nked S —>Ga1—>G1cNAc—> The preferred oligosaccharide modification scheme consists of the following steps wherein all but the -Asn-1inked GlcNAc of the N-linked oligosaccharide chains are enzymatically or chemically removed from the protein and a trisaccharide constructed in its place: Step 1. Generation of GlcNAc—>Asn(proteiπ) . The initial step is cleavage of the glycosidic bond connecting tne two innermost core GlcNAc residues of some or all N-linked o gosaccharide chains of a giycoprotein with an appropriate endo-β-N-acetylglucosaminidase such as Endo H or Endo F. Endo H cleaves the high-mannose and hybrid ol gosaccharide chains of glycoproteins produced in eukaryotic cells as well as the mannans produced in yeast such as Saccharomyces C're- v siae, removing all but a single GlcNAc residue attached to each glycosylated Asn residue of the polypeptide backbone. Endo ? can cleave both high-mannose and biantennary complex chains of N-linked oligosaccharides, again leaving a single GlcNAc residue attached at each glycosylation site. If a given giycoprotein contains complex oligosaccharides such as tri- or tetraantenπary chains which are inefficiently cleaved by known endoglycosidases, these chains can be trim ed with exoglycosidases such as sialidase, β- and α-galactosi- dase, α-fucosidase and β-hexosaminidase. The innermost GlcNAc residue of the resul ing core can be then be exposed by any of several proce¬ dures. One procedure is digestion with Endo F or other endo-B-N- acetylglucosaminidases such as Endo D. A second procedure is diges¬ tion with α-mannosidase followed by digestion with either Endo L or with β-mannosidase and β-hexosaminidase. Alternatively, glycoproteins normally bearing complex Asn-linked oligosaccharides can be produced in mammalian cell culture in the presence of a processing inhibitor such as swainsonine or deoxymanno- jirimycin. The resulting giycoprotein will bear hybrid or high- mannose chains susceptible to cleavage by Endo H, thereby eliminating the need for an initial treatment of the giycoprotein with exoglyco¬ sidases. In a related variation, the giycoprotein may be produced in a mutant cell line tnat is incapable of synthesizing complex N-linked chains resistant to endoglycosidases such as Endo H or Endo F. All sugars other tnan the N-linked GlcNAc residues may also be removed chemically rather than enzymatically by treatment with trifluoromethanesulfonic acid or hydrofluoric acid. In general, chemical cleavage can be expected to be less useful than enzymatic methods because of the denaturing effects of the relatively harsh conditions used. Step 2. Attachment of Gal to GlcNAc—>Asn(protein) . The second step is tne enzymatic addition of a Gal residue to the residual GlcNAc on the protein by the action of a galactosyltransferase. The pre¬ ferred galactosyltransferase is a bovine milk enzyme which transfers Gal to GlcNAc in the presence of the sugar donor UDP-Gal to form a βl—>4 linkage. In another variation, galactose can be added to the GlcNAc residue with a βl—>3 linkage by the use of a galactosyltrans¬ ferase from a source such as pig trachea. Step 3. Attachment of SA to Gal—>GlcNAc—>Asn(protein) . The final step is the enzymatic addition of a sialic acid residue to Galβl—>4(3)GlcNAc-->Asn(protein) . This reaction can be carried out with an α2—>6-sialyltransferase isolated, for example, from bovine colostrum or rat liver, which transfers SA from CMP-SA to form an α2—>6 linkage to the terminal galactose residue of Galβl—>4(3)- GlcNAc—>Asn(protein). Alternatively, an α2—>3-sialyltransferase may be used to form an α2—>3 linkage to each terminal Gal residue. Although the preferred sialic acid is N-acetylneuraminic acid (NeuAc), any naturally occurring or chemically synthesized sialic acid which the sialyltransferase can transfer from the CPM-SA derivative to galactose may be used, for exapmole, N-glycolyl neuraminic acid, 9-0- acetyl-N-acetyl neuraminic acid, and 4-0-acetyl-N-acetyl neuraminic acid. Generation of glycoproteins containing Asn-1inked SA—>Gal—>- GlcNAc—>G1cNAc—> In a second embodiment, the oligosaccharide chains of the giyco¬ protein, whether natural or produced in the presence of a processing inhibitor or in a mutant cell line, are trimmed back to the two, rather than one, innermost core GlcNAc residues by the use of approp¬ riate exoglycosidases. For example, α and β-mannosidase would be used to trim a high-mannose oligosaccharide. The product of this treat¬ ment, GlcNAcβl—>4GlcNAc—>Asn(protein) , is then converted to the tetrasaccharide SAo2—>6(3)Galβl—>4(3)GlcNAcBl—>4G1cNAc—>Asn- (protein) by sequential treatment with galactosyl- and sialyltransfer¬ ases. Attachaent of oligosaccharides to non-glycosylated amino acid residues of proteins. In a third embodiment, an oligosaccharide such as the trisac¬ charide SA—>Gal—>GlcNAc—> or disaccharide SA—>Gal—> is attached at non-glycosylated amino acid residues of a protein expressed eitner in a eukarykotic system or in a bacterial system. For example, to attach the trisaccharide SA—>Gal—>GlcN'Ac, the protein is treated with a chemically reactive glycoside derivative of GlcNAc—>, Gal- —>GlcNAc—>, or SA—>Gal—>GlcNAc—>. In the first two cases, the mono- or disaccharide is then extended to the trisaccharide by tne appropriate glycosyltransferase(s) . The initial carbohydrate moieties can be attached to tne protein by a chemical reaction between a suit¬ able amino acid and a glycoside derivative of the carbohydrate containing an appropriately activated chemical group. Depending on the activation group present in the glycoside, the carbohydrate will be attached to amino acids with free amino groups, carboxyl groups, sulfhydryl groups, or hydroxyl groups or to aromatic amino acids. Generation of other oligosaccharides by elongation of prote n-linked GlcNAc residues. Variations of the disclosed procedures can be used to produce glycoproteins with oligosaccharides other than the tri- or tetrasac- charides described above. For example, extended oligosaccharide chains consisting of SAα2~>6(3)Galβl-->4(GlcNAcβl">3Galβl-->4)nGlcNAc—> or SAα2—>6(3)GalSl—>4(GlcNAcβl—>3GalBl—>4) n GlcNAcβl—>4GlcNAc—> ; where n is 1-10, can be constructed by subjecting a giycoprotein carrying one or two core GlcNAc residues to alternate rounds of βl—>4 galactosyltransferase and βl—>3 N-acetylglucosaminyltransferase treatments. The resulting extended oligosaccharide chain can be useful for increasing solubility or masking protease-sensitive or ant genie sites of tne Dolypeptide. Many other useful oligosaccharide structures can be constructed Dy elongation of protein-linked monosaccharides or disacchariαes with the use of appropriate glycosyl transferases. An example is the branched fucosylated trisaccharide Galβl—>4(Fucαl—>3)G1cNAc—>. These and other structures could be useful in preferentially "tar¬ geting" a giycoprotein to a specific tissue known to contain receptors for a specific mono- or oligosaccharide. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the structures of (A), the lipid-1inked precursor oligosaccharide, Glc 3 Man g GlcNAc 2 ; (B), a high-mannose Asn-1inked oligosaccharide, Man g GlcNAc 2 ; and (C), a typical triantennary complex Asn-1inked oligosaccharide. The anomeric configurations and linkage positions of the sugar residues are indicated, and dotted lines en¬ close the invariant pentasaccharide core shared by all known eukary¬ otic Asn-1inked oligosaccharides. Fig. 2 is a simplified biosyπthetic pathway for Asn-linked oligosaccharide biosynthesis in yeast and higher organisms. For clarity, anomeric configurations and linkage positions are not shown, but the arrangement of the branches is tne same as in Fig. 1. Fig. 3 is a Coo assie blue-stained gel prepared by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of yeast external invertase before and after treatment with glycosidases. The acrylamide concentration was 6%. (A) untreated invertase; (B) inver¬ tase after treatment with Endo H under non-denaturing conditions; (C) invertase after Endo H treatment under denaturing conditions (0.7% SDS); and (D) an aliαuot of a sample first treated with Endo H under non-denaturing conditions and subseαuently treated with jack bean α- mannosidase. Fig. 4 is a fluorogram of a 6% SDS-PAGE gel of samples of yeast external invertase removed at intervals (5 min, 1 hr, 2 hr, 3 hr, 5 hr, 9 hr and 19 hr) during galactosylation of Endo H-treated, SDS- denatured invertase (Fig. 3B) with UD?-[°H]Gal and bovine milk SI—>4 galactosyltransferase. Fig. 5 shows the rate of incorporation of acid-precipitable radio¬ activity into Endo H-treated, SDS-denatured yeast external invertase during treatment with UDP-[ H]Gal and bovine milk βl—>4 galactosyl- transferase. Fig. 6 is an autoradiogra of a 5% SDS-PAGE gel of various yeast external invertase derivatives that have been sialylated using CMP- L rl4 CJNeuAc and bovine col ostrum α2— >6 sialyl transferase . .