Insights into the regulation of cellular Mn2+ homeostasis via TMEM165.

Golgi cation homeostasis is known to be crucial for many cellular processes including vesicular fusion events, protein secretion, as well as for the activity of Golgi glycosyltransferases and glycosidases. TMEM165 was identified in 2012 as the first cation transporter related to human glycosylation diseases, namely the Congenital Disorders of Glycosylation (CDG). Interestingly, divalent manganese (Mn) supplementation has been shown to suppress the observed glycosylation defects in TMEM165-deficient cell lines, thus suggesting that TMEM165 is involved in cellular Mn homeostasis. This paper demonstrates that the origin of the Golgi glycosylation defects arises from impaired Golgi Mn homeostasis in TMEM165-depleted cells. We show that Mn supplementation fully rescues the Mn content in the secretory pathway/organelles of TMEM165-depleted cells and hence the glycosylation process. Strong cytosolic and organellar Mn accumulations can also be observed in TMEM165- and SPCA1-depleted cells upon incubation with increasing Mn concentrations, thus demonstrating the crucial involvement of these two proteins in cellular Mn homeostasis. Interestingly, our results show that the cellular Mn homeostasis maintenance in control cells is correlated with the presence of TMEM165 and that the Mn-detoxifying capacities of cells, through the activity of SPCA1, rely on the Mn-induced degradation mechanism of TMEM165. Finally, this paper highlights that TMEM165 is essential in secretory pathway/organelles Mn homeostasis maintenance to ensure both Golgi glycosylation enzyme activities and cytosolic Mn detoxification.

N-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sda Synthase B4GALNT2.

The Sda carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human B4GALNT2 gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sda and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual N-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type N-glycan. We explored the influence of this N-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an N-glycan on each monomer corroborated these findings and suggested that N-glycosylation of each B4GALNT2 isoform controlled their biological activity.

Analysis of the proximal promoter of the human colon-specific B4GALNT2 (Sda synthase) gene: B4GALNT2 is transcriptionally regulated by ETS1.

BACKGROUND: The Sda antigen and corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in normal colonic mucosa and are down-regulated to a variable degree in colon cancer tissues. Although their expression profile is well studied, little is known about the underlying regulatory mechanisms. METHODS: To clarify the molecular basis of Sda expression in the human gastrointestinal tract, we investigated the transcriptional regulation of the human B4GALNT2 gene. The proximal promoter region was delineated using luciferase assays and essential trans-acting factors were identified through transient overexpression and silencing of several transcription factors. RESULTS: A short cis-regulatory region restricted to the -72 to +12 area upstream of the B4GALNT2 short-type transcript variant contained the essential promoter activity that drives the expression of the human B4GALNT2 regardless of the cell type. We further showed that B4GALNT2 transcriptional activation mostly requires ETS1 and to a lesser extent SP1. CONCLUSIONS: Results presented herein are expected to provide clues to better understand B4GALNT2 regulatory mechanisms.

B4GALNT2 Controls Sda and SLex Antigen Biosynthesis in Healthy and Cancer Human Colon.

The Sda carbohydrate antigen and the corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in human normal colonic mucosa and are down-regulated to variable degrees in colon cancer. On the other hand, the tumor associated antigen SLex is not detected in the healthy colon and is upregulated in colon cancer. High level of B4GALNT2 gene expression appears to be a good marker of prognosis in colon cancer; however, the molecular mechanisms regulating these carbohydrate antigens' expression are still poorly understood. We review here the most recent progress made towards understanding this balanced expression of blood group carbohydrate epitopes Sda and SLex . In particular in recent years, we have attained a better understanding of genetic and epigenetic regulation of the B4GALNT2 gene and of the subcellular fate of B4GALNT2 isoforms.

Role of Sialyl-O-Acetyltransferase CASD1 on GD2 Ganglioside O-Acetylation in Breast Cancer Cells.

