# Patent

## Title
PmST2 enzyme for chemoenzymatic synthesis of α-2-3-sialylglycolipids

## Abstract

The present invention provides novel methods for preparing glycolipid products. Novel sialyltransferases are also disclosed.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 61/585,376, filed Jan. 11, 2012, which is incorporated in its entirety herein for all purposes.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with Government support under Grant Nos. R01GM076360 and R01HD065122, awarded by the National Institutes of Health. The Government has certain rights in this invention.


REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
The Sequence Listing written in file -2108-1.TXT, created on Mar. 26, 2013, 28,672 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.
BACKGROUND OF THE INVENTION
Glycosyltransferase-catalyzed reactions have gained increasing attention and application for the synthesis of complex carbohydrates and glycoconjugates. Sialyltransferases, in particular, are the key enzymes that catalyze the transfer of a sialic acid residue from cytidine 5′-monophosphate-sialic acid (CMP-sialic acid) to an acceptor. Resulting sialic acid-containing products have been implicated in various biological and pathological processes, including cell-cell recognition, cell growth and differentiation, cancer metastasis, immunological regulation, as well as bacterial and viral infection. Besides being prevalent in mammals, sialyltransferases have been found in some pathogenic bacteria. They are mainly involved in the formation of sialic acid-containing capsular polysaccharides (CPS) and lipooligo(poly)saccharides (LOS/LPS), serving as virulence factors, preventing recognition by host's immune system, and modulating interactions with the environment. Sialyltransferases have been used for the synthesis of sialic acid-containing molecules with or without CMP-sialic acid biosynthetic enzymes.
Cloning of sialyltransferases from various sources, including mammalian tissues, bacteria, and viruses has been reported. Bacterial sialyltransferases have been cloned from several Gram-negative bacteria belonging to Escherichia, Campylobacter, Neisseria, Photobacterium, Haemophilus, and Pasteurella genera. The genera Pasteurellaand Haemophilus, both belong to the Haemophilus-Actinobacillus-Pasteurella (HAP) group, generally produced negatively charged outer cell surface and contain multiple genes encoding functional sialyltransferases. Two functional α2,3-sialyltransferases encoded by 1st and Hd0053 have been identified from Haemophilus ducreyi. Lic3A, SiaA, LsgB, and Lic3B are four sialyltransferases involved in the complex process of lipopolysaccharide sialylation in Haemophilus influenzae. 
Most mammalian glycosyltransferases—including sialyltransferases—suffer from no or low expression in E. coli systems and more restricted substrate specificity. In comparison, bacterial glycosyltransferases are generally easier to access using E. coli expression systems and have more promiscuous substrate flexibility. Although certain wild-type bacterial glycosyltransferases with promiscuities for both donor and acceptor substrates have been discovered, readily obtainable enzymes with a wider substrate tolerance are needed to further the application of glycosyltransferases. The present invention meets this and other needs, providing surprisingly useful sialyltransferases for synthesis of glycoconjugates.
BRIEF SUMMARY OF THE INVENTION
In a first aspect, the invention provides a method of preparing a glycolipid product. The method includes forming a reaction mixture containing an acceptor glycolipid, a donor substrate having a sugar moiety and a nucleotide, and a sialyltransferase selected from PmST2 (SEQ ID NO: 4) and certain variants thereof. In some embodiments, the donor substrate is formed via conversion of a suitable hexosamine derivative to a cytidine 5′-monophosphate (CMP)-sialic acid in a one-pot reaction mixture containing a sialic acid aldolase and a CMP-sialic acid synthetase.
In a second aspect, the invention provides an isolated or purified polynucleotide comprising a nucleotide sequence that is substantially identical to SEQ ID NO: 1 (PmST2) or certain variants thereof.
In a third aspect, the invention provides an isolated or purified polypeptide comprising an amino acid sequence selected from SEQ ID NO:4 (PmST2) and certain variants thereof.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the amino acid sequence alignment of PmST2 (a Pm0508protein homolog) (SEQ ID NO:4), HiLsgB from Haemophilus influenzae 86-028NP (GeneBank accession no. AAX88755) (SEQ ID NO:14), and NmLst from Neisseria meningitidis MC58(GeneBank accession no. AAC44541) (SEQ ID NO:15). Black boxes indicate identical or similar amino acid residues shared by all three sequences, while grey boxes indicate identical or similar amino acid residues shared by two of three sequences. In PmST2, the amino acid residue differing from the protein encoded by reported Pm0508 gene is marked with an asterisk (*) above the residue.
FIG. 2 shows the SDS-PAGE (12% Tris-Glycine gel) analysis of MBP-PmST2-His6 expression and purification. Lanes: PS, protein standards (Precision Plus Protein Standards, Bio-Rad); 1, whole cell extraction, before IPTG induction; 2, whole cell extraction, after induction; 3, lysate after induction; and 4, Ni2+-NTA column purified protein.
FIG. 3 shows the structures of LacβPro2AA and LacβPro-triazole-C14 used as acceptors for PmST2.
FIG. 4 shows the time course analysis of the α-2,3-sialyltransferase activity of PmST1(open circle, dashed line) and MBP-PmST2-His6(filled diamond, solid line) using LacβPro2AA (A) or LacβPro-triazole-C14(B; 0.3% Triton X-100 was added) as the sialyltransferase acceptor.
FIG. 5 shows the pH profile of MBP-PmST2-His6-catalyzed α2-3-sialyltransferase reaction when LacβPro2AA (dashed line with open circles) or LacβPro-triazole-C14 (solid line with filled diamonds, 0.3% Triton X-100 was added) was used as the acceptor substrate. Buffers (200 mM) used: sodium acetate (pH 4.0-6.0), Tris-HCl (pH 7.0-9.0), and CHES (pH 10.0).
FIG. 6 shows the effects of divalent metal concentrations, EDTA, and 2-mercaptoethanol (2-ME) on the α2,3-sialyltransferase activity of MBP-PmST2-His6 using LacβPro-triazole-C14 as the acceptor substrate in the presence of 0.3% of Triton X-100.
FIG. 7 shows a schematic diagram for the one-pot, two-enzyme synthesis of lyso-GM3 from lactosyl sphingosine and CTP using a recombinant N. meningitidis CMP-sialic acid synthetase (NmCSS) and PmST2 in the presence of Mg2+.
FIG. 8 shows schematic diagrams for the chemical synthesis of various PmST2 acceptor substrates.