(A) Sialyl ation product derived from gal actosyl ated, Endo H-treated, SDS- denatured invertase; (B) sialyl ation product derived from a ga actosy- l ated sampl e of Endo H- and jack bean α-mannosidase-treated, non- denatured invertase; (C) sialylation product derived from untreated invertase. Fig. 7 is a Coomassie blue-stained 6% SDS-PAGE gel of (A) untrea¬ ted bovine serum albumin (BSA); (B) BSA converted to GlcNAc-BSA containing approximately 48 GlcNAc residues per molecule of protein by incubation with 2-imino-2-methoxyethyl-l-thio-N-acetylglucosaminide in 0.25 M sodium borate pH 8.5 for 24 hr at room temperature; (C) gal ac¬ tosyl ated BSA formed by treatment of GlcNAc-BSA with UDP-[ 3 H]Gal and bovine milk βl— >4 gal actosyl transferase; and (D) sialylated BSA formed by treatment of Gal~>GlcNAc-BSA with CMP-[ C]NeuAc and bovine colostrum α2— >6 sialyl transferase. ς Fig. 8 is a graph of specific uptake (ng/2 x 10 cells) of Gal— >GlcNAc-[ 125 I]BSA (Q) and GlcNAc-[ 125 I]BSA (■) by the an/GlcNAc receptor of thioglycollate-eli cited mouse peritoneal macrophages as a function of the concentration of glycosylated BSA (ug/ml), where specific uptake is equal to total uptake (uptake in the absence of mannan) minus non-specific uptake (value obtained in the presence of mannan) . Fig. 9 is a graph of specific uptake (ng/mg cellular protein) of Gal— >GlcNAc[ 125 I]BSA (■) and NeuAc— >Gal— >GlcNAc-[ 125 I]BSA ( •) by the Gal/GalNAc receptor of HepG2 cells vs. protein concentration (0.5 to 7.5 μg protein/ml), where specific uptake is equal to total uptake (uptake in 'the absence of asialo-orosomucoid) minus non-specific uptake (value obtained in the presence of asialo-orosomucoid). Fig. 10 Analysis of [ H]Gal— >GlcNAc-RNase by fast protein liquid chromatography (FPLC) on a Mono S column before (O o ) and after (^ ) sialylation with CMP-NeuAc and rat liver α2— >6 sialyltrans- ferase, where the column was eluted with a linear gradient as des¬ cribed below. DETAILED DESCRIPTION OF THE INVENTION The present invention is a method for modifying proteins wherein oligosaccharide chains are bound to the protein to enhance ϋ vivo stability or to target the protein to cells having specific receptors for an exposed saccharide in the attached oligosaccharide chaiπ(s). The method has two principal embodiments. The first is to cleave the existing Asn-1 inked oligosaccharide chains on a giycoprotein to leave one or two GlcNAc residues attached to the protein at Asn and then enzymatically extend the terminal GlcNAc to attach Gal and SA. The second is to chemically or enzymatically attach a GlcNAc or Gal resi¬ due to the protein at any of a number of different amino acids and then enzymatically extend the terminal GlcNAc or Gal to form an oligo¬ saccharide chain capped with sialic acid. There are a number of variations of the methods and enzymes used at each step of the methods, depending on the substrate and desired oligosaccharide struc¬ ture. A. Generation of glycoproteins containing S —>Ga1—>61cNAc—>Asn- ( rotein) Step 1. Generation of GlcNAc—>Asn(protein). There are several methods for preparing glycoproteins containing a single GlcNAc residue attached to glycosylated asparagine residues. Six methods are as fol1ows. a. Cleavage by Endo H. To generate GlcNAc—>£sn(protein) enzyma¬ tically on gl coproteins having one or more oligosaccharides of the high-mannose or mannan type, the giycoprotein is incubated with an endo-β-N-acetylglucosaminidase capable of cleaving these oligosaccha¬ ride structures. The enzyme hydrol zes the bond between the two core GlcNAc residues of susceptible N-linked oligosaccharides, leaving behind a single GlcNAc residue attached to the glycosylated Asn resi¬ dues. The preferred enzyme for this purpose is Endo H, which has been isolated from Streptomyces plicatus. The enzyme is available either as the naturally occurring protein or as the recombinant DNA product expressed in Z^ coli or Streptomyces lividans. Endo H cleaves all susceptible oligosaccharide structures of denatured glycoproteins and many of those on native glycoproteins. However, in native glycoproteins the GlcNAc^ cores of some high- mannose glycans may be protected from cleavage by Endo H due to steric factors such as polypeptide folding. This can frequently be overcome by the use of one of several mild denaturing agents that promote par¬ tial polypeptide unfolding. Examples of such mild denaturants include detergent such as Triton X-100, NP-40, octyl glucoside, deoxycholate and dilute sodium dodecyl sulfate; disulfide bond reducing agents such as dithiothreitol and β-mercaptoethanol ; chaotropic agents such as urea, guanidinium hydrochloride and sodium isothiocyanate; and low concentrations of organic solvents such as alcohols (methanol , etha- nol , propanol or butanol), DMSO or acetone. Endo H is a very stable enzyme, active over a pH range of about 5 to 6.5, in low- or high- ionic strength buffers, and in the presence of the above-mentioned denaturing agents or protease inhibitors such as phenylmethanesul fonyl fluoride, EDTA, aprotinin, leupeptide and pepstatin. Protocols for the use of Endo H have been published by Trimble and Maley in Ana . Biochem. 141, 515-522 (1984). The precise set of reaction conditions which will optimize the cleavage of oligosaccharides by Endo H while preserving biological activity will most likely vary depending on the giycoprotein being modified and can be determined routinely by someone of ordinary skill in this field. In situations where one or more intact high-mannose glycans per¬ sist even after incubation under the most stringent Endo H reaction conditions judged safe to use, exposed mannose residues can be trimmed away by the use of an α-mannosidase such as the cornmercially available α-mannosidase from jack bean. While high-mannose oligosaccharides modified in this way will not serve as substrates for the further modification reactions described below, this treatment should reduce the possibility that mannose-specific receptors on macrophages or other cells might bind to residual high-mannose glycan(s) on the giycoprotein and cause its premature clearance from the circulation. As mentioned earlier, yeast glycoproteins sometimes contain 0-1 inked oligosaccharides consisting of one to four α-1 inked mannose residues. Because these could bind to a mannose-specific receptor and shorten the serum lifetime of a giycoprotein, it is advisable to treat any protein found to contain such ol iσosaccharides with an α-mannosi- dase such as the enzyme from jack bean. This would remove all but the innermost, protein-linked mannose residue from the 0-1 inked chains. Because α-mannosidase treatment could interfere with subsequent cleavage by Endo H or Endo C,-, it should be performed after digestion with these enzymes. A common 0-1 inked oligosaccharide in animal cells is Gal — >GalNAc- — >Ser/Thr( protein) . These glycans can be removed with the enzyme endo-α-N-acetyl galactosaminidase, which is commercially available from Genzy e Corp., Boston MA. Many other mammalian 0-1 inked oligosaccha¬ rides can be converted to Gal — >GalNAc— >Ser/Thr( protein) by treatment with exoglycosidases such as sialidase, β-hexosaminiαase and α-fucosi- dase. The resulting protein-linked disaccharides could then be removed from tne polypeptide with endo-α-N-acetyl galactosaminidase. b. Cleavage by other endo-8-N-acetyl glucosaminidases. Several other endo-β-N-acetyl glucosa inidases are also capable of cleaving between the two innermost GlcNAc residues of various N-linked oligo¬ saccharides. The oligosaccharide specificities of these enzymes vary and are summarized in Table I. Two of these endoglycosidases, Endo C ττ and Endo F, can be used in place of Endo H to cleave high-mannose glycans. Unlike Endo H, however, Endo F is also active with biaπten- nary complex N-linked oligosaccnarides. Although the N-linked oligosaccharides of vertebrates are not substrates for Endo D, this enzyme would be active with glycoproteins produced oy insect cells, which produce significant quantities cf N-linked Man.,GlcNAc 2 in addition to high-mannose oligosaccharides, as reported by Hsieh and Robbins in OL Biol . C e . 