GLUT1 is a highly efficient L-fucose transporter.

Understanding L-fucose metabolism is important because it is used as a therapy for several congenital disorders of glycosylation. Exogenous L-fucose can be activated and incorporated directly into multiple N- and O-glycans via the fucose salvage/recycling pathway. However, unlike for other monosaccharides, no mammalian L-fucose transporter has been identified. Here, we functionally screened nearly 140 annotated transporters and identified GLUT1 (SLC2A1) as an L-fucose transporter. We confirmed this assignment using multiple approaches to alter GLUT1 function, including chemical inhibition, siRNA knockdown, and gene KO. Collectively, all methods demonstrate that GLUT1 contributes significantly to L-fucose uptake and its utilization at low micromolar levels. Surprisingly, millimolar levels of D-glucose do not compete with L-fucose uptake. We also show macropinocytosis, but not other endocytic pathways, can contribute to L-fucose uptake and utilization. In conclusion, we determined that GLUT1 functions as the previously missing transporter component in mammalian L-fucose metabolism.

A mutation in SLC37A4 causes a dominantly inherited congenital disorder of glycosylation characterized by liver dysfunction.

SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423∗), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423∗) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans.

Homozygous truncating variant in MAN2A2 causes a novel congenital disorder of glycosylation with neurological involvement.

BACKGROUND: Enzymes of the Golgi implicated in N-glycan processing are critical for brain development, and defects in many are defined as congenital disorders of glycosylation (CDG). Involvement of the Golgi mannosidase, MAN2A2 has not been identified previously as causing glycosylation defects. METHODS: Exome sequencing of affected individuals was performed with Sanger sequencing of the MAN2A2 transcript to confirm the variant. N-glycans were analysed in patient-derived lymphoblasts to determine the functional effects of the variant. A cell-based complementation assay was designed to assess the pathogenicity of identified variants using MAN2A1/MAN2A2 double knock out HEK293 cell lines. RESULTS: We identified a multiplex consanguineous family with a homozygous truncating variant p.Val1101Ter in MAN2A2. Lymphoblasts from two affected brothers carrying the same truncating variant showed decreases in complex N-glycans and accumulation of hybrid N-glycans. On testing of this variant in the developed complementation assay, we see the complete lack of complex N-glycans. CONCLUSION: Our findings show that pathogenic variants in MAN2A2 cause a novel autosomal recessive CDG with neurological involvement and facial dysmorphism. Here, we also present the development of a cell-based complementation assay to assess the pathogenicity of MAN2A2 variants, which can also be extended to MAN2A1 variants for future diagnosis.

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

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.

Therapeutic Monosaccharides: Looking Back, Moving Forward.

In this review, we focus on the metabolism of mammalian glycan-associated monosaccharides, where the vast majority of our current knowledge comes from research done during the 1960s and 1970s. Most monosaccharides enter the cell using distinct, often tissue specific transporters from the SLC2A family. If not catabolized, these monosaccharides can be activated to donor nucleotide sugars and used for glycan synthesis. Apart from exogenous and dietary sources, all monosaccharides and their associated nucleotide sugars can be synthesized de novo, using mostly glucose to produce all nine nucleotide sugars present in human cells. Today, monosaccharides are used as treatment options for a small number of rare genetic disorders and even some common conditions. Here, we cover therapeutic applications of these sugars and highlight biochemical gaps that must be revisited as we go forward.

Analysis of homologous and heterologous interactions between UDP-galactose transporter and beta-1,4-galactosyltransferase 1 using NanoBiT.

Split luciferase complementation assay is one of the approaches enabling identification and analysis of protein-protein interactions in vivo. The NanoBiT technology is the most recent improvement of this strategy. Nucleotide sugar transporters and glycosyltransferases of the Golgi apparatus are the key players in glycosylation. Here we demonstrate the applicability of the NanoBiT system for studying homooligomerization of these proteins. We also report and discuss a novel heterologous interaction between UDP-galactose transporter and beta-1,4-galactosyltransferase 1.

N-acetylglucosaminyltransferases and nucleotide sugar transporters form multi-enzyme-multi-transporter assemblies in golgi membranes in vivo.

