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. Our idea was to force equivalent stoichiometry of the two proteins in the cells in order to reproduce the behavior of the SLC35A2/SLC35A3 complexes in the Golgi membrane. The hybrid proteins were able to fully restore glycosylation in the double knockout. In contrast, the expression of SLC35A3 alone in these cells improved galactosylation only to a limited extent. Our study shows that the proper glycosylation requires a balanced cooperation between SLC35A2 and SLC35A3.

SLC35A2 deficiency reduces protein levels of core 1 ß-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. Here we report that UDP-galactose transporter (UGT; SLC35A2) associates with core 1 ß-1,3-galactosyltransferase 1 (C1GalT1; T-synthase). This provides the first example of an interaction between an enzyme that acts exclusively in the O-glycosylation pathway and an NST. We also found that SLC35A2 associated with the C1GalT1-specific chaperone Cosmc, and that the endogenous Cosmc was localized in both the endoplasmic reticulum and Golgi apparatus of wild-type HEK293T cells. Furthermore, in SLC35A2-deficient cells protein levels of C1GalT1 and Cosmc were decreased and their Golgi localization was less pronounced. Finally, we identified SLC35A2 as a novel molecular target for the antifungal agent itraconazole. Based on our findings we propose that NSTs may contribute to the stabilization of their interaction partners and help them to achieve target localization in the cell, most likely by facilitating their assembly into larger functional units.

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. Mutations in the SLC35A1 gene cause a subtype of congenital disorder of glycosylation (CDG). Over the past several years, heterologous complexes formed in the Golgi membrane by some SLC35A subfamily members and functionally related glycosyltransferases have been reported. However, to date no interaction between SLC35A1 and a sialyltransferase has been identified. In this study we attempted to clarify the role of SLC35A1 in α2,3 sialylation of N-glycans. We showed that SLC35A1 associates with ST3Gal4, the main α2,3-sialyltransferase acting on N-glycans. This phenomenon is compromised by the E196K (but not T156R) mutation in the SLC35A1 gene. We also demonstrated that the E196K mutant is less efficient in restoring N-glycan sialylation upon expression in the SLC35A1 knockout cells. On the basis of our findings, we propose that the interaction between SLC35A1 and ST3Gal4 may be important for proper sialylation.

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. 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.

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. According to a number of reports, impaired glycosylation may trigger the process of epithelial-to-mesenchymal transition (EMT). We therefore examined whether the loss of SLC35A2 activity would promote EMT in a non-malignant epithelial cell line. For this purpose, we knocked out the SLC35A2 gene in Madin-Darby canine kidney (MDCK) cells. The resulting clones adopted an elongated, spindle-shaped morphology and showed impaired cell-cell adhesion. Using qPCR and western blotting, we revealed down-regulation of E-cadherin in the knockouts, while the fibronectin and vimentin levels were elevated. Moreover, the knockout cells displayed reorganization of vimentin intermediate filaments and altered subcellular distribution of a vimentin-binding protein, formiminotransferase cyclodeaminase (FTCD). Furthermore, depletion of SLC35A2 triggered Golgi compaction. Finally, the SLC35A2 knockouts displayed increased motility and invasiveness. In conclusion, SLC35A2-deficient MDCK cells showed several hallmarks of EMT. Our findings point to a novel role for SLC35A2 as a gatekeeper of the epithelial phenotype.

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. The resulting cells were cultured in the presence/absence of exogenous fucose and mannose, followed by examination of fucosylation potential and nucleotide sugar levels. We found that cells displayed low but detectable levels of fucosylation in the absence of SLC35C1. Strikingly, we show that defects in fucosylation were almost completely reversed upon treatment with millimolar concentrations of fucose. Furthermore, we show that even if fucose was supplemented at nanomolar concentrations, it was still incorporated into glycans by these knockout cells. We also found that the SLC35C1-independent transport preferentially utilized GDP-fucose from the salvage pathway over the de novo biogenesis pathway as a source of this substrate. Taken together, our results imply that the Golgi systems of GDP-fucose transport discriminate between substrate pools obtained from different metabolic pathways, which suggests a functional connection between nucleotide sugar transporters and nucleotide sugar synthases.

The Solute Carrier MFSD1 Decreases the Activation Status of ß1 Integrin and Thus Tumor Metastasis.

Solute carriers are increasingly recognized as participating in a plethora of pathologies, including cancer. We describe here the involvement of the orphan solute carrier Major Facilitator Superfamily Domain-containing protein 1 (MFSD1) in the regulation of tumor cell migration. Loss of MFSD1 enabled higher levels of metastasis in experimental and spontaneous metastasis mouse models. We identified an increased migratory potential in MFSD1-/- tumor cells which was mediated by increased focal adhesion turnover, reduced stability of mature inactive ß1 integrin, and the resulting increased integrin activation index. We show that MFSD1 promoted recycling to the cell surface of endocytosed inactive ß1 integrin and thereby protected ß1 integrin from proteolytic degradation; this led to dampening of the integrin activation index. Furthermore, downregulation of MFSD1 expression was observed during the early steps of tumorigenesis, and higher MFSD1 expression levels correlate with a better cancer patient prognosis. In sum, we describe a requirement for endolysosomal MFSD1 in efficient ß1 integrin recycling to suppress tumor cell dissemination.