259, 2375-82 (1984). In situations where the target giycoprotein contains multiple oligosaccharides sensitive to different endo-β-N-acetyl glucosaminidases, the giycoprotein can De incubated with tne enzymes either sequentially or in combination to maximize cleavage. c. Cleavage by Endo H after incubation of cul ured cells with oligosaccharide processing inhibitors. Mammalian cells often syntne- size glycoproteins carrying oligosaccharides with structures that are resistant to all of the above-mentioned endo-β-N-acetyl glucosamini¬ dases, e.g., tri- or tetraantennary complex ol gosaccharides. If such a giycoprotein is being produced in a cultured cell system, it is possible to block the later stages of oligosaccharide processing by addiπg oligosaccharide processing inhibitors to the culture medium. Two preferred processing inhibitors are deoxymannojirimycin and swain- soπine. Cells treated with one of these inhibitors will preferen¬ tially synthesize N-linked oligosaccharides with Endo H-sensitive structures. Deoxymannojirimycin inhibits Mannosidase I, thereby blocking further modification of high-mannose N-linked oligosaccha¬ rides. Swainsonine is a Mannosidase II inhibitor, blocking the removal of the two α-1 inked mannose residues on the αl— >6-l nked mannose residue of the Man 3 GlcNAc 2 core (i.e., conversion of structure M-d to structure M-e in Fig. 2). As a result, glycosylated Asn resi¬ dues which would normally carry Endo H-resistant complex type glycans will carry Endo H-sensitive "hybrid" oligosaccharides instead. Swain¬ sonine and deoxymannojirimycin are both cornercially available, for example from Genzy e Corp., Boston MA, or Boehringer Mannheim, India¬ napolis IN . In most cases, the altered glycoproteins produced in tne presence of deoxymannojirimycin or swainsonine will still be secreted in biologically active form. The use and properties of swainsonine and deoxymannoji imycin, as well as those of other oligosaccnari e processing inhibitors, have been reviewed by Schwartz and Datema, Aαv . Carbohyd. Chem. B ocnem. 40, 287-379 (1982) and by Fuhrmann et aj_., Biochim. Biophys. Acta 825, 95-110 (1985). Oligosaccharide processing inhibitors that block Glucosidases I or II, such as deoxynojirimycin or castanospermine, which a^e bcth available from Genzyme Corp., Boston MA, will also generate Endo h- sensitive structures, but these inhibitors are less preferred because they sometimes block secretion. Many other oligosaccharide processing inhibitors, described in the two reviews cited in the previous para¬ graph, will also serve the same purpose. d. Cleavage by endo-β-N-acetyl lucosaminidases after production of a giycoprotein in a mutant cell line. Another approach for mani¬ pulating the structures of the N-linked oligosaccharides of a giyco¬ protein is to express it in cells with one or more mutations in tne oligosaccharide processing pathways. Such mutations are readily selected for in mammalian cells. A number of techniques have been used to generate processing mutants, but selection for resistance or hypersensitivity to one or more of a variety of lectins, as an indi¬ cator of the presence of a processing mutation, has been one useful approach. DNA coding for a glycoprotein(s) can be introduced into such a mutant cell line using conventional methods (e.g., transforma¬ tion with an expression vector containing the DNA). Alternatively, a mutant subline with defective processing can be selected from a line already capable of producing a desired giycoprotein. Depending on the desired phenotype, any of a wide variety of mutant cell lines can be used. For example, there are perfectly viable, fast-growing GlcNAc transferase I mutants of both CHO cells (an established Chinese hamster ovary cell line long used for muta- tioπal studies and mammalian protein expression) and BHK-21 cells (an established line of baby hamster kidney origin). Both CHO and BHK-21 cells are available from the American Type Culture Collection, Rock- ville MD. Because of the missing enzyme activity, the mutant cells are unable to synthesize any complex or hybrid N-linked oligosac¬ charides; glycosylated Asn residues which would normally carry sucn glycans carry Man ^ GlcNAc 2 instead. Thus, glycosylated Asn residues carry only Man ς Q GlcNAc 2 , all structures which are sensitive to Endo H. Many other mutant cell lines have also been characterized, exam¬ ples of which include lines with various defects in fucosyl ation, a defect in gal actosyl ation resulting in failure to extend the outer branches past the GlcNAc residues, an inability to add extra branches to produce tri- and tetraantennary complex oligosaccharides, and various defects in Ser/Thr-1 inked gl can synthesis. The subject of processing-defective animal cell mutants has been reviewed by Stanley, in The Biochemistry of Glycoproteins and Proteogl eans, edited by Lennarz, Plenum Press, New York, 1980. A series of yeast mutants with various defects in mannan synthesis has also been produced, as described by Ballou, in The Molecular Biology of the Yeast Saccharomyces, edited by Stratherπ et a_., Cold Spring Harbor Laboratory, 1982. Thus, it is possible to produce a giycoprotein in a mutant S_. cerevisiae strain which cannot elongate high-mannose oligosaccharides into large mannans. e. Sequential exoglycosidase digestion with or without subsequent cleavage by Endo L or Endo D. An alternative, but less preferred method for generating GlcNAc— >Asn( protein) in cases where the giyco¬ protein contains high-mannose or mannan-type oligosaccharides is to remove monosaccharide units by exoglycosidase digestion with or without subsequent use of Endo L. The first step is digestion with an α-mannosidase to remove all α-linked mannose residues. In the case of mannans from some yeast strains, it may be desirable to include other exoglycosidases or phosphatases if other sugars or phosphate residues are present in the outer portion of the mannan structure. In the second digestion step, the last mannose residue is removed with a β- mannosidase. The product, GlcNAc 2 —>Asn(protein), is then subjected to the third digestion step, which is carried out with β-hexosamini¬ dase. This enzyme removes the terminal GlcNAc residue to generate GlcNAc—>Asn(protein) ; since the last GlcNAc is linked to the protein by an amide rather than a glycosidic bond, the hexosaminidase cannot remove the innermost GlcNAc residue from the asparagine. Alternatively, α-mannosidase treatment of high-mannose or mannan- type oligosaccharides can be followed by incubation with Endo L, which can be purified from Streptomyces plicatus. This enzyme can cleave between the GlcNAc residues of Manβl—>4GlcNAcβl—>4GlcNAc. In the case of a giycoprotein containing complex or hybrid-type oligosaccharides, sequential (or, when the requirements of the enzymes make it possible, simultaneous) incubation with the appropriate exoglycosidases, such as sialidase, β- and/or α-galactosidase, β- hexosaminidase, and α-fucosidase, will trim the o igosaccharides back to Man^GlcNAc,. This oligosaccharide can be cleaved by Endo D or Endo F. Alternatively, it can be treated with α-mannosidase to generate protein-linked Manβl—>4GlcNAcβi—>4GlcNAc. This can be cleaved either with Endo L or with digestions with α-mannosidase, β-mannosi- dase, and β-hexosaminidase. Sialidase can be purified from a variety of sources, including E. col , Clostrid um perfringens, V brio cnolerae, and Arthrobacter urefaciens, and is commercially available from a number of sources such as Calbiochem-Behring, San Diego CA, or Sigma Chemical Corp., St. Louis MO. β-Galactosidase can be purified from AsDerail!us niger, _. perfringens, jack bean, or other suitable sources and is commercially available from Sigma Chemical Corp., St. Louis MO. α-Galactosi ase from E_. col i or green coffee beans is available from Boehringer Mannheim, Indianapolis IN. β-Hexosaminidase can be purifed from jack bean, bovine liver or testis, or other suitable sources and is also commercially available from Sigma Chemical Corp., St. Louis MO. β- Mannosidase has been purified from the snail Achatina fulica, as described by Sugahara and Yamashima in Meth. Enzy ol . 28, 769-772 (1972), and from hen oviduct, as described by Sukeno e_ _al_. in Meth. Enzymol . 28, 777-782 (1972). α-Mannosidase from jack bean is pre¬ ferred and is commercially available from Sigma Chem. Corp., St. Louis MO. Endo H, Endo D, and Endo F are commercially available from Gen¬ zyme Corp., Boston MA; from New England Nuclear, Boston MA; from Miles Scientific, Naperville IL; or from Boehringer Mannheim, Indianapolis IN. Conditions for the use of these and the other endo-β-N-acetyl glu¬ cosaminidases Endo CJJ and Endo L are described in the publications cited in Table I. f. Chemical removal of all sugars except N-linked GlcNAc. It is also possible to generate protein-linked GlcNAc chemically. For example, as described by Kalyan and Bah! in J_. Biol . Chem. 258, 67-74 (1983), hydrolysis with trifluoromethane sulfonic acid (TFMS) has been used to remove all sugars except the M-l inked GlcNAc residues while leaving the protein backbone intact. Similar results have been obtained using hydrofluoric acid, as described by Mort and Lamport in Anal. Biochem. 