The transmembrane domain of N –acetylglucosaminyltransferase I is the key determinant for its Golgi subcompartmentation

Golgi‐resident type–II membrane proteins are asymmetrically distributed across the Golgi stack. The intrinsic features of the protein that determine its subcompartment‐specific concentration are still largely unknown. Here, we used a series of chimeric proteins to investigate the contribution of the cytoplasmic, transmembrane and stem region of Nicotiana benthamiana N–acetylglucosaminyltransferase I (GnTI) for its cis/medial‐Golgi localization and for protein–protein interaction in the Golgi. The individual GnTI protein domains were replaced with those from the well‐known trans‐Golgi enzyme α2,6–sialyltransferase (ST) and transiently expressed in Nicotiana benthamiana. Using co‐localization analysis and N–glycan profiling, we show that the transmembrane domain of GnTI is the major determinant for its cis/medial‐Golgi localization. By contrast, the stem region of GnTI contributes predominately to homomeric and heteromeric protein complex formation. Importantly, in transgenic Arabidopsis thaliana, a chimeric GnTI variant with altered sub‐Golgi localization was not able to complement the GnTI‐dependent glycosylation defect. Our results suggest that sequence‐specific features in the transmembrane domain of GnTI account for its steady‐state distribution in the cis/medial‐Golgi in plants, which is a prerequisite for efficient N–glycan processing in vivo.     

Differential effects of lobe A and lobe B of the Conserved Oligomeric Golgi complex on the stability of {beta}1,4-galactosyltransferase 1 and {alpha}2,6-sialyltransferase 1

Initially described by Jaeken et al. in 1980, congenital disorders of glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the conserved oligomeric Golgi (COG) complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of the total serum N-glycans of COG-deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small-interfering RNAs strategy in HeLa cells stably expressing green fluorescent protein (GFP)-tagged β1,4-galactosyltransferase 1 (B4GALT1) and α2,6-sialyltransferase 1 (ST6GAL1), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, whereas lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1- and ST6GAL1-induced degradation are likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, whereas lobe A appears to be essential for maintaining the overall Golgi structure.     

Inhibition of Liver Golgi Glycosylation Activities by Carbonyl Products of Lipid

The present report deals with the investigation of the effect of 4-hydroxy-/ram 2,3-nonenal (HNE), hexanal (HEX) and malondialdehyde (MDA), the major products of lipid peroxidation, on the glycosylation pathway of rat liver Golgi apparatus. Defined concentrations of the aldehydes were added to isolated fractions of formative (F,) and secretory (F, + F,) Golgi compartments, then incubated at 37°C for lOmin. At the end of the incubation the activity of galactosyl-(GT) and sialyl-(ST)transferases, the main enzymes of the terminal protein and lipoprotein glycosylation, was evalued. A significant impairment of both these activities was observed with HNE and HEX but not with MDA.

These data suggest that aldehydes generated during peroxidation reactions are able to impair the protein and lipoprotein maturation mechanism which is normally achieved through a complete glycosylation.     

Spatial Regulation of Golgi Phosphatidylinositol‐4‐Phosphate is Required for Enzyme Localization and Glycosylation Fidelity

The enrichment of phosphatidylinositol‐4‐phosphate (PI(4)P) at the trans Golgi network (TGN) is instrumental for proper protein and lipid sorting, yet how the restricted distribution of PI(4)P is achieved remains unknown. Here, we show that lipid phosphatase Suppressor of actin mutations 1 (SAC1) is crucial for the spatial regulation of Golgi PI(4)P. Ultrastructural analysis revealed that SAC1 is predominantly located at cisternal Golgi membranes but is absent from the TGN, thus confining PI(4)P to the TGN. RNAi‐mediated knockdown of SAC1 caused changes in Golgi morphology and mislocalization of Golgi enzymes. Enzymes involved in glycan processing such as mannosidase‐II (Man‐II) and N‐acetylglucosamine transferase‐I (GnT‐I) redistributed to aberrant intracellular structures and to the cell surface in SAC1 knockdown cells. SAC1 depletion also induced a unique pattern of Golgi‐specific defects in N‐and O‐linked glycosylation. These results indicate that SAC1 organizes PI(4)P distribution between the Golgi complex and the TGN, which is instrumental for resident enzyme partitioning and Golgi morphology.     

