Abstract
β-1,4-Galactosyltransferase 1 (B4GALT1) and ST6 β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) catalyze the successive addition of terminal β-1,4-linked galactose and α-2,6-linked sialic acid to N-glycans. Their exclusive interaction in the Golgi compartment is a prerequisite for their full catalytic activity, whereas a lack of this interaction is associated with cancers and hypoxia. To date, no structural information exists that shows how glycosyltransferases functionally assemble with each other. Using molecular docking simulations to predict interaction surfaces, along with mutagenesis screens and high-throughput FRET analyses in live cells to validate these predictions, we show here that B4GALT1 and ST6GAL1 interact via highly charged noncatalytic surfaces, leaving the active sites exposed and accessible for donor and acceptor substrate binding. Moreover, we found that the assembly of ST6GAL1 homomers in the endoplasmic reticulum before ST6GAL1 activation in the Golgi utilizes the same noncatalytic surface, whereas B4GALT1 uses its active-site surface for assembly, which silences its catalytic activity. Last, we show that the homomeric and heteromeric B4GALT1/ST6GAL1 complexes can assemble laterally in the Golgi membranes without forming cross-cisternal contacts between enzyme molecules residing in the opposite membranes of each Golgi cisterna. Our results provide detailed mechanistic insights into the regulation of glycosyltransferase interactions, the transitions between B4GALT1 and ST6GAL1 homo- and heteromers in the Golgi, and cooperative B4GALT1/ST6GAL1 function in N-glycan synthesis.
Highlights
-1,4-Galactosyltransferase 1 (B4GALT1) and ST6 -galactoside ␣-2,6-sialyltransferase 1 (ST6GAL1) catalyze the successive addition of terminal -1,4 –linked galactose and ␣-2,6 – linked sialic acid to N-glycans
Using molecular docking simulations to predict interaction surfaces, along with mutagenesis screens and highthroughput FRET analyses in live cells to validate these predictions, we show here that B4GALT1 and ST6GAL1 interact via highly charged noncatalytic surfaces, leaving the active sites exposed and accessible for donor and acceptor substrate binding
Glycosyltransferases and glycosidases in the endoplasmic reticulum (ER)2 and the Golgi apparatus are responsible for enzymatic addition and removal of sugar moieties to generate linear or branched glycan chains found in proteins and lipids [1]
Summary
We presented the first human homodimeric crystal structure of B4GALT1 and showed that the interaction takes place at the surface surrounding the active site of the enzyme through concerted loop movements involving amino acids Met340 and His343 [13]. The heterodimer and homodimer docking models were overlapping and involved several shared salt bridges on the same surface (Fig. 2C) Based on these models, a mutated construct was designed (Fig. 2D) to remove seven charged amino acids. Cells expressing the interaction mutants (B4GALT1.Ncat and ST6GAL1.Ncat) did not show such an increase, suggesting either that the mutations rendered the B4GALT1 enzyme inactive despite not localizing in the active site or that the low activity may be caused by the loss of its ability to interact with ST6GAL1 To investigate whether B4GALT1/ST6GAL1 heteromers utilize the cis or trans configuration for interaction, we expressed truncated enzyme constructs separately in COS-7 cells and measured the FRET signals for comparison. We show negatively and positively charged residues, respectively, and in yellow shared residues between the homodimeric (model I) and the heterodimeric (model II)
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