82, 289-309 (1977). Step 2. Attachment of galactose to GlcNAc — >Asπ( protein) . In Step 2, the terminal GlcNAc residue generated in Step 1 serves as a site for tne attachment of gal ctose. Either of two gal actosyl - transferases may be used: UDP-Gal :GlcNAc-R βl — >4 gal actosyl trans¬ ferase or UDP-Gal :GlcNAc-R βl— >3 gal actosyl transferase. In tne first variation of tnis step, a βl— >4-l inked galactose residue is added to GlcNAc— >Asn( protein). UDP-Gal :GlcNAc-R Bl — >4 gal actosyl transferase can be obtained from a variety of sources, the most common and cost- effective one being bovine milk. Enzyme from this source is commerci- ally available from Sigma Chem. Corp., St. Louis MC. The reaction conditions for using the bovine milk gal actosyl transferase to transfer galactose from UDP-Gal to GlcNAc— >Asn( protein) are similar to those described by Trayer and Hill in J_. Biol . Chem. 246, 6666-75 (1971) for natural substrates. The preferred reaction pH is 6.0 to 6.5. Most buffers can be used with the exception of phosphate, which inhibits enyzme activity, and a broad range of salt concentrations can be used. It is preferable to have 5-20 mM Mn +2 or Mg +2 present. Peptidase inhibitors such as phenylmethanesulfonyl fluoride, TPCK, aprotinin, leupeptin, and pepstatin and exoglycosidase inhibitors such as galac- tono-l,4-lactone can be added without interfering with the activity of the gal actosyl transferase. Since the removal of the carbohydrate from the protein can cause solubility problems, it is sometimes necessary to use relatively high concentrations of a non-ionic detergent such as 2-3% Triton X-10C, other suitable solubilizers such as DMSO, or denaturing agents such as 2-3 M urea to keep the protein in solution. We have found that this does not interfere with the gal actosyl ation step, the bovine milk gl — >4 gal actosyl transferase apparently remaining sufficiently active under these conditions. In the second variation of this step, a βl— >3-l inked galactose residue is transferred to GlcNAc— >Asn(protein). UDP-Gal : GlcNAc -R βl— >3 gal actosyl transferase has been purified from pig trachea. Conditions for the use of this enzyme to transfer galactose from UDP-Gal to GlcNAc-R have been described by Sheares and Carlson in J_. Biol . Chem. 258, 9893-98 (1983). Step 3. Attachment of sialic acid to GalBl— >4(3)G1cNAc— >Asn- ( protein) The term "sialic acid" (SA) includes any naturally occurring or chemically synthesized sialic acid or sialic acid derivative. The preferred naturally occurring sialic acid is N-acetyl neuram nic acid (NeuAc). As discussed by Schauer in Ad_. Carb. Chem. Biochem. 40_, 131-234 (1982), other sialic acids can also be transferred from CMP-SA to galactose, for example, N-glycolyl neuraminic acid, 5-O-acetyl neuraminic acid, and 4-O-acetyl-N-acetyl neuraminic acid. Many other sialic acids such as those described in Sialic Acids: Chemis¬ try, Metabolism and Function, edited by R. Schauer (Springer-Verlag, New York, (1982), are potential substrates. There are two variations of the method for attaching sialic acid tc the substrate generated in Steps 1 and 2, Galβl— >4(3)G!cNAc— >Asn(protein). In the first of the two variations, the sialic acid is attached to Galβl— >4GlcNAc— >Asn(protein) in an α2— >6 linkage. The CMP-SA:- Gal β l— >4GlcNAc-R α2— >6 sialyl transferase used in this step can be obtained from a variety of sources, the more usual ones being bovine colostrum and rat liver. The rat liver enzyme has recently become commerciany available from Genzyme Corp., Boston MA. The reaction conditions for using the bovine colostrum and rat liver α2— >6 sialyl transferases to transfer sialic acid from CMP-SA to Galβl— >4GlcNAc— >Asn(protein) are similar to those described by Paulson et al_. in <J. Biol. Chem. 252, 2356-62 (1977) for natural sub¬ strates, except that it may be desirable to add additional enzyme to accelerate the rate of the reaction. The preferred pH is 6.5-7.0. Although most buffers, with the exception of phosphate, can be em¬ ployed, preferred buffers are Tris-maleate or cacodylate. The enzyme is functional in the presence of mild detergents such as NP-40 and Triton X-100; peptidase inhibitors such as phenylmethanesulfonyl fluo¬ ride, TPCK, aprotinin, leupeptin and pepstatiπ; and exoglycosidase inhibitors such as galactono-l,4-lactone. In the second variation of this step, the sialic acid is attached to the Galβl— >4(3)GlcNAc— >Asn(protein) by an α2— >3 linkage. Two sialyl transferases producing this linkage have been described. The first, CMP-SA: al βi~ >4GlcNAc α2— >3 sialyl transferase, has been identified in human placenta by van den Eijnden and Schiphorst as described in JL Biol. Chem. 256, 3159-3162 (1981). This enzyme, although not yet purified, can be purified using conventional methods. The second enzyme, CMP-SA:GalBl— >3(4)GlcNAc α2— >3 sialyl transferase, has been purified from rat liver by Weinstein e_t _aj_. as described in J_. Biol . Chem. 257, 13835-^4 (1982). Tne rat liver enzyme has a somewhat relaxed specificity and is able to transfer sialic acid from CMP-sialic acid to- the C-3 position of galactose in both Galβl— >4- GlcNAc and Galβl— >3GlcNAc sequences. Conditions for the use of the α2— >3 sialyl transferases are described in the two publications just c ted. B. Method for preparing glycoproteins containing SA — >Gal — >- G1 cNAc— >G1 cNAc— >Asn ( rotei n ) The method used to generate SA— >Gal— >GlcNAc— >GlcNAc— >Asn- ( protein) is similar to the method described above for generating modified glycoproteins containing the trisaccharide sequence SA— >Gal— >GlcNAc-->Asn(protein). In the preferred embodiment, both core GlcNAc residues of the original N-linked oligosaccharide are left attached to the protein and a tetrasaccharide sequence, SA— >Gal— >- GlcNAc— >GlcNAc--> is constructed enzymatically. Step 1. Generation of GlcNAcSl— >4G1cNAc— >Asn( protein) The intact N-linked oligosaccharide cnain is treated with exoglycosidases selected to remove all carbohydrate exterior to the two innermost GlcNAc residues. In tne case of high-mannose or mannan-type oligosaccharides, α- and β-mannosidase are used. In the case of complex or hybrid-type oligosaccharides, additional exoglyco¬ sidases are required, the specific enzymes used depending on the structures of the carbohydrate chains being modified. In most cases, treatments with sialidase, β- and/or α-galactosidase, β-hexosamini¬ dase, and if necessary, α-fucosidase, are car r ied out in addition to treatment with α- and β-mannosioase. Tne 6-hexcsaminidase treatment is intended to remove GlcNAc residues only from the outer branches of the oligosaccharides, not from the core, and care snould be taken that no β-hexosaminidase is present during or after β-mannosidase treat¬ ment. The reaction conditions and sources of the exoglycosidases are identical to those described above for Step 1 in the generation of SA— >Gal — >G1 cNAc— >Asn( protei n ) . The methods used to attach galactose to GlcNAcBl— >4GlcNAc— >Asn- (protein) and sialic acid to Galβl— >4(3)GlcNAcβl— >4GlcNAc— >Asn- (protein) are the same as those described earlier for the preparation of modified glycoproteins containing N-linked SAα2— >3(6)GalSl— >4(3)- Gl cNAc— >Asn( protei n ) . C. Method for attaching oligosaccharides to nonglycosyl ated amino acid residues of proteins The principal method for attaching oligosaccharides such as SA—>Gal—>GlcNAc—> to non-glycosylated amino acid residues is to react an activated glycoside derivative of what is to be the innermost sugar residue, in this case GlcNAc, with the protein and then to use glycosyltransferases to extend the oligosaccharide chain. Chemical and/or enzymatic coupling of glycosides to proteins can be accom¬ plished using a variety of activated groups, for example, as described by Aplin and Wriston in CRC Crit. Rev. Biochem., pp. 259-306 (1981). The advantages of the chemical coupling techniques are that they are relatively simple and do not need the complicated enzymatic machinery required for natural N-linked glycosylation. Depending on the coup¬ ling mode used, the sugar(s) can be attach arginine, histidine, or the amino-terminal amino acid of the polypeptide; (b) free carboxyl groups, such as those of glutamic acid or aspartic acid or the carboxy- terminal amino acid of the polypeptide; (c) free sulfhydryl groups, such as those of cysteine; (d) free hydroxyl groups, such as those of serine, threonine, or hydroxyprol ine; (e) aromatic residues such as those of phenylalanine. tyrosine, or tryptophan; or (f) the amide group of glutamine. As shown below, the aglycone, R, is the chemical moiety that com¬ bines with the sugar to form a glycoside and which is reacted with the amino acid to bind tne sugar to the protein. R 3 = -S( CH 2 ) n NH 2 ; R 4 = -0(CH 2 ) p NH 2 ; R 5 = -S(CH 2 ) n NHC-CH=CH 2 ; R g = -0(CH 2 ) NHC-CH*CH 2 ; R 7 = -S ( CH 2 ) n -< -N 2 +cr ; and R g . GlcNAc residues can be attached to the β-amino groups of lysine residues of a nonglycosylated protein by treating the protein with 2- imino-2-methyoxyethyl-l-thio-β-N-acetylglucosaminide as described by Stowell and Lee in Meth. Enzymol . 