Assembly of B4GALT1/ST6GAL1 heteromers in the Golgi membranes involves lateral interactions via highly charged surface domains

Core fucose is an N-glycan structure synthesized by α1,6-fucosyltransferase 8 (FUT8) localized to the Golgi apparatus and critically regulates the functions of various glycoproteins. However, how FUT8 activity is regulated in cells remains largely unclear. At the luminal side and uncommon for Golgi proteins, FUT8 has an Src homology 3 (SH3) domain, which is usually found in cytosolic signal transduction molecules and generally mediates protein-protein interactions in the cytosol. However, the SH3 domain has not been identified in other glycosyltransferases, suggesting that FUT8's functions are selectively regulated by this domain. In this study, using truncated FUT8 constructs, immunofluorescence staining, FACS analysis, cell-surface biotinylation, proteomics, and LC-electrospray ionization MS analyses, we reveal that the SH3 domain is essential for FUT8 activity both in cells and in vitro and identified His-535 in the SH3 domain as the critical residue for enzymatic activity of FUT8. Furthermore, we found that although FUT8 is mainly localized to the Golgi, it also partially localizes to the cell surface in an SH3-dependent manner, indicating that the SH3 domain is also involved in FUT8 trafficking. Finally, we identified ribophorin I (RPN1), a subunit of the oligosaccharyltransferase complex, as an SH3-dependent binding protein of FUT8. RPN1 knockdown decreased both FUT8 activity and core fucose levels, indicating that RPN1 stimulates FUT8 activity. Our findings indicate that the SH3 domain critically controls FUT8 catalytic activity and localization and is required for binding by RPN1, which promotes FUT8 activity and core fucosylation.

Section: Roles Of the Fut8 Sh3 Domainmentioning

confidence: 99%

The SH3 domain in the fucosyltransferase FUT8 controls FUT8 activity and localization and is essential for core fucosylation

Tomida

Takata

Hirata

et al. 2020

“…Formation of such glycosyltransferase complexes has been well documented but often underappreciated, as many researchers tend to study glycosyltransferases individually ( 67 ). For example, human β1,4-galactosyltransferase 1 ( B4GALT1 ) and β-galactoside α2,6-sialyltransferase ( ST6GAL1 ) form heterodimers in the Golgi using lateral interactions by highly charged surface domains ( 68 ). Using Bimolecular Fluorescence Complementation Assay (BiFC), we found before that the human Gb3/CD77 synthase forms heterodimers with β1,4-galactosyltransferase 6 ( B4GALT6 ), which is specific toward GSLs (unpublished results).…”

Section: Discussionmentioning

confidence: 99%

Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry

Szymczak-Kulus

Weidler

Bereznicka

et al. 2021

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, P k ) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli . A single amino acid substitution (p.Q211E) ramps up the enzyme’s promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galβ1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.

“…In general, on N-Cdh from ΔPOMGnT1, N -glycans with a lower degree of galactosylation and sialylation, along with a slight increase in the abundance of bisecting GlcNAc, are observed. These changes are driven by the transcriptional regulation of specific glycosyltransferases, such as B4GALT1 and ST6GAL1, which act as heteromeric complex in the successive addition of terminal ß1,4-linked galactose and α2,6-linked sialic acid to N -glycans ( 42 ); or probably ST3GAL3 and ST3GAL4, which most likely contribute to the addition of α2,3-linked sialic acid to N -glycans of N-Cdh ( 24 ). Most interestingly, the changes in N -glycosylation are site-specific and especially site N402 in the EC3 domain contains less branched complex glycans, which have been reported to favor cell–cell adhesion.…”

Section: Discussionmentioning

confidence: 99%

Glycosyltransferase POMGNT1 deficiency strengthens N-cadherin-mediated cell–cell adhesion

Noor

Hoffmann

Rinis

et al. 2021

Defects in protein O -mannosylation lead to severe congenital muscular dystrophies collectively known as α-dystroglycanopathy. A hallmark of these diseases is the loss of the O -mannose-bound matriglycan on α-dystroglycan, which reduces cell adhesion to the extracellular matrix. Mutations in protein O -mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O -mannosyl glycans, have mainly been associated with muscle–eye–brain (MEB) disease. In addition to defects in cell–extracellular matrix adhesion, aberrant cell–cell adhesion has occasionally been observed in response to defects in POMGNT1. However, specific molecular consequences of POMGNT1 deficiency on cell–cell adhesion are largely unknown. We used POMGNT1 knockout HEK293T cells and fibroblasts from an MEB patient to gain deeper insight into the molecular changes in POMGNT1 deficiency. Biochemical and molecular biological techniques combined with proteomics, glycoproteomics, and glycomics revealed that a lack of POMGNT1 activity strengthens cell–cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N- glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. Moreover, in POMGNT1-deficient cells, ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable with the epithelial–mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell–cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or α-dystroglycanopathy in general and suggests that the impact of changes in O -mannosylation on N -glycosylation has been underestimated.