Human glycosylation enzymes

Details Human glycosylation enzymes Human glycosylation enzymes

2000-03-01

2001-12-25

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

C435S325000, C435S252300, C435S320100, C435S006120, C435S193000, C435S440000, C536S023100, C536S023200

Reexamination Certificate

active

06333182

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to novel human glycosylation enzymes. More specifically, isolated nucleic acid molecules are provided encoding three enzymes involved in post-translational glycosylation: CMP Sialic Acid Synthetase; Sialic Acid Synthetase and Aldolase, collectively “glycosylation enzymes.” Amino acid sequences comprising the glycosylation enzymes are also provided. The present invention further relates to methods for treating physiologic and pathologic disease conditions, antibodies, and detection methods.
BACKGROUND OF THE INVENTION
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.
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, lepidopteran insect cells are a common host cell used in producing a wide variety of proteins in a baculovirus system. However, such lepidopteran cells do not contain all of the cellular glycosylation machinery present in mammalian cells required to attach certain carbohydrate groups to the proteins it manufactures.
From the above, it is apparent that there is a need to identify human polypeptides which can be used to alter the glycosylation machinery of non-human 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
The present invention includes isolated nucleic acid molecules comprising polynucleotides encoding a glycosylation enzyme polypeptide. The present invention further includes glycosylation enzyme polypeptides encoded by these polynucleotides. The present invention further provides for isolated nucleic acid molecules encoding portions (fragments) and/or variants of full length glycosylation enzyme polypeptides and the polypeptides encoded thereby.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a glycosylation enzyme polypeptide having an amino acid sequence as shown in the sequence listing; (b) a nucleotide sequence encoding a mature glycosylation enzyme polypeptide having the amino acid sequences as shown in the sequence listing and described in Table 1; (c) a nucleotide sequence encoding a biologically active fragment of a glycosylation enzyme polypeptide having an amino acid sequence shown in the sequence listing and described in Table 1; (d) a nucleotide sequence encoding an antigenic fragment of a glycosylation enzyme polypeptide having an amino acid sequence shown in the sequence listing and described in Table 1; (e) a nucleotide sequence encoding a glycosylation enzyme polypeptide comprising the complete amino acid sequence encoded by a human cDNA clone contained in the ATCC Deposit and described in Table 1; (f) a nucleotide sequence encoding a mature glycosylation enzyme polypeptide having an amino acid sequence encoded by a human cDNA clones contained in the ATCC Deposit and described in Table 1; (g) a nucleotide squence encoding a biologically active fragment of a glycosylation enzyme polypeptide having an amino acid sequence encoded by a human cDNA clone contained in the ATCC Deposit and described in Table 1; (h) a nucleotide sequence encoding an antigenic fragment of a glycosylation enzyme polypeptide having an amino acid sequence encoded by a human cDNA clone contained in the ATCC Deposit and described in Table 1; and (i) a nucleotide sequence complementary to any of the nucleotide sequences in (a) through (h), above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise, or alternatively consist of, a polynucleotide having a nucleotide sequence at least 80% identical, and more preferably at least 85%, 90%, 95%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i), above. Polypeptides encoded by these nucleic acids or polynucleotides are also encompassed by the invention. In specific embodiments, polynucleotide which hybridizes to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a glycosylation enzyme polypeptide having an amino acid sequence in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polypeptides encoded by these nucleic acids are also encompassed by the invention.
The present invention also relates to recombinant vectors, which include the nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of glycosylation enzyme polypeptides or peptides by recombinant techniques. Polypeptides produced by such methods are also provided.
In another embodiment, the invention provides isolated polypeptides comprising a polypeptide having an amino acid sequence described in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polypeptide portions (fragments) or variants of such glycosylation enzyme polypeptides are also provided.
In a specific embodiment, the invention relates to a peptide or polypeptide which comprises or alternatively consists of, the amino acid sequence of an epitope-bearing portion of a glycosylation enzyme polypeptide having an amino acid sequence described above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a glycosylation enzyme polypeptide of the invention include portions of such polypeptides. In another embodiment, the invention provides an isolated antibody that specifically binds a glycosylation enzyme polypeptide having an amino acid sequence described above.
For a number of applications the level of glycosylation enzyme gene expression can be detected in a sample of tissue or bodily fluid. The presence of glycosylation enzyme gene expression or an increased or decreased level of glycosylation enzyme gene expression can be measured. Thus, the present invention provides for methods useful for detection of glycosylation enzymes and for the diagnosis of applicable disorders. The diagnosis of disorders involves assaying the expression level of the gene encoding the glycosylation enzyme protein i

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