83, 278-288 (1982). Other coupling procedures can be used as well, such as treatment of the protein with a glycoside or thioglycoside derivative of GlcNAc in which the agly¬ cone contains an activated carboxylic acid, for example R. or R 2 . GlcNAc residues can be attached to the carboxyl groups of aspartic acid and glutamic acid residues of a nonglycosyl ated protein by treatment of the protein with a glycoside or thioglycoside derivative of GlcNAc in which the aglycone contains a free amino group, for example R-, or R., in the presence of a coupling reagent such as a carbodiimide. Compounds which contain free amino groups, for example GlcNAc derivatives containing the aglycones R 3 or R., can also be used to derivatize the amide groups of glutamine through the action of transglutaminase as described by Yan and Wold in Biochemistry 23, 3759-3765 (1984). Attachment of GlcNAc residues to the thiol groups of the cysteine residues of a nonglycosylated protein can be accomplished by treating the protein with a GlcNAc glycoside or thioglycoside in which the aglycone contains an electrophilic site such as an acrylate unit, for example the aglycones Rg or R g . The glycosylation of aromatic amino acid residues of a protein with a monosaccharide such as GlcNAc can be accomplished by treatment with a glycoside or thioglycoside in which the aglycone contains a diazo group, for example aglycones R-, or R g . A large number of other coupling methods and aglycone structures can be employed to derivatize a protein with a GlcNAc derivative. After chemical derivatization of the protein with GlcNAc residues, the trisaccharide sequence SAα2—>3(6)Galβl—>4(3)GlcNAc—> is con¬ structed by seαuential enzymatic attachment of galactose and sialic acid residues, as described for Asn-1 nked GlcNAc residues. In other variations, the protein is derivatized with: Galβl—>4(3)GlcNAc-X, Galβl—>4(3)GlcNAcβl—>4GlcNAc-X, SAα2—>3(6)Galβl—>4(3)GlcNAc-X, or SAα2—>3(6)Galβl—>4(3)GlcNAcβl—>4GlcNAc-X, where X is an aglycone containing a free amino group, an activated ester of a carboxylic acid, a diazo group, or other groups described above. The same procedures may be used to chemically attach galactose, rather than GlcNAc, directly to an amino acid. The galactose may then be enzymatically extended or capped with sialic acid, as previously described. D. Generation of additional protein-linked oligosaccharides by elon¬ gation of GlcNAc-protein or GlcNAc — >GlcNAc-protein. Procedures similar to those used to extend GlcNAc-protein or GlcNAc— >GlcNAc-protein to a protein-linked oligosaccharide resembling tne outer branch of a complex oligosaccharide can be employed to construct other carbohydrate structures found on GlcNAc residues attached to the terminal mannose units of the core pentasaccharide. Example 1. Generation of proteins containing repeating units of (GlcNAcβl— >3Galβl— >4). After preparation of either GlcNAc-protein or GlcNAcβl— >4GlcNAc-protein using the methods described above, a long carbohydrate chain may be generated by several rounds of alternating UDP-Gal :G!cNAc-R 01 — >4 gal actosyl transferase and UDP-GlcNAc: Galβl— >4GlcNAc-R βl— >3 -acetyl glucosaminyl transferase incubations. This will generate a pol lactosaminyl-type structure cf the type (GlcNAcβi— >3GalBl— >4) n attached to the GlcNAc-protein or GlcNAc β l— >4GlcNAc-protein starting material. Kaur, Turco and Laine reported in Biochem stry Internationa! , 345-351 (1982) thai bovine milk UDP-Gal : GlcNAc βl— >4 gal actosyl transferase can transfer the βl— >4 -linked gal actosyl residues to ool ylactosaminyl oligosaccha¬ rides, and a βl— >3 N-acetyl glucosaminyl transferase has Deen identi¬ fied in Novikoff ascites tumor cells by van αen Eijnαen e_ ___., J_. Biol ♦ Chem. 258, 3435-37 (1983). The number of repeating GlcNAc— >Gal units in the structure can be varied depending on the desired length; 1-10 such units should suffice for most applications. The essential element is that, after attachment of the disaccharide units, an exposed galactose residue is present so that the carbohydrate chain can be capped with α2— >3- or α2—>6-l inked sialic acid, as described above. Thus, the final structure would be SAα2— >6(3)Galβl— >4[GlcNAcβl— >3Gal β l— >4] n GlcNAc-protein, or SAα2 — >6(3) Galβl — >4[GlcNAcβl— >3Galβl~>4 GlcNAcβl— >4GlcNAc- n protein, where n is 1-10. The advantages of introducing such a polyTactosaminyl structure would be to increase solubility or to better mask the protein backbone to protect it from recognition by the immune system or from degrada¬ tion by proteases. Example 2. Generation of glycoproteins containing terminal Galβl— >4(3)[Fucαl— >3(4)GlcNAc or SAα2— >3Galβl— >3(Fucαl — >4)GlcNAc structures. After preparation of Galβl— >4(3)GlcNAc-protein, Gal βl— >3 (4 ) [Gl cNAcSl— >3Gal 61-->4] p Gl cNAc-protei n , Galβl— >4(3)GlcNAcβl— >4GlcNAc-protein, Galβl— >3(4)[GlcNAcSl— >3GalSl— >4] n GlcNAcβl— >4G!cNAc-protein, SAα2— >3Gal βl— >3G! cNAc-protei n , SAα2— >3Galβi— >3[GlcNAcβl— >3GalBl— >4] π GlcNAc-protein, SAα2— >3Galβl— >3GlcNAcBl— >4GlcNAc-protein or SAα2— >3Galβl— >3[GlcNAcSl— >3Galβl — >4] n GlcNAcβl— >4GlcNAc-protein where n is between 1 and 10, using the methods described above, a fucose can be attached to any of the acceptor GlcNAc residues by treatment with GDF--UC and a GDP-Fuc: GlcNAc αl— >3(4) fucosyl trans¬ ferase. The purification of this fucosyl transferase, its substrate specificity and preferred reaction conditions have been reported by Prieels et al in J_. Biol. Chem. 256, 104456-63 (1981). The activity of this enzyme with sialyl ateα substrates has been described by Jonnson and Watkiπs in Proc. Vlllth Int. Syr?,-. Glycocoπjugates (1985), eds. E.A. Davidson, .C. Williams and .M. Di Ferrante. If it is desired to attach fucose only in an αl— >3 linkage to the appropriate acceptor GlcNAc residues, the GD C -Fuc: GlcNAc αl— >3 fucosyl transferase can be used. This enzyme has been described by Johnson and Watkiπs in Proc. VIHth Int. Sy p. Glycoconjugates (1985), eds. E.A. Davidson, J.C. Williams and N.M. Di Ferrante. E. Targeting of glycosylated proteins to specific cells Cells with sugar-specific cell surface receptors are able to recognize and internalize glycoproteins bearing appropriate carbohydrate structures. The best characterized sugar-specific eel! surface receptors are the Gal receptor of hepatocytes, the Man/GlcNAc receptor of reticulo-endothelial cells and the fucose receptor found on hepatocytes, lymphocytes and teratocarcinoma cells. The subject of sugar-specific cell surface receptors has been reviewed by Neufeld and Ashwell in The Biochemistry of Glycoproteins and Proteoglycans, edited by Lennarz, Plenum Press, New York (1980), pp. 241-266. Proteins can be targeted to cells with sugar-specific cell surface receptors by generating glycoproteins that contain the appropriate sugar at nonreducing terminal positions. Several procedures are used to expose the desired terminal sugars. One procedure, in general, involves the treatment of a native giycoprotein with exoglycosidases, as described by Ashwell and Morel! in Adv. Enzymol . 43., 99-128 (1974). Another procedure is the attachment of monosaccharides to the protein, as described by Stahl e_t a _. in Proc. Nat! . Acad. Sc . USA 75, 1399- 1403 (1978). A third approach is the attachment of derivatives of oligosaccharides isolated from natural sources such as ovalbumin, as reported by Yan and Wold in Biochemistry 23, 3759-3765 (1984). The glycosylated proteins tnat are the subject of the present invention can be targeted to specific cells, depending on the specific sugars attached. Gal—>G!cNAc-protein, Gal—>G1cNAc—>G1cNAc-protein, (Gal—>G1cNAc)—>Gal—>G1cNAc-protein and (Gal—>G1cNAc) —>Gal—>G1cNAc—>G1cNAc-protein, where n is 1-10, are directed to hepatocytes. GlcNAc-protein, GlcNAc—>G1cNAc-protein, (GlcNAc—>Gal ') n—>GlcNAc-protein and (Gl cNAc— >Ga! ) n — >Gl cNAc— >Gl cNAc-protein, where n i s 1-10, are targeted to macrophages . Fi nal ly , Gal — > (Fuc— > )G1 cNAc-protei n , Gal — > ( Fuc— > ) Gl cNAc— >G1 cNAc-protei n , Gal — >(Fuc— > )Gl cNAc— >[Gal— > (Fuc— > ) Gl cNAc] -Drotei n , and m π • Gal— > ( Fuc— > ) Gl cNAc— >[Gal — > ( Fuc— > )m Gl cNAc]n — >Gl cNAc-protei n, where n is 1-10 and m is 0 or 1, are targeted to hepatocytes, lympho¬ cytes and teratocarcinoma cells. One application of targeting is for enzyme replacement therapy. For example, glucocerebrosidase can be targeted to macrophages for the treatment of Gaucher's disease. A second application is to target drugs or toxins to teratocarcinoma cells. The following non-limiting example demonstrates the method of the present invention on a yeast giycoprotein possessing multiple high- mannose and mannan oligosaccharides. Step 1. Endo H treatment of yeast external invertase. Yeast external invertase is a giycoprotein containing approxi¬ mately two high mannose and seven mannan oligosaccharides. External invertase of a commercial preparation from Saccharomyces cerevisiae, obtained from Sigma Chem. Corp, St. Louis MO, was purified as des¬ cribed by Trimble and Ma ley in J_. Biol . Chem. 252, 4409-12 (1977), and treated with Endo H essentially as described by Trimble e_t al_. in J_. Bi ol . Chem. 258, 2562-67 (1983). The purified invertase was denatured by placing a 1% SDS solution of the giycoprotein in a boiling water bath for 5 minutes. The denatured invertase (250 μg) was then incu¬ bated with Endo H (C.3 μg, from Miles Scientific, Naperville ID for 20 hours at 37°C in 175 ul of 0.1 M sodium citrate buffer, pH 5.5. After Endo H treatment, the reaction mixture was desalted on a Bio-Gel P-4 column (1 x 10 cm) eσuilibrated and eluted with 50 mM ammonium acetate, pH 6.5. The method of desalting