Process for controlling intracellular glycosylation of proteins

A process for controlling the glycosylation of protein in a cell wherein the cell is genetically engineered to produce one or more enzymes which provide internal control of the cell's glycosylation mechanism. A Chinese hamster ovary (CHO) cell line is genetically engineered to produce a sialyltransferase. This supplemental sialyltransferase modifies the CHO glycosylation machinery to produce glycoproteins having carbohydrate structures which more closely resemble naturally occurring human glycoproteins.

BACKGROUND OF THE INVENTION 
1. Field of the Invention. 
The present invention relates generally to the cellular mechanisms and 
machinery involved in the glycosylation of proteins manufactured by the 
cell. More particularly, the present invention involves altering the 
glycosylation capabilities of a cell in order to control the structure of 
carbohydrate groups attached during glycosylation. 
2. Description of Related Art. 
The publications and other reference materials referred to herein to 
describe the background of the invention and to provide additional detail 
regarding its practice are hereby incorporated by reference. For 
convenience, the reference materials are numerically referenced and 
grouped in the appended bibliography. 
During the last decade, numerous processes and procedures have been 
developed for genetically engineering cells in order to produce a wide 
variety of proteins and glycoproteins. These procedures involve utilizing 
recombinant DNA technology to prepare a vector which includes genetic 
material that codes for a specific protein or glycoprotein. Upon 
introduction of the vector into the host cell, the inserted genetic 
material instructs the host cell's biochemical machinery to manufacture a 
specific protein or glycoprotein. 
Problems have been experienced with the production of glycoproteins by 
genetically engineering host cells. Glycoproteins are proteins having 
carbohydrate groups attached at various points along the protein's amino 
acid backbone. The carbohydrate groups are commonly attached to 
asparagine, serine or threonine. The genetic sequence introduced into the 
host cell usually includes instructions with respect to the amino acid 
sequence of the protein and the location and structure of the carbohydrate 
groups. Most of the cell lines which are commonly used as host cells are 
capable of following the vector's instructions with respect to preparing a 
protein having a specific amino acid sequence. However, many host cells 
are not capable of following instructions with respect to glycosylation of 
the protein. For example, E. coli is a common host cell used in producing 
a wide variety of proteins. However, E. coli does not contain the cellular 
glycosylation machinery required to attach carbohydrate groups to the 
proteins it manufactures. 
Unlike E. coli, many other host cells do have varying capabilities with 
respect to protein glycosylation. However, even though these cells have 
glycosylation capabilities, the glycosylation machinery is not controlled 
by the recombinant DNA vector. Accordingly, the glycoprotein produced by 
such host cells may differ in carbohydrate structure from the natural 
glycoprotein coded for by the vector.(1, 2) 
Chinese hamster ovary (CHO) cells are a standard cell line used 
commercially for the high yield expression of glycoproteins from vectors 
engineered through recombinant DNA technology. The protein sequence of the 
glycoprotein expressed by CHO comes from the DNA transinfected into the 
cell while the structure of the carbohydrate portion of the glycoprotein 
is determined by the cellular machinery of the CHO cells. While most 
glycoproteins normally contain a mixture of NeuAc-.alpha.-2,6Gal and 
NeuAc-.alpha.-2,3Gal linkages on their N-linked oligosaccharides, CHO 
cells only make asparagine linked carbohydrate chains with terminal sialic 
acids in the NeuAc-.alpha.-2,3Gal linkage.(1,2) 
For example, erythropoietin is a glycoprotein naturally occurring in humans 
which has N-linked carbohydrate groups with both the NeuAc-.alpha.-2,6Gal 
and NeuAc-.alpha. -2,3Gal linkages. CHO cells which are genetically 
engineered to produce erythropoietin can only produce this protein with 
the NeuAc-.alpha.-2,3Gal linkages.(1) Although a number of mutant CHO cell 
lines have been developed which have altered capabilities for protein 
glycosylation,(3) they are not suitable for the production of 
glycoproteins intended for use in animals. Indeed, the carbohydrate groups 
produced by the cells are truncated, resulting in the rapid clearance of 
the recombinant glycoproteins from the blood followed by degradation. 
Thus, while the glycoproteins produced by these mutant CHO cell lines do 
display in vitro biological activity, they are inactive in vivo because of 
the rapid clearance from the blood stream. 
From the above, it is apparent that there is a need to develop a process 
which can be used to alter the glycosylation machinery of host cells in 
order to control the structure of carbohydrates attached during 
glycosylation. Such a process for controlling host cell glycosylation 
would be useful not only in expressing glycoproteins which accurately 
mimic naturally occurring proteins, but would also be useful in preparing 
glycoproteins having selected altered carbohydrate structures for 
diagnostic and research uses. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, a process is disclosed which 
provides for control of cellular glycosylation. The invention is based 
upon the discovery that the glycosylation machinery of host cells can be 
altered and controlled by introducing a gene into the host cell which 
codes for at least one enzyme which is capable of affecting glycosylation 
of a protein in the cell. 