DETAILED DESCRIPTION OF THE INVENTION
I. General
The present invention provides α2,3-sialyltransferases useful for the preparation of glycosylated molecules. In particular, the second sialyltransferase from Pasteurella multocida strain P-1059 (PmST2; encoded by gene Pm0508) is a sialidase-free monofunctional α2,3-sialyltransferase. Certain variants of soluble, active PmST2 can be obtained in high yield, making this enzyme desirable for large-scale synthesis of glycosylated products. The surprising monofunctionality of PmST2 is particularly advantageous, allowing for the preparation of a variety of sialic acid containing glycolipids.
II. Definitions
“Glycosyltransferase” refers to a polypeptide that catalyzes the formation of a glycoside or an oligosaccharide from a donor substrate and an acceptor or acceptor sugar. In general, a glycosyltransferase catalyzes the transfer of the monosaccharide moiety of the donor substrate to a hydroxyl group of the acceptor. The covalent linkage between the monosaccharide and the acceptor sugar can be a 1-4 linkage, a 1-3 linkage, a 1-6-linkage, a 1-2 linkage, a 2-3-linkage, a 2-4-linkage, a 2-6-linkage, a 2-8-linkage, or a 2-9-linkage. The linkage may be in the α- or β-configuration with respect to the anomeric carbon of the monosaccharide. Other types of linkages may be formed by the glycosyltransferases in the methods of the invention. Glycosyltransferases include, but are not limited to, sialyltransferases, heparosan synthases (HSs), glucosaminyltransferases, N-acetylglucosaminyltransferases, glucosyltransferases, glucuronyltransferases, N-acetylgalactosaminyltransferases, galactosyltransferases, galacturonyltransferases, fucosyltransferases, mannosyltransferases, xylosyltransferases. Sialyltransferases are enzymes that catalyze the transfer of sialic acid, or analogs thereof, to a monosaccharide, an oligosaccharide, or a glycoconjugate. In some embodiments, the glycosyltransferases useful in the present invention include those in Glycosyltransferase family 52 (GT52 using Carbohydrate-Active enZYme database (CAZy) nomenclature), and includes beta-galactoside alpha-2,3-sialyltransferases that catalyze the following conversion: CMP-sialic acid+β-D-galactosyl-R=CMP+α-sialic acid-(2→3)-β-D-galactosyl-R, where the acceptor is GalβOR, where R is H, a monosaccharide, an oligosaccharide, a polysaccharide, a glycopeptide, a glycoprotein, a glycolipid, or a hydroxyl-containing compound. GT80 family sialyltransferases also include galactoside or N-acetylgalactosaminide alpha-2,6-sialyltransferases that catalyze the following conversion: CMP-sialic acid+galactosyl/GalNAcOR→CMP+α-sialic acid-(2→6)-D-galactosyl/GalNAcOR, where the acceptor is GalOR or GalNAcOR, where R is H, serine or threonine on a peptide or protein, a monosaccharide, an oligosaccharide, a polysaccharide, a glycopeptide, a glycoprotein, a glycolipid, or a hydroxyl-containing compound.
“Sialidase” refers to an enzyme that catalyzes the hydrolysis of glycosidic linkages of terminal sialic acids on glycosylated molecules.
“Donor substrate hydrolysis” refers to hydrolysis of O-glycosidic bond of the sugar and the phosphate in the nucleotide-sugar donor substrate.
“Amino acid” refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. As used herein, the term “amino acid” includes the following twenty natural or genetically encoded alpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (H is or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). In cases where “X” residues are undefined, these should be defined as “any amino acid.” The structures of these twenty natural amino acids are shown in, e.g., Stryer et al., Biochemistry, 5th ed., Freeman and Company (2002), which is incorporated by reference. Additional amino acids, such as selenocysteine and pyrrolysine, can also be genetically coded for (Stadtman (1996) “Selenocysteine,” Annu Rev Biochem. 65:83-100 and Ibba et al. (2002) “Genetic code: introducing pyrrolysine,” Curr Biol. 12(13):R464-R466, which are both incorporated by reference). The term “amino acid” also includes unnatural amino acids, modified amino acids (e.g., having modified side chains and/or backbones), and amino acid analogs.
“Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three teams apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-natural amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
“Mutant,” in the context of glycosyltransferases of the present invention, means a polypeptide, typically recombinant, that comprises one or more amino acid substitutions relative to a corresponding, naturally-occurring or unmodified glycosyltransferase, such as an alpha2-3 sialyltransferase.
A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
“Percent sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
“Identical” or “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Sequences are “substantially identical” to each other if they have a specified percentage of nucleotides or amino acid residues that are the same (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. These definitions also refer to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more typically over a region that is 100 to 500 or 1000 or more nucleotides in length.
“Similarity” or “percent similarity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of amino acid residues that are either the same or similar as defined by a conservative amino acid substitutions (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Sequences are “substantially similar” to each other if they are at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% similar to each other. Optionally, this similarly exists over a region that is at least about 50 amino acids in length, or more typically over a region that is at least about 100 to 500 or 1000 or more amino acids in length.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are commonly used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities or similarities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).
Algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977), and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001.
“Recombinant,” as used herein, refers to an amino acid sequence or a nucleotide sequence that has been intentionally modified by recombinant methods. By the term “recombinant nucleic acid” herein is meant a nucleic acid, originally formed in vitro, in general, by the manipulation of a nucleic acid by endonucleases, in a form not normally found in nature. Thus an isolated, mutant glycosyltransferase nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention. A “recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.
“Vector” refers to a piece of DNA, typically double-stranded, which may have inserted into it a piece of foreign DNA. The vector may be, for example, of plasmid origin. Vectors contain “replicon” polynucleotide sequences that facilitate the autonomous replication of the vector in a host cell. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell, which, for example, replicates the vector molecule, encodes a selectable or screenable marker, or encodes a transgene. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of or coincidental with the host chromosomal DNA, and several copies of the vector and its inserted DNA can be generated. In addition, the vector can also contain the necessary elements that permit transcription of the inserted DNA into an mRNA molecule or otherwise cause replication of the inserted DNA into multiple copies of RNA. Some expression vectors additionally contain sequence elements adjacent to the inserted DNA that increase the half-life of the expressed mRNA and/or allow translation of the mRNA into a protein molecule. Many molecules of mRNA and polypeptide encoded by the inserted DNA can thus be rapidly synthesized.
“Nucleotide,” in addition to referring to the naturally occurring ribonucleotide or deoxyribonucleotide monomers, shall herein be understood to refer to related structural variants thereof, including derivatives and analogs, that are functionally equivalent with respect to the particular context in which the nucleotide is being used (e.g., hybridization to a complementary base), unless the context clearly indicates otherwise.
“Nucleic acid” or “polynucleotide” refers to a polymer that can be corresponded to a ribose nucleic acid (RNA) or deoxyribose nucleic acid (DNA) polymer, or an analog thereof. This includes polymers of nucleotides such as RNA and DNA, as well as synthetic forms, modified (e.g., chemically or biochemically modified) forms thereof, and mixed polymers (e.g., including both RNA and DNA subunits). Exemplary modifications include methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, and the like), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids and the like). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Typically, the nucleotide monomers are linked via phosphodiester bonds, although synthetic forms of nucleic acids can comprise other linkages (e.g., peptide nucleic acids as described in Nielsen et al. (Science 254:1497-1500, 1991). A nucleic acid can be or can include, e.g., a chromosome or chromosomal segment, a vector (e.g., an expression vector), an expression cassette, a naked DNA or RNA polymer, the product of a polymerase chain reaction (PCR), an oligonucleotide, a probe, and a primer. A nucleic acid can be, e.g., single-stranded, double-stranded, or triple-stranded and is not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence comprises or encodes complementary sequences, in addition to any sequence explicitly indicated.
“Forming a reaction mixture” refers to the process of bringing into contact at least two distinct species such that they mix together and can react, either modifying one of the initial reactants or forming a third, distinct, species, a product. It should be appreciated, however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.
“Donor substrate” refers to a compound having a nucleotide and a sugar moiety that is added to an acceptor, where the sugar moiety and nucleotide are covalently bound together. In general, the sugar moiety is characterized by monosaccharide core having a linear formula of H(CHOH)n(CO)(CHOH)mH, wherein the sum of n and m is at least 2. In certain embodiments, the sum of n and m is 5. In certain embodiments, n is 5 and m is 0. Any H or OH group in the monosaccharide core can be replaced by an amine group NHR′, wherein R′ is selected from H, alkyl, and acyl. One of skill in the art will appreciate that the monosaccharide core can be in the linear form or in the cyclic, hemiacetal form. The hemiacetal can be a pyranose (i.e., a six-membered ring) or a furanose (i.e., a five-membered ring). In general, the hydroxyl group at the anomeric carbon of the hemiacetal is the point of connection between the sugar moiety and the nucleotide in the donor substrate. The monosaccharide core of the sugar moiety can be substituted with various functional groups as described herein. In certain embodiments, the monosaccharide core of the sugar moiety is substituted with pyruvate. In certain embodiments, the sugar moiety is sialic acid or analog thereof. The nucleotide in the donor substrate can be any suitable nucleotide, such as cytidine monophosphate (CMP).
“Acceptor glycolipid” refers to a lipid containing a sugar that accepts the sugar moiety from the donor substrate. The sugar of the glycolipid can be a monosaccharide or an oligosaccharide as defined herein. In certain embodiments, the acceptor glycolipid contains a galactoside moiety, wherein the hydroxyl group at the anomeric carbon of the galactopyranose ring is the point of connection to the remainder of the glycolipid. In some embodiments, the galactoside moiety is a β1-4 linked galactoside moiety or a β1-3 linked galactoside moiety. In certain embodiments, the acceptor glycolipid contains a lactoside moiety having a 4-O-β-D-galactopyranosyl-D-glucopyranose disaccharide unit. The hydroxyl group at the anomic carbon of the glucopyranose ring is the point of connection between the lactoside moiety and the remainder of the glycolipid. In some embodiments, the acceptor glycolipid comprises an N-acetyl lactoside moiety, a Galβ1-3GlcNAc moiety, or a Galβ1-3GalNAc moiety.
“Oligosaccharide” refers to a compound containing at least two sugars covalently linked together. Oligosaccharides include disaccharides, trisaccharides, tetrasachharides, pentasaccharides, hexasaccharides, heptasaccharides, octasaccharides, and the like. Covalent linkages for linking sugars generally consist of glycosidic linkages (i.e., C—O—C bonds) formed from the hydroxyl groups of adjacent sugars. Linkages can occur between the 1-carbon (the anomeric carbon) and the 4-carbon of adjacent sugars (i.e., a 1-4 linkage), the 1-carbon (the anomeric carbon) and the 3-carbon of adjacent sugars (i.e., a 1-3 linkage), the 1-carbon (the anomeric carbon) and the 6-carbon of adjacent sugars (i.e., a 1-6 linkage), or the 1-carbon (the anomeric carbon) and the 2-carbon of adjacent sugars (i.e., a 1-2 linkage). A sugar can be linked within an oligosaccharide such that the anomeric carbon is in the α- or β-configuration. The oligosaccharides prepared according to the methods of the invention can also include linkages between carbon atoms other than the 1-, 2-, 3-, 4-, and 6-carbons.
“Glycolipid” refers to a lipid containing a sugar moiety or an oligosaccharide moiety. Examples of glycolipids include, but are not limited to, glycoglycerolipids, glycosphingolipids, glycosyl polyisoprenol pyrophosphates, and glycosylphosphatidylinositols. A “glycolipid product” is a glycolipid formed by an enzymatic reaction, such as a PmST-catalyzed reaction.
“CMP-sialic acid synthetase” refers to a polypeptide that catalyzes the synthesis of cytidine monophosphate sialic acid (CMP-sialic acid) from cytidine triphosphate (CTP) and sialic acid.
“Sialic acid aldolase” refers to an aldolase that catalyzes a reversible reaction that converts a suitable hexosamine, hexose, pentose, or derivative (such as N-acetyl mannosamine) to sialic acid via reaction with pyruvate.
III. Sialyltransferases
Sialyltransferases are one class of glycosyltransferases, enzymes that catalyze the transfer of a sugar from a nucleotide-sugar (donor substrate) to an acceptor (e.g., a natural product, a monosaccharide, an oligosaccharide, a glycolipid, a glycoprotein, or a hydroxyl-containing compounds). Specifically, sialyltransferases catalyze the transfer of sialic acid, or analogs thereof, from a sialic acid-nucleotide donor substrate to the terminal sugar of an acceptor substrate. Representative sialyltransferases include, but are not limited to, sialyltransferases in family EC 2.4.99, such as beta-galactosaminide alpha-2,6-sialyltransferase (EC 2.4.99.1), alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase (EC 2.4.99.3), beta-galactoside alpha-2,3-sialyltransferase (EC 2.4.99.4), N-acetyllactosaminide alpha-2,3-sialyltransferase (EC 2.4.99.6), alpha-sialyl alpha-2,8-sialyltransferase (EC 2.4.99.8), and lactosylceramide alpha-2,3-sialyltransferase (EC 2.4.99.9). The sialyltransferases of the present invention also include those of the CAZy GT52 family, or EC 2.4.99.4 and EC 2.4.99.1, made up of alpha2-3 and alpha2-6 sialyltransferases, as well as sialyltransferases in the GT4, GT29, GT30, GT38, GT42, GT73, and GT80 families. Representative GT52 sialyltransferases include, but are not limited to, PmST2, Salmonella enterica WaaH, Neisseria meningitidis Lst, Neisseria gonorrhoeae Lst, and NST. (See Glycobiology 2011, 21(6), 716; J. Mol. Biol. 2003, 328, 307; Annu. Rev. Biochem. 2008, 77, 521; Appl. Microbiol. Biotechnol. 2012, 94, 887 for review of sialyltransferases.) PmST2 is a preferred sialyltransferase in some embodiments of the invention.
In general, the sialyltransferases of the present invention are α-2,3-sialyltransferases. The α2,3-sialyltransferases of the present invention can include sialyltransferases of Pasteurella multocida. The sialyltransferases include those having decreased α2,3-sialidase activity compared to a control glycosyltransferase. α2,3-sialidase activity, in particular, refers to the cleavage of the glycosidic bond between the sialic acid from the donor substrate and the sugar of the acceptor molecule, which results in free sialic acid and the acceptor. For certain sialyltransferases of the invention, this activity is essentially absent.
The sialyltransferases of the present invention can include a polypeptide having any suitable percent identity to a reference sequence (e.g., SEQ ID NO: 4). For example, the glycosyltransferases of the present invention can include a polypeptide having a percent sequence identity to the control glycosyltransferase sequence of at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99%. In some embodiments, percent sequence identity can be at least 80%. In some embodiments, percent sequence identity can be at least 90%. In some embodiments, percent sequence identity can be at least 95%.