is not critical . Dialysis or protein precipitation can also be used. The material eluting in the void volume of the column was pooled and lyophiϋzed. Analysis of the Enαo H-treated preparation of SDS-denatured inver¬ tase by SDS-PAGE, snown in Fig. 3c, indicated that the giycoprotein had been converted to a form consistent with an invertase possessing only a single GlcNAc residue at each glycosylation site. In a parallel experiment, native invertase was treated with Endo H in the same manner as the SDS-oenatured invertase. Analysis of the desalted reaction product by SDS-PAGE, shown in Fig. 3b, indicated that 2-3 oligosaccnaride chains of native invertase were resistant to cleavage by Endo H. To remove exposed mannose residues on the resistant chains, 250 μg Endo H-treated invertase was desalted, lyophilized, and incubated in 100 μl of 50 M sodium acetate, pH 5.C, containing 50 mM NaCl, 4 mM ZnC . and 20 mU of jack bean α-mannosi¬ dase (a gift from Dr. R. Trimble at State University of New York, Albany NY) for 17 hours at 37°C. Analysis of the reaction mixture by SDS-PAGE, shown in Fig. 3d, demonstrated through a shift to lower molecular weight that the α-mannosidase treatment removed additional annose residues. Step 2. Gal actosyl ation of the Endo H-treated samples of native and denatured yeast external invertase. An Endo H-treated sample of denatured yeast external invertase [85 μg, containing approximately 15 nmol of GlcNAc— >Asn( protein) sites] was incubated at 37 β C in 180 μl of 50 M 2-(N-morpho! ino)ethanesul- fonic acid (MES), pH 6.3, containing 0.8? Triton X-100, 25 mM MnC!~, - - 1.25 mM UDP-C H]Gal (specific activity, 8 Ci/mol) and bovine milk UDP-Gal : GlcNAc βl — >4 gal actosyl transferase (100 U, Sigma Chem. Corp., St. Louis MO). Aliquots were removed at selected times and analyzed by SDS-PAGE, as shown in Fig. 4. A gradual increase in appa¬ rent molecular weight was apparent up to a reaction time of one hour. This result was confirmed by measuring the incorporation of tritium into material precipitable by 0.5 M HCl/1 * phosphotungstic acid, which gave the result shown in Fig. 5. Nonradiolabeled gal actosyl ated samples of native and denatured yeast external invertase were prepared as substrates for the sialyla¬ tion reaction. Endo H-treated denatured invertase and Endo H plus α-mannosidase- treated native invertase were gal actosyl ated with noπradioactive UDP-Gal using the procedures described above. Step 3. Sialylation of the gal actosyl ated samples of native and denatured yeast external invertase. The native and denatured samples of nonradioactive gal actosyl ated yeast external invertase (50 μg of protein) were incubated at 37 e C for 17 hours in 70 μl of 0.1 M Tris-maleate, pH 6.7, containing 0.7 % Triton X-100, 2 mM CMP-[ 14 C]NeuAc (specific activity, 1.1 Ci/mmol) and bovine colostrum CMP-SA:GalSl— >4G!cNAc-R α2— >6 sialyl transferase [1.1 mU, purified according to Paulson et _a]_. in _. Biol . Chem. 252, 2356-2362 (1977)]. The reaction mixtures were analyzed by SDS-PAGE and autoradiography, as shown in Fig. 6. The radioactivity associated with the invertase band demonstrates that sialic acid has been at¬ tached to the galactose residues of the invertase by the α2 — >6 si aly! transferase. The following non-limiting example demonstrates the method of the present invention using chemical and enzymatic techniques on a protein that is not glycosylated in its native form. Step 1. Chemical attachment of a thioglycoside derivative of GlcNAc to bovine serum albumin (BSA). BSA was derivati zed by treatment with 2-imino-2-methoxyethyl-l- thio-N-acetylglucosaminide by Dr. R. Schnaar at Johns Hopkins University according to the procedure described by Lee et al_. in Biochemistry 15, 3956-63 (1976). The glycosylated BSA contained, on the average, 48 lysine-1 inked GlcNAc residues per molecule. Step 2. Gal actosyl ation of GlcNAc .g-BSA. GlcNAc 4 g-BSA (0.9 mg) was incubated at 37°C for 17 hours in 600- μl of 0.12 M MFS, pH 6.3, containing 0.6% Triton X-100, 20 mM MnCl ? _ 5 M - - UDP-[ H]Gal (specific activity, 1 Ci/mol), 1 M galactono-l,4-lactone, 1 M phenyl ethanesulfonyl fluoride, TPCK (21 μg), aprotinin (12 μTIU), leupeptin (0.6 μg), oepstatiπ (0.6 μg) and bovine milk UDP- Gal :G!cNAc-R SI— >4 gal actosyl transferase. The glycosylated BSA was partially purified from other reaction components by Bio-Gel P-4 gel filtration. After measurement of the amount of radioactivity incor¬ porated into the BSA, it was calculated that 46% of the available GlcNAc residues were gal actosyl ated. A second incubation of tne gal actosyl ated BSA under identical conditions increased the extent of reaction from 46 to 51%. The gal actosyl ated BSA was Durifiec with an anti-BSA antibody column obtained from Cooper Biomedical, Malvern PA. Step 3. Sialylation of gal actosyl ated BSA. The gal actosyl ated BSA (240 μg) was incubated for 16 hours at 37°C in 120 μl of 0.1 M Tris-maleate, pH 6.7, containing 3 mM CMP- [ C]NeuAc (specific activity 0.55 Ci/mol) and bovine colostrum CMP-SA: Gal βl— >4GlcNAc-R α2— >6 sialyl transferase (2.1 mU). The glycosylated BSA was partially purified from other reaction components by gel filtration. After measurement of the ratio of 14 C to ύ H radio¬ activity incorporated into the samples, it was calculated that 42% of the Gal — >GlcNAc — >protein residues were sialylated. A second incuba¬ tion of the sialylated BSA with 25 mU of sialyl transferase increased the extent of sialylation to 51%. The giycoprotein was isolated by immunoaffinity chromatography on an anti-BSA antibody column. Analysis of the three glycosylated forms of BSA by SDS-PAGE demon¬ strated a significant increase in apparent molecular weight after each step of the procedure, as shown in Fig. 7. This evidence confirms that SA— >Gal— >G!cNAc— > moieties have been constructed on the protein. The following nonlimiting example demonstrates the differential uptake of GlcNAc-BSA and Galβl— >4GlcNAc-BSA by GlcNAc/Man-specific receptors of macrophages. Mouse peritoneal macrophages, which possess cell surface receptors that recognize terminal GlcNAc and Man residues, were obtained from mice 4-5 days after intraperitoneal injection of thioglycollate broth (1.5 ml per mouse). The peritoneal cells were washed with Dulbecco's modified minimal essential medium (DME) containing 10% fetal calf serum (FCS) and plated in 96-well tissue cul ure trays at a density of 2 x 10 cells per well. After 4 hours the wells were washed twice with phosphate-buffered saline (PBS) to remove nonadherent cells. The adherent cells remaining in the wells were used for uptake experiments with GlcNAc-[ 125 I]BSA and Galβl— >4GlcNAc-: i2:' IjESA which had been radiolabeled with " " ""I Dy the chloramine T metnod. The radio! abeled protein preparations were added at a concentration of C.1-1.2 μg/ml to 100 μ! of DME containing 10% FCS and 10 mM HEPES [4-(2-nydroxyethyl )- 1-piperazine ethanesul fonic acid], pH 7.4. Parallel exDeriments were run in the presence of yeast mannan (1 mg/ml ) to measure nonsDecific uptake of the glycosylated BSA samples. The cells were incubated with the samples for 30 min at 37°C and then washed *ive times with PBS to remove residual protein not taken up by tne cells. The washed cells were dissolved in 200 μl of 1% SDS and the radioactivity determined. Nonspecific uptake (CPM in the presence of yeast mannan) was subtrac¬ ted from the total uptake (CPM in the absence of yeast mannan) to determine Maπ/GlcNAc receptor-specific uptake by the mouse peritoneal macrophages. The specific u p take of GlcNAc-[ 1 5 I]BSA and Galβl— >4GlcNAc- 125 C I]BSA is presented as a function of ESA concentration in Fig. 8. The results demonstrate that GlcNAc-BSA, Dut not GalBl— >4G!cNAc-BSA, is recognized and endocytosed by mouse peritoneal macrophages. The following non-limiting example demonstrates the differential uptake of Galβl— >4GlcNAc-BSA and SAα2— >6Galβl— >4GlcNAc-BSA by galactose-specific receptors of hepatoma cell line HepG2. Samples of GlcNAc-BSA and Gal— >GlcNAc-BSA were radiolabeled with 12 ^I by the chloramine T method. HepG2 cells were cultured in DME containing 10% fetal calf serum. Uptake experiments were performed on cells plated in 35 mm tissue culture dishes at approximately 70% confluency. The cells were washed with protein-free medium and incubated with 1 ml of DME containing 20 M HEPES, pH 7.3, containing cytochrome c (0.2 mg/ml ) and 0.5-7.5 μg of Galβl— >4GlcNAc-[ i£3 I]BSA ι ?