Subcellular imaging of MGAT1/MGAT2 homo- and heteromers in living cells using bioluminescence microscopy.

Protein-protein interactions (PPIs) play fundamental roles in many biological processes including the functioning of glycosylation machineries present in the endoplasmic reticulum (ER) and Golgi apparatus of mammalian cells. For the last couple of years, we have been successfully employing the most advanced version of the split luciferase complementation assay, termed NanoBiT, to demonstrate PPIs between solute carrier 35 (SLC35) family members with nucleotide sugar transporting activity and functionally related glycosyltransferases. NanoBiT has several unmatched advantages as compared with other strategies for studying PPIs.

The glycosylation defect in solute carrier SLC35A2/SLC35A3 double knockout cells is rescued by SLC35A2-SLC35A3 and SLC35A3-SLC35A2 hybrids.

SLC35A2 and SLC35A3 are homologous proteins with postulated nucleotide sugar transporting activities. Unlike SLC35A2, whose specificity for UDP-Gal is well-established, the UDP-GlcNAc transporting activity initially attributed to SLC35A3 has recently been put into question. In this study, we constructed two hybrid proteins (SLC35A2-SLC35A3 and SLC35A3-SLC35A2) and expressed them in a previously generated SLC35A2/SLC35A3 double knockout HEK293T cell line.

SLC35A2 deficiency reduces protein levels of core 1 β-1,3-galactosyltransferase 1 (C1GalT1) and its chaperone Cosmc and affects their subcellular localization.

Nucleotide sugar transporters (NSTs) are multitransmembrane proteins, localized in the Golgi apparatus and/or endoplasmic reticulum, which provide glycosylation enzymes with their substrates. It has been demonstrated that NSTs may form complexes with functionally related glycosyltransferases, especially in the N-glycosylation pathway. However, potential interactions of NSTs with enzymes mediating the biosynthesis of mucin-type O-glycans have not been addressed to date.

An interaction between SLC35A1 and ST3Gal4 is differentially affected by CDG-causing mutations in the SLC35A1 gene.

The sialylation of glycoconjugates is performed by a variety of sialyltransferases using CMP-sialic acid (CMP-Sia) as a substrate. Sialylation requires the translocation of CMP-Sia across the Golgi membranes. This function has been assigned to SLC35A1, the only CMP-Sia transporter identified to date.

Delivery of Nucleotide Sugars to the Mammalian Golgi: A Very Well (un)Explained Story.

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.

SLC35A2 Deficiency Promotes an Epithelial-to-Mesenchymal Transition-like Phenotype in Madin-Darby Canine Kidney Cells.

In mammalian cells, SLC35A2 delivers UDP-galactose for galactosylation reactions that take place predominantly in the Golgi lumen. Mutations in the corresponding gene cause a subtype of a congenital disorder of glycosylation (SLC35A2-CDG). Although more and more patients are diagnosed with SLC35A2-CDG, the link between defective galactosylation and disease symptoms is not fully understood.

Incorporation of fucose into glycans independent of the GDP-fucose transporter SLC35C1 preferentially utilizes salvaged over de novo GDP-fucose.

Mutations in the SLC35C1 gene encoding the Golgi GDP-fucose transporter are known to cause leukocyte adhesion deficiency II. However, improvement of fucosylation in leukocyte adhesion deficiency II patients treated with exogenous fucose suggests the existence of an SLC35C1-independent route of GDP-fucose transport, which remains a mystery. To investigate this phenomenon, we developed and characterized a human cell-based model deficient in SLC35C1 activity.

Identification of novel potential interaction partners of UDP-galactose (SLC35A2), UDP-N-acetylglucosamine (SLC35A3) and an orphan (SLC35A4) nucleotide sugar transporters.

Nucleotide sugar transporters (NSTs) are ER and Golgi-resident members of the solute carrier 35 (SLC35) family which supply substrates for glycosylation by exchanging lumenal nucleotide monophosphates for cytosolic nucleotide sugars. Defective NSTs have been associated with congenital disorders of glycosylation (CDG), however, molecular basis of many types of CDG remains poorly characterized. To better understand the biology of NSTs, we identified potential interaction partners of UDP-galactose transporter (SLC35A2), UDP-N-acetylglucosamine transporter (SLC35A3) and an orphan nucleotide sugar transporter SLC35A4 of to date unassigned specificity.