Branching and processing of N-glycans in the medial-Golgi rely both on the transport of the donor UDP-N-acetylglucosamine (UDP-GlcNAc) to the Golgi lumen by the SLC35A3 nucleotide sugar transporter (NST) as well as on the addition of the GlcNAc residue to terminal mannoses in nascent N-glycans by several linkage-specific N-acetyl-glucosaminyltransferases (MGAT1-MGAT5). Previous data indicate that the MGATs and NSTs both form higher order assemblies in the Golgi membranes. Here, we investigate their specific and mutual interactions using high-throughput FRET- and BiFC-based interaction screens. We show that MGAT1, MGAT2, MGAT3, MGAT4B (but not MGAT5) and Golgi alpha-mannosidase IIX (MAN2A2) form several distinct molecular assemblies with each other and that the MAN2A2 acts as a central hub for the interactions. Similar assemblies were also detected between the NSTs SLC35A2, SLC35A3, and SLC35A4. Using in vivo BiFC-based FRET interaction screens, we also identified novel ternary complexes between the MGATs themselves or between the MGATs and the NSTs. These findings suggest that the MGATs and the NSTs self-assemble into multi-enzyme/multi-transporter complexes in the Golgi membranes in vivo to facilitate efficient synthesis of complex N-glycans.

SLC35A2-CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals.

Pathogenic de novo variants in the X-linked gene SLC35A2 encoding the major Golgi-localized UDP-galactose transporter required for proper protein and lipid glycosylation cause a rare type of congenital disorder of glycosylation known as SLC35A2-congenital disorders of glycosylation (CDG; formerly CDG-IIm). To date, 29 unique de novo variants from 32 unrelated individuals have been described in the literature. The majority of affected individuals are primarily characterized by varying degrees of neurological impairments with or without skeletal abnormalities. Surprisingly, most affected individuals do not show abnormalities in serum transferrin N-glycosylation, a common biomarker for most types of CDG. Here we present data characterizing 30 individuals and add 26 new variants, the single largest study involving SLC35A2-CDG. The great majority of these individuals had normal transferrin glycosylation. In addition, expanding the molecular and clinical spectrum of this rare disorder, we developed a robust and reliable biochemical assay to assess SLC35A2-dependent UDP-galactose transport activity in primary fibroblasts. Finally, we show that transport activity is directly correlated to the ratio of wild-type to mutant alleles in fibroblasts from affected individuals.

SLC35A5 Protein-A Golgi Complex Member with Putative Nucleotide Sugar Transport Activity.

Solute carrier family 35 member A5 (SLC35A5) is a member of the SLC35A protein subfamily comprising nucleotide sugar transporters. However, the function of SLC35A5 is yet to be experimentally determined. In this study, we inactivated the SLC35A5 gene in the HepG2 cell line to study a potential role of this protein in glycosylation. Introduced modification affected neither N- nor O-glycans. There was also no influence of the gene knock-out on glycolipid synthesis. However, inactivation of the SLC35A5 gene caused a slight increase in the level of chondroitin sulfate proteoglycans. Moreover, inactivation of the SLC35A5 gene resulted in the decrease of the uridine diphosphate (UDP)-glucuronic acid, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine Golgi uptake, with no influence on the UDP-galactose transport activity. Further studies demonstrated that SLC35A5 localized exclusively to the Golgi apparatus. Careful insight into the protein sequence revealed that the C-terminus of this protein is extremely acidic and contains distinctive motifs, namely DXEE, DXD, and DXXD. Our studies show that the C-terminus is directed toward the cytosol. We also demonstrated that SLC35A5 formed homomers, as well as heteromers with other members of the SLC35A protein subfamily. In conclusion, the SLC35A5 protein might be a Golgi-resident multiprotein complex member engaged in nucleotide sugar transport.

An insight into the orphan nucleotide sugar transporter SLC35A4.