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. For this purpose, each of the SLC35A2-A4 proteins was used as a bait in four independent pull-down experiments and the identity of the immunoprecipitated material was discovered using MS techniques. From the candidate list obtained, we selected a few for which the interaction was confirmed in vitro using the NanoBiT system, a split luciferase-based luminescent technique. NSTs have been shown to interact with two ATPases (ATP2A2, ATP2C1), Golgi pH regulator B (GPR89B) and calcium channel (TMCO1), which may reflect the regulation of glycosylation by ion homeostasis, and with basigin (BSG). Our findings provide a starting point for the NST interaction network discovery in order to better understand how glycosylation is regulated and linked to other cellular processes. SIGNIFICANCE: Despite the facts that nucleotide sugar transporters are a key component of the protein glycosylation machinery, and deficiencies in their activity underlie serious metabolic diseases, biology, function and regulation of these essential proteins remain enigmatic. In this study we have advanced the field by identifying sets of new potential interaction partners for UDP-galactose transporter (SLC35A2), UDP-N-acetylglucosamine transporter (SLC35A3) and an orphan transporter SLC35A4 of yet undefined role. Several of these new interactions were additionally confirmed in vitro using the NanoBiT system, a split luciferase complementation assay. This work is also significant in that it addresses the overall challenge of discovering membrane protein interaction partners by a detailed comparison of 4 different co-immunoprecipitation strategies and by custom sample preparation and data processing workflows.

Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase is expendable.

N-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the effects of N-glycans on the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Galα1→4Gal (Gb3 and the P1 antigen) and Galα1→4GalNAc disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and its absence did not reduce, but, surprisingly, even increased the activity of the enzyme. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localized in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the endoplasmic reticulum. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants resulted from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling the biological activity of proteins.

Modified secreted alkaline phosphatase as an improved reporter protein for N-glycosylation analysis.

N-glycosylation is a common posttranslational modification of proteins in eukaryotic cells. The modification is often analyzed in cells which are able to produce extracellular, glycosylated proteins. Here we report an improved method of the use of genetically modified, secreted alkaline phosphatase (SEAP) as a reporter glycoprotein which may be used for glycoanalysis. Additional N-glycosylation sites introduced by site-directed mutagenesis significantly increased secretion of the protein. An improved purification protocol of recombinant SEAP from serum or serum-free media is also proposed. The method enables fast and efficient separation of reporter glycoprotein from a relatively small amount of medium (0.5-10 ml) with a high recovery level. As a result, purified SEAP was ready for enzymatic de-glycosylation without buffer exchange, sample volume reductions or other procedures, which are usually time-consuming and may cause partial loss of the reporter glycoprotein.

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

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, Pk) 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→4Galß1→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.

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. Heterologous interactions between different NSTs have also been reported. To verify whether the technique is suitable for studying the phenomenon of NST heteromerization, we tested it against a combination of the two Golgi-resident NSTs that have been previously shown to associate by several other means. The luciferase complementation assay appears to be particularly suitable for studying interactions between Golgi-resident membrane proteins, as it does not require high expression levels, which often trigger protein mislocalization and increase the risk of false positives.

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.

N-glycosylation of the human ß1,4-galactosyltransferase 4 is crucial for its activity and Golgi localization.

ß1,4-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.

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.

Prevotella intermedia produces two proteins homologous to Porphyromonas gingivalis HmuY but with different heme coordination mode.

As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensable for life and enables the microorganism to survive and multiply at the infection site. This oral pathogenic bacterium uses a newly discovered novel hmu heme uptake system with a leading role played by the HmuY hemophore-like protein, responsible for acquiring heme and increasing virulence of this periodontopathogen. We demonstrated that Prevotella intermedia produces two HmuY homologs, termed PinO and PinA. Both proteins were produced at higher mRNA and protein levels when the bacterium grew under low-iron/heme conditions. PinO and PinA bound heme, but preferentially under reducing conditions, and in a manner different from that of the P. gingivalis HmuY. The analysis of the three-dimensional structures confirmed differences between apo-PinO and apo-HmuY, mainly in the fold forming the heme-binding pocket. Instead of two histidine residues coordinating heme iron in P. gingivalis HmuY, PinO and PinA could use one methionine residue to fulfill this function, with potential support of additional methionine residue/s. The P. intermedia proteins sequestered heme only from the host albumin-heme complex under reducing conditions. Our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme coordination modes. The newer data presented in this manuscript on HmuY homologs produced by P. intermedia sheds more light on the novel mechanism of heme uptake, could be helpful in discovering their biological function, and in developing novel therapeutic approaches.