The O-acetylated form of GD2, almost exclusively expressed in cancerous tissues, is considered to be a promising therapeutic target for neuroectoderm-derived tumors, especially for breast cancer. Our recent data have shown that 9-O-acetylated GD2 (9-OAcGD2) is the major O-acetylated ganglioside species in breast cancer cells. In 2015, Baumann et al. proposed that Cas 1 domain containing 1 (CASD1), which is the only known human sialyl-O-acetyltransferase, plays a role in GD3 O-acetylation. However, the mechanisms of ganglioside O-acetylation remain poorly understood. The aim of this study was to determine the involvement of CASD1 in GD2 O-acetylation in breast cancer. The role of CASD1 in OAcGD2 synthesis was first demonstrated using wild type CHO and CHOΔCasd1 cells as cellular models. Overexpression using plasmid transfection and siRNA strategies was used to modulate CASD1 expression in SUM159PT breast cancer cell line. Our results showed that OAcGD2 expression was reduced in SUM159PT that was transiently depleted for CASD1 expression. Additionally, OAcGD2 expression was increased in SUM159PT cells transiently overexpressing CASD1. The modulation of CASD1 expression using transient transfection strategies provided interesting insights into the role of CASD1 in OAcGD2 and OAcGD3 biosynthesis, and it highlights the importance of further studies on O-acetylation mechanisms.

Fetal bovine serum impacts the observed N-glycosylation defects in TMEM165 KO HEK cells.

TMEM165 is involved in a rare genetic human disease named TMEM165-CDG (congenital disorders of glycosylation). It is Golgi localized, highly conserved through evolution and belongs to the uncharacterized protein family 0016 (UPF0016). The use of isogenic TMEM165 KO HEK cells was crucial in deciphering the function of TMEM165 in Golgi manganese homeostasis. Manganese is a major cofactor of many glycosylation enzymes. Severe Golgi glycosylation defects are observed in TMEM165 Knock Out Human Embryonic Kidney (KO HEK) cells and are rescued by exogenous manganese supplementation. Intriguingly, we demonstrate in this study that the observed Golgi glycosylation defect mainly depends on fetal bovine serum, particularly its manganese level. Our results also demonstrate that iron and/or galactose can modulate the observed glycosylation defects in TMEM165 KO HEK cells. While isogenic cultured cells are widely used to study the impact of gene defects on proteins' glycosylation patterns, these results emphasize the importance of the use of validated fetal bovine serum in glycomics studies.

Investigating the functional link between TMEM165 and SPCA1.

TMEM165 was highlighted in 2012 as the first member of the Uncharacterized Protein Family 0016 (UPF0016) related to human glycosylation diseases. Defects in TMEM165 are associated with strong Golgi glycosylation abnormalities. Our previous work has shown that TMEM165 rapidly degrades with supraphysiological manganese supplementation. In this paper, we establish a functional link between TMEM165 and SPCA1, the Golgi Ca2+/Mn2+ P-type ATPase pump. A nearly complete loss of TMEM165 was observed in SPCA1-deficient Hap1 cells. We demonstrate that TMEM165 was constitutively degraded in lysosomes in the absence of SPCA1. Complementation studies showed that TMEM165 abundance was directly dependent on SPCA1's function and more specifically its capacity to pump Mn2+ from the cytosol into the Golgi lumen. Among SPCA1 mutants that differentially impair Mn2+ and Ca2+ transport, only the Q747A mutant that favors Mn2+ pumping rescues the abundance and Golgi subcellular localization of TMEM165. Interestingly, the overexpression of SERCA2b also rescues the expression of TMEM165. Finally, this paper highlights that TMEM165 expression is linked to the function of SPCA1.

The extended cytoplasmic tail of the human B4GALNT2 is critical for its Golgi targeting and post-Golgi sorting.

The Sda /Cad antigen reported on glycoconjugates of human tissues has an increasingly recognized wide impact on the physio-pathology of different biological systems. The last step of its biosynthesis relies on the enzymatic activity of the ß1,4-N-acetylgalactosaminyltransferase-II (B4GALNT2), which shows the highest expression level in healthy colon. Previous studies reported the occurrence in human colonic cells of two B4GALNT2 protein isoforms that differ in the length of their cytoplasmic tail, the long isoform showing an extended 66-amino acid tail. We examined here, the subcellular distribution of the two B4GALNT2 protein isoforms in stably transfected colonic LS174T cells and in transiently transfected HeLa cells using fluorescence microscopy. While a similar subcellular distribution at the trans-Golgi cisternae level was observed for the two isoforms, our study pointed to an atypical subcellular localization of the long B4GALNT2 isoform into dynamic vesicles. We demonstrated a critical role of its extended cytoplasmic tail for its Golgi targeting and post-Golgi sorting and highlighted the existence of a newly described post-Golgi sorting signal as well as a previously undescribed fate of a Golgi glycosyltransferase. DATABASE: The proteins ß1,4GalNAcT II, ß1,4-GalT1, FucT I, FucT VI and ST3Gal IV are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4, whereas the corresponding human genes are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4 according to the HUGO nomenclature.