*. or SAα2— >6Galβl— >4GlcNAc-[ I]BSA. Parallel experiments were performed in the presence of nonradioactive asialo-orosomucoid (0.2 mg/ml) to determine nonspecific uptake. The cells were incubated with the radiolabeled protein solutions for 2.5 hours at 37°C in a 5% C0 2 atmosphere, and then rinsed five times with chilled PBS containing 1.7 mM Ca " * " * " . The washed cells were solubilized with 1 ml of 1 M NaOH/10% SDS. Separate aϋαuots were used to measure radioactivity and the amount of protein per culture dish. It is assumed that the amount of protein in each disn is proportional tc the number of cells. Non¬ specific uptate (CPM in tne presence of as alo-orosomucoid) was subtracted from tne tota! uptake (CPM in tne absence of asialo- orosomucoid) to determine the galactose receptor-specific uptake by the HepG2 cells. The galactose receptor-specific uptake is shown as a function of glycosylated BSA concentration in Fig. 9. The results demonstrate that Galβl— >4G!cNAc-BSA, but not SAα2— >6Galβl— >4GlcNAc-BSA, is recognized and endocytosed by HepG2 cells. The following non-limiting example demonstrates the method of the present invention on a mammalian giycoprotein having one oligosaccha¬ ride chain of the high-mannose type. Step 1. Deglycosyl ation of ribonuclease B, a giycoprotein having a single high-mannose oligosaccharide. Native ribonuclease B (490 μg), obtained from Sigma Chem. Corp., St. Louis MO, and further purified by concanavalin A affinity chroma- tography as described by Baynes and Wold in J_. Bi ol . Chem. 251, 6016- 24 (1976) was incubated with Endo H (50 mU, obtained from Genzyme Corp., Boston MA) in 100 μl of 50 M sodium acetate, pH 5.5, for 24 hours at 37°C. SDS-PAGE indicated complete conversion of the giyco¬ protein to a form containing a single GlcNAc residue. The modified ribonuclease B was desalted on a Bio-Gel P6DG column and the ribonu¬ clease fractions were freeze-dried. Step 2. Gal actosyl ation of Endo H-treated ribonuclease B. Endo H-treated ribonuclease B (400 μg) was incubated for 3 hours at 37° in 250 μ! of 0.1 M MES, pH 6.3, containing 0.1% Triton X-100, 0.01 M MnCl 2 , 100 mU bovine milk UDP-Gal :GlcNAc-R βl — >4 galactosyl- transferase and 300 nmol UDP-f 3 H]Ga! (specific activity 17.3 Ci/mmol). The ga! actosyl ated ribonuclease was analyzed by FPLC on a Mono S column. A linear gradient from 20 mM sodium phosphate, pH 7.95 to 20 mM sodium phosphate containing 1 M NaCl was run. The gal actosyl ated ribonuclease eluted at a NaCl concentration of 0.13 M. The protein peak measured by UV absorbance (A 280 ) coincided with a peak of radioactivity, as shown in Fig. 10 ( o o ). The protein peak eluting at 0.13 M NaC! was collected and analyzed by SDS-PAGE. The only protein band detected after staining with Coo assie blue co-migrated with Endo H-treated ribonuclease B (not shown). Step 3. Sialylation of gal actosy! ated ribonuclease. A 40 μl aliquot of the reaction mixture from Step 2 was mixed with 10 μl of 6.5 mM CMP-NeuAc and 10 ul of rat liver CMP-NeuAc :Ga " i-R α2— >6 sialy! transferase (1.6 mU, obtained from Genzyme Corp., Boston MA) and incubated at 37°C for 18 hours. The sialylated ribonuclease was analyzed by FPLC on a Mono S column using the conditions described in Step 2. The sialylated ribonuclease eluted at a NaCl concentration of 0.18 M, as judged by the profiles of both A 280 and radioactivity. The profile of radioactivity is shown in Fig 10, (*- — *-*) . The con¬ version of Gal— >GlcNAc-RNAse to SA— >Gal— >RNAse appeared to be quantitative. Although this invention has been described with reference to specific embodiments, it is understood that modifications and varia ¬ tions of the methods for modifying or glycosylating proteins, and the glycosylated proteins, may occur to those skilled in the art. It is intended that all such modifications and variations be included within the scope of the appended claims. Table I. Oligosaccharide Specificities of Endo-β-N-acetyl gl ucosami nidases Enzyme and Susceptible N-linked oligosaccharides (Ref.) Source Endo H Yeast mannans, all high-mannose oligosac- (1,2) (Streptomyces charides, and hybrid oligosaccharides. piicams} (The enzyme requires an αl—>3-linked mannose residue attached to the αl—>6 mannose residue of the Man.,G!cNAc 2 core and we have found that the enzyme is not inhibited by an αl—>6 linked fucose attached to the innermost GlcNAc residue.) Endo C Certain high-mannose oligosaccharides. (3) (Clostr IiIdium (Similar to Endo H except that it will not perfringens) cleave substrates if the mannose linked αl—>3 to the β-1inked mannose is substituted at-C-4 with another sugar or if the β-1 inked mannose residue is substituted with a βl—>4- 1inked GlcNAc residue.) Endo D Man 3 -_ 5 GlcNAc with or without a fucose (4) (Diplococcus res idue link d αi— >6 to the innermost pπeumoniae) Gl c Ac residue Endo L ManGlcNAc, (5) (S. olicatus) Endo F High-mannose and bianteππary complex (5) (Flavobacterium oligosaccharides meningosepticum) References: 1. Tarentino et aj_. , Meth. Enzymol . 50, 574-580 (1978). 2. Tai and KoData, Biocne . " Biopnys . "" Res . Commun. 78, 434-441 (1977) 3. Kobata, Meth. Enzymol. 50, 567-574~ T97o r T ~ 4. Muramatsϋ7~fietπ . Enzy oTT j>0 , 555-559 (1978). 5. Trimble et al., C. Biol Chem. 254, 9708-13 (1979). 6. Plummer et IT., 7J. IToT. cTiem. 7 10700-4 (1984)

Claims: We claim: 1. A method for modifying a giycoprotein comprising: attaching a gal actose residue to a core N-acetyl gl ucosamine to form a Gal -GlcNAc sequence; and attaching a sial ic acid residue to the galactose to form a S_A- Gal -GlcNAc sequence. 2. The method of cl aim 1 further comprising fi rst cleaving asparagine-1 inked ol gosaccharide chai ns of the giycoprotein to remove all sugars other than core N-acetyl gl ucosamine residues bound to the giycoprotein. .3. The method of claim 2 wherein the ol igosaccharide chains are cleaved by an endoglycosidase. 4. The method of cl aim 3 wherein the endoglycosidase is sel ected from the group consisting of endo-β-N-acetyl gl ucosamiπidase H, endo-β- N-acetyl glucosaminidase F, endo-β-N-acetyl gl ucosaminidase C τ τ , endo-β- N-acetyl glucosaminidase D, endo-β-N-acetyl l ucosaminidase L, and combinations thereof. 5. The method of claim 4 further comprising cleaving 0-1 inked ol igosaccharide chains with an enzyme selected from the group consisting of α-manπosidase, endo-α-N-acetyl gal actosaminidase, and combinations thereof. 6. The method of cl aim 1 further comprising cleaving 0-1 inked ol igosaccharide chains with an enzyme selected from the group consisting of α-mannosidase, eπdo-α-N-acetyl gal actosaminidase, and combinations thereof. 7. The method of claim 2 wherein the oligosaccharide chain is cleaved by digestion with an exoglycosidase. 8. The method of claim 7 wherein the exoglycosidase is selected from the group consisting of sialidase, α-mannosidase, β-mannosidase, α-galactosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, and combi nati ons thereof . 9. The method of claim 8 wherein the oligosaccharide chains are sequentially cleaved by: 1) digesting the giycoprotein with α-mannosidase to re ov α-manπose residues; 2) digesting the product of step 1) with β-mannosidase to remove β-mannose residues. 10. The method of claim 9 further comprising digesting the giycoprotein with an additional enzyme selected from the group consisting of exoglycosidases and phosphatases. 11. The method of claim 10 wherein the product of step 2 is digested with β-hexosaminidase. 12. The method of claim 2 wherein the oligosaccharide chains are cleaved by sequentially digesting the giycoprotein first with an exoglycosidase and secondly with an endoglycosidase. 13. The method of claim 12 wherein the oligosaccharide chains are cleaved by 1) digesting the giycoprotein with α-mannosidase to remove α-mannose residues; and 2) digesting the product of step 1) with an endoglycosidase selected from the group consisting of endo-β-N-acetyl glucosaminidase L and eπdo-S-N-acetyl glucosaminidase D. 14. The method of claim 12 for modifying glycoproteins wherein the oligosaccharide chains are cleaved by digesting the giycoprotein with an enzyme selected from the group consisting of sialidase, α-galactosidase, β-galactosidase, β-hexosaminidase, α-fucosidase, and combinations thereof followed by digestion with an enzyme selected from the group consisting of endo-β-N-acetyl glucosaminidase D and endo-β-N-acetyl glucosaminidase F. 15. The method of claim 2 further comprising cleaving high-mannose ol gosaccharide chains with α-mannosidase to remove mannose residues. 16. The method of claim 2 wherein the oligosaccharide chains are cleaved by chemical treatment. 17. The method of claim 16 wherein the oligosaccharide chains are cleaved with a compound selected from the group consisting of trifluo- romethane sulfonic acid and hydrofluoric acid. 18. The method of claim 1 further comprising first producing the glycoproteins in cells in the presence of a glycosidase inhibitor. .19. The method of claim 18 wherein the glycosidase inhibitor is selected from the group consisting of deoxymannojirimycin, swainsonine, castanospermine and deoxynoji imyciπ. 20. The method of claim 1 further comprising first producing the giycoprotein in cells with one or more mutations in the oligosaccha¬ ride processing pathway. 