Novel Insights into Selected Disease-Causing Mutations within the SLC35A1 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 SLC35A1 gene encoding the CMP-sialic acid transporter (CST) have been reported to date. In this study we examined how specific mutations in the SLC35A1 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). The effects of single mutations and the combination of T156R and E196K mutations on the CST's functionality was examined separately in CST-deficient HEK293T cells. As shown by microscopic studies, none of the CDG-causing mutations affected the protein's proper localization in the Golgi apparatus. Cellular glycophenotypes were characterized using lectins, structural assignment of N- and O-glycans and analysis of glycolipids. Single Q101H, T156R and E196K mutants were able to partially restore sialylation in CST-deficient cells, and the deleterious effect of a single T156R or E196K mutation on the CST functionality was strongly enhanced upon their combination. We also revealed differences in the ability of CST variants to form dimers. The results of this study improve our understanding of the molecular background of SLC35A1-CDG cases.

Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function.

Porphyromonas gingivalis, a keystone pathogen of chronic periodontitis, uses ferric uptake regulator homolog (PgFur) to regulate production of virulence factors. This study aimed to characterize PgFur protein in regard to its structure-function relationship. We experimentally identified the 5' mRNA sequence encoding the 171-amino-acid-long PgFur protein in the A7436 strain and examined this PgFur version as a full-length protein. PgFur protein did not bind to the canonical Escherichia coli Fur box, but the wild-type phenotype of the mutant Δpgfur strain was restored partially when expression of the ecfur gene was induced from the native pgfur promoter. The full-length PgFur protein contained one zinc atom per protein monomer, but did not bind iron, manganese, or heme. Single cysteine substitutions of CXXC motifs resulted in phenotypes similar to the mutant Δpgfur strain. The modified proteins were produced in E. coli at significantly lower levels, were highly unstable, and did not bind zinc. The pgfur gene was expressed at the highest levels in bacteria cultured for 24 h in the absence of iron and heme or at higher levels in bacteria cultured for 10 h in the presence of protoporphyrin IX source. No influence of high availability of Fe2+, Zn2+, or Mn2+ on pgfur gene expression was observed. Two chromosomal mutant strains producing protein lacking 4 (pgfurΔ4aa) or 13 (pgfurΔ13aa) C-terminal amino acid residues were examined in regard to importance of the C-terminal lysine-rich region. The pgfurΔ13aa strain showed a phenotype typical for the mutant Δpgfur strain, but both the wild-type PgFur protein and its truncated version bound zinc with similar ability. The Δpgfur mutant strain produced higher amounts of HmuY protein compared with the wild-type strain, suggesting compromised regulation of its expression. Potential PgFur ligands, Fe2+, Mn2+, Zn2+, PPIX, or serum components, did not influence HmuY production in the Δpgfur mutant strain. The mutant pgfurΔ4aa and pgfurΔ13aa strains exhibited affected HmuY protein production. PgFur, regardless of the presence of the C-terminal lysine-rich region, bound to the hmu operon promoter. Our data suggest that cooperation of PgFur with partners/cofactors and/or protein/DNA modifications would be required to accomplish its role played in an in vivo multilayer regulatory network.

PgFur participates differentially in expression of virulence factors in more virulent A7436 and less virulent ATCC 33277 Porphyromonas gingivalis strains.

BACKGROUND: Porphyromonas gingivalis is considered a keystone pathogen responsible for chronic periodontitis. Although several virulence factors produced by this bacterium are quite well characterized, very little is known about regulatory mechanisms that allow different strains of P. gingivalis to efficiently survive in the hostile environment of the oral cavity, a typical habitat characterized by low iron and heme concentrations. The aim of this study was to characterize P. gingivalis Fur homolog (PgFur) in terms of its role in production of virulence factors in more (A7436) and less (ATCC 33277) virulent strains. RESULTS: Expression of a pgfur depends on the growth phase and iron/heme concentration. To better understand the role played by the PgFur protein in P. gingivalis virulence under low- and high-iron/heme conditions, a pgfur-deficient ATCC 33277 strain (TO16) was constructed and its phenotype compared with that of a pgfur A7436-derived mutant strain (TO6). In contrast to the TO6 strain, the TO16 strain did not differ in the growth rate and hemolytic activity compared with the ATCC 33277 strain. However, both mutant strains were more sensitive to oxidative stress and they demonstrated changes in the production of lysine- (Kgp) and arginine-specific (Rgp) gingipains. In contrast to the wild-type strains, TO6 and TO16 mutant strains produced larger amounts of HmuY protein under high iron/heme conditions. We also demonstrated differences in production of glycoconjugates between the A7436 and ATCC 33277 strains and we found evidence that PgFur protein might regulate glycosylation process. Moreover, we revealed that PgFur protein plays a role in interactions with other periodontopathogens and is important for P. gingivalis infection of THP-1-derived macrophages and survival inside the cells. Deletion of the pgfur gene influences expression of many transcription factors, including two not yet characterized transcription factors from the Crp/Fnr family. We also observed lower expression of the CRISPR/Cas genes. CONCLUSIONS: We show here for the first time that inactivation of the pgfur gene exerts a different influence on the phenotype of the A7436 and ATCC 33277 strains. Our findings further support the hypothesis that PgFur regulates expression of genes encoding surface virulence factors and/or genes involved in their maturation.

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.