Bovine β 1 4 galactosyltransferase

Cytidine 5′-diphosphate (CDP), cytidine, adenosine 5′-diphosphate (ADP), and 6`-sialyllactose were purchased from GeneChem (Daejeon, Korea). Peptide-N-glycosidase F (PNGase F) and graphitized carbon column were obtained from New England Biolabs (Ipswich, MA, USA) and Alltech (Lexington, MA, USA) respectively. Asialo, galactosylated, biantennary oligosaccharide (G2 glycan), calf intestinal alkaline phosphatase, and biotinylated Sambucus nigra (SNA) lectin were purchased from Prozyme (Hayward, CA), Takara (Tokyo, Japan), and Ey Laboratories (San Mateo, CA, USA) respectively. Solvents for high-performance liquid chromatography (HPLC) including acetonitrile and water were purchased from Burdick and Jackson (Muskegon, MI, USA). 2-Aminobenzoic acid (AA), sodium cyanoborohydride, acetic acid, tetrahydrofuran, triethylamine, trifluoroacetic acid (TFA), CMP-NeuAc, adenosine 5′-triphosphate (ATP), cytidine 5′-triphosphate (CTP), cytidine 5′-monophosphate (CMP), GDP-galactose, bovine β(1,4)-galactosyltransferase, asialofetuin and other reagents (unless stated otherwise) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

UDP-¹³C₆-galactose was prepared by incubation of ¹³C₆-galactose (Cambridge Isotope Laboratories, Tewksbury, MA, USA) with galactokinase (Sigma-Aldrich, Vienna, Austria). The galactose 1-phosphate was converted to the nucleotide sugar in the presence of UPD-glucose by human galactose 1-phosphate uridylyltransferase, which was recombinantly expressed in Escherichia coli BL21 and purified via its His₆-tag (a yeast enzyme with this activity is now commercially available from Sigma-Aldrich ). The UDP-¹³C₆-galactose was finally purified by PGC chromatography with use of a slightly alkaline buffer as described in [41 (link)].
This UDP-¹³C₆-galactose was then used to regalactosylate fibrin glycans previously degalactosylated by Aspergillus oryzae β-galactosidase [33 (link)]. Bovine β-1,4-galactosyltransferase (Sigma-Aldrich, Vienna, Austria) was used in 50 mM tris(hydroxymethyl)aminomethane–HCl buffer for transfer of ¹³C₆-galactose. The thus produced ^C13A⁴A⁴ (^C13G2) was sialylated with α-2,6-sialyltransferase (see earlier) to arrive at singly and doubly sialylated (^C13G2S2) standards with a 12-Da increment compared with the natural versions.
A trisialylated standard was prepared by partial degalactosylation of fetuin asialo N-glycans, isolation of the G2 form, followed by incorporation of just one ¹³C₆-galactose and α-2,6-sialylation.