The present invention involves controlling the glycosylation of a protein 
in a host cell wherein attachment of the carbohydrate moiety to proteins 
during glycosylation is dependent upon a number of naturally occurring 
enzymes which are present in the cell. In accordance with the present 
invention, at least one gene is introduced into the cell which is capable 
of expressing at least one supplemental enzyme which is capable of 
affecting the glycosylation mechanism. The expression of the supplemental 
enzyme in the cell produces a cell having both naturally occurring and 
supplemental enzymes wherein the presence of the supplemental enzyme 
alters the cell's glycosylation mechanism. 
As a feature of the present invention, it was discovered that transfection 
of CHO cells with cDNA coding for a glycosyltransferase resulted in 
production of the glycosyltransferase enzyme in the CHO cell and 
subsequent alteration of the carbohydrate structure of glycoproteins 
produced by the CHO cell. 
A feature of the present invention is that the glycosylation machinery of a 
host cell can be controlled to produce glycoproteins wherein the location 
and structure of carbohydrates is equivalent to a given naturally 
occurring glycoprotein. As an additional feature of the present invention, 
the glycosylation process of the host cell can also be controlled to 
produce glycoproteins wherein the carbohydrate structure is changed 
slightly from the naturally occurring glycoprotein. Such purposely altered 
glycoproteins are useful as both diagnostic and research tools in studying 
the biochemistry of various naturally occurring glycoproteins. 
The above discussed and many other features and attendant advantages of the 
present invention will become better understood by reference to the 
following detailed description when taken in conjunction with the 
accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention involves controlling the glycosylation machinery of a 
cell by using genetic engineering to instruct the cell to produce various 
enzymes upon which glycosylation in the cell is dependent. The process for 
controlling glycosylation is based on the well-documented fact that a 
given cell will synthesize carbohydrate groups whose structures are 
determined by the specificities of the glycosyltransferases produced by 
that cell. (2) The most bioactive terminal sugars are attached to common 
core structures by "terminal" glycosyltransferases.(5) When two terminal 
enzymes compete with each other, the ultimate carbohydrate structure is 
determined by the specificity of the enzyme that acts first. The invention 
relies on the concept that the introduction and over expression of a 
terminal (or branching) glycosyltransferase, not normally produced by a 
cell, will result in the successful competition with the endogenous 
enzymes, and will produce carbohydrate groups with a structure specified 
by the new enzyme. 
The basic procedure involves transfection of a host cell with a vector 
carrying a gene which expresses at least one enzyme upon which 
glycosylation in the host is dependent. The resultant enzyme(s) which is 
expressed in the cell provides internal control of the glycosylation 
machinery of the cell. Accordingly, the invention provides a useful 
procedure for controlling the structure of carbohydrates attached during 
glycosylation to more closely resemble naturally occurring glycolipids. In 
addition, one can use the present invention to alter the carbohydrate 
structure of a glycoprotein produced in a host cell for investigational 
purposes. 
The invention has wide application to host cells which are naturally 
capable of glycosylation. Exemplary cell lines to which the present 
invention is amenable include Chinese hamster ovary (CHO) cells, mouse L 
cells, mouse A9 cells, baby hamster kidney cells, C127 cells, PC8 cells 
and other eukaryotic cell lines capable of the expression of recombinant 
glycoproteins. 
The particular procedure used to introduce genetic material into the host 
cell for expression of the glycosyl transferase is not particularly 
critical. Any of the well-known procedures for introducing foreign 
nucleotide sequences into host cells may be used. These include the use of 
plasmid vectors, viral vectors and any of the other well-known methods for 
introducing cDNA or other foreign genetic material into a host cell. It is 
only necessary that the particular genetic engineering procedure utilized 
be capable of successfully introducing at least one gene into the host 
cell which is capable of expressing at least one enzyme which is known to 
be involved in glycosylation. Further, the genetic material must be 
introduced in such a way that the host cell expresses the enzyme coded for 
by the inserted genetic material so that, upon expression, the enzyme 
alters the glycosylation capabilities of the cell. 