In some embodiments, the invention provides an isolated or purified polypeptide including an amino acid sequence selected from SEQ ID NO: 4 (PmST2); SEQ ID NO: 5 (PmST2-His6); and SEQ ID NO: 6 (MBP-PmST2-His6). In some embodiments, the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 7 (sialyltransferase motif A), and SEQ ID NO: 8 (sialyltransferase motif B).
The precise length of the sialyltransferases can vary, and certain variants can be advantageous for expression and purification of the enzymes with high yields. For example, removal of certain peptide subunits from the overall polypeptide sequence of a sialyltransferase can improve solubility of the enzyme and increase expression levels. Alternatively, addition of certain peptide or protein subunits to a sialyltransferase polypeptide sequence can modulate expression, solubility, activity, or other properties. The sialyltransferases of the present invention can include point mutations at any position of the PmST2 wild type sequence or a PmST2 variant (e.g., a fusion protein or a truncated form). The mutants can include any suitable amino acid other than the native amino acid. For example, the amino acid can be V, I, L, M, F, W, P, S, T, A, G, C, Y, N, Q, D, E, K, R, or H. Amino acid and nucleic acid sequence alignment programs are readily available (see, e.g., those referred to supra) and, given the particular motifs identified herein, serve to assist in the identification of the exact amino acids (and corresponding codons) for modification in accordance with the present invention.
The sialyltransferases of the present invention can be constructed by mutating the DNA sequences that encode the corresponding unmodified sialyltransferase (e.g., a wild-type sialyltransferase or a corresponding variant), such as by using techniques commonly referred to as site-directed mutagenesis. Nucleic acid molecules encoding the unmodified form of the sialyltransferase can be mutated by a variety of techniques well-known to one of ordinary skill in the art. (See, e.g., PCR Strategies (M. A. Innis, D. H. Gelfand, and J. J. Sninsky eds., 1995, Academic Press, San Diego, Calif.) at Chapter 14; PCR Protocols: A Guide to Methods and Applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White eds., Academic Press, NY, 1990).
By way of non-limiting example, the two primer system, utilized in the Transformer Site-Directed Mutagenesis kit from Clontech, may be employed for introducing site-directed mutants into a polynucleotide encoding an unmodified form of the sialyltransferase. Following denaturation of the target plasmid in this system, two primers are simultaneously annealed to the plasmid; one of these primers contains the desired site-directed mutation, the other contains a mutation at another point in the plasmid resulting in elimination of a restriction site. Second strand synthesis is then carried out, tightly linking these two mutations, and the resulting plasmids are transformed into a mutS strain of E. coli. Plasmid DNA is isolated from the transformed bacteria, restricted with the relevant restriction enzyme (thereby linearizing the unmutated plasmids), and then retransformed into E. coli. This system allows for generation of mutations directly in an expression plasmid, without the necessity of subcloning or generation of single-stranded phagemids. The tight linkage of the two mutations and the subsequent linearization of unmutated plasmids result in high mutation efficiency and allow minimal screening. Following synthesis of the initial restriction site primer, this method requires the use of only one new primer type per mutation site. Rather than prepare each positional mutant separately, a set of “designed degenerate” oligonucleotide primers can be synthesized in order to introduce all of the desired mutations at a given site simultaneously. Mutagenesis can also be conducted using a QuikChange multisite-directed mutagenesis kit (Stratagene) and the like. Transformants can be screened by sequencing the plasmid DNA through the mutagenized region to identify and sort mutant clones. Each mutant DNA can then be restricted and analyzed by electrophoresis, such as for example, on a Mutation Detection Enhancement gel (Mallinckrodt Baker, Inc., Phillipsburg, N.J.) to confirm that no other alterations in the sequence have occurred (by band shift comparison to the unmutagenized control). Alternatively, the entire DNA region can be sequenced to confirm that no additional mutational events have occurred outside of the targeted region.
Verified mutant duplexes in pET (or other) overexpression vectors can be employed to transform E. coli such as, e.g., strain E. coli BL21 (DE3) or strain E. coli BL21 (DE3) pLysS, for high level production of the mutant protein, and purification by standard protocols. The method of FAB-MS mapping, for example, can be employed to rapidly check the fidelity of mutant expression. This technique provides for sequencing segments throughout the whole protein and provides the necessary confidence in the sequence assignment. In a mapping experiment of this type, protein is digested with a protease (the choice will depend on the specific region to be modified since this segment is of prime interest and the remaining map should be identical to the map of unmutated protein). The set of cleavage fragments is fractionated by, for example, HPLC (reversed phase or ion exchange, again depending on the specific region to be modified) to provide several peptides in each fraction, and the molecular weights of the peptides are determined by standard methods, such as FAB-MS. The determined mass of each fragment are then compared to the molecular weights of peptides expected from the digestion of the predicted sequence, and the correctness of the sequence quickly ascertained. Since this mutagenesis approach to protein modification is directed, sequencing of the altered peptide should not be necessary if the MS data agrees with prediction. If necessary to verify a changed residue, CAD-tandem MS/MS can be employed to sequence the peptides of the mixture in question, or the target peptide can be purified for subtractive Edman degradation or carboxypeptidase Y digestion depending on the location of the modification.
Recombinant Nucleic Acids
Sialyltransferase variants can be generated in various ways. In the case of amino acids located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If however, the amino acids are located some distance from each other (separated by more than ten amino acids, for example) it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed. In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions. An alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mutants: DNA encoding the unmodified sialyltransferase is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations. The oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis. This resultant DNA can be used as a template in a third round of mutagenesis, and so on. Alternatively, the multi-site mutagenesis method of Seyfang & Jin (Anal. Biochem. 324:285-291. 2004) may be utilized.
Accordingly, also provided are recombinant nucleic acids, optionally isolated, encoding any of the sialyltransferases of the present invention. In some embodiments, the invention provides an isolated or purified polynucleotide including a nucleotide sequence that is substantially identical to a sequence selected from SEQ ID NO:1 (PmST2), SEQ ID NO:2 (PmST2-His6), and SEQ ID NO:3 (MBP-PmST2-His6), or complements thereof. In some embodiments, the polynucleotide includes a nucleotide sequence that is substantially identical to a sequence selected from SEQ ID NO:1 (PmST2), SEQ ID NO:2 (PmST2-His6), and SEQ ID NO:3 (MBP-PmST2-His6), or complements thereof. In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding SEQ ID NO: 7 (sialyltransferase motif A) or SEQ ID NO: 8 (sialyltransferase motif B), or the complement of a sequence that encodes SEQ ID NO: 7 or 8. In general, the polynucleotide has at least 50% sequence identity to a sequence selected from SEQ ID NOS: 1, 2, and 3, and complements thereof. The sequence identity can be, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, a given polynucleotide can be optimized for expression in yeast. In some embodiments, the polynucleotide contains a sequence selected from SEQ ID NOS: 1, 2, and 3, and complements thereof.
Using a nucleic acid of the present invention, encoding a sialyltransferase of the invention, a variety of vectors can be made. Any vector containing replicon and control sequences that are derived from a species compatible with the host cell can be used in the practice of the invention. Generally, expression vectors include transcriptional and translational regulatory nucleic acid regions operably linked to the nucleic acid encoding the mutant sialyltransferase. The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. In addition, the vector may contain a Positive Retroregulatory Element (PRE) to enhance the half-life of the transcribed mRNA (see Gelfand et al. U.S. Pat. No. 4,666,848). The transcriptional and translational regulatory nucleic acid regions will generally be appropriate to the host cell used to express the sialyltransferase. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells. In general, the transcriptional and translational regulatory sequences may include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In typical embodiments, the regulatory sequences include a promoter and transcriptional start and stop sequences. Vectors also typically include a polylinker region containing several restriction sites for insertion of foreign DNA. In certain embodiments, “fusion flags” are used to facilitate purification and, if desired, subsequent removal of tag/flag sequence, e.g., “His-Tag”. However, these are generally unnecessary when purifying an thermoactive and/or thermostable protein from a mesophilic host (e.g., E. coli) where a “heat-step” may be employed. The construction of suitable vectors containing DNA encoding replication sequences, regulatory sequences, phenotypic selection genes, and the mutant sialyltransferase of interest are prepared using standard recombinant DNA procedures. Isolated plasmids, viral vectors, and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well-known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, N.Y., 2nd ed. 1989)). In some embodiments, the present invention provides a recombinant nucleic acid encoding an isolated sialyltransferase of the present invention.
Host Cells
In certain embodiments, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used. Suitable selection genes can include, for example, genes coding for ampicillin and/or tetracycline resistance, which enables cells transformed with these vectors to grow in the presence of these antibiotics.
In one aspect of the present invention, a nucleic acid encoding a sialyltransferase of the invention is introduced into a cell, either alone or in combination with a vector. By “introduced into” or grammatical equivalents herein is meant that the nucleic acids enter the cells in a manner suitable for subsequent integration, amplification, and/or expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type. Exemplary methods include CaPO4 precipitation, liposome fusion, LIPOFECTIN®, electroporation, viral infection, and the like.
In some embodiments, prokaryotes are used as host cells for the initial cloning steps of the present invention. Other host cells include, but are not limited to, eukaryotic (e.g., mammalian, plant and insect cells), or prokaryotic (bacterial) cells. Exemplary host cells include, but are not limited to, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Sf9 insect cells, and CHO cells. They are particularly useful for rapid production of large amounts of DNA, for production of single-stranded DNA templates used for site-directed mutagenesis, for screening many mutants simultaneously, and for DNA sequencing of the mutants generated. Suitable prokaryotic host cells include E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325), E. coli K12 strain DG116 (ATCC No. 53,606), E. coli X1776 (ATCC No. 31,537), and E. coli B; however many other strains of E. coli, such as HB101, JM101, NM522, NM538, NM539, and many other species and genera of prokaryotes including bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species can all be used as hosts. Prokaryotic host cells or other host cells with rigid cell walls are typically transformed using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation can be used for transformation of these cells. Prokaryote transformation techniques are set forth in, for example Dower, in Genetic Engineering, Principles and Methods 12:275-296 (Plenum Publishing Corp., 1990); Hanahan et al., Meth. Enzymol., 204:63, 1991. Plasmids typically used for transformation of E. coli include pBR322, pUCI8, pUCI9, pUCI18, pUCI19, and Bluescript M13, all of which are described in sections 1.12-1.20 of Sambrook et al., supra. However, many other suitable vectors are available as well.
In some embodiments, the sialyltransferases of the present invention are produced by culturing a host cell transformed with an expression vector containing a nucleic acid encoding the sialyltransferase, under the appropriate conditions to induce or cause expression of the sialyltransferase. Methods of culturing transformed host cells under conditions suitable for protein expression are well-known in the art (see, e.g., Sambrook et al., supra). Suitable host cells for production of the sialyltransferases from lambda pL promoter-containing plasmid vectors include E. coli strain DG116 (ATCC No. 53606) (see U.S. Pat. No. 5,079,352 and Lawyer, F. C. et al., PCR Methods and Applications 2:275-87, 1993, which are both incorporated herein by reference). Following expression, the sialyltransferase can be harvested and isolated. Methods for purifying thermostable glycosyltransferases are described in, for example, Lawyer et al., supra. In some embodiments, the present invention provides a cell including a recombinant nucleic acid of the present invention. In some embodiments, the cell can be prokaryotes, eukaryotes, mammalian, plant, bacteria or insect cells.
IV. Methods of Making Oligosaccharides
The sialyltransferases of the present invention can be used to prepare oligosaccharides, specifically to add N-acetylneuraminic acid (Neu5Ac), other sialic acids, and analogs thereof, to a glycolipid. For example, PmST2 can catalyze the addition of CMP-Neu5Ac to a lactosyl glycolipid by transferring the Neu5Ac to the lactoside moiety of the lactosyl glycolipid.
Accordingly, some embodiments of the present invention provide a method of preparing a glycolipid product. The method includes forming a reaction mixture containing an acceptor glycolipid, a donor substrate having a sugar moiety and a nucleotide, and a glycosyltransferase of the present invention. The glycosyltransferase includes a polypeptide having a sequence that is selected from SEQ ID NO:4 (PmST2), SEQ ID NO:5 (PmST2-His6), and SEQ Ill NO:6 (MBP-PmST2-His6). The reaction mixture is formed under conditions sufficient to transfer the sugar moiety from the donor substrate to the acceptor glycolipid, thereby forming the glycolipid product.
Any suitable acceptor glycolipid can be used in the methods of the invention. Suitable acceptor glycolipids include, but are not limited to, glycoglycerolipids (such as monogalactosyldiacylglycerols, digalactosylmonoacylglycerols, and sulfoquinovosyl diacylglycerols), glycosphingolipids (such as lacto-, neolacto-, ganglio-, globo-, and iso-globo-series glycosphingolipids), and glycosylphosphatidylinositols (e.g., 1-phosphatidyl-L-myo-inositol 2,6-di-O-α-D-mannopyranoside.). In some embodiments, the acceptor glycolipid comprises a galactoside moiety. In some embodiments, the galactoside moiety is selected from the group consisting of a β1-4 linked galactoside moiety and a β1-3 linked galactoside moiety. In some embodiments, the acceptor glycolipid comprises a lactoside moiety or an N-acetyl lactoside moiety. In some embodiments, the acceptor glycolipid comprises a Galβ1-3GlcNAc moiety or a Galβ1-3GalNAc moiety.
The donor substrate of the present invention includes a nucleotide and sugar. Suitable nucleotides include, but are not limited to, adenine, guanine, cytosine, uracil and thymine nucleotides with one, two or three phosphate groups. In some embodiments, the nucleotide can be cytidine monophosphate (CMP). The sugar can be any suitable sugar. For example, the sugar can be N-acetylneuraminic acid (Neu5Ac) or other sialic acids and analogs thereof. Sialic acid is a general term for N- and O-substituted derivatives of neuraminic acid, and includes, but is not limited to, N-acetyl (Neu5Ac) or N-glycolyl (Neu5Gc) derivatives, as well as O-acetyl, O-lactyl, O-methyl, O-sulfate and O-phosphate derivatives. In some embodiments, the sialic acid can be a compound of the formula:




wherein R1 is selected from H, OH, N3, NHC(O)Me, NHC(O)CH2OH, NHC(O)CH2N3, NHC(O)OCH2C≡CH, NHC(O)CH2F, NHC(O)CH2NHCbz, NHC(O)CH2OC(O)Me, and NHC(O)CH2OBn; and R2, R3, R4, and R5 are independently selected from H, OH, N3, OMe, F, OSO3−, OPO3H−, and OC(O)Me. In some embodiments, the donor substrate is a cytidine 5′-monophosphate (CMP)-sialic acid. In some embodiments, the CMP-sialic acid is cytidine 5′-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) or a CMP-Neu5Ac analog. Other donor substrates are useful in the methods of the present invention. In other embodiments, the sialic acid can be a compound of the formula:





In some embodiments, the sialic acid donor can be a compound of the formula:





The methods of the invention include providing reaction mixtures that contain the sialyltransferases described herein. The sialyltransferases can be, for example, purified prior to addition to the reaction mixture or secreted by a cell present in the reaction mixture. Alternatively, a sialyltransferase can catalyze the reaction within a cell expressing the sialyltransferase.
Reaction mixtures can contain additional reagents for use in glycosylation techniques. For example, in certain embodiments, the reaction mixtures can contain buffers (e.g., 2-(N-morpholino)ethanesulfonic acid (MES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 3-morpholinopropane-1-sulfonic acid (MOPS), 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), potassium phosphate, sodium phosphate, phosphate-buffered saline, sodium citrate, sodium acetate, and sodium borate), cosolvents (e.g., dimethylsulfoxide, dimethylformamide, ethanol, methanol, tetrahydrofuran, acetone, and acetic acid), salts (e.g., NaCl, KCl, CaCl2, and salts of Mn2+ and Mg2+), chelators (e.g., ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 2-({2-[Bis(carboxymethyl)amino]ethyl}(carboxymethyl)amino)acetic acid (EDTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)), reducing agents (e.g., dithiothreitol (DTT), β-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP)), and labels (e.g., fluorophores, radiolabels, and spin labels). Buffers, cosolvents, salts, chelators, reducing agents, and labels can be used at any suitable concentration, which can be readily determined by one of skill in the art. In general, buffers, cosolvents, salts, chelators, reducing agents, and labels are included in reaction mixtures at concentrations ranging from about 1 μM to about 1 M. For example, a buffer, a cosolvent, a salt, a chelator, a reducing agent, or a label can be included in a reaction mixture at a concentration of about 1 μM, or about 10 μM, or about 100 μM, or about 1 mM, or about 10 mM, or about 25 mM, or about 50 mM, or about 100 mM, or about 250 mM, or about 500 mM, or about 1 M.
Reactions are conducted under conditions sufficient to transfer the sugar moiety from a donor substrate to an glycolipid. The reactions can be conducted at any suitable temperature. In general, the reactions are conducted at a temperature of from about 4° C. to about 40° C. The reactions can be conducted, for example, at about 25° C. or about 37° C. The reactions can be conducted at any suitable pH. In general, the reactions are conducted at a pH of from about 4.5 to about 10. The reactions can be conducted, for example, at a pH of from about 5 to about 9. The reactions can be conducted for any suitable length of time. In general, the reaction mixtures are incubated under suitable conditions for anywhere between about 1 minute and several hours. The reactions can be conducted, for example, for about 1 minute, or about 5 minutes, or about 10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, or about 4 hours, or about 8 hours, or about 12 hours, or about 24 hours, or about 48 hours, or about 72 hours. Other reaction conditions may be employed in the methods of the invention, depending on the identity of a particular sialyltransferase, donor substrate, or acceptor molecule.
The donor substrate can be prepared prior to preparation of the oligosaccharide, or prepared in situ immediately prior to preparation of the oligosaccharide. In some embodiments, the method of the present invention also includes forming a reaction mixture including a CMP-sialic acid synthetase, cytidine triphosphate, and N-acetylneuraminic acid (Neu5Ac) or a Neu5Ac analog, under conditions suitable to form CMP-Neu5Ac or a CMP-Neu5Ac analog. Any suitable CMP-sialic acid synthetase (i.e., N-acylneuraminate cytidylyltransferase, EC 2.7.7.43) can be used in the methods of the invention. For example, CMP-sialic acid synthetases from E. coli, C. thermocellum, S. agalactiae, P. multocida, H ducreyi, or N. meningitidis can be used. In some embodiments, the step of forming the donor substrate and the step of forming the oligosaccharide are performed in one pot.
In some embodiments, the sugar moiety of the donor substrate is prepared separately prior to use in the methods of the present invention. Alternatively, the sugar moiety can be prepared in situ immediately prior to use in the methods of the present invention. In some embodiments, the method also includes forming a reaction mixture including a sialic acid aldolase, pyruvic acid or derivatives thereof, and N-acetylmannosamine or derivatives thereof, under conditions suitable to form Neu5Ac or a Neu5Ac analog. Any suitable sialic acid aldolase (i.e., N-acetylneuraminate pyruvate lyase, EC 4.1.3.3) can be used in the methods of the invention. For example, sialic acid aldolases from E. coli, L. plantarum, P. multocida, or N. meningitidis can be used. In some embodiments, the step of forming the sugar moiety, the step of forming the donor substrate, and the step of forming the oligosaccharide are performed in one pot.
The products prepared by the method of the present invention can include a variety of glycolipid products. In some embodiments, the glycolipid product is an 2,3-linked sialylglycolipid. In some embodiments, the α2,3-linked sialylglycolipid is Neu5Acα2-3lactosyl sphingosine (lyso-GM3) or a derivative thereof.
V. EXAMPLES
General Materials and Methods
Chemicals and Reagents. T4 DNA ligase, 1 kb DNA ladder, and BamHI restriction enzyme were obtained from Promega (Madison, Wis.). Herculase enhanced DNA polymerase was from Stratagene (La Jolla, Calif.). DNeasy Tissue kit, QIAprep spin miniprep kit, and QIAEX II gel extraction kit were bought from Qiagen (Valencia, Calif.). Nickel-nitrilotriacetic acid (Ni2+-NTA) agarose was obtained from Fisher Scientific (Tustin, Calif.). Precision Plus Protein Standards and BioGel P-2 fine resin were from Bio-Rad (Hercules, Calif.). Bicinchoninic acid (BCA) protein assay kit was from Pierce Biotechnology, Inc. (Rockford, Ill.). Gel filtration LMW calibration kit and Superdex 75 10/300 GL column were from Amersham Biosciences (Piscataway, N.J.). Cytidine 5′-triphosphate (CTP), N-acetylmannosamine (ManNAc), and pyruvate were purchased from Sigma (St. Louis, Mo.). The sialyltransferase sugar nucleotide donor cytidine 5′-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) was synthesized enzymatically from ManNAc, pyruvate (5 equiv.), and CTP using a one-pot two-enzyme system containing a recombinant sialic acid aldolase cloned from E. coli K12 and a recombinant N. meningitidis CMP-sialic acid synthetase (NmCSS) as described previously. Lactosyl sphingosine and lactosyl ceramide were purchased from Avanti Polar Lipids Inc. (Alabaster, Ala.).
Bacterial strains and plasmids. Electrocompetent E. coli DH5α cells and chemically competent E. coli BL21(DE3) cells were from Invitrogen (Carlsbad, Calif.). Genomic DNA was prepared from Pasteurella multocida P-1059 from American Type Culture Collection (ATCC, Manassas, Va.) (ATCC#15742). Restriction enzymes XhoI, BamHI, EcoRI, and HindIII, and the expression vector pMal-c4X were purchased from New England Biolabs (Ipswich/Beverly, Mass.). Expression vector pET22b(+) was purchased from Novagen (EMD Biosciences, Inc. Madison, Wis.).
Analytical Methods. DNA sequencing was performed by Davis Sequencing Facility in the University of California-Davis. High resolution electrospray ionization (ESI) mass spectra were obtained at the Mass Spectrometry Facility in the University of California, Davis. All NMR experiments were carried out at 26° C. in D2O or CD3OD on Varian VNMRS 600 MHz or Bruker 800 MHz spectrometry. LacβPro2AA, LacβPro-triazole-C14, Galβ1-4GlcNAcβPro-triazole-C14 (LacNAcβPro-triazole-C14), Galβ1-3GlcNAcβPro-triazole-C14, Galβ1-3GlcNAcαPro-triazole-C14, Galβ1-3GalNAcαPro-triazole-C14, and GalβPro-triazole-C14 were synthesized as described in the Supporting Information.
Example 1
Cloning, Expression, and Purification of Pm0508 Homolog from Pm Strain P-1059 (ATCC 15742)
Methods
Cloning. The Pm0508 gene locus (GenBank accession no. AAK02592) was amplified by polymer chain reaction (PCR) from Pasteurella multocida P-1059(ATCC 15742) genomic DNA. Full-length Pm0508 gene was cloned as either a C-His6 (SEQ ID NO:9) tagged or an N-terminal Maltose Binding Protein (MBP)-tagged and C-terminal His6 (SEQ ID NO:9) tagged fusion protein. A forward primer 5′-CGC GGATCCATGAATTTGATTATTTGTTGTACACCG-3′ (SEQ ID NO:10) (BamHI restriction site is underlined) and a reverse primer 5′-CCGCTCGAGCTCTCTTATATCAATAACGTTAAC-3′ (SEQ ID NO:11) (Xhol restriction site is underlined) were used to clone the C-His6 (SEQ ID NO:9) tagged fusion protein PmST2-His6 in pET22b(+) vector. A forward primer 5′-GACCGAATTCATGAATTTGATTATTTGTTGTACACCG-3′ (SEQ ID NO:12) (EcoRI restriction site is underlined) and a reverse primer 5′-GATCAAGCTTTTAGTGGTGGTGGTGGTGGTGCTCTCTTATATCAATAACG-3′ (SEQ ID NO:13) (HindIII restriction site is underline and the codons for the C-His6 (SEQ ID NO:9) tag are in italics) were used to clone of the full-length MBP-PmST2-His6 fusion protein in pMa1-c4X vector. The C-His6 (SEQ ID NO:9) tag was introduced to simplify purification using Ni2+-NTA resin. The resulting PCR product was purified, digested, and inserted into the corresponding pre-digested vector DNAs by ligation. The ligation product was transformed into electrocompetent E. coli DH5α cells. Positive plasmids were selected and subsequently transformed into BL21(DE3) chemically competent cells.
Expression and Purification of MBP-PmST2-His6. Positive recombinant plasmid was transformed into E. coli BL21(DE3) for overexpression. E. coli strain bearing the recombinant plasmid was grown in LB medium supplemented with ampicillin (100 μg ml−1) until OD600 nm of 0.8-1.0 was reached. Overexpression of the protein was achieved by inducing the E. coli culture with 0.1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) at 20° C. for 18-20 h. The bacterial cells were harvested by centrifugation at 4° C. in a Sorvall Legend RT centrifuge with a hanging bucket rotor at 3,696×g for 2 h. Harvested cells were resuspended in 20 ml lysis buffer (pH 8.0, Tris-HCl containing 0.1% Triton X-100) for cells collected from one liter cell culture. Lysozyme (100 μg ml−1) and DNaseI (5 μg ml−1) were added to the cell resuspension. The resulting mixture was then incubated at 37° C. for 1 h with shaking at 210 rpm. Cell lysate (supernatant) was obtained by centrifugation at 14,905×g for 45 min. Purification was carried out by loading the supernatant onto a Ni2+-NTA column pre-equilibrated with 10 column volumes of binding buffer (10 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 8.0). The column was wash with 10 column volumes of binding buffer and 10 column volumes of washing buffer (50 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 8.0). The target protein was eluted with Tris-HCl buffer (50 mM, pH 8.0) containing imidazole (200 mM) and NaCl (0.5 M). The amount of protein obtained was analyzed by BCA method. The fractions containing the purified enzymes were collected and dialyzed against Tris-HCl buffer (20 mM, pH 8.0) containing 10% glycerol. Dialyzed proteins were stored at 4° C.
Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). SDS-PAGE was performed in 12% Tris-glycine gels using Bio-Rad Mini-protein III cell gel electrophoresis unit (Bio-Rad, Hercules, Calif.) at DC=150 V. Bio-Rad Low range SDS-PAGE standards or Precision Plus Protein Standards were used as molecular weight standards. Gels were stained with Coomassie Blue.
Quantification of Purified Protein. Protein concentration was determined in a 96-well plate using a Bicinchoninic acid (BCA) Protein Assay Kit (Pierce Biotechnology, Rockford, Ill.) with bovine serum albumin as a protein standard. The absorbance of each sample was measured at 562 nm on a multiple-well plate reader (BioTek Synergy HT RDR Multidetection Plate Reader).
Protein Encoded by the Pm0508 Gene Homolog from Pm Strain P-1059 (ATCC 15742)
Protein encoded by Pm0508 gene from Pasteurella multocida(strain Pm70), designated as PmST2, was identified by BLAST search as a hypothetic sialyltransferase due to its protein sequence homology (31% sequence identity) to a lipooligosaccharide α2,3-sialyltransferase from Neisseria meningitidis encoded by 1st gene. DNA sequencing of the cloned Pm0508 gene homolog from Pm strain P-1059(ATCC15742) indicates that it has four base differences (t74c, c87t, c306t, t867c) compared to the published Pm0508 gene sequence of the genomic strain Pm70, resulting in only one amino acid difference (M25T) in the deduced protein. Amino acid sequence alignment using ClustalW multiple alignment program (FIG. 1) shows the homology shared by protein sequences encoded by the Pm0508 gene homolog, Haemophilus influenzae LsgB (40% identity), and N. meningitidis Lst (30% identity). Hydropathy plot (TMpred) indicated that there are two possible transmembrane helices in PmST2. One spanned from 1-19 amino acid residues and the other spanned from 85-107 amino acid residues. The first possible transmembrane helix (1-19 aa) is shared among all members of GT52 family and the second transmembrane helix (85-107 aa) is shared among some GT52 family members.
Expression and Purification of MBP-PmST2-His6 
Initially, the full-length Pm0508 gene from Pasteurella multocida P-1059was cloned into pET22b(+) and expressed as a C-His6 (SEQ ID NO:9) tagged fusion protein. Expression in E. coli BL21 (DE3) at 20 ° C. for 20 hrs with vigorous shaking (250 rpm) after induction with isopropyl-1-thio-β-D-galactopyranoside (IPTG, 0.1 mM) yielded  ̃3 mg of soluble protein that can be purified from cell lysate obtained from one liter cell culture by Ni2+-NTA affinity column. The yield of soluble enzyme was improved by introducing an N-terminal maltose binding protein (MBP) to the fusion protein. Under the same expression conditions, active and soluble recombinant protein MBP-PmST2-His6 can be routinely purified at a level of 21 mg from cell lysate obtained from one liter E. coli culture by Ni2+-NTA affinity column. As shown in FIG. 2, the recombinant protein showed a molecular weight of around 75 kD by SDS-PAGE, which was close to its calculated molecular weight of 79.5 kDa.
Example 2
Time-Course Studies for MBP-PmST2-His6 and PmST1
Methods.
Enzymatic reactions were performed in a total volume of 20 μl (when LacβPro2AA was used as an acceptor) or 40 μl (when LacβPro-triazole-C 14 was used as an acceptor) in a Tris-HCl buffer (200 mM, pH 8.0) containing an acceptor (2 mM of LacβPro2AA or LacβPro-triazole-C14), CMP-Neu5Ac (8 mM), 0.3% Triton X-100, and a sialyltransferase (2.4 MBP-PmST2-His6 or PmST1) at 37° C. At different time points (0, 1, 5, 10, 15, 20, 30, and 60 min), 1.5 μl aliquots were taken from the LacβPro2AA reaction and pre-chilled 12% acetonitrile aqueous solution (148.5 μl) was added to each aliquot. Similarly, 3.5 μl aliquots were taken from the LacβPro-triazole-C14 reaction at different time points and pre-chilled 95% ethanol (7 μl) was added to each aliquot. All samples were kept on ice for 10 min and then centrifuged for 5 min at 13,000 rpm. Supernatants from LacβPro2AA reactions were analyzed by a Shimadzu LC-2010A HPLC system equipped with a membrane on-line degasser, a temperature control unit, and a fluorescence detector (the excitation wavelength was set at 315 nm and the emission wavelength was set at 400 nm) using a reverse phase Premier C18 column (250×4.6 mm I.D., 5 μm particle size, Shimadzu) protected with a C18 guard column cartridge. Mobile phase used was 12% acetonitrile in water. Supernatants from LacβPro-triazole-C14 reactions were analyzed by a Beckman P/ACE MDQ capillary electrophoresis (CE) system (60 cm×75 μm i.d.) equipped with a PDA detector (optimal absorption at 214 nm). The running buffer used was sodium borate buffer (50 mM, pH 10.2) containing β-cyclcodextrin (20 mM).
Results.
Both LacβPro2AA and LacβPro-triazole-C14 (FIG. 3) were used as acceptor substrates for the time course studies of the sialyltransferase activity of MPB-PmST2-His6 and PmST1. The reactions were carried out under same conditions with the same molar concentrations of enzymes and substrates. As shown in FIG. 4, PmST1 was very active towards LacβPro2AA (FIG. 4A, dashed line with open circles), the sialyltransferase reaction reached optimum at 1 min. The sialidase activity of the PmST1 was then responsible for cleaving the product formed. In comparison, LacβPro-triazole-C14 was not a good acceptor for PmST1 (FIG. 4B, dashed line with open circles). The sialyltransferase reaction reached to an optimum at 15 min with less than 5% product formation. The sialidase activity then started to hydrolyze the product formed. In contrast, LacβPro-triazole-C14 (FIG. 4B, solid line with filled diamonds) was a much better acceptor substrate for MBP-PmST2-His6 than LacβPro2AA (FIG. 4A, solid line with filled diamonds). A 60% yield of sialylation was achieved in 1 hour by MBP-PmST2-His6 when LacβPro-triazole-C14 was used as an acceptor, while only 15% sialylation was achieved in the same time frame when LacβPro2AA was used as an acceptor.
Example 3
pH Profiles of MBP-PmST2-His6 
Methods.
Each reaction was carried out in duplicate at 37° C. for 15 min in a buffer (200 mM) with a total volume of 15 μl containing an acceptor (2 mM, LacβPro2AA or LacβPro-triazole-C14), CMP-Neu5Ac (8 mM), 0.3% Triton X-100, and the enzyme MBP-PmST2-His6 (2.4 μg μl−1). Buffers used were: NaOAc-HOAc (pH 4.0-6.0), Tris-HCl (pH 7.0-9.0), and CHES (pH 10.0). Enzymatic reactions were terminated by adding 15 μl of pre-chilled 95% ethanol. All samples were kept on ice for 10 min and then centrifuged at 13,000 rpm for 5 min. Supernatants were analyzed by HPLC (for LacβPro2AA reaction) or CE (for LacβPro-triazole-C14 reaction) as described above for the time course studies.
Results.
The pH profile studies were carried out for MBP-PmST2-His6 using either LacβPro2AA (FIG. 5, dashed line with open circles) or LacβPro-triazole-C14 (FIG. 5, solid line with filled diamonds) as the acceptor substrate. The α-2,3-sialyltransferase activity of MBP-PmST2-His6 was active in a broad pH range of 4.5-10.0. The optimal pH was 6.0. When LacβPro2AA was used as the acceptor, pH of the buffer in the range of 4.5 to 9.0 did not change the α2,3-sialyltransferase activity of the enzyme significantly. In comparison, when LacβPro-triazole-C14 was used as the acceptor, the optimal pH range is 5.0-8.0 and the α-2,3-sialyltransferase activity of the enzyme decreased modestly at pH 4.5 and 9.0. Low activity was found at pH 10.0 and no significant activity was found at pH 4.0.
Example 4
Effects of Metal Ions, EDTA, and a Reducing Reagent on MBP-PmST2-His6 Activity
Methods.
Reactions were carried out in duplicate at 37° C. for 15 min in a total volume of 15 μl in a Tris-HCl buffer (200 mM, pH 8.0) containing LacβPro-triazole-C14 (2 mM), CMP-Neu5Ac (8 mM), 0.3% Triton X-100, and the enzyme (2.4 μg μl−1). For metal effects, various concentrations (1, 5, 10, or 20 mM) of MgCl2 or MnCl2 were added and ethylenediaminetetraacetic acid (EDTA as chelating agent was used at two concentrations (1 or 10 mM). A reducing reagent 2-mercaptoethanol (2-ME) was used at two concentrations (1 or 10 mM). Reaction without metal ions, EDTA, or 2-ME was used as a control. All reactions were stopped by adding 15 μl of pre-chilled 95% ethanol and the reaction mixtures were kept on ice. The samples were centrifuged at 13,000 rpm for 5 min before the supernatants were analyzed by CE as described above.
Results.
Similar to other sialyltransferases reported before, divalent metal cations are not required by the α2,3-sialyltransferase activity of MBP-PmST2-His6 as the addition of different concentrations of MnC12 or MgC12 (1, 5, 10 or 20 mM) or EDTA (1 mM or 10 mM) did not affect the activity significantly (26-32% conversions) (FIG. 6). Different from the effect of MnC12 at high concentration in decreasing the activity of PmST1 and a truncated Photobacterium damselae α2,6-sialyltransferase (Pd2,6ST), the presence of MnC12 at a concentration up to 20 mM did not decrease the activity of PmST2. Although MBP-PmST2-His6 has six cysteine residues in the PmST2 protein sequence and among which three cysteine residues near the N-terminus are highly conserved among homologous bacterial sialyltransferases, the addition of a reducing reagent 2-mercaptoethanol at 1 mM or 10 mM did not affect the α2,3-sialyltransferase activity of MBP-PmST2-His6. This indicates that disulfide bonds are not required for the enzymatic activity.
Example 5
Kinetic Studies
Methods.
Reactions were carried out at 37° C. for 10 mM in a total volume of 15 μl in Tris-HCl buffer (200 mM, pH 8.0) containing 2-mercaptoethanol (1 mM), 0.3% Triton X-100, enzyme (14 μM), with varied CMP-Neu5Ac concentrations (0.25, 0.5, 1.0, 2.0, 5.0, and 10.0 mM) and a fixed LacβPro-triazole-C14 (2.0 mM) concentration, or varied LacβPro-triazole-C14 concentrations (0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 mM) or varied LacβPro2AA concentrations (0.5, 1.0, 2.0, 5.0, 10.0, and 25.0 mM) and a fixed CMP-Neu5Ac concentration (4.0 mM). HPLC assays were used for reactions with LacβPro2AA as the sialyltransferase acceptor and CE assays were used for reactions with LacβPro-triazole-C14 as the sialyltransferase acceptor. Apparent kinetic parameters were obtained by fitting the data (the average values of duplicate assay results) into the Michaelis-Menten equation using Grafit 5.0.
Results.
As shown in Table 1, the binding of MBP-PmST2-His6 to a β-lactoside with a long hydrocarbon chain (LacβPro-triazole-C14, KM =4.1±0.3 mM) was stronger (7-fold difference in KM values) than its binding to a lactoside without the long hydrocarbon chain (LacβPro2AA, KM =28±3 mM). In addition, the turn over number was higher when LacβPro-triazole-C14 (kcat =12±1 min −1) was used as the acceptor for the α2,3-sialyltransferase activity of MBP-PmST2-His6 compared to LacβPro2AA (kcat =7.9±0.7 min −1) as the acceptor. The differences in both KM and kcat lead to a 10-fold higher catalytic activity when LacβPro-triazole-C14 was used as the acceptor substrate.







