scholarly journals Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3

2020 ◽  
Vol 295 (48) ◽  
pp. 16445-16463 ◽  
Author(s):  
Bozena Szulc ◽  
Paulina Sosicka ◽  
Dorota Maszczak-Seneczko ◽  
Edyta Skurska ◽  
Auhen Shauchuk ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Hepg2 Cells ◽  
Double Knockout ◽  
Nucleotide Sugar ◽  
Sugar Transporters ◽  
Nucleotide Sugars ◽  
Nucleotide Sugar Transporters ◽  
Hepg2 Cell Lines ◽  
Nucleotide Sugar Transporter ◽  
Qualitative Changes

Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, 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 WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout 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-knockout 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-knockout 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 WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout 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.

scholarly journals Structural basis for substrate specificity and regulation of nucleotide sugar transporters in the lipid bilayer

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Joanne L. Parker ◽  
Robin A. Corey ◽  
Phillip J. Stansfeld ◽  
Simon Newstead
Keyword(s):  
Secretory Pathway ◽  
Membrane Lipids ◽  
Regulatory Elements ◽  
Structural Basis ◽  
Nucleotide Sugar ◽  
Dimer Interface ◽  
Sugar Transporters ◽  
Nucleotide Sugars ◽  
Nucleotide Sugar Transporters ◽  
Insight Into

Abstract Nucleotide sugars are the activated form of monosaccharides used by glycosyltransferases during glycosylation. In eukaryotes the SLC35 family of solute carriers are responsible for their selective uptake into the Endoplasmic Reticulum or Golgi apparatus. The structure of the yeast GDP-mannose transporter, Vrg4, revealed a requirement for short chain lipids and a marked difference in transport rate between the nucleotide sugar and nucleoside monophosphate, suggesting a complex network of regulatory elements control transport into these organelles. Here we report the crystal structure of the GMP bound complex of Vrg4, revealing the molecular basis for GMP recognition and transport. Molecular dynamics, combined with biochemical analysis, reveal a lipid mediated dimer interface and mechanism for coordinating structural rearrangements during transport. Together these results provide further insight into how SLC35 family transporters function within the secretory pathway and sheds light onto the role that membrane lipids play in regulating transport across the membrane.

scholarly journals Functional Redundancy between Two Caenorhabditis elegans Nucleotide Sugar Transporters with a Novel Transport Mechanism

2007 ◽  
Vol 282 (38) ◽  
pp. 27970-27975 ◽  
Author(s):  
Carolina E. Caffaro ◽  
Carlos B. Hirschberg ◽  
Patricia M. Berninsone
Keyword(s):  
Amino Acid ◽  
Caenorhabditis Elegans ◽  
Transport Mechanism ◽  
Functional Redundancy ◽  
Nucleotide Sugar ◽  
Sugar Transporters ◽  
Amino Acid Divergence ◽  
Nucleotide Sugars ◽  
Nucleotide Sugar Transporters ◽  
Multicellular Organisms

Transporters of nucleotide sugars regulate the availability of these substrates required for glycosylation reactions in the lumen of the Golgi apparatus and play an important role in the development of multicellular organisms. Caenorhabditis elegans has seven different sugars in its glycoconjugates, although 18 putative nucleotide sugar transporters are encoded in the genome. Among these, SQV-7, SRF-3, and CO3H5.2 exhibit partially overlapping substrate specificity and expression patterns. We now report evidence of functional redundancy between transporters CO3H5.2 and SRF-3. Reducing the activity of the CO3H5.2 gene product by RNA interference (RNAi) in SRF-3 mutants results in oocyte accumulation and abnormal gonad morphology, whereas comparable RNAi treatment of wild type or RNAi hypersensitive C. elegans strains does not cause detectable defects. We hypothesize this genetic enhancement to be a mechanism to ensure adequate glycoconjugate biosynthesis required for normal tissue development in multicellular organisms. Furthermore, we show that transporters SRF-3 and CO3H5.2, which are closely related in the phylogenetic tree, share a simultaneous and independent substrate transport mechanism that is different from the competitive one previously demonstrated for transporter SQV-7, which shares a lower amino acid sequence identity with CO3H5.2 and SRF-3. Therefore, different mechanisms for transporting multiple nucleotide sugars may have evolved parallel to transporter amino acid divergence.

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