Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-07-17
2003-05-06
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S183000, C435S069100, C435S252300, C435S320100, C435S325000, C536S023200
Reexamination Certificate
active
06558934
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to the biosynthesis of glycans found as free oligosaccharides or covalently bound to proteins and glycosphingolipids. This invention is more particularly related to a family of nucleic acids encoding UDP-D-galactose: &bgr;-N-acetylglucosamine &bgr;-1,4-galactosyltransferases (&bgr;4Gal-transferases), which add galactose to the hydroxy group at carbon 4 of 2-acetamido-2-deoxy-D-glucose (GlcNAc). This invention is more particularly related to a gene encoding the second member of the family of &bgr;4Gal-transferases, termed &bgr;4Gal-T2, probes to the DNA encoding &bgr;4Gal-T2, DNA constructs comprising DNA encoding &bgr;4Gal-T2, recombinant plasmids and recombinant methods for producing &bgr;4Gal-T2, recombinant methods for stably transfecting cells for expression of &bgr;4Gal-T2, and methods for identification of DNA polymorphism in patients.
BACKGROUND OF THE INVENTION
The UDP-galactose: &bgr;-N-acetyl-glucosamine &bgr;-1,4-galactosyltransferase (&bgr;4Gal-T1) was the first animal glycosyltransferase to be isolated and cloned (Narimatsu et al., 1986; Shaper et al., 1986; Nakazawa et al, 1988; Shaper et al., 1988; D'Agostaro et al., 1989), and early searches for homologous genes by low stringency Southern hybridisation suggested that this gene was unique. Characterisation of &bgr;4Gal-transferase activities from different sources, however, indicate that distinct activities exist (Sheares and Carlson, 1984; Furukawa et al., 1990). Emerging evidence now reveal that several &bgr;4galactosyltransferase genes may exist. Shaper and colleagues (Shaper et al., 1995) have identified two different chick cDNA sequences, which have 65% and 48% sequence similarity to human &bgr;4Gal-T1. Both chick cDNAs were shown to encode catalytically active p4Gal-transferases (Shaper et al., 1997). Two independent groups have analysed &bgr;4Gal-transferase activities in mice homozygously deficient for &bgr;4Gal-T1 (Asano et al., 1997; Lu et al., 1997). Both studies showed residual &bgr;4Gal-transferase activity, providing clear evidence for the existence of additional &bgr;4Gal-transferases. Thus, the &bgr;4Gal-T1 gene is likely to be part of a homologous gene family with recognisable sequence motifs, and this is supported by a large number of human ESTs with sequence similarities to &bgr;4Gal-T1 in EST databases (National Center for Biotechnology Information).
&bgr;-1,4-Galactosyltransferase activities add galactose to different acceptor substrates including free oligosaccharides, N- and O-linked glycoproteins, and glycosphingolipids (Kobata, 1992). In addition, &bgr;4Gal-T1 is modulated by &agr;-lactalbumin to function as lactose synthase and hence has a major role in lactation (Brew et al., 1968). Given the diverse functions of &bgr;-1,4-galactosyltransferase activities and the evidence that multiple &bgr;4Gal-transferases exist, it is likely that these enzymes may have different kinetic properties. Furukawa et al (Furukawa et al., 1990) showed that liver &bgr;4Gal-transferase activity was near 20-fold higher with asialo-agalacto-transferrin compared to asialo-agalacto-IgG, whereas the activity found in T and B cells only showed a 4 to 5-fold difference with the two substrates. The &bgr;4Gal-transferase activity in B cells of rheumatoid arthritis patients appear to be similar to B cells from healthy controls with several substrates including asialo-agalacto-transferrin (Furukawa et al., 1990) and &bgr;GlcNAc-pITC-BSA (Keusch et al., 1995), but different with asialo-agalacto-IgG (Furukawa et al., 1990). Furthermore, the Km for UDP-Gal of &bgr;4Gal-transferase activity from B cells of rheumatoid arthritis patients were 2-fold higher (35.6 mM) than normal B cells (17.6 mM) (Furukawa et al., 1990). Finally, the activity in B cells for asialo-agalacto-transferrin was more sensitive to &agr;-lactalbumin inhibition than the activity with asialo-agalacto-IgG. A number of studies have concluded that there was no change in &bgr;4Gal-transferase activity in B cells of rheumatoid arthritis patients (Wilson et al., 1993; Axford et al., 1994). However, if multiple &bgr;4Gal-transferases exist, it is possible that the contradictory findings of Furukawa et al. (Furukawa et al., 1990) can be explained by a model with two &bgr;4Gal-transferases with different kinetic parameters expressed in normal B cells, and a selective down regulation of one in B cells of rheumatoid arthritis patients.