TABLE 1







Apparent kinetic parameters for the α-2,3-sialyltransferase activity of


MBP-PmST2-His6.












LacβPro-triazole-



Substrates
CMP-Neu5Ac
C14
LacβPro2AA





KM (mM)
1.3 ± 0.1
4.1 ± 0.3
28 ± 3


Vmax
(7.4 ± 0.5) × 10−2
(1.7 ± 0.1) × 10−1
(1.1 ± 0.1) × 10−1


(mM min−1)





kcat (min−1)
5.3 ± 0.4
12 ± 1 
7.9 ± 0.7


kcat/KM (min−1
4.1
2.9
0.28


mM−1)









Example 6
Enzymatic Synthesis of Characterization of Sialosides
Methods
Neu5Acα2-3LacβPro-triazole-C14. LacβPro-triazole-C14 (34 mg, 0.052 mmol), N-acetylneuraminic acid (Neu5Ac) (24 mg, 0.079 mmol), CTP (44 mg, 0.079 mmol), and MgCl2 (43 mg, 21 mM) were dissolved in 10 ml of Tris-HCl buffer (100 mM, pH 8.5, 1 ml). After the addition of N. meningitidis CMP-sialic acid synthetase (NmCSS, 1.4 mg) and MBP-PmST2-His6 (5.8 mg), the reaction was carried out by incubating the solution in an incubator shaker overnight at 37° C. The reaction was then quenched by adding cold EtOH (10 ml) and the mixture was centrifuged to remove the precipitates. The filtrate was concentrated and purified by a BioGel P-2 filtration column (elute with water) and a silica gel column (EtOAc:MeOH:H2O, 7:2:1) to afford Neu5Acα2-3LacβPro-triazole-C14 (36 mg, 73%).
Sialyl lactosyl sphingosine (lyso-GM3). Lactosyl sphingosine (15 mg, 0.026 mmol), Neu5Ac (12 mg, 0.039 mmol), CTP (22 mg, 0.039 mmol), and MgCl2 (22 mg, 22 mM) were dissolved in 5 ml of Tris-HCl buffer (100 mM, pH 8.5) in the presence of 0.2% Triton X-100. After the addition of NmCSS (0.8 mg) and MBP-PmST2-His6 (2.9 mg), the reaction was carried out by incubating the solution in an incubator shaker overnight at 37° C. The reaction was then quenched by adding cold EtOH (5 ml) and the mixture was centrifuged to remove the precipitates. The filtrate was concentrated and purified by a BioGel P-2 filtration column (elute with water) and a silica gel column (EtOAc:MeOH:H2O=6:2:1 by volume) to afford Neu5Acα2-3Lactosyl sphingosine (15 mg, 68%).
Results.
Preparative enzymatic synthesis of α2,3-linked sialosides Neu5Acα2-3LacβPro-triazole-C14 and Neu5Acα2-3lactosyl sphingosine (lyso-GM3) using a one-pot two-enzyme system containing a recombinant N. meningitidis CMP-sialic acid synthetase (NmCSS) and MBP-PmST2-His6 (FIG. 7) led to 73% and 68% yields, respectively. Nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (ESI-HRMS) studies confirmed the structures of both sialosides Neu5Acα2-3LacβPro-triazole-C14 and Lyso-GM3. The α-2,3-sialyl linkage formed in Neu5Acα2-3LacβPro-triazole-C14 was determined by comparing the chemical shift values of the product and the acceptor LacβPro-triazole-C14. As shown in Table 2, a significant downfield shift on the C3 of the Gal in the sialoside products compared to LacβPro-triazole-C14 (72.66 ppm in the acceptor compared to 77.76 ppm in the product) and lactosyl sphingosine (72.64 ppm in the acceptor compared to 77.84 ppm in the product) acceptors and small upfield chemical shifts on the neighboring C2 indicated that the sialylation occurred at the C3 of the Gal in the acceptors. High resolution mass spectrometry (HRMS) spectra obtained using electrospray ionization (ESI) method showed the desired m/z for molecular ions. For Neu5Acα2-3LacβPro-triazole-C14, the m/z (937.4845) matched well to the calculated m/z value (937.4874) for C42H73N4O19 (M-H). For Lyso-GM3, the m/z (913.4726) matched well to the calculated value (913.4762) for C41H73N2O20 (M-H).







TABLE 2







13C NMR chemical shifts assignement of LacβPro-triazole-C14, Neu5Acα2-3LacβPro-triazole-C14,


Lactosyl sphingosine, and Lyso-GM3.









Chemical shift (ppm)