21. A method for modifying proteins comprising: derivatizing amino acids on the protein with a glycoside or thioglycoside S-X, wherein S is a first saccharide selected from the group consisting of N-acetylglucosamine and galactose and X is an aglycone, and enzymatically attaching a second saccharide selected from the group consisting of galactose, N-acetylglucosamine, fucose, and sialic acid. 22. The method of claim 21 wherein said aglycone comprises an activation group selected from the group consisting of an activated carboxylic acid; a free amino group; an electrophilic site; and a diazo grouping. 23. Tne method of claim 21 comprising reacting a glycoside or thioglycoside having an aglycone containing an activated carboxylic acid with an amino acid of the protein selected from the group consisting of lysine, arginine, histidine, the amino-terminal amino acid of the protein; and other amino acids containing free amino groups. 24. The method of claim 21 comprising reacting a glycoside or thioglycoside having an aglycone containing a free amino group with an amino acid of the protein selected from the group consisting of gluta ic acid, aspartic acid, the carboxy-terminal amino acid of the protein, and other amino acids containing free carboxy! groups. 25. The method of claim 21 comprising reacting a glycoside or thioglycoside having an aglycone containing an electrophilic site with an amino acid of the protein selected from the group consisting of cysteine and other amino acids containing free sulfhydryl groups. 26. The method of claim 21 comprising reacting a glycoside or thioglycoside having an aglycone containing a free amino group with an amino acid of the protein selected from the group consisting of hydroxyproline, serine, threonine, and other amino acids with free hydroxy! groups. 27. The method of claim 21 comprising reacting a glycoside or thioglycoside having an aglycone containing a diazo grouping with an amino acid of the protein selected from the group consisting of phenylalanine, tyrosine, tryptophan, and other aromatic amino acids. 28. The method of claim 22 comprising reacting a glycoside or thioglycoside having an aglycone containing a free amino group with glutamine using a transglutaminase. 29. The method of claim 21, wherein the first saccharide is GlcNAc, and wherein said enzymatic attachment comprises: attaching a galactose residue to the N-acetylglucosamine to form a Gal -GlcNAc— > sequence; and attaching a sialic acid residue to the galactose to form a SA-Gal -GlcNAc— > sequence. 30. The method of claim 21, wherein the first saccharide is galactose, and wherein said enzymatic attachment comprises: attaching a sialic acid residue to tne galactose to form a SA-Gal sequence. 31. The method of claim 1 or 29 wherein the galactose residue is attached to the N-acetylglucosamine residue by a gal actosyl transferase . 32. The method of claim 31 wherein the gal actosyl transferase is selected from the group consisting of UDP-Gal :G ' ιcNAc-R βl — >4 galac- tosyl transf erase and UDP-Gal :GlcNAc-R SI— >3 gal actosyl transferase. 33. The method of claim 1 or 29 wherein the galactose is attached to the N-acetylglucosamine by: 1) incubating UDP-Gal :GlcNAc-R βl— >4 gal actosyl transferase with the derivatized protein to attach a terminal galactose to the GlcNAc; 2) incubating the product of step 1 with UDP-GlcNAc:- Galβl— >4GlcNAc-R βl— >3 N-acetyl gl ucosaminyl transferase to attach a terminal GlcNAc to the terminal galactose; 3) incubating the product of step 2 with UDP-Gal : GlcNAc -R 1— >4 gal actosyl transferase to attach a terminal galactose to the terminal GlcNAc; and 4) repeating steps 2 and 3 until an oligosaccharide chain (Galβi— >4GlcNAcβl— >3) units, wherein n is between 1 and IC, is pro¬ duced. 34. The method of claim 1 or 29 or 33 further comprising attaching fucose to a Gal— >GlcNAc— > sequence. 35. The method of claim 34 wherein the fucose is attached to the Gal -GlcNAc— > sequence with GDP-Fuc: GlcNAc αl— >3 fucosyl transferase. 36. The method of claim 1 or 29 further comprising attaching the galactose residues to the N-acetylglucosamine in a solution containing a non-ionic detergent, a chaotropic agent, an organic solvent, urea, a protease inhibitor, an exoglycosidase inhibitor, a disulfide bond reducing agent, or a combination thereof. 37. The method of claim 1 wherein the sialic acid residue is attached to the Gal -GlcNAc sequence in an α linkage by a si aly! transferase. 38. The method of claim 37 wherein the sialyl transferase is selected from the group consisting of CMP-SA: Gal βl— >4G!cNAc-R β2— >6 si aly! transferase, CMP-SA:Galβl— >3(4)GlcNAc α2— >3 sialyl transferase, and CMP-SA:GalSl— >4GlcNAc α2— >3 sialyl transferase 39. A method for targeting a protein to a eel! having a specific surface receptor for a saccnaride, said metnod comprising: attaching to tne protein an oligosaccharide chain, said oligosaccharide chain having an exposed saccharide and a Gal— >GlcNAc sequence, wherein said exposed saccharide is recognized by the eel! surface receptor and said oligosaccharide chain is attached to the protein with a Gal— >GlcNAc sequence. 40. The method of claim 39 wherein the oligosaccharide chain is a disaccharide consisting of GalBl— >4 GlcNAc. 41. The method of claim 39 wherein the oligosaccharide chain is a branched oligosaccharide selected from the group of Galβl— >3(4)[Fucαl— >4(3)]GlcNAc, SAα2— >3Galβl— >3(Fucαl— >4)GlcNAc, Galβl— >3(4)[Fucαl— >4(3)]G!cNAcβl— >4GlcNAc, and SAα2— >3Galβl— >3(Fucαl— >4)G!cNAcβl— >4GlcNAc. 42. A protein comprising an oligosaccharide sequence consisting of any of Galβl— >4GlcNAc— >; Galβl— >3GlcNAc— >: SAα2— >6Gal βl— >4G1 cNAc--> ; SAα2— >3Galβl— >4GlcNAc— >; SAα2— >3Ga!βl~>3GlcNAc-->: Gal βl— >4( Fucαl— >3 ) Gl cNAc— > ; Galβl— 3(Fucαl— >4)GlcNAc-->; SAα2— >3Galβl—>3 (Fucαl— >4)GlcMAc—>; Galβl— >4GlcNAcSl— >4GlcNAc— >; Gal βl— >3G1 cNAcβl — >4G1 cNAc— >; SAα2— >6GalBl— >4G1 cNAcβl— >4G! cNAc— > : SAα2— >3Gal Sl— >4G1 cNAcβl— >4G1 cNAc— > ; SAα2— >3Ga!βl— >3G1 cNAcβl— >4GlcNAc— >: Gal βl— >4 ( Fucαl— >31 GLcNAcSl — >4G1 cNAc— > ; Galβl— >3 (Fucαl— >4)G1 cNAcβl— >4GlcNAc—>; SAα2— >3Ga!βl— >3( Fucαl— >4)GlcNAcβl— >4GlcNAc— >; [GlcNAc β l— >3Galβl— >4] π ιcNAc— >, wherein π is between 1 and 10; [GlcNAcβl— >3Sa1βl— >4] GlcNAcSl— >4GlcNAc-->_ wherein n is between 1 and 10; Gal β l— >4[GlcNAcBl— >3Galβl— >4J π GlcNAc— >, wherein n is between 1 and 10; Gal β l— >3[G!cNAc β l— 3Gal β l— >4] r GlcNAc— >, wherein n is between 1 and 10: SAα2—>6Ga! β l—>4[GlcNAcβi—>3Ga!βl—>4] n GlcNAc—>, wherein n is between 1 and 10: SAα2—>3Galβl—>4[G!cNAcβl—>3Galβl-->4]_π,GlcNAc-->- wherein n is between 1 and 10; SAα2—>3Gal β l—>3[GlcNAcβl—>3GalβI—>4] n GlcNAc—>, wherein n is between 1 and 10; Gal β l—>4(FucαI—>3)G!cNAcβl—>3[Galβl—>4(FucαI—>3) m G!cNAcβl—>3] - Galβl—>4GlcNAc—>, wherein m is between 0 and 1, and n is between 1 and 10; Galβl— >3(Fucαl—>4)Gl cNAcβl—>3[Gal βl—>4(Fucαl—>3 ) m G! cNAcβl— >3] n - Galβl— >4GlcNAc-->, wherein is between 0 and 1, and n is between 1 and 10; SAα2— >3GalSl— >3( Galβl — >4GlcNAc— >, wherein n is between 1 and 10; Galβl— >4[G1 cNAcβl— >3Galβl—>4] rι G!cNAcβl—>4GlcNAc—>, wherein n is between 1 and 10; Galβl— >3[G!cNAcBl— >3Galβl—>4] n G! cNAcβl— >4GlcNAc—>, wherein n is between 1 and 10; SAα2— >6Ga!βl— >4[GlcNAcβi— >3Galβl— >4] n Gl cNAcβl— >4GlcNAc— >, wherein n is between 1 and 10; SAα2—>3Galβl—>4[Gl cNAcβl— >3GalBl—>4] n G! cNAcβl— >4GlcNAc—>, wherein n is between 1 and 10; SAα2— >3GalSl— >3[G!cNAcβl— >3GalBl— >4] n G!cNAcβl— >4GlcNAc— >, wherein n is between 1 and 10; Galβl— >4(Fucαi—>3)Gl cNAcβl— >3[GalBl—>4(Fucαl—>3) m G!cNAcSl—>3] π - Gal βl— >4G1 cNAcSl — >4G1 cNAc— > , wherein m is oetweεn 0 and 1, and n is between 1 and 10; GaTβl— >3(FucαI— >4)G1 cNAcβl— >3[Gal Si— >4(Fucαl— >3 )__ 1 cNAcβl— >3] n - Galβl— >4GlcNAcSi— 4GlcNAc— >, wherein m is between 0 and 1, and n is between 1 and 10; SAα2 — >3Ga!βl — >3( Fucαl— >4)GlcNAcβi— >3 [Galβl — >4GlcNAcβ— >3] n - GalBl— >4GlcNAc81— >4GlcNAc— >, wherein n is between 1 and 10; 43. A protein comprising an oligosaccnaride sequence consisting of any of: (GlcNAcβl— >3Galβl—>4) n l cNAcβl— >3Gal—>, wherein n is between 1 and 10; SAα2— >6Gal— >; SAα2—>6Gal β l—>4(G! cNAcβl— >3Galβl—>4) n G! cNAcβl— >3Gal, wherein π is between 1 and 10; SAα2— >3Gal— >; and SAα2—>3Gal β l—>4(G! cNAc β l— >3Galβl—>i; n G! cNAcβl— >3Gal—>, wherein n is between 1 and 10. 44. A glycosylated protein comprising Gal -GlcNAc-protein, wherein the Gal is attached to the GlcNAc by an enzyme selected from the group consisting of UDP-Gal :GlcNAc-R βl— >4 gal actosyl transferase and UDP- Gal :GlcNAc-R βl— >3 gal actosyl transferase. 45. A glycosylated protein comprising SA-Gal -GlcNAc-protein, wherein the SA is attached to the Gal by an enzyme selected from tne group consisting of CMP-SA:Galβl— >4GlcNAc-R α2~>6 sialyl transferase; CMP-SA:Galβl— >3(4)GlcNAc-R α2— >3 sialyl transferase; and CMP-SA:- Galβl— >4GlcNAc-R α2— >3 sialyl transferase.

Inventors: BERGH MICHEL LOUIS EUGENE, HUBBARD CATHERINE S, RASMUSSEN JAMES R, BERGH, MICHEL, LOUIS, EUGENE, HUBBARD, CATHERINE, S., RASMUSSEN, JAMES, R.
Applicants: BERGH MICHEL LOUIS EUGENE, HUBBARD CATHERINE S, RASMUSSEN JAMES R, BERGH, MICHEL, LOUIS, EUGENE, HUBBARD, CATHERINE, S., RASMUSSEN, JAMES, R.
Application year: 1986
Main classification: C12P21
IPC codes: C07K1, C07K14, C12P21