Role of the Lectin VIP36 in Post‐ER Quality Control of Human α1‐Antitrypsin

The leguminous‐type (L‐type) lectin VIP36 localizes to the Golgi apparatus and cycles early in the secretory pathway. In vitro, VIP36 binds high‐mannose glycans with a pH optimum of 6.5, a value similar to the luminal pH of the Golgi apparatus. Although the sugar‐binding properties of VIP36 in vitro have been characterized in detail, the function of VIP36 in the intact cell remains unclear as no convincing glycoprotein cargo has been identified. Here, we used yellow fluorescent protein (YFP) fragment complementation to identify luminal interaction partners of VIP36. By screening a human liver cDNA library, we identified the glycoprotein α1‐antitrypsin (α1‐AT) as a cargo of VIP36. The VIP36/α1‐AT complex localized to Golgi and endoplasmic reticulum (ER). In the living cell, VIP36 bound exclusively to the high‐mannose form of α1‐AT. The binding was increased when complex glycosylation was prevented by kifunensine and abolished when the glycosylation sites of α1‐AT were inactivated by mutagenesis. Silencing VIP36 accelerated α1‐AT transport, arguing against a role of VIP36 in anterograde traffic. The complex formed by VIP36 and α1‐AT in the Golgi recycled back to the ER. The combined data are most consistent with a function of VIP36 in post‐ER quality control of α1‐AT.     

Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression

N‐Glycans of human proteins possess both α2,6‐ and α2,3‐linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3‐linkage due to the absence of α2,6‐sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)‐producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC‐Sambucus nigra (SNA) lectin that preferentially binds α2,6‐linked SA. The presence of α2,6‐linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2‐fold compared to the control. For host cell engineering, the CHOZN^® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single‐cell clones were derived from the enriched population and selected based on FITC‐SNA staining and St6gal1 expression. Two clones (“ST6GAL1 OE Clone 31 and 32”) were confirmed for the presence of α2,6‐linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6‐linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human‐like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of “bio‐better” protein therapeutics and cell culture vaccine production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:334–346, 2015     

Integrated Genome and Protein Editing Swaps α‐2,6 Sialylation for α‐2,3 Sialic Acid on Recombinant Antibodies from CHO

Immunoglobin G with α‐2,6 sialylation has been reported to have an impact on antibody‐dependent cellular cytotoxicity and anti‐inflammatory efficacy. However, production of antibodies with α‐2,6 sialylation from Chinese hamster ovary cells is challenging due to the inaccessibility of sialyltransferases for the heavy chain N‐glycan site and the presence of exclusively α‐2,3 sialyltransferases. In this study, combining mutations on the Fc regions to allow sialyltransferase accessibility with overexpression of α‐2,6 sialyltransferase produced IgG with significant levels of both α‐2,6 and α‐2,3 sialylation. Therefore, ST3GAL4 and ST3GAL6 genes were disrupted by CRISPR/Cas9 to minimize the α‐2,3 sialylation. Sialidase treatment and SNA lectin blot indicated greatly increased α‐2,6 sialylation level relative to α‐2,3 sialylation for the α‐2,3 sialyltransferase knockouts when combined with α‐2,6 sialyltransferase overexpression. Indeed, α‐2,3 linked sialic acids were not detected on IgG produced from the α‐2,3 sialyltransferase knockout‐α‐2,6 sialyltransferase overexpression pools. Finally, glycoprofiling of IgG with four amino acid substitutions expressed from an α‐2,3 sialyltransferase knockout‐α‐2,6 sialyltransferase stable clone resulted in more than 77% sialylated glycans and more than 62% biantennary disialylated glycans as indicated by both MALDI‐TOF and LC‐ESI‐MS. Engineered antibodies from these modified Chinese hamster ovary cell lines will provide biotechnologists with IgGs containing N‐glycans with different structural variations for examining the role of glycosylation on protein performance.     

An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation

Recently, the Golgi phosphoprotein 3 (GOLPH3) and its yeast homolog Vps74p have been characterized as essential for the Golgi localization of glycosyltransferase in yeast. GOLPH3 has been identified as a new oncogene that is commonly amplified in human cancers to modulate mammalian target of rapamycin signaling. However, the molecular mechanisms of the carcinogenic signaling pathway remain largely unclear. To investigate whether the expression of GOLPH3 was involved in the glycosylation processes in mammalian cells, and whether it affected cell behavior, we performed a loss-of-function study. Cell migration was suppressed in GOLPH3 knockdown (KD) cells, and the suppression was restored by a re-introduction of the GOLPH3 gene. HPLC and LC/MS analysis showed that the sialylation of N-glycans was specifically decreased in KD cells. The specific interaction between sialyltransferases and GOLPH3 was important for the sialylation. Furthermore, overexpression of α2,6-sialyltransferase-I rescued cell migration and cellular signaling, both of which were blocked in GOLPH3 knockdown cells. These results are the first direct demonstration of the role of GOLPH3 in N-glycosylation to regulate cell biological functions.     