Access to additional existing &bgr;4Gal-transferase genes encoding &bgr;4Gal-transferases with better kinetic properties than &bgr;4Gal-T1 would allow production of more efficient enzymes for use in galactosylation of oligosaccharides, glycoproteins, and glycosphingolipids. Such enzymes could be used, for example, in pharmaceutical or other commercial applications that require synthetic galactosylation of these or other substrates that are not or poorly acted upon by &bgr;4Gal-T1, in order to produce appropriately glycosylated glycoconjugates having particular enzymatic, immunogenic, or other biological and/or physical properties.
Consequently, there exists a need in the art for additional UDP-galactose: &bgr;-N-acetyl-glucosamine &bgr;-1,4-galactosyltransferases and the primary structure of the genes encoding these enzymes. The present invention meets this need, and further presents other related advantages.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acids encoding human UDP-galactose: &bgr;-N-ace-tylglucosamine &bgr;1,4-galactosyltransferase (&bgr;4Gal-T2), including cDNA and genomic DNA. &bgr;4Gal-T2 has better kinetic parameters than &bgr;4Gal-T1, as exemplified by its lower Km for UDP-Gal and its better activity with saccharide derivatives, glycoprotein substrates, and &bgr;GlcNAc-glycopeptides. The complete nucleotide sequence of &bgr;4Gal-T2, SEQ ID NO:1, is set forth in FIG.
2
.
In one aspect, the invention encompasses isolated nucleic acids comprising the nucleotide sequence of nucleotides 1-1116 as set forth in SEQ ID NO:1 or sequence-conservative or function-conservative variants thereof Also provided are isolated nucleic acids hybridizable with nucleic acids having the sequence of SEQ ID NO:1 or fragments thereof or sequence-conservative or function-conservative variants thereof; preferably, the nucleic acids are hybridizable with &bgr;4Gal-T2 sequences under conditions of intermediate stringency, and, most preferably, under conditions of high stringency. In one embodiment, the DNA sequence encodes the amino acid sequence, SEQ ID NO:2, also shown in
FIG. 2
, from methionine (amino acid no. 1) to glycine (amino acid no. 372). In another embodiment, the DNA sequence encodes an amino acid sequence comprising a sequence from tyrosine (no. 31) to glycine (no. 372) of SEQ ID NO:3.
In a related aspect, the invention provides nucleic acid vectors comprising &bgr;4Gal-T2 DNA sequences, including but not limited to those vectors in which the &bgr;4Gal-T2 DNA sequence is operably linked to a transcriptional regulatory element, with or without a polyadenylation sequence. Cells comprising these vectors are also provided, including without limitation transiently and stably expressing cells. Viruses, including bacteriophages, comprising &bgr;4Gal-T2-derived DNA sequences are also provided. The invention also encompasses methods for producing &bgr;4Gal-T2 polypeptides. Cell-based methods include without limitation those comprising: introducing into a host cell an isolated DNA molecule encoding &bgr;4Gal-T2, or a DNA construct comprising a DNA sequence encoding &bgr;4Gal-T2; growing the host cell under conditions suitable for &bgr;4Gal-T2 expression; and isolating &bgr;4Gal-T2 produced by the host cell. A method for generating a host cell with de novo stable expression of &bgr;4Gal-T2 comprises: introducing into a host cell an isolated DNA molecule encoding &bgr;4Gal-T2 or an enzymatically active fragment thereof (such as, for example, a polypeptide comprising amino acids 31-372 of SEQ ID NO:2), or a DNA construct comprising a DNA sequence encodi
Bennett Eric Paul
Clausen Henrik
Darby & Darby
Glycozm APS
Prouty Rebecca E.
Rao Manjunath N.
LandOfFree
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