The preferred enzymes for production within a host cell are 
glycosyltransferases such as sialyltransferase. Other possible enzymes 
which may be coded for and produced within the cell to alter the 
glycosylation machinery include fucosyltransferases, 
galactosyltransferases, .beta.-acteylgalactosaminyltransferases, 
N-acetylglycosaminyltransferases, and sulfotransferases.(5) 
The particular vector used is also not particularly critical. Any of the 
conventional vectors used for expression of recombinant glycoproteins in 
eukaryotic cells may be used. Exemplary vectors include pMSG, 
pAV009/A.sup.+, pMT010/A.sup.+ and any other vector allowing expression of 
glycoproteins under direction of the SV-40 early promotor, metallothionein 
promotor, murine mammary tumor virus promotor, Rous sarcoma virus promotor 
or other promotors shown effective for expression in eukaryotic cells. A 
suitable vector is the pECE vector which is described in Ellis, et al.(4) 
The types of glycoprotein which would be expressed having modified 
carbohydrate structural forms include erythropoietin, insulin, plasminogen 
activator (TPA), interferon and various glycopeptide hormones. The 
nucleotide sequences for various cDNA coding for these proteins are known. 
The following portion of this detailed description is limited to alteration 
of the glycosylation machinery of CHO cells. However, it is understood 
that the principles disclosed with respect to the CHO cell line also apply 
to the other various host cells previously mentioned. 
In this specific example, a CHO cell line is produced which produces 
modified terminal sialic acid groupings on its N-linked oligosaccharides. 
Normally these cells make N-linked carbohydrate groups that contain sialic 
acid exclusively in the NeuAcc.alpha. 2,3Gal sequence. By expression of a 
.beta.-galactosideo.alpha. 2,6 sialyltransferase cDNA in these cells, the 
N-linked carbohydrate groups are directed to produce the NeuAc.alpha. 
2,6Gal sequence commonly found on many glycoproteins. 
The expression vector used to transfect the CHO cells is shown in FIG. 1. 
The vector was constructed as follows. ST3, a 1.6 kb cDNA encompassing the 
complete amino acid coding sequences for the .beta.-galactoside .alpha. 
2,6 sialyltransferase, was shotgun subcloned from an EcoRI digest of ST3 
into M13mp19 as described by Weinstein, et al.(6). Although an internal 
EcoRI site was present, the two fragments were correctly oriented as 
determined by dideoxy sequencing(7). Site directed mutagenesis by the 
procedure of Zoller and Smith(8) eliminated the internal EcoRI site using 
the primer GCCAAGGAGTTCCAGAT which binds to nucleotides 115-132. An A to G 
transition abolished the EcoRI recognition site, GGAATTC, but preserved 
the native amino acid coding sequence. The mutation in ST3 was confirmed 
by the dideoxy chain termination DNA sequencing method (7). 
The nucleotide sequence of the sialyltransferase cDNA prior to mutagenesis 
at the EcoRI site is set forth in FIGS. 3a and 3b. Also shown is the 
complete amino acid sequence inferred from the nucleotide sequences. 
Peptide sequence overlaps (black boxes) include the NH.sub.2 -terminal 
sequence of the purified sialyltransferase (arrow). Stippled areas 
indicate residues that were not identified. Potential glycosylation sites 
with the sequence Asn-X-Thr/Ser are boxed. The proposed signal-anchor 
sequence is underscored with the cross-hatched box bordered at either end 
by open boxes highlighting charged lysine residues. 
The altered cDNA was subcloned from M13mp19 into the EcoRI site of the 
polylinker of bluescript (bs-ST3) and subsequently into the EcoRI site of 
pECE (pECE-ST3) for expression of the sialyltransferase as shown in FIG. 
1. In this vector the sialyltransferase cDNA is under the direction of 
SV-40 virus early promotor, allowing the sialyltransferase to be expressed 
in a wide variety of eukaryotic cell lines. The vector was obtained from 
William J. Rutter (University of California at San Francisco School of 
Medicine-Department of Biochemistry and Biophysics), and is described in 
Ellis, et al.(4) 
CHO cells were transfected with pECE-ST3 according to the method of Graham 
and Van der Eb(9). Cells at 50% confluency were transfected with 20 ug 
supercoiled pECEST3 and 2 ug supercoiled pSV2neo per 100 mm dish. After 48 
hours the cells were split 1:5 and replated in 75 cm.sup.2 flasks with 
selection medium containing the antibiotic G418. After six weeks in 
selection medium, resistant cells were presumed to have the transfected 
DNA stably integrated in the genome, and the cells were then maintained in 
the absence of G418. 
To select clonal cell lines expressing the sialyltransferase, advantage was 
taken of a newly described plant lectin, Sambucus nigra agglutinin (SNA), 
which recognizes the product of the sialyltransferase, NeuAc.alpha. 
2,6Gal, with 50-100 fold higher avidity than the NeuAc.alpha. 2,3Gal 
sequence normally produced by the CHO cells.(10) Accordingly, cells 
producing the sialyltransferase were found to bind the fluorescent labeled 
lectin (FITC-SNA), but not the wild type cells. Following automated 
fluorescence activated cell sorting (FACS), clonal cell lines expressing 
the .beta.-galactoside .alpha. 2,6 sialyltransferase were readily isolated 
and amplified. 