Integrating glycomics and genomics uncovers SLC10A7 as essential factor for bone mineralization by regulating post-Golgi protein transport and glycosylation.

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix.

Ets-1 interacts through a similar binding interface with Ku70 and Poly (ADP-Ribose) Polymerase-1.

The Ets-1 transcription factor plays an important role in various physiological and pathological processes. These diverse roles of Ets-1 are likely to depend on its interaction proteins. We have previously showed that Ets-1 interacted with DNA-dependent protein kinase (DNA-PK) complex including its regulatory subunits, Ku70 and Ku86 and with poly (ADP-ribose) polymerase-1 (PARP-1). In this study, the binding domains for the interaction between Ets-1 and these proteins were reported. We demonstrated that the interaction of Ets-1 with DNA-PK was mediated through the Ku70 subunit and was mapped to the C-terminal region of Ets-1 and the C-terminal part of Ku70 including SAP domain. The interactive domains between Ets-1 and PARP-1 have been mapped to the C-terminal region of Ets-1 and the BRCA1 carboxy-terminal (BRCT) domain of PARP-1. The results presented in this study may advance our understanding of the functional link between Ets-1 and its interaction partners, DNA-PK and PARP-1.

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2) displays an unexpected subcellular localization in the plasma membrane.

BACKGROUND: Heparan sulfate (HS) 3-O-sulfation can be catalysed by seven 3-O-sulfotransferases (HS3STs) in humans, still it is the rarest modification in HS and its biological function is yet misunderstood. HS3ST2 and HS3ST3B exhibit the same activity in vitro. They are however differently expressed in macrophages depending on cell environment, which suggests that they may be involved in distinct cellular processes. Here, we hypothesized that both isozymes might also display distinct subcellular localizations. METHODS: The subcellular distribution of HS3ST2 and HS3ST3B was analysed by using overexpression systems in HeLa cells. The localization of endogenous HS3ST2 was confirmed by immunostaining in primary macrophages. RESULTS: We found that HS3ST3B was only localized in the Golgi apparatus and no difference between full-length enzyme and truncated construct depleted of its catalytic domain was observed. In contrast, HS3ST2 was clearly visualized at the plasma membrane. Its truncated form remained in the Golgi apparatus, meaning that the catalytic domain might support correct addressing of HS3ST2 to cell surface. Moreover, we found a partial co-localization of HS3ST2 with syndecan-2 in HeLa cells and primary macrophages. Silencing the expression of this proteoglycan altered the localization of HS3ST2, which suggests that syndecan-2 is required to address the isozyme outside of the Golgi apparatus. CONCLUSIONS: We demonstrated that HS3ST3B is a Golgi-resident isozyme, while HS3ST2 is addressed to the plasma membrane with syndecan-2. GENERAL SIGNIFICANCE: The membrane localization of HS3ST2 suggests that this enzyme may participate in discrete processes that occur at the cell surface.

Chemical glycomics enrichment: imaging the recycling of sialic acid in living cells.

The development of metabolic oligosaccharide engineering (MOE) over the past two decades enabled the bioimaging studies of glycosylation processes in physio-pathological contexts. Herein, we successfully applied the chemical reporter strategy to image the fate of sialylated glycoconjugates in healthy and sialin-deficient patient fibroblasts. This chemical glycomics enrichment is a powerful tool for tracking sialylated glycoconjugates and probing lysosomal recycling capacities. Thus, such strategies appear fundamental for the characterization of lysosomal storage diseases.

Probing the CMP-Sialic Acid Donor Specificity of Two Human ß-d-Galactoside Sialyltransferases (ST3Gal†I and ST6Gal†I) Selectively Acting on O- and N-Glycosylproteins.

Sialylation of glycoproteins and glycolipids is catalyzed by sialyltransferases in the Golgi of mammalian cells, whereby sialic acid residues are added at the nonreducing ends of oligosaccharides. Because sialylated glycans play critical roles in a number of human physio-pathological processes, the past two decades have witnessed the development of modified sialic acid derivatives for a better understanding of sialic acid biology and for the development of new therapeutic targets. However, nothing is known about how individual mammalian sialyltransferases tolerate and behave towards these unnatural CMP-sialic acid donors. In this study, we devised several approaches to investigate the donor specificity of the human ß-d-galactoside sialyltransferases ST6Gal I and ST3Gal I by using two CMP-sialic acids: CMP-Neu5Ac, and CMP-Neu5N-(4pentynoyl)neuraminic acid (CMP-SiaNAl), an unnatural CMP-sialic acid donor with an extended and functionalized N-acyl moiety.