Golgi α1,4‐fucosyltransferase of Arabidopsis thaliana partially localizes at the nuclear envelope

We analyzed plant‐derived α1,4‐fucosyltransferase (FucTc) homologs by reporter fusions and focused on representatives of the Brassicaceae and Solanaceae. Arabidopsis thaliana AtFucTc‐green fluorescent protein (GFP) or tomato LeFucTc‐GFP restored Lewis‐a formation in a fuctc mutant, confirming functionality in the trans‐Golgi. AtFucTc‐GFP partly accumulated at the nuclear envelope (NE) not observed for other homologs or truncated AtFucTc lacking the N‐terminus or catalytic domain. Analysis of At/LeFucTc‐GFP swap constructs with exchanged cytosolic, transmembrane and stalk (CTS), or only the CT regions, revealed that sorting information resides in the membrane anchor. Other domains of AtFuctc also contribute, since amino‐acid changes in the CT region strongly reduced but did not abolish NE localization. By contrast, two N‐terminal GFP copies did, indicating localization at the inner nuclear membrane (INM). Tunicamycin treatment of AtFucTc‐GFP abolished NE localization and enhanced overlap with an endosomal marker, suggesting involvement of N‐glycosylation. Yet neither expression in protoplasts of Arabidopsis N‐glycosylation mutants nor elimination of the N‐glycosylation site in AtFucTc prevented perinuclear accumulation. Disruption of endoplasmic reticulum (ER)‐to‐Golgi transport by co‐expression of Sar1(H74L) trapped tunicamycin‐released AtFucTc‐GFP in the ER, however, without NE localization. Since recovery after tunicamycin‐washout required de novo‐protein synthesis, our analyses suggest that AtFucTc localizes to the NE/INM due to interaction with an unknown (glyco)protein.      

Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery

Cell surface lectin staining, examination of Golgi glycosyltransferases stability and localization, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were employed to investigate conserved oligomeric Golgi (COG)-dependent glycosylation defects in HeLa cells. Both Griffonia simplicifolia lectin-II and Galanthus nivalus lectins were specifically bound to the plasma membrane glycoconjugates of COG-depleted cells, indicating defects in activity of medial- and trans-Golgi-localized enzymes. In response to siRNA-induced depletion of COG complex subunits, several key components of Golgi glycosylation machinery, including MAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mislocalized. MALDI-TOF analysis of total N-linked glycoconjugates indicated a decrease in the relative amount of sialylated glycans in both COG3 KD and COG4 KD cells. In agreement to a proposed role of the COG complex in retrograde membrane trafficking, all types of COG-depleted HeLa cells were deficient in the Brefeldin A- and Sar1 DN-induced redistribution of Golgi resident glycosyltransferases to the endoplasmic reticulum. The retrograde trafficking of medial- and trans-Golgi-localized glycosylation enzymes was affected to a larger extent, strongly indicating that the COG complex regulates the intra-Golgi protein movement. COG complex-deficient cells were not defective in Golgi re-assembly after the Brefeldin A washout, confirming specificity in the retrograde trafficking block. The lobe B COG subcomplex subunits COG6 and COG8 were localized on trafficking intermediates that carry Golgi glycosyltransferases, indicating that the COG complex is directly involved in trafficking and maintenance of Golgi glycosylation machinery.     

The 17-residue transmembrane domain of beta-galactoside alpha 2,6- sialyltransferase is sufficient for Golgi retention

beta-Galactoside alpha 2,6-sialyltransferase (ST) is a type II integral membrane protein of the Golgi apparatus involved in the sialylation of N-linked glycans. A series of experiments has shown that the 17-residue transmembrane domain of ST is sufficient to confer localization to the Golgi apparatus when transferred to the corresponding region of a cell surface type II integral membrane protein. Lectin affinity chromatography of chimeric proteins bearing this 17-residue sequence suggests that these chimeric proteins are localized in the trans-Golgi cisternae and/or trans-Golgi network. Further experiments suggest that this 17-residue sequence functions as a retention signal for the Golgi apparatus.     

ST6Gal-I regulates macrophage apoptosis via α2-6 sialylation of the TNFR1 death receptor