Neu5Acα2-




Residue
Carbon atom
LacβPro-
3LacβPro-
Lactosyl



βDGlc
C
triazole-C14
triazole-C14
sphingosine
Lyso-GM3
















1
104.34
104.36
104.34
104.00



2
74.90
74.84
74.80
74.67



3
74.97
75.05
74.91
75.10



4
77.19
77.17
77.19
77.26



5
76.51
76.45
76.37
76.38



6
62.07
62.05
61.92
62.00


βDGal(1-4)
1
105.26
105.22
105.21
105.30



2
74.87
73.95
74.73
73.96



3
72.66
77.76
72.64
77.84



4
70.46
70.22
70.39
70.35



5
76.63
76.60
76.60
76.38



6
62.63
62.83
62.59
62.87


αDNeu5Ac(2-3)
1

175.02

174.91



2

101.25

101.22



3

42.21

42.33



4

69.10

69.12



5

54.08

54.13



6

73.07

73.08



7

69.46

69.44



8

70.94

70.92



9

61.80

61.89



C═O

175.59

175.64



CH3

22.69

22.68


Pro-triazole-C14
OCH2CH2CH2(N3CH═C)CH2CH2(CH2)10CH2CH3
67.13
67.02





OCH2CH2CH2(N3CH═C)CH2CH2(CH2)10CH2CH3
26.38
26.37





OCH2CH2CH2(N3CH═C)CH2CH2(CH2)10CH2CH3
48.19
48.16





OCH2CH2CH2(N3CH═C)CH2CH2(CH2)10CH2CH3
123.75
123.86





OCH2CH2CH2(N3CH═C)CH2CH2(CH2)10CH2CH3
149.34
149.26





OCH2CH2CH2(N3CH═C)CH2CH2(CH2)10CH2CH3
33.11
33.15





OCH2CH2CH2(N3CH═C)CH2CH2(CH2)10CH2CH3
31.61
31.62





OCH2CH2CH2(N3CH═C)CH2CH2(CH2)10CH2CH3
30.32-30.79
30.35-30.84





OCH2CH2CH2(N3CH═C)CH2CH2(CH2)10CH2CH3
23.76
23.81





OCH2CH2CH2(N3CH═C)CH2CH2(CH2)10CH2CH3
14.46
14.52




Sphingosine
OCH2CHNH2CHOHCH═CHCH2CH2(CH2)9CH2CH3


71.73
71.61



OCH2CHNH2CHOHCH═CHCH2CH2(CH2)9CH2CH3


56.30
56.87



OCH2CHNH2CHOHCH═CHCH2CH2(CH2)9CH2CH3


80.61
80.92



OCH2CHNH2CHOHCH═CHCH2CH2(CH2)9CH2CH3


135.79
136.68



OCH2CHNH2CHOHCH═CHCH2CH2(CH2)9CH2CH3


130.92
128.76



OCH2CHNH2CHOHCH═CHCH2CH2(CH2)9CH2CH3


33.54
33.44



OCH2CHNH2CHOHCH═CHCH2CH2(CH2)9CH2CH3


33.18
33.12



OCH2CHNH2CHOHCH═CHCH2CH2(CH2)9CH2CH3


30.47-30.90
30.28-30.83



OCH2CHNH2CHOHCH═CHCH2CH2(CH2)9CH2CH3


23.84
23.78



OCH2CHNH2CHOHCH═CHCH2CH2(CH2)9CH2CH3


14.55
14.48









Example 7
Acceptor Substrate Specificity Studies of MBP-PmST2-His6 
Methods.
General Synthetic Methods. Chemicals were purchased and used without further purification. 1H NMR (600 MHz) and 13C NMR (150 MHz) spectra were recorded on a Varian VNMRS 600 MHz spectrometer for Lacβ2AA. All other 1H and 13C NMR spectra were recorded on a Bruker 800 MHz spectrometer. High resolution electrospray ionization (ESI) mass spectra were obtained at the Mass Spectrometry Facility in the University of California, Davis. Silica gel 60 Å was used for flash column chromatography. Thin-layer chromatography (TLC) was performed on silica gel plates using anisaldehyde sugar stain or 5% sulfuric acid in ethanol stain for detection. Gel filtration chromatography was performed with a column (100 cm×2.5 cm) packed with BioGel P-2 Fine resins. N. meningitidis CMP-sialic acid synthetase (NmCSS) was expressed in E. coli and purified as described previously.
Synthesis of LacβPro2AA. LacβPro2AA was prepared according to FIG. 8A. To a solution of LacβProNH2 (30 mg, 0.075 mmol) in 6 ml anhydrous DMF, dry triethylamine (50 μl) was added under argon atmosphere. Two equivalents of N-hydroxy succinamide (NHS) activated 4-((2-(methoxycarbonyl)phenyl)amino)-4-oxobutanoic acid (2AA-OSu) (52 mg, 0.15 mmol) were then added at 0° C. The resulted solution was stirred at room temperature for overnight. The reaction mixture was concentrated and the residue was purified by flash column chromatography (EtOAc:MeOH:H2O=9:2:1 by volume) to afford pure LacβPro2AA (40 mg, 84%). 1H NMR (600 MHz, D2O): δ 8.01 (d, 1H, J=7.8 Hz), 7.88 (d, 1H, J=8.4 Hz), 7.67 (t, 1H, J=7.8 Hz), 7.35 (t, 1H, J=7.8 Hz), 4.45 (d, 1H, J=7.8 Hz), 4.37 (d, 1H, J=8.4 Hz), 3.96-3.55 (m, 15H), 3.51-3.49 (m, 1H), 3.35-3.29 (m, 3H), 2.79 (t, 2H, J=6.6 Hz), 2.64 (t, 2H, J=6.6 Hz), 1.82 (m, 2H); 13C NMR (150 MHz, D2O): δ 174.66, 173.63, 169.27, 137.24, 134.16, 131.07, 125.50, 123.63, 121.05, 103.11, 102.23, 78.59, 75.52, 74.88, 74.52, 72.96, 72.73, 71.12, 68.71, 67.83, 61.18, 60.24, 52.95, 36.35, 32.54, 31.17, 28.55. HRMS (ESI) m/z calcd for C27H40N2O15Na (M-Na) 655.2326. Found 655.2352.
Synthesis of LacβPro-triazole-C14. LacβPro-triazole-C14 was synthesized according to FIG. 8B. To a solution of LacβProN3(80 mg, 0.19 mmol) in 10 ml anhydrous DMF, hexadecyne (126 mg, 0.57 mmol) and DIPEA (74 mg, 0.57 mmol) were added. CuI (25 mg, 0.13 mmol) was then added into the reaction mixture and the resulted solution was stirred at room temperature for overnight. The reaction mixture was concentrated and the residue was purified by flash column chromatography (EtOAc:MeOH:H2O, 12:2:1) to afford pure LacβPro-triazole-C14 (76 mg, 62%). The detailed NMR data is shown in Table 2. HRMS (ESI) m/z calcd for C31H57N3O11Na (M-Na) 670.3891. Found 670.3904.
Synthesis of LacNAcβPro-triazole-C14. LacNAcβPro-triazole-C14 was synthesize according to FIG. 8c. To a solution of LacNAcβProN3(30 mg, 0.064 mmol) in 5 ml anhydrous MeOH, hexadecyne (72 mg, 0.32 mmol) and DIPEA (25 mg, 0.19 mmol) were added. CuI (12 mg, 0.062 mmol) was then added into the reaction mixture and the resulted solution was stirred at room temperature for 2 days. The reaction mixture was concentrated and the residue was purified by flash column chromatography (EtOAc:MeOH:H2O, 12:2:1) to afford pure LacNAcβPro-triazole-C14 (35 mg, 78%). 1H NMR (800 MHz, MeOD) δ 7.83 (s, 1H), 4.58-4.56 (m, 2H), 4.53 (d, J=8 Hz, 1H), 4.50 (d, J=8 Hz, 1H), 4.01-3.94 (m, 4H), 3.89-3.87 (m, 2H), 3.82 (dd, J=4.8 and 11.2 Hz, 1H), 3.76-3.70 (m, 3H), 3.67-3.52 (m, 4H), 2.78 (t, J=8 Hz, 2H), 2.24-2.19 (m, 2H), 2.11 (s, 3H), 1.78-1.76 (m, 2H), 1.45-1.39 (m, 22), 1.00 (t, J=7.2 Hz, 3H) 13C HMR (200 MHz, MeOD) δ 171.27, 147.83, 121.93, 103.65, 101.32, 79.48, 75.66, 75.10, 73.36, 72.81, 71.12, 68.93, 65.32, 61.10, 60.44, 55.23, 46.50, 31.56, 30.01, 29.23-38.77 (10C), 24.82, 22.21, 21.61, 12.91. HRMS (ESI) m/z calculated for C17H31N4O11 (M+H) 467.1989, measured 467.1988. HRMS (ESI) m/z calcd for C33H60N4O11Na (M-Na) 711.4156. Found 711.4141.
Synthesis of Galβ1-3GlcNAcβPro-triazole-C14. Galβ1-3GlcNAcβPro-triazole-C14 was prepared according to FIG. 8D. To the solution of Galβ1-3GlcNAcβProN3 (40 mg, 0.086 mmol) in 5 ml anhydrous MeOH, hexadecyne (95 mg, 0.43 mmol) and DIPEA (33 mg, 0.26 mmol) were added. CuI (17 mg, 0.088 mmol) was then added into the reaction mixture and the resulted solution was stirred at room temperature for 2 days. The reaction mixture was concentrated and the residue was purified by flash column chromatography (EtOAc:MeOH:H2O, 12:2:1) to afford pure Galβ1-3GlcNAcβPro-triazole-C14 (44 mg, 74%). 1H NMR (800 MHz, MeOD) δ 7.84 (s, 1H), 4.60 (d, J=8.8 Hz, 1H), 4.58-4.52 (m, 2H), 4.43 (d, J=8 Hz, 1H), 4.00-3.86 (m, 5H), 3.83-3.80 (m, 3H), 3.70-3.60 (m, 4H), 3.54 (t, J=8.8 Hz, 1H), 3.45-3.43 (m, 1H), 2.78 (t, J=8 Hz, 2H), 2.24-2.20 (m, 2H), 2.12 (s, 3H), 1.78-1.75 (m, 2H), 1.45-1.39 (m, 22), 1.00 (t, J=7.2 Hz, 3H) 13C HMR (200 MHz, MeOH) δ 172.91, 147.85, 121.96, 104.08, 101.00, 76.13, 75.60, 73.19, 70.91, 69.08, 68.82, 68.80, 65.37, 61.21, 61.02, 57.77, 46.54, 31.56, 30.02, 29.27-28.78 (10C), 24.83, 22.22, 21.83, 12.93. HRMS (ESI) m/z calcd for C33H60N4O11Na (M-Na) 711.4156. Found 711.4142.
Synthesis of Galβ1-3GlcNAcαPro-triazole-C14. Galβ1-3GlcNAcαPro-triazole-C14 was prepared according to FIG. 8E. To the solution of Galβ1-3GlcNAcαProN3(50 mg, 0.11 mmol) in 5 ml anhydrous MeOH, hexadecyne (119 mg, 0.54 mmol) and DIPEA (42 mg, 0.32 mmol) were added. CuI (21 mg, 0.11 mmol) was then added into the reaction mixture and the resulted solution was stirred at room temperature for 2 days. The reaction mixture was concentrated and the residue was purified by flash column chromatography (EtOAc:MeOH:H2O, 12:2:1) to afford pure Galβ1-3GlcNAcαPro-triazole-C14 (53 mg, 71%). 1H NMR (800 MHz, MeOD) δ 7.91 (s, 1H), 4.87 (d, J=3.2 Hz, 1H), 4.72-4.63 (m, 2H), 4.58 (d, J=6.4 Hz, 1H), 4.21 (dd, J=3.2 Hz and 10.4 Hz, 1H), 3.98-3.82 (m, 7H), 3.76-3.73 (m, 2H), 3.65-3.64 (m, 2H), 3.58 (t, J=9.6 Hz, 1H), 3.48-3.45 (m, 1H), 2.79 (t, J=7.2 Hz, 2H), 2.34-2.30 (m, 2H), 2.15 (s, 3H), 1.78-1.76 (m, 2H), 1.45-1.39 (m, 22), 1.00 (t, J=7.2 Hz, 3H) 13C HMR (200 MHz, MeOD) δ 172.82, 147.93, 121.90, 103.69, 97.51, 75.51, 73.18, 72.25, 70.95, 69.04, 68.92, 68.89, 63.97, 61.10, 61.04, 52.62, 46.81, 31.59, 29.74, 29.29-28.39 (10C), 24.87, 22.26, 21.62, 13.00. HRMS (ESI) m/z calcd for C33H60N4O11Na (M-Na) 711.4156. Found 711.4141.
Synthesis of Galβ1-3GalNAcαPro-triazole-C14. Galβ1-3GalNAcαPro-triazole-C14 was prepared according to FIG. 8F. To a solution of Galβ1-3GalNAcαProN3 (30 mg, 0.064 mmol) in 5 ml anhydrous MeOH, hexadecyne (72 mg, 0.32 mmol) and DIPEA (25 mg, 0.19 mmol) were added. CuI (12 mg, 0.062 mmol) was then added into the reaction mixture and the resulted solution was stirred at room temperature for 2 days. The reaction mixture was concentrated and the residue was purified by flash column chromatography (EtOAc:MeOH:H2O, 12:2:1) to afford pure Galβ1-3GalNAcαPro-triazole-C14 (31 mg, 69%). 1H NMR (800 MHz, MeOD) δ 7.87 (s, 1H), 4.92 (d, J=4 Hz, 1H), 4.69-4.61 (m, 2H), 4.57 (d, J=8 Hz, 1H), 4.53 (dd, J=3.2 Hz and 10.4 Hz, 1H), 4.29 (m, 1H), 4.03 (dd, J=2.4 Hz and 10.4 Hz, 1H), 3.96 (m, 2H), 3.88-3.82 (m, 5H), 3.68-3.60 (m, 3H), 3.48-3.46 (m, 1H), 2.79 (t, J=7.2 Hz, 2H), 2.34-2.28 (m, 2H), 2.13 (s, 3H), 1.78-1.76 (m, 2H), 1.45-1.39 (m, 22), 1.00 (t, J=7.2 Hz, 3H) 13C HMR (200 MHz, MeOD) δ 172.70, 147.00, 121.76, 104.74, 97.74, 77.56, 75.25, 73.28, 71.12, 70.69, 68.95, 68.73, 64.03, 61.46, 61.16, 48.76, 46.82, 31.56, 29.74, 29.26-28.77 (10C), 24.85, 22.21, 21.52, 12.92. HRMS (ESI) m/z calcd for C33H60N4O11Na (M-Na) 711.4156. Found 711.4154.
Synthesis of GalβPro-triazole-C14. GalβPro-triazole-C14 was synthesized according to FIG. 8G. To the solution of GalβProN3 (30 mg, 0.11 mmol) in 5 ml anhydrous MeOH, hexadecyne (127 mg, 0.57 mmol) and DIPEA (44 mg, 0.34 mmol) were added. CuI (22 mg, 0.11 mmol) was then added into the reaction mixture and the resulted solution was stirred at room temperature for 2 days. The reaction mixture was concentrated and the residue was purified by flash column chromatography (EtOAc:MeOH, 13:2) to afford pure GalβPro-triazole-C 14 (35 mg, 64%). NMR (800 MHz, MeOD) δ 7.89 (s, 1H), 4.64-4.62 (m, 2H), 4.32 (d, J=7.2 Hz, 1H), 4.01-3.98 (m, 1H), 3.95 (d, J=2.4 Hz, 1H), 3.87-3.83 (m, 2H), 3.66-3.58 (m, 4H), 2.78 (t, J=7.2 Hz, 2H), 2.28-2.27 (m, 2H), 1.78-1.76 (m, 2H), 1.45-1.39 (m, 22), 1.00 (t, J=7.2 Hz, 3H). 13C HMR (200 MHz, MeOD) δ 172.70, 147.71, 122.26, 103.57, 75.25, 73.55, 71.11, 68.87, 61.08, 46.59, 31.56, 30.09, 29.24-27.86 (10C), 24.82, 22.22, 12.91. HRMS (ESI) m/z calcd for C25H47N3O6Na (M-Na) 508.3363. Found 508.3342.
Enzymatic Reactions. All reactions were carried out in duplicate for 10 min at 37° C. in a total volume of 10 μl in Tris-HCl (200 mM, pH 8.0) containing an acceptor substrate (2 mM), CMP-Neu5Ac (4 mM), 0.4% Triton X-100, and enzyme (3.6 μg μl−1). Addition of 15 μl of cold 95% ethanol was used to terminate each reaction. All reactions were incubated on ice for 10 min and were then centrifuged for 5 min at 13,000 rpm. The supernatant was transferred to a new vial for CE analysis.
Results.
To test the acceptor substrate specificity of MBP-PmST2-His6, a list of glycosides containing a long hydrocarbon tail (Pro-triazole-C14) including Galβ1-4GlcNAcβPro-triazole-C14 (LacNAcβPro-triazole-C14), Galβ1-3GlcNAcβPro-triazole-C14, Galβ1-3GlcNAcαPro-triazole-C14, Galβ1-3GalNAcαPro-triazole-C14, and GalβPro-triazole-C14 in addition to Galβ1-4GlcβPro-triazole-C14 (LacβPro-triazole-C14) were chemically synthesized and used as potential acceptors for PmST2. As shown in Table 3, β1-4-linked galactosides such as LacNAcβPro-triazole-C14 and LacβPro-triazole-C14 were better acceptor substrates for PmST2 than β1-3-linked galactosides such as Galβ1-3GlcNAcβPro-triazole-C14 (lacto-N-biose or LNB-type structure), Galβ1-3GlcNAcαPro-triazole-C14, Galβ1-3GalNAcαPro-triazole-C14 (galacto-N-biose or GNB-type structure). In addition, the β-galactosylmonosaccharide lipid was a worse PmST2 acceptor than β1-4-linked galactosyldisaccharide lipids but a better acceptor than β1-3-linked galactosyldisaccharide lipids. While GNB is generally found in mucin-type O-GalNAc glycans representing one of the core antigens (core 1 or T antigen), LNB is a well-known component of human milk oligosaccharides, glycoproteins or glycolipids. On the other hand, LacNAc and Lac are commonly presented as the glycan components of bacterial glycolipids such as in Neisseria and Haemophilus. Among all Pro-triazole-C14-containing galactosides tested, LacβPro-triazole-C14 was the best acceptor substrate for PmST2. This means that the natural acceptor of PmST2 may resembles lactosyl lipid the best. Commercially available lactosyl ceramide was also tested but was found not an acceptor for PmST2.