Manganese-induced turnover of TMEM165.

TMEM165 deficiencies lead to one of the congenital disorders of glycosylation (CDG), a group of inherited diseases where the glycosylation process is altered. We recently demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from a Golgi manganese homeostasis defect and that Mn2+ supplementation was sufficient to rescue normal glycosylation. In the present paper, we highlight TMEM165 as a novel Golgi protein sensitive to manganese. When cells were exposed to high Mn2+ concentrations, TMEM165 was degraded in lysosomes. Remarkably, while the variant R126H was sensitive upon manganese exposure, the variant E108G, recently identified in a novel TMEM165-CDG patient, was found to be insensitive. We also showed that the E108G mutation did not abolish the function of TMEM165 in Golgi glycosylation. Altogether, the present study identified the Golgi protein TMEM165 as a novel Mn2+-sensitive protein in mammalian cells and pointed to the crucial importance of the glutamic acid (E108) in the cytosolic ELGDK motif in Mn2+-induced degradation of TMEM165.

Evidence for splice transcript variants of TMEM165, a gene involved in CDG.

BACKGROUND: Defects in TMEM165 gene cause a type-II Congenital Disorder of Glycosylation affecting Golgi glycosylation processes. TMEM165 patients exhibit psychomotor retardation, important osteoporosis, scoliosis, irregular epiphyses and thin bone cortex. TMEM165 protein is highly conserved in evolution and belongs to the family of UPF0016 membrane proteins which could be an unique group of Ca2+/H+ antiporters regulating Ca2+ and pH homeostasis and mainly localized in the Golgi apparatus. METHODS: RT-PCR from human brain tissues revealed TMEM165 splice-transcript variants. mRNA expression was analyzed by RT-Q-PCR. Expression plasmids allowed us to visualize isoform proteins and their subcellular localization. Their functions on glycosylation were achieved by looking at the gel mobility of highly glycosylated proteins in cells overexpressing isoforms. RESULTS: In this study, we highlight, as previously shown for other ion channels, the existence of TMEM165 splice-transcripts isoforms, in particular the Short-Form (SF) and the Long-Form (LF) transcripts, leading to a 129 aa and 259 aa protein isoform, respectively. These proteins both localize in the endoplasmic reticulum and have different effects on glycosylation compared to the wild-type protein (324 aa). We also point out that the SF is expressed at low levels in all human cells and tissues checked, excepted in brain, and forms homodimer. The LF was only expressed in the temporal lobe of human brain. GENERAL SIGNIFICANCE: The finding of numerous splice variants could lead to a family of TMEM165 isoforms. This family of TMEM165 splice transcripts could participate in the fine regulation of TMEM165 isoforms' functions and localizations.

Glycosylation abnormalities in Gdt1p/TMEM165 deficient cells result from a defect in Golgi manganese homeostasis.

Congenital disorders of glycosylation (CDG) are severe inherited diseases in which aberrant protein glycosylation is a hallmark. From this genetically and clinically heterogenous group, a significant subgroup due to Golgi homeostasis defects is emerging. We previously identified TMEM165 as a Golgi protein involved in CDG. Extremely conserved in the eukaryotic reign, the molecular mechanism by which TMEM165 deficiencies lead to Golgi glycosylation abnormalities is enigmatic. AsGDT1 is the ortholog of TMEM165 in yeast, both gdt1Δ null mutant yeasts and TMEM165 depleted cells were used. We highlighted that the observed Golgi glycosylation defects due to Gdt1p/TMEM165 deficiency result from Golgi manganese homeostasis defect. We discovered that in both yeasts and mammalian Gdt1p/TMEM165-deficient cells, Mn(2+) supplementation could restore a normal glycosylation. We also showed that the GPP130 Mn(2+) sensitivity was altered in TMEM165 depleted cells. This study not only provides novel insights into the molecular causes of glycosylation defects observed in TMEM165-deficient cells but also suggest that TMEM165 is a key determinant for the regulation of Golgi Mn(2+) homeostasis.

TMEM199 Deficiency Is a Disorder of Golgi Homeostasis Characterized by Elevated Aminotransferases, Alkaline Phosphatase, and Cholesterol and Abnormal Glycosylation.