Detailed analysis of one cell line showed that the .beta.-galactoside 
.alpha. 2,6 sialyltransferase is expressed at equivalent levels to the 
endogenous .beta.-galactoside .alpha. 2,3 sialyltransferase normally 
present in CHO cells. The result is that 20-25% of the total cell surface 
carbohydrate groups contain the NeuAc.alpha. 2,6Gal sequence instead of 
the NeuAc.alpha. 2,3Gal sequence. Thus, this cell line produced the 
mixture of sialic acid linkages found on many naturally produced 
glycoproteins, rather than only the NeuAc.alpha. 2,3Gal sequence produced 
by wild type CHO cells. This invention is therefore useful in the 
expression of recombinant glycoproteins such as erythropoietin, where the 
natural mixture of sialic acid linkages differs from that of the 
recombinant glycoprotein produced in CHO cells. 
A comparison of the carbohydrate structures which result from addition of 
terminal sialyl acid groups in endogenous CHO cells and CHO cells 
transfected with the cDNA clone expressing the .alpha. 2,6 
sialyltransferase is set forth in FIG. 2. In FIG. 2, 
GlcNAc=N-acetylglucosamine; Gal=galactose; Man=mannose; SA=sialic acid 
(N-acetyl neuraminic acid); and Asn=asparagine. As can be seen, the 
terminal SA groups in the endogenous CHO cells are only attached by 
.alpha. 2,3 linkages. However, the CHO cells which are modified in 
accordance with the present invention also produces .alpha. 2,6 terminal 
SA linkages. 
The ratio of the NeuAc.alpha. 2,6Gal and NeuAc alpha 2,3Gal linkages can 
now be controlled by controlling the level of expression of the 
.beta.-galactoside .alpha. 2,6 sialyltransferase. To this end an 
expression vector has been constructed placing the sialyltransferase under 
the control of the metallothionein promotor in a plasmid (pMT010/A.sup.+) 
containing the DHFR gene.(11) When transfected into cells, this vector 
allows a twofold control on the level of expression. The first is by 
induction of the metallothionein promotor with metal ions, and the second 
by amplification of the gene by selection with methotrexate. Such vectors 
will allow amplification of expression 100 fold over that obtained in the 
transfected cell lines examined to date.(11) Thus, a simple alteration of 
the procedure described above, will allow additional control over the 
terminal sialic acid linkages of CHO cells. 
The general applicability of the procedure of the present invention is 
limited only by the availability of cDNA's coding for glycosyltransferases 
making terminal structures not normally found on the target cells, and the 
availability of an appropriate lectin or carbohydrate specific antibody 
capable of recognizing the newly expressed carbohydrate structure on the 
surface of the transfected cells. Numerous specific carbohydrate lectins 
and monoclonal antibodies suitable for this purpose have been reported and 
are available.(12,13) 
Having thus described exemplary embodiments of the present invention, it 
should be noted by those skilled in the art that the within disclosures 
are exemplary only and that various other alternatives, adaptations and 
modifications may be made within the scope of the present invention. 
Accordingly, the present invention is not limited to the specific 
embodiments as illustrated herein, but is only limited by the following 
claims. 
BIBLIOGRAPHY 
(1) Takeuchi, M., et al. (1988) J. Biol. Chem 263, 3657-3663. 
(2) Kagawa, Y., et al. (1988) J. Biol. Chem. 263, 17508-17515. 
(3) Stanley, P. (1987) Meth. Enzymol. 138, 443-470. 
(4) Ellis, L., et al. (1986) Cell 45, 721-732. 
(5) Beyer, T., et al. (1981) Advances in Enzymol. 52, 24-175. 
(6) Weinstein, et al., (1987) J. Biol. Chem. 17735-17743. 
(7) Sanger, F. (1977) Proc. Nat. Acad. Sci. USA 74, 5463-5467. 
(8) Zoller, M.J. and Smith, M. (1984) DNA 3, 479-488. 
(9) Graham, F.L. and Van der Eb, A.J. (1973) Virol. 52, 456-467. 
(10) Shibuya, N. (1987) J. Biol. Chem. 262, 1596-1601. 
(11) Choo, K.H. et al. (1986) DNA 5, 529-537. 
(12) Product catalogs of Sigma Chemical Co. and E.Y. Labs, San Mateo, 
Calif. 
(13) Hakomori, S. (1984) In "Monoclonal antibodies and functional cell 
lines" (R.H. Kennett, K.B. Bechtol and T.J. McKearn) Plenum Pub. Corp. New 
York, pp. 67-100.