SLC35A4 has been classified in the SLC35A subfamily based on amino acid sequence homology. Most of the proteins belonging to the SLC35 family act as transporters of nucleotide sugars. In this study, the subcellular localization of endogenous SLC35A4 was determined via immunofluorescence staining, and it was demonstrated that SLC35A4 localizes mainly to the Golgi apparatus. In silico topology prediction suggests that SLC35A4 has an uneven number of transmembrane domains and its N-terminus is directed towards the Golgi lumen. However, an experimental assay refuted this prediction: SLC35A4 has an even number of transmembrane regions with both termini facing the cytosol. In vivo interaction analysis using the FLIM-FRET approach revealed that SLC35A4 neither forms homomers nor associates with other members of the SLC35A subfamily except SLC35A5. Additional assays demonstrated that endogenous SLC35A4 is 10 to 40nm proximal to SLC35A2 and SLC35A3. To determine SLC35A4 function SLC35A4 knock-out cells were generated with the CRISPR-Cas9 approach. Although no significant changes in glycosylation were observed, the introduced mutation influenced the subcellular distribution of the SLC35A2/SLC35A3 complexes. Additional FLIM-FRET experiments revealed that overexpression of SLC35A4-BFP together with SLC35A3 and the SLC35A2-Golgi splice variant negatively affects the interaction between the two latter proteins. The results presented here strongly indicate a modulatory role for SLC35A4 in intracellular trafficking of SLC35A2/SLC35A3 complexes.

UDP-galactose (SLC35A2) and UDP-N-acetylglucosamine (SLC35A3) Transporters Form Glycosylation-related Complexes with Mannoside Acetylglucosaminyltransferases (Mgats).

UDP-galactose transporter (UGT; SLC35A2) and UDP-N-acetylglucosamine transporter (NGT; SLC35A3) form heterologous complexes in the Golgi membrane. NGT occurs in close proximity to mannosyl (α-1,6-)-glycoprotein ß-1,6-N-acetylglucosaminyltransferase (Mgat5). In this study we analyzed whether NGT and both splice variants of UGT (UGT1 and UGT2) are able to interact with four different mannoside acetylglucosaminyltransferases (Mgat1, Mgat2, Mgat4B, and Mgat5). Using an in situ proximity ligation assay, we found that all examined glycosyltransferases are in the vicinity of these UDP-sugar transporters both at the endogenous level and upon overexpression. This observation was confirmed via the FLIM-FRET approach for both NGT and UGT1 complexes with Mgats. This study reports for the first time close proximity between endogenous nucleotide sugar transporters and glycosyltransferases. We also observed that among all analyzed Mgats, only Mgat4B occurs in close proximity to UGT2, whereas the other three Mgats are more distant from UGT2, and it was only possible to visualize their vicinity using proximity ligation assay. This strongly suggests that the distance between these protein pairs is longer than 10 nm but at the same time shorter than 40 nm. This study adds to the understanding of glycosylation, one of the most important post-translational modifications, which affects the majority of macromolecules. Our research shows that complex formation between nucleotide sugar transporters and glycosyltransferases might be a more common phenomenon than previously thought.

HmuY is an important virulence factor for Porphyromonas gingivalis growth in the heme-limited host environment and infection of macrophages.

Porphyromonas gingivalis, the main etiologic agent and key pathogen responsible for initiation and progression of chronic periodontitis, is a haem auxotroph, and the uptake of this compound is essential for its survival and the ability to establish an infection. The aim of this study was to examine the role of a hemophore-like HmuY protein in P. gingivalis growth and infection of macrophages. Inactivation of the hmuY gene caused reduced P. gingivalis growth in vitro in the presence of serum as a heme sole source, as well as in vivo co-cultures with THP-1-derived macrophages. This resulted in diminished invasion efficiency of macrophages by live bacteria lacking functional hmuY gene. Both features were partially restored after addition of the purified HmuY protein, which was internalized when added either together with the hmuY mutant strain or alone to macrophage cultures. We conclude that HmuY is an important virulence factor of P. gingivalis for infection of macrophages in a heme-limited host environment.

UDP-N-acetylglucosamine transporter (SLC35A3) regulates biosynthesis of highly branched N-glycans and keratan sulfate.

