Mutational analysis of the cytoplasmic domain of β1,4-galactosyltransferase I: influence of phosphorylation on cell surface expression

Abstract

β1,4-Galactosyltransferase I (GalT I) exists in two subcellular compartments where it performs two distinct functions. The majority of GalT I is localized in the Golgi complex where it participates in glycoprotein biosynthesis; however, a small portion of GalT I is expressed on the cell surface where it functions as a matrix receptor by binding terminal N-acetylglucosamine residues on extracellular glycoside ligands. The GalT I polypeptide occurs in two alternate forms that differ only in the length of their cytoplasmic domains. It is thought that the longer cytoplasmic domain is responsible for GalT I function as a cell surface receptor because of its ability to associate with the detergent-insoluble cytoskeleton. In this study, we demonstrate that the long GalT I cytoplasmic and transmembrane domains are capable of targeting a reporter protein to the plasma membrane, whereas the short cytoplasmic and transmembrane domains do not have this property. The surface-localized GalT I reporter protein partitions with the detergent-insoluble pool, a portion of which co-fractionates with caveolin-containing lipid rafts. Site-directed mutagenesis of the cytoplasmic domain identified a requirement for serine and threonine residues for cell surface expression and function. Replacing either the serine or threonine with aspartic acid reduces surface expression and function, whereas substitution with neutral alanine has no effect on surface expression or function. These results suggest that phosphorylation negatively regulates GalT I function as a surface receptor. Consistent with this, phosphorylation of the endogenous, full-length GalT I inhibits its stable expression on the cell surface. Thus, the 13 amino acid extension unique to the long GalT I isoform is required for GalT I expression on the cell surface, the function of which is regulated by phosphorylation.

Sugar-free frosting, a homolog of SAD kinase, drives neural-specific glycan expression in the Drosophila embryo

“…Finally, glycosyltransferase phosphorylation has been described in vitro, but evidence for a functional consequence of this modification is currently lacking (Gallagher et al, 2001;Hathaway et al, 2003;Ma et al, 1999). The hypothesis most consistent with our results is that Sff modulates glycan processing by influencing the compartmentation of glycan processing within the Golgi apparatus.…”

Mouse oviduct-specific glycoprotein is an egg-associated ZP3-independent sperm-adhesion ligand

“…Several studies have reported alterations in lipid raft composition during sperm capacitation that are thought to be a prerequisite for sperm binding to the egg coat (Cross, 2004;Bou Khalil et al, 2006). GalT1 has been shown to locate to lipid rafts in somatic cells (Hathaway et al, 2003), although it is still unclear whether GalT1 is present within lipid rafts on sperm as well. If so, then the loss of GalT1 might disrupt or alter the presentation of the EBM components within the lipid raft, leading to reduced affinity for egg coat ligands, including ZP3 and OGP.…”

Characterization of a Novel Ubiquitin-Conjugating Enzyme That Regulates β1,4-Galactosyltransferase-1 in Embryonic Stem Cells

“…These data suggest that GalT1-associated GTAP may instead affect the internalization of cell surface GalT1 through ubiquitination of surrounding membrane proteins in coated pits or caveolae. Partial support for this hypothesis is that a small fraction of cell surface GalT1 has been cofractionated with caveolin in lipid rafts [21]. Furthermore, long GalT1 has been shown to associate with Src-suppressed C kinase substrate (SSeCKS), a previously defined kinase and cytoskeleton scaffolding protein important for agonist-induced internalization and resensitization of G-protein-linked receptors [20,35].…”

“…A HindIII/SacI fragment containing the gene of interest was then ligated into the multiple cloning site of pAcGFP1-C2 expression vector (BD Biosciences). Expression vectors harboring cDNA coding for GFP fusion proteins of GalT1 long and short cytoplasmic domains, GFP-24-amino-acid-long cytoplasmic domain (LCD 24 ) and GFP-SCD 11 were produced, as previously described [21].…”