Novel Insights into Selected Disease-Causing Mutations within the Gene Encoding the CMP-Sialic Acid Transporter.

Congenital disorders of glycosylation (CDG) are a group of rare genetic and metabolic diseases caused by alterations in glycosylation pathways. Five patients bearing CDG-causing mutations in the gene encoding the CMP-sialic acid transporter (CST) have been reported to date. In this study we examined how specific mutations in the gene affect the protein's properties in two previously described SLC35A1-CDG cases: one caused by a substitution (Q101H) and another involving a compound heterozygous mutation (T156R/E196K).

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay.

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous complexes formed by nucleotide sugar transporters (NSTs). These ER- and Golgi-resident multitransmembrane proteins carry the cytoplasmically synthesized nucleotide sugars across organelle membranes to supply enzymes that mediate glycosylation with their substrates. NSTs exist as dimers and/or higher oligomers.

Biosynthesis of GlcNAc-rich - and -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.

N-glycosylation of the human β1,4-galactosyltransferase 4 is crucial for its activity and Golgi localization.

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.

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.

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 gene in the HepG2 cell line to study a potential role of this protein in glycosylation.

Lysine at position 329 within a C-terminal dilysine motif is crucial for the ER localization of human SLC35B4.

SLC35B4 belongs to the solute carrier 35 (SLC35) family whose best-characterized members display a nucleotide sugar transporting activity. Using an experimental model of HepG2 cells and indirect immunofluorescent staining, we verified that SLC35B4 was localized to the endoplasmic reticulum (ER). We demonstrated that dilysine motif, especially lysine at position 329, is crucial for the ER localization of this protein in human cells and therefore one should use protein C-tagging with caution.

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 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.

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 β-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).

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.

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.

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.

Gallium(III), cobalt(III) and copper(II) protoporphyrin IX exhibit antimicrobial activity against Porphyromonas gingivalis by reducing planktonic and biofilm growth and invasion of host epithelial cells.

Porphyromonas gingivalis acquires heme for growth, and initiation and progression of periodontal diseases. One of its heme acquisition systems consists of the HmuR and HmuY proteins. This study analyzed the antimicrobial activity of non-iron metalloporphyrins against P.

Subcellular localization of UDP-GlcNAc, UDP-Gal and SLC35B4 transporters.

The mechanisms of transport and distribution of nucleotide sugars in the cell remain unclear. In an attempt to further characterize nucleotide sugar transporters (NSTs), we determined the subcellular localization of overexpressed epitope-tagged canine UDP-GlcNAc transporter, human UDP-Gal transporter splice variants (UGT1 and UGT2), and human SLC35B4 transporter splice variants (longer and shorter version) by indirect immunofluorescence using an experimental model of MDCK wild-type and MDCK-RCA(r) mutant cells. Our studies confirmed that the UDP-GlcNAc transporter was localized to the Golgi apparatus only and its localization was independent of the presence of endogenous UDP-Gal transporter.

Comparative analysis of involvement of UGT1 and UGT2 splice variants of UDP-galactose transporter in glycosylation of macromolecules in MDCK and CHO cell lines.

Nucleotide sugar transporters deliver nucleotide sugars into the Golgi apparatus and endoplasmic reticulum. This study aimed to further characterize mammalian UDP-galactose transporter (UGT) in MDCK and CHO cell lines. MDCK-RCA(r) and CHO-Lec8 mutant cell lines are defective in UGT transporter, although they exhibit some level of galactosylation.

Overexpression of UDP-GlcNAc transporter partially corrects galactosylation defect caused by UDP-Gal transporter mutation.

Nucleotide sugar transporters deliver substrates for glycosyltransferases into the endoplasmic reticulum and the Golgi apparatus. We demonstrated that overexpression of UDP-GlcNAc transporter (NGT) in MDCK-RCA(r) and CHO-Lec8 mutant cells defective in UDP-Gal transporter (UGT) restored galactosylation of N-glycans. NGT overexpression resulted in decreased transport of UDP-GlcNAc into the Golgi vesicles.