Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. N-acetylglucosamine, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T and HepG2 cell lines including wild type cells, SLC35A2 knockouts, SLC35A3 knockouts, and double knock-out cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knock-out CHO cells, only limited changes were observed - GlcNAc was still incorporated into N-glycans but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knock-out HEK293T cells, UDP-GlcNAc transport was significantly decreased, but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knock-out HEK293T and HepG2 cells GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between wild type and SLC35A3 knock-out cells, as well as between SLC35A2 knock-out and double knock-out cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.
β1,4-galactosyltransferase 4 (B4GalT4) is one of seven B4GalTs that belong to CAZy glycosyltransferase family 7 and transfer galactose to growing sugar moieties of proteins, glycolipids, glycosaminoglycans as well as single sugar for lactose synthesis. Herein, we identify two asparagine-linked glycosylation sites in B4GalT4. We found that mutation of one site (Asn220) had greater impact on enzymatic activity while another (Asn335) on Golgi localization and presence of N-glycans at both sites is required for production of stable and enzymatically active protein and its secretion. Additionally, we confirm B4GalT4 involvement in synthesis of keratan sulfate (KS) by generating A375 B4GalT4 knock-out cell lines that show drastic decrease in the amount of KS proteoglycans and no significant structural changes in N- and O-glycans. We show that KS decrease in A375 cells deficient in B4GalT4 activity can be rescued by overproduction of either partially or fully glycosylated B4GalT4 but not with N-glycan-depleted B4GalT4 version.
Nucleotide sugars (NSs) serve as substrates for glycosylation reactions. The majority of these compounds are synthesized in the cytoplasm, whereas glycosylation occurs in the endoplasmic reticulum (ER) and Golgi lumens, where catalytic domains of glycosyltransferases (GTs) are located. Therefore, translocation of NS across the organelle membranes is a prerequisite. This process is thought to be mediated by a group of multi-transmembrane proteins from the SLC35 family, i.e., nucleotide sugar transporters (NSTs). Despite many years of research, some uncertainties/inconsistencies related with the mechanisms of NS transport and the substrate specificities of NSTs remain. Here we present a comprehensive review of the NS import into the mammalian Golgi, which consists of three major parts. In the first part, we provide a historical view of the experimental approaches used to study NS transport and evaluate the most important achievements. The second part summarizes various aspects of knowledge concerning NSTs, ranging from subcellular localization up to the pathologies related with their defective function. In the third part, we present the outcomes of our research performed using mammalian cell-based models and discuss its relevance in relation to the general context.
Mutations in the SLC35C1 gene, encoding the Golgi GDP-fucose transporter, cause leukocyte adhesion deficiency II (LADII). Fucosylation improvement in LADII patients treated with fucose suggests the existence of an SLC35C1-independent route of GDP-fucose transport, which still remains a mystery. Here, we developed and characterized a human cell-based model deficient in the SLC35C1 activity. The knockout cells displayed low but detectable levels of fucosylation. Strikingly, the fucosylation defect was almost completely reversed upon treatment with millimolar concentrations of fucose. Even if fucose was supplemented at nanomolar concentrations, it was still incorporated into glycans by the knockout cells. We also found that the SLC35C1-independent transport preferred the salvage pathway over the de novo pathway as a source of GDP-fucose. Our results imply that the Golgi systems of GDP-fucose transport discriminate between the substrate pools obtained from different metabolic pathways, which suggests a functional connection between nucleotide sugar transporters and nucleotide sugar synthetases.
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