Congenital disorders of glycosylation (CDGs) form a genetically and clinically heterogeneous group of diseases with aberrant protein glycosylation as a hallmark. A subgroup of CDGs can be attributed to disturbed Golgi homeostasis. However, identification of pathogenic variants is seriously complicated by the large number of proteins involved. As part of a strategy to identify human homologs of yeast proteins that are known to be involved in Golgi homeostasis, we identified uncharacterized transmembrane protein 199 (TMEM199, previously called C17orf32) as a human homolog of yeast V-ATPase assembly factor Vph2p (also known as Vma12p). Subsequently, we analyzed raw exome-sequencing data from families affected by genetically unsolved CDGs and identified four individuals with different mutations in TMEM199. The adolescent individuals presented with a mild phenotype of hepatic steatosis, elevated aminotransferases and alkaline phosphatase, and hypercholesterolemia, as well as low serum ceruloplasmin. Affected individuals showed abnormal N- and mucin-type O-glycosylation, and mass spectrometry indicated reduced incorporation of galactose and sialic acid, as seen in other Golgi homeostasis defects. Metabolic labeling of sialic acids in fibroblasts confirmed deficient Golgi glycosylation, which was restored by lentiviral transduction with wild-type TMEM199. V5-tagged TMEM199 localized with ERGIC and COPI markers in HeLa cells, and electron microscopy of a liver biopsy showed dilated organelles suggestive of the endoplasmic reticulum and Golgi apparatus. In conclusion, we have identified TMEM199 as a protein involved in Golgi homeostasis and show that TMEM199 deficiency results in a hepatic phenotype with abnormal glycosylation.

CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation.

Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal glycosylation in plasma.

Integrative view of α2,3-sialyltransferases (ST3Gal) molecular and functional evolution in deuterostomes: significance of lineage-specific losses.

Sialyltransferases are responsible for the synthesis of a diverse range of sialoglycoconjugates predicted to be pivotal to deuterostomes' evolution. In this work, we reconstructed the evolutionary history of the metazoan α2,3-sialyltransferases family (ST3Gal), a subset of sialyltransferases encompassing six subfamilies (ST3Gal I-ST3Gal VI) functionally characterized in mammals. Exploration of genomic and expressed sequence tag databases and search of conserved sialylmotifs led to the identification of a large data set of st3gal-related gene sequences. Molecular phylogeny and large scale sequence similarity network analysis identified four new vertebrate subfamilies called ST3Gal III-r, ST3Gal VII, ST3Gal VIII, and ST3Gal IX. To address the issue of the origin and evolutionary relationships of the st3gal-related genes, we performed comparative syntenic mapping of st3gal gene loci combined to ancestral genome reconstruction. The ten vertebrate ST3Gal subfamilies originated from genome duplication events at the base of vertebrates and are organized in three distinct and ancient groups of genes predating the early deuterostomes. Inferring st3gal gene family history identified also several lineage-specific gene losses, the significance of which was explored in a functional context. Toward this aim, spatiotemporal distribution of st3gal genes was analyzed in zebrafish and bovine tissues. In addition, molecular evolutionary analyses using specificity determining position and coevolved amino acid predictions led to the identification of amino acid residues with potential implication in functional divergence of vertebrate ST3Gal. We propose a detailed scenario of the evolutionary relationships of st3gal genes coupled to a conceptual framework of the evolution of ST3Gal functions.

Assessing ER and Golgi N-glycosylation process using metabolic labeling in mammalian cultured cells.

Modifications of N-glycosylation in disease states are common and illustrate the crucial requirement of glycosylation in human biology. Mainly based on glycan permethylation and the use of mass spectrometry analysis, we can easily understand that many different methods to analyze the N-glycome have seen the day. While extremely powerful, these methods are mainly used to analyze qualitative variations of N-glycosylation of human serum proteins and do not necessarily reflect the glycosylation status of derived mammalian cultured cells. This chapter summarizes two methods that we are routinely using in our laboratory to assess the ER and Golgi N-glycosylation process. The proposed methodology allows pinpointing ER as well as Golgi glycosylation deficiencies in mammalian cultured cells. The first approach is based on direct metabolic labeling of cultured mammalian cells with [2-(3)H] mannose followed by sequential extraction and HPLC analysis of the purified oligosaccharides. The second one is based on the copper-catalyzed azide alkyne cycloaddition (CuAAC) strategy. We propose the use of alkyne-tagged sialic acid (SialNAl) to visualize the Golgi glycosylation efficiency. Their metabolic incorporation into newly synthesized glycoproteins can then be chemoselectively coupled to complementary azide-functionalized fluorophores, and visualized by using confocal laser scanning microscopy. To summarize, we present here a detailed description of our know-how in the field of ER and Golgi N-glycosylation.