SLC35A3 is considered the main UDP-N-acetylglucosamine transporter (NGT) in mammals. Detailed analysis of NGT is restricted because mammalian mutant cells defective in this activity have not been isolated. Therefore, using the siRNA approach, we developed and characterized several NGT-deficient mammalian cell lines. CHO, CHO-Lec8, and HeLa cells deficient in NGT activity displayed a decrease in the amount of highly branched tri- and tetraantennary N-glycans, whereas monoantennary and diantennary ones remained unchanged or even were accumulated. Silencing the expression of NGT in Madin-Darby canine kidney II cells resulted in a dramatic decrease in the keratan sulfate content, whereas no changes in biosynthesis of heparan sulfate were observed. We also demonstrated for the first time close proximity between NGT and mannosyl (α-1,6-)-glycoprotein ß-1,6-N-acetylglucosaminyltransferase (Mgat5) in the Golgi membrane. We conclude that NGT may be important for the biosynthesis of highly branched, multiantennary complex N-glycans and keratan sulfate. We hypothesize that NGT may specifically supply ß-1,3-N-acetylglucosaminyl-transferase 7 (ß3GnT7), Mgat5, and possibly mannosyl (α-1,3-)-glycoprotein ß-1,4-N-acetylglucosaminyltransferase (Mgat4) with UDP-GlcNAc.

Short N-terminal region of UDP-galactose transporter (SLC35A2) is crucial for galactosylation of N-glycans.

UDP-galactose transporter (UGT) and UDP-N-acetylglucosamine transporter (NGT) form heterologous complexes in the Golgi apparatus (GA) membrane. We aimed to identify UGT region responsible for galactosylation of N-glycans. Chimeric proteins composed of human UGT and either NGT or CMP-sialic acid transporter (CST) localized to the GA, and all but UGT/CST chimera corrected galactosylation defect in UGT-deficient cell lines, although at different efficiency. Importantly, short N-terminal region composed of 35 N-terminal amino-acid residues of UGT was crucial for galactosylation of N-glycans. The remaining molecule must be derived from NGT not CST, confirming that the role played by UGT and NGT is coupled.

UDP-Gal/UDP-GlcNAc chimeric transporter complements mutation defect in mammalian cells deficient in UDP-Gal transporter.

The role of UDP-galactose transporter (UGT; SLC35A2) and UDP-N-acetylglucosamine transporter (NGT; SLC35A3) in glycosylation of macromolecules may be coupled and either of the transporters may partially replace the function played by its partner. The aim of this study was to construct chimeric transporters composed of the N-terminal portion of human NGT and the C-terminal portion of human UGT1 or UGT2 (NGT-UGT1 or NGT-UGT2, respectively), as well as of the N-terminal portion of UGT and C-terminal portion of NGT (UGT-NGT), overexpress them stably in UGT-deficient CHO-Lec8 and MDCK-RCA(r) cells, and characterize their involvement in glycosylation. Two chimeric proteins, NGT-UGT1 and NGT-UGT2, did not overexpress properly. In contrast, UGT-NGT chimeric protein was successfully overexpressed and localized properly to the Golgi apparatus. UGT-NGT chimeric transporter delivered UDP-Gal to the Golgi vesicles of UGT-deficient cells, which resulted in the increased content of galactosylated structures to such an extent that the wild-type phenotypes were completely restored. Our data further support our hypothesis that UGT and NGT cooperate in the UDP-Gal delivery for glycosyltransferases located in the Golgi apparatus. In a wider context, the results gained in this study add to our understanding of glycosylation, one of the basic posttranslational modifications, which affects the majority of macromolecules.

Chemical Therapies for Congenital Disorders of Glycosylation.

Congenital disorders of glycosylation (CDG) are ultrarare, genetically and clinically heterogeneous metabolic disorders. Although the number of identified CDG is growing rapidly, there are few therapeutic options. Most treatments involve dietary supplementation with monosaccharides or other precursors. These approaches are relatively safe, but in many cases, the molecular and biochemical underpinnings are incomplete. Recent studies demonstrate that yeast, worm, fly, and zebrafish models of CDG are powerful tools in screening repurposed drugs, ushering a new avenue to search for novel therapeutic options. Here we present a perspective on compounds that are currently in use for CDG treatment or have a potential to be applied as therapeutics in the near future.

Origin of cytoplasmic GDP-fucose determines its contribution to glycosylation reactions.

