UDP-N-ACETYLGLUCOSAMINE: GALACTOSE-β 1,3-N-ACETYLGALACTOSAMINE-α-R / N-ACETYLGLUCOSAMINE-β 1,3,-N-ACETYLGALACTOSAMINE-α-R (GLCNAC TO GALNAC) β1,6-N-ACETYLGLUCOSAMINYLTRANSFERASE, C2/4GNT
A novel gene defining a novel human UDP-GlcNAc: Gal/Gl cNAcβ 1-3GalNAc αβ1, 6GlcNAc-transferase, termed C2/4GnT, with unique enzymatic properties is disclosed. The enzymatic activity of C2/4GnT is shown to be distinct from that of previously identified enzymes of this gene family. The invention discloses isolated DNA molecules and DNA constructs encoding C2/4GnT and derivatives thereof by way of amino acid deletion, substitution or insertion exhibiting C2/4GnT activity, as well as cloning and expression vectors including such DNA, cells transfected with the vectors, and recombinant methods for providing C2/4GnT. The enzyme C2/4GnT and C2/4GnT-active derivatives thereof are disclosed, in particular soluble derivatives comprising the catalytically active domain of C2/4GnT. Further, the invention discloses methods of obtaining 1,6-N-acetyl glucosaminyl glycosylated saccharides, glycopeptides or glycoproteins by use of an enzymically active C2/4GnT protein or fusion protein thereof or by using cells stably transfected with a vector including DNA encoding an enzymatically active C2/4GnT protein as an expression system for recombinant production of such glycopeptides or glycoproteins. Also a method for the identification for the identification of DNA sequence variations in the C2/4GnT gene by isolating DNA from a patient, amplifying C2/4GnT-coding exons by PCR, and detecting the presence of DNA sequence variation are disclosed.
1. An isolated nucleic acid encoding UDP-N-acetylglucosamine: galactose-β1,3-N-acetylgalactosamine-α-R/N-acetylglucosainine-β1,3-N-acetylgalactosamine-α-R β1,6-N-acetylglucosaminyltransferase (C2/4GnT) having the amino acid sequence SEQ ID NO: 2 or an enzymatically active fragment thereof.
2. An isolated nucleic acid as defined in claim 1 , wherein said nucleic acid is DNA.
3. An isolated nucleic acid as defined in claim 2 , wherein said DNA is cDNA.
4. An isolated nucleic acid as defined in claim 2 , wherein said DNA is genomic DNA.
5. An isolated nucleic acid encoding UDP-N-acetylglucosamine: galactose-β1,3-N-acetylgalactosamine-α-R/N-acetylglucosamine-β1,3-N-acetylgalactosamine-α-R β1,6-N-acetylglucosaminyl-transferase (C2/4GnT), wherein said nucleic acid comprises the sequence of nucleotides 1-2319 in SEQ ID NO:1 or sequence-conservative variants thereof.
6. A nucleic acid vector comprising the nucleic acid of claim 1 .
7. A vector as defined in claim 6 , wherein said nucleic acid comprises the nucleotide sequence of nucleotides 1-2319 in SEQ ID NO: 1 or sequence-conservative variants thereof.
8. A vector as defined in claim 7 , wherein said sequence encoding C2/4GnT is operably linked to a transcriptional regulatory element.
9. A cell comprising a vector as defined in claim 6 .
10. A cell comprising a vector as defined in claim 8 .
11. A cell as defined in claim 10 , wherein said cell is stably transfected with said vector.
12. A cell as defined in claim 9 , wherein said cell produces enzymatically active C2/4GnT.
13. A cell as defined in claim 9 , wherein said cell is selected from the group consisting of bacterial, yeast, insect, avian, and mammalian cells.
14. A cell as defined in claim 12 , wherein said cell is selected from the group consisting of bacterial, yeast, insect, avian, and mammalian cells.
15. A cell as defined in claim 14 , wherein said cell is Sf9.
16. A cell as defined in claim 14 , wherein said cell is CHO.
17. A method for the identification of DNA sequence variations in a region of the C2/4GnT gene, comprising the steps of:
(i) isolating DNA from a patient;
(ii) amplifying by PCR a segment of the patient's DNA using amplification primers capable of amplifying a subsequence of SEQ ID NO: 1, said sequence consisting of a fragment of a nucleotide sequence selected from the group consisting of nucleotides 1-245 , nucleotides 246-435 , and nucleotides 436-2319 SEQ ID NO: 1; and
(iii) detecting the presence of DNA sequence variation in the amplified segment compared to said subsequence of SEQ ID NO: 1 by DNA sequencing or single strand conformational polymorphism (SSCP).
18. An isolated nucleic acid as defined in claim 1 , wherein said nucleic acid comprises the nucleotide sequence of nucleotides 496-1812 in SEQ ID NO:1 or sequence-conservative variants thereof.
19. An isolated nucleic acid as defined in claim 1 , wherein said nucleic acid comprises the nucleotide sequence of nucleotides 634-1812 in SEQ ID NO:1 or sequence-conservative variants thereof.
20. A method for screening for DNA sequence variations in a region of the human C2/4GnT gene comprising the steps of:
(i) amplifying a segment of genomic DNA obtained from a human subject, using amplification primers capable of amplifying a subsequence of SEQ ID NO: 1, said subsequence consisting of a fragment of a nucleotide sequence selected from the group consisting of nucleotides 1-245, nucleotides 246-435, and nucleotides 436-2319 of SEQ ID NO: 1; and
(ii) comparing the sequence of the amplified segment with said subsequence of SEQ ID NO: 1 and identifying the differences between the sequence of said segment and said subsequence of SEQ ID NO:1.
21. A nucleic acid vector comprising the nucleic acid of claim 18 .
22. A nucleic acid vector comprising the nucleic acid of claim 19 .
23. A cell comprising a vector as defined in claim 21 .
24. A cell comprising a vector as defined in claim 22 .