Recombinant soluble beta-1,4-galactosyltransferases expressed in Saccharomyces cerevisiae. Purification, characterization and comparison with human enzyme.
beta-1,4-Galactosyltransferase (Gal-T, EC 2.4.1.38) transfers galactose (Gal) from UDP-Gal to N-acetyl-D-glucosamine or a derivative GlcNAc-R. Soluble Gal-T, purified from human breast milk, was shown to be very heterogeneous by isoelectric focusing (IEF). In order to produce sufficient homogeneous enzyme for three-dimensional analysis, the human enzyme (hGal-T) has been expressed in Saccharomyces cerevisiae, production scaled up to 187 U recombinant Gal-T (rGal-T) and purified. The purification protocol was based on chromatography on concanavalin-A-Sepharose followed by affinity chromatographies on GlcNAc-Sepharose and alpha-lactalbumin-Sepharose. Analysis by SDS/PAGE revealed hyperglycosylation at the single N-glycosylation site, preventing recognition by antibodies. Analysis by IEF revealed considerable heterogeneity of rGal-T. The N-glycan could be removed by treatment with endoglycosidase H (endo H). The N-deglycosylated form of rGal-T retained full activity and showed only three isoforms by IEF analysis. Then we abolished the single N-glycosylation consensus sequence by site-directed mutagenesis changing Asn69-->Asp. The soluble mutated enzyme (N-deglycosylated rGal-T) was expressed in S. cerevisiae and its production scaled up to 60 U.N-deglycosylated rGal-T was purified to electrophoretic homogeneity. When analyzed by IEF, N-deglycosylated rGal-T was resolved in two bands. The O-glycans could be removed by jack bean alpha-mannosidase treatment and the completely deglycosylated Gal-T appeared homogeneous by IEF. The kinetic parameters of N-deglycosylated rGal-T were shown not to differ to any significant extent from those of the hGal-T. No significant changes in CD spectra were observed between hGal-T and N-deglycosylated rGal-T. Light-scattering analysis revealed dimerization of both enzymes. These data indicate that N-deglycosylated rGal-T was correctly folded, homogeneous and thus suitable for crystallization experiments.
Malissard
M
M
Institute of Physiology, University of Zürich, Switzerland.
Borsig
L
L
Di Marco
S
S
Grütter
M G
MG
Kragl
U
U
Wandrey
C
C
Berger
E G
EG
D001665:Binding Sites; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005285:Fermentation; D006031:Glycosylation; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D007768:Lactalbumin; D008895:Milk, Human / Q000201:enzymology*; D008970:Molecular Weight; D009237:N-Acetyllactosamine Synthase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D007768:Lactalbumin; D009237:N-Acetyllactosamine Synthase