TABLE 3







Acceptor substrate specificity of MBP-PmST2-His6.












% product
%



Acceptor Substrate
formationa
Relativeb














LacβPro-triazole-C14
36.1 ± 1.5
100



LacNAcβPro-triazole-C14
18.5 ± 2.7
51.2



GalβPro-triazole-C14
 9.2 ± 1.3
25.5



Galβ1-3GlcNAcβPro-triazole-C14
<4
<11



(LNBβ)





Galβ1-3GlcNAcαPro-triazole-C14
<2
<6



(LNBα)





Galβ1-3GalNAcαPro-triazole-C14
<2
<6



(GNBα)









All sialyltransferases identified to date have been classified into six glycosyltransferase families GT4, GT29, GT38, GT42, GT52, and GT80 by the Carbohydrate-Active enZymes (CAZy) (http://www.cazy.org/) database based on protein sequence homology. All eukaryotic sialyltransferases and some viral sialyltransferases have been grouped into GT29. Polysialyltransferase (SiaD) from Neisseria meningitidis W135 responsible for the synthesis of capsular polysaccharide belongs to GT4 along with other glycosyltransferases, while α-2,8-polysialyltransferases from E. coli (NeuS) and N meningitides serogroup B strains (SiaD) are grouped into GT38. GT42 includes α-2,3-sialyltransferases (CstI and CstIII) from Campylobacter jejuni, as well as multifunctional α2,3/8-sialyltransferases from Campylobacter jejuni (CstII) or Haemophilus influenzae (Lic3B). An 2,3-sialyltransferase encoded by Haemophilus influenzae lic3A gene and a hypothetic sialyltransferase encoded by Pasteurella multocida Pm1174 gene are also grouped into this GT42 family. Unlike PmST1 encoded by Pm0188 gene analog which belongs to CAZy GT80 family containing multifunctional bacterial α2,3- and or α-2,6-sialyltransferases, PmST2 encoded by Pm0508 gene analog belongs to CAZy GT52 family. This GT52 family also contains characterized α-2,3/6-sialyltransferases from Neisseria meningitides (Lst), Neisseria gonorrhoeae (Lst), Haemophilus influenzae (LsgB), as well as a Salmonella enterica α1,2-glucosyltransferase (WaaH). CpsK, another member of GT52 family and a homolog to the Lst of Haemophilus ducreyi, has also been identified as a putative α2,3-sialylatransferase for the synthesis of sialic acid-terminated capsular polysaccharide of Streptococcus agalactiae (GBS or Group B Streptococcus).
PmST2encoded by gene Pm0508 is in the midst of a locus (Pm0506 to Pm0512) of putative glycosyltransferases. It lines up very well with the so-called lipooligosaccharide synthesis genes (lsg) locus from both H. influenzae and H. ducreyi. Here we demonstrate that PmST2, the Lst from Pasteurella multocida encoded by Pm0508 gene analog, is a novel α2,3-sialyltransferase that can be used for synthesizing sialylglycolipids. Therefore, two of the three potential sialyltransferase gene products of Pasteurella multocida strain Pm70 have now been confirmed to be functional sialyltransferases.
Although sialylated capsular polysaccharide or lipooligosaccharide (LOS) structures have yet been reported for Pasteurella multocida, it appears that Pasteurella multocida has invested significantly for sialic acid metabolism and has been shown to be able to use sialic acid as the sole carbon source. Two sialidases, one sialic acid aldolase/lyase (encoded by gene Pm1715), at least one CMP-sialic acid synthetase (encoded by gene Pm0187) (gene Pm1710 may also encode another putative CMP-sialic acid synthetase), and a possible tripartite ATP-dependent periplasmic (TRAP) sialic acid transporter have been identified in Pm. Furthermore, membrane-associated sialyltransferase activity has been detected in multiple Pm strains and a relatively low molecular weight product resembling LOS may be the possible native acceptor. Sialylation of cell surface oligosaccharides has also been demonstrated vital for the virulence of Pasteurella multocida. Nevertheless, sialylated structures have not been isolated from Pasteurella multocida yet. Therefore, the natural acceptor for PmST2 is currently unknown. Our substrate specificity studies of PmST2 showed that LacβPro-triazole-C14 was the best acceptor among all galactosyl lipids tested. This indicate that the natural acceptor substrate of PmST2 may resembles lactosyl lipid. Two possible transmembrane helices have been identified in PmST2 by hydropathy plot (TMpred). The first one spanning from 1-19 amino acid residues is conserved among all members of GT52 family and the second one spanning from 85-107 amino acid residues has only been found in some GT52 family members. It has yet to be determined whether one of these transmembrane helices or both are involved in the binding to the lipid portion of the glycosyl lipid substrates.
It seems that the presence of multiple sialyltransferase genes is a common feature for members of the Haemophilus-Actinobacillus-Pasteurella (HAP). For example, four sialyltransferase genes (siaA, lic3A, lic3B, and lsgB) have been identified in H influenzae. While Lic3A is an α2,3-sialyltransferase in all H influenzae strains, Lic3B has been confirmed to be a bifunctional α2,3/8-sialyltransferase which only exist in some of the H influenzae strains. Haemophilus ducreyi 35000HP has at least two functional sialyltransferases and at least two functional sialyltransferases have now been confirmed in Pasteurella multocida. 
Compared with H. influenzae (an obligate human microparasite), Pm has a broader host range and has continued to cause a wide range of diseases in animals and humans. Nevertheless, similar to that described for other HAP members such as H. influenzae, H. ducreyi, and H. somnus, Pasteurella multocida seems to acquire sialic acids from the environment or the host through the precursor scavenging sialylation mechanism since it lacks the genes for early steps of de novo sialic acid synthesis. This precursor scavenging pathway could be a common sialylation mechanism for the HAP group.
A previous report indicates that N. meningitidis (MC58 and 406Y) Lst has no stringent metal requirement although the activity can be stimulated by Mg2+(2-fold) or Mn2+(3-fold). Similarly, metal ions are not required for PmST2 and the addition of either MnC12 or MgC12 does not affect the sialyltransferase activity of PmST2 significantly. In comparison, although the α2-3-sialyltransferase activity of PmST1 does not require metal ions and the addition of Mg2+does not stimulate or change the sialyltransferase activity, the activity decreases with the addition of Mn2+. Unlike PmST1 which has multiple functions such as α2,3-sialyltransferase, α2,6-sialyltransferase, α2,3-sialidase, and α2,3-trans-sialidase activities, PmST2 seems to be monofunctional α2,3-sialyltransferase without sialidase activity. This monofunctionality of PmST2 allows efficient synthesis of glycolipids without worrying about product hydrolysis. PmST2 has been used successfully in preparative scale synthesis of sialyllactosyl sphingosine (lyso-GM3). PmST2 thus joins a list of bacterial and mammalian α2,3-sialyltransferases including Neisseria meningitidis α2,3-sialyltransferase, Campylobacter jejuniα2,3-sialyltransferase, porcine submaxillary gland α2,3-sialyltransferase, porcine liver α2,3-sialyltransferase, recombinant rat liver Ga1β1-4G1cNAcα2-3-(N)-sialyltransferase, and several sialyltransferases from marine bacteria for efficient synthesis of lyso-GM3 and its derivatives, which are themselves important probes while can also be used as intermediates for synthesizing more complex gangliosides. With a good expression level in E. coli and the superior α2,3-sialyltransferase activity using lactosyl lipids including lactosyl sphingosine as acceptors, PmST2 is an efficient catalyst for large scale chemoenzymatic synthesis of α2,3-linked sialylglycolipids for elucidating their important biological functions.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.

## Claims

1. A method of preparing a glycolipid product, the method comprising:
forming a reaction mixture comprising an acceptor glycolipid, a donor substrate comprising a sugar moiety and a nucleotide, and a polypeptide selected from the group consisting of:
SEQ ID NO:4 (PmST2),
SEQ ID NO:5 (PmST2-His6), and
SEQ ID NO:6 (MBP-PmST2-His6),
wherein the reaction mixture is formed under conditions sufficient to transfer the sugar moiety from the donor substrate to the acceptor glycolipid, thereby forming the glycolipid product.

2. The method of claim 1, wherein the acceptor glycolipid comprises a galactoside moiety.

3. The method of claim 2, wherein the galactoside moiety is selected from the group consisting of a β1-4 linked galactoside moiety and a β1-3 linked galactoside moiety.

4. The method of claim 2, wherein the acceptor glycolipid comprises a lactoside moiety or an N-acetyl lactosaminide moiety.

5. The method of claim 2, wherein the acceptor glycolipid comprises a Galβ1-3GlcNAc moiety or a Galβ1-3GalNAc moiety.

6. The method of claim 1, wherein the donor substrate comprises a cytidine 5′-monophosphate (CMP)-sialic acid.

7. The method of claim 6, wherein the CMP-sialic acid comprises cytidine 5′-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) or a CMP-Neu5Ac analog.

8. The method of claim 7, wherein the CMP-Neu5Ac or CMP- Neu5Ac analong is prepared prepared by a process comprising forming a reaction mixture comprising a CMP-sialic acid synthetase, cytidine triphosphate, and N-acetylneuraminic acid (Neu5Ac) or a Neu5Ac analog under conditions sufficient to form the CMP-Neu5Ac or CMP-Neu5Ac analog.

9. The method of claim 8, wherein preparing the CMP-Neu5Ac or CMP- Neu5Ac analong and preparing the glycolipid product are performed in one pot.

10. The method of claim 8, wherein the Neu b5c or Neu5analog is prepared by a process comprising forming a reaction mixture comprising a sialic acid aldolase, pyruvic acid or derivatives thereof, and N-acetylmannosamine or derivatives thereof under conditions sufficient to form the Neu5Ac or Neu5Ac analog.

11. The method of claim 10, wherein preparing the Neu5Ac or Neu5Ac anlalog, preparing the CMP-Neu5c or CMP-Neu5Ac analog, and preparing the glycolipid product are performed in one pot.

12. The method of any of claims 1-11, wherein the glycolipid product is an α-2,3-linked sialylglycolipid.

13. The method of claim 12, wherein the α-2,3-linked sialylglycolipid is Neu5Acα2-3lactosyl sphingosine (lyso-GM3) or a derivative thereof.