Compositions and methods for the identification and synthesis of sialyltransferases

#This invention relates to the sialyltransferase gene family, a group of glycosyltransferases responsible for the terminal sialylation of carbohydrate groups of glycoproteins, glycolypids and oligosaccharides which contain a conserved region of homology in the catalytic domain. Members of the sialyltransferase gene family comprise Gal.beta.1,3GalNAc .alpha.2,3 sialyltransferase and Gal1,3(4)GlcNAc .alpha.2,3 sialyltransferase. The invention further relates to novel forms and compositions thereof and particularly to the means and methods for the identification and production of members of the sialyltransferase gene family to homogeneity in significant useful quantities. This invention also relates to preparation of isolated deoxyribonucleic acid (DNA) coding for the production of sialyltransferases; to methods of obtaining DNA molecules which code for sialyltransferases; to the expression of human and mammalian sialyltransferases utilizing such DNA, as well as to novel compounds, including novel nucleic acids encoding sialyltransferases or fragments thereof. This invention is also directed to sialyltransferase derivatives, particularly derivatives lacking cytoplasmic and/or transmembrane portions of the protein, and their production by recombinant DNA techniques.

#Sialyltransferases are a family of enzymes that catalyze the transfer of sialic acid (SA) to terminal portions on the carbohydrate groups of glycolipids and oligosaccharides in the general reaction:

#Cytididine 5 monophosphate-sialic acid (CMP-SA)+HO-acceptor.fwdarw.CMP+SA-O-Acceptor (Beyer, T. A. et. Adv. Enzynol. 52, 23-175 ›1981!). Sialyltransferases are found primarily in the Golgi apparatus of cells where they participate in post-translational glycosylation pathways. (Fleischer, B. J. Cell Biol. 89, 246-255 ›1981!). They are also found in body fluids, such as breast milk, colostrum and blood. At least 10-12 different sialyltransferases are required to synthesize all the sialyloligossacharide sequences known. Four sialyltransferases have been purified. (Weinstein, J. et al., J. Biol. Chem. 257, 13835-13844 ›1982!; Miagi, T and Tsuiki, S. Eur. J. Biochem. 125, 253-261 ›1982!; and Joziasse, D. H. et al., J. Biol. Chem. 260, 4941-4951 ›1985!). More specifically, a Gal.beta.1,4GlcNAc .alpha.2-6 sialyltransferase and a Gal.beta.1,3(4)GlcNAc .alpha.2-3 sialyltransferase have been purified from rat liver membranes. (Weinstein et al. Ibid)

#FIG. 1 Nucleotide and amino acid sequence of porcine Gal.beta.1,3GalNAc .alpha.2,3 sialyltransferase (".alpha.2,3-0"). The nucleotide sequence of the porcine .alpha.2,3-0 mRNA was determined from DNA sequence analysis of two overlapping clones, .lambda.ST1 and .lambda.ST2. Predicted amino acids of the .alpha.2,3-0 polypeptide are shown above the DNA sequence and are numbered from the first residue of the N-terminal of the analagous purified protein. The proposed signal-anchor sequence is indicated (light box). Potential glycosylation sites with the sequence Asn-X-Thr are marked with an asterisk (*). The sequence corresponds to the long form of the .alpha.2,3 sialyltransferase, encoded by the overlapping clones .lambda.ST1 and .lambda.ST2. See Sequence ID Nos. 1 and 2.

#FIG. 2 Nucleotide and amino acid sequence of rat Gal.beta.1,3(4)GlcNAc .alpha.2,3-sialyltransferase (".alpha.2,3-N"). The nucleotide sequence of the rat .alpha.2,3-N mRNA was determined from DNA sequence analysis. Predicted amino acids of the sialyltransferase polypeptide are shown above the DNA sequence and are numbered from the first residue of the mature protein as determined by N-terminal protein sequencing. See Sequence ID Nos.3 and 4.

#FIG. 3 Purification of the .alpha.2,3-0 sialyltransferase on CDP-hexanolamine agarose (KCl elution). Homogenate from 2 kg porcine liver were loaded onto a CDP-hexanolamine agarose column and eluted with a linear gradient of KCl (see Experimental Procedures). Protein concentration and sialyltransferase activity using lactose as an acceptor substrate were determined for individual fractions. The two peaks of enzyme activity were separated into pools A and B, as indicated.

#As used herein, sialyltransferase or sialyltransferase derivatives refer to sialyltransferase enzymes other than rat Gal.beta.1,4GlcNAc .alpha.2,6 sialyltransferase which contain a conserved region of homology in the catalytic domain and are enzymatically active in transferring sialic acid to a terminal position on sugar chains of glycoproteins, glycolipids, oligosaccharides and the like. Examples of enzymatically functional sialyltransferases are those capable of transferring sialic acid from CMP-sialic acid to an acceptor oligosaccharide, where the oligosaccharide acceptor varies depending upon the particular sialyltransferase.

#"Conserved region of homology" refers to a series of amino acids in one sialyltransferase which is essentially identical to the same series of amino acids in another sialyltransferase enzyme once the sequences of the two enzymes have been aligned. In the sialyltransferase gene family of this invention the conserved region of homology is in the catalytic domain and extends over at least about 7 contiguous amino acids, preferably at least about 20 amino acids and most preferably over at least about 55 amino acids having the amino acid sequence of residues 156-210 of FIG. 2 or residues 142-196 of FIG. 1. Once having identified the conserved region of homology, amino acid sequence variants of the conserved region may be made and fall into one or more of three classes: substitutional, insertional, or deletional variants. These variants ordinarily are prepared by site-specific mutagenesis nucleotides in the DNA encoding the sialyltransferase, thereby producing DNA encoding sialyltransferase comprising a conserved region of homology variant.

#The sialyltransferases in accordance with the present invention include: rat Gal.beta.1,3(4)GlcNAc .alpha.2,3 sialyltransferase (herein referred to as ".alpha.2,3-N" and identified as Seq. ID No.3) which forms the NeuNAc .alpha.2,3Gal.beta.1,3GlcNAc and NeuAc.alpha.2,3Gal.beta.1,4GlcNAc sequences which often terminate complex N-linked oligosaccharides; porcine Gal.beta.1,3GalNAc .alpha.2,3 sialyltransferase (herein referred to as ".alpha.2,3-0" and identified as Seq. ID No.1) which forms NeuAc.alpha.2,3Gal.beta.1,3GalNAc found on sugar chains O-linked to threonine or serine as well as a terminal sequence on certain gangliosides; human Ga1.beta.1,3(4)G1cNAc.alpha.2,3 sialyltransferase (Seq. ID No.10); and the sialyltransferases identified as ST3 and set forth in Seq. ID No.12.