Biosynthesis of macromolecules requires precursors such as sugars or amino acids, originating from exogenous/dietary sources, reutilization/salvage of degraded molecules, or de novo synthesis. Since these sources are assumed to contribute to one homogenous pool, their individual contributions are often overlooked. Protein glycosylation uses monosaccharides from all the above sources to produce nucleotide sugars required to assemble hundreds of distinct glycans. Here, we demonstrate that cells identify the origin/heritage of the monosaccharide, fucose, for glycosylation. We measured the contribution of GDP-fucose from each of these sources for glycan synthesis and found that different fucosyltransferases, individual glycoproteins, and linkage-specific fucose residues identify and select different GDP-fucose pools dependent on their heritage. This supports the hypothesis that GDP-fucose exists in multiple, distinct pools, not as a single homogenous pool. The selection is tightly regulated since the overall pool size remains constant. We present novel perspectives on monosaccharide metabolism, which may have a general applicability.

Golgi Acidification by NHE7 Regulates Cytosolic pH Homeostasis in Pancreatic Cancer Cells.

Cancer cells reprogram their metabolism to meet elevated energy demands and favor glycolysis for energy production. This boost in glycolytic flux supports proliferation, but also generates acid in the form of hydrogen ions that must be eliminated from the cytoplasm to maintain the alkaline intracellular pH (pHi) associated with transformation. To cope with acid production, tumor cells employ ion transport systems, including the family of sodium-hydrogen exchangers (NHE). Here, we identify NHE7 as a novel regulator of pHi in pancreatic ductal adenocarcinoma (PDAC). We determine that NHE7 suppression causes alkalinization of the Golgi, leading to a buildup of cytosolic acid that diminishes tumor cell fitness mainly through the dysregulation of actin. Importantly, NHE7 knockdown in vivo leads to the abrogation of tumor growth. These results identify Golgi acidification as a mechanism to control pHi and point to the regulation of pHi as a possible therapeutic vulnerability in PDAC. SIGNIFICANCE: NHE7 regulates cytosolic pH through Golgi acidification, which points to the Golgi as a "proton sink" for metabolic acid. Disruption of cytosolic pH homeostasis via NHE7 suppression compromises PDAC cell viability and tumor growth.See related commentary by Ward and DeNicola, p. 768.This article is highlighted in the In This Issue feature, p. 747.

In Situ Proximity Ligation Assay (PLA) Analysis of Protein Complexes Formed Between Golgi-Resident, Glycosylation-Related Transporters and Transferases in Adherent Mammalian Cell Cultures.

In situ proximity ligation assay (PLA) is a novel, revolutionary technique that can be employed to visualize protein complexes in fixed cells and tissues. This approach enables demonstration of close (i.e., up to 40 nm) proximity between any two proteins of interest that can be detected using a pair of specific antibodies that have been raised in distinct species. Primary antibodies bound to the target proteins are subsequently recognized by two PLA probes, i.e., secondary antibodies conjugated with oligonucleotides that anneal when brought into close proximity in the presence of two connector oligonucleotides and a DNA ligase forming a circular DNA molecule. In the next step, the resulting circular DNA is amplified by a rolling circle polymerase. Finally, fluorescent oligonucleotide probes hybridize to complementary fragments of the amplified DNA molecule, forming a typical, spot-like pattern of PLA signal that reflects subcellular localization of protein complexes. Here we describe the use of in situ PLA in adherent cultures of mammalian cells in order to visualize interactions between Golgi-resident, functionally related glycosyltransferases and nucleotide sugar transporters relevant to N-glycan biosynthesis.

UDP-N-acetylglucosamine transporter and UDP-galactose transporter form heterologous complexes in the Golgi membrane.

UDP-galactose transporter (UGT; SLC35A2) and UDP-N-acetylglucosamine transporter (NGT; SLC35A3) are evolutionarily related. We hypothesize that their role in glycosylation may be coupled through heterologous complex formation. Coimmunoprecipitation analysis and FLIM-FRET measurements performed on living cells showed that NGT and UGT form complexes when overexpressed in MDCK-RCA(r) cells. We also postulate that the interaction of NGT and UGT may explain the dual localization of UGT2. For the first time we demonstrated in vivo homodimerization of the NGT nucleotide sugar transporter. In conclusion, we suggest that NGT and UGT function in glycosylation is combined via their mutual interaction.