Macrophages play a central role in innate immunity, however mechanisms regulating macrophage survival are not fully understood. Herein we describe a novel apoptotic pathway involving α2-6 sialylation of the TNFR1 death receptor by the ST6Gal-I sialyltransferase. Variant glycosylation of TNFR1 has not previously been implicated in TNFR1 function, and little is known regarding the TNFR1 glycan composition. To study sialylation in macrophages, we treated U937 monocytic cells with PMA, which stimulates both macrophage differentiation and apoptosis. Interestingly, macrophage differentiation induces ST6Gal-I down-regulation, leading to reduced α2-6 sialylation of selected receptors. To prevent loss of α2-6 sialylation, we forced constitutive expression of ST6Gal-I, and found that this strongly inhibited PMA-induced apoptosis. Given that PMA-mediated apoptosis is thought to result from up-regulation of TNFα, which then activates TNFR1, we next evaluated the α2-6 sialylation of TNFR1. U937 cells with forced ST6Gal-I displayed TNFR1 with elevated α2-6 sialylation, and this was associated with diminished TNFα-stimulated apoptosis. Correspondingly, removal of α2-6 sialylation from TNFR1 through either neuraminidase treatment or expression of ST6Gal-I shRNA markedly enhanced TNFα-mediated apoptosis. To confirm the physiologic importance of TNFR1 sialylation, we generated overexpressing ST6Gal-I transgenic mice. Peritoneal macrophages from transgenic lines displayed TNFR1 with elevated α2-6 sialylation, and these cells were significantly protected against TNFα-stimulated apoptosis. Moreover, greater numbers of thioglycollate-induced peritoneal cells were observed in transgenic mice. These collective results highlight a new mechanism of TNFR1 regulation, and further intimate that loss of α2-6 sialylation during macrophage differentiation may limit macrophage lifespan by sensitizing cells to TNFα-stimulated apoptosis.     

A Role for the Lumenal Domain in Golgi Localization of the Saccharomyces cerevisiae Guanosine Diphosphatase

Integral membrane proteins (IMPs) contain localization signals necessary for targeting to their resident subcellular compartments. To define signals that mediate localization to the Golgi complex, we have analyzed a resident IMP of the Saccharomyces cerevisiae Golgi complex, guanosine diphosphatase (GDPase). GDPase, which is necessary for Golgi-specific glycosylation reactions, is a type II IMP with a short amino-terminal cytoplasmic domain, a single transmembrane domain (TMD), and a large catalytic lumenal domain. Regions specifying Golgi localization were identified by analyzing recombinant proteins either lacking GDPase domains or containing corresponding domains from type II vacuolar IMPs. Neither deletion nor substitution of the GDPase cytoplasmic domain perturbed Golgi localization. Exchanging the GDPase TMD with vacuolar protein TMDs only marginally affected Golgi localization. Replacement of the lumenal domain resulted in mislocalization of the chimeric protein from the Golgi to the vacuole, but a similar substitution leaving 34 amino acids of the GDPase lumenal domain intact was properly localized. These results identify a major Golgi localization determinant in the membrane-adjacent lumenal region (stem) of GDPase. Although necessary, the stem domain is not sufficient to mediate localization; in addition, a membrane-anchoring domain and either the cytoplasmic or full-length lumenal domain must be present to maintain Golgi residence. The importance of lumenal domain sequences in GDPase Golgi localization and the requirement for multiple hydrophilic protein domains support a model for Golgi localization invoking protein–protein interactions rather than interactions between the TMD and the lipid bilayer.     

Organizational Interplay of Golgi N-Glycosyltransferases Involves Organelle Microenvironment-Dependent Transitions between Enzyme Homo- and Heteromers

Glycosylation of proteins and lipids takes place in the Golgi apparatus by the consecutive actions of functionally distinct glycosidases and glycosyltransferases. Current evidence indicates that they function as enzyme homomers and/or heteromers in the living cell. Here we investigate their organizational interplay and show that glycosyltransferase homomers are assembled in the endoplasmic reticulum. Upon transport to the Golgi, the majority of homomers are disassembled to allow the formation of enzyme heteromers between sequentially acting medial-Golgi enzymes GnT-I and GnT-II or trans-Golgi enzymes GalT-I and ST6Gal-I. This transition is driven by the acidic Golgi environment, as it was markedly inhibited by raising Golgi luminal pH with chloroquine. Our FRAP (fluorescence recovery after photobleaching) measurements showed that the complexes remain mobile Golgi membrane constituents that can relocate to the endoplasmic reticulum or to the scattered Golgi mini-stacks upon brefeldin A or nocodazole treatment, respectively. During this relocation, heteromers undergo a reverse transition back to enzyme homomers. These data unveil an unprecedented organizational interplay between Golgi N-glycosyltransferases that involves dynamic and organelle microenvironment-driven transitions between enzyme homomers and heteromers during their trafficking within the early secretory compartments.     

Syntaxin 17 cycles between the ER and ERGIC and is required to maintain the architecture of ERGIC and Golgi

Background information. Syntaxin 17 is a SNARE (soluble N‐ethylmaleimide‐sensitive‐factor‐attachment protein receptor) protein that predominantly localizes to the ER (endoplasmic reticulum) and to some extent in the ERGIC (ER—Golgi intermediate compartment). Syntaxin 17 has been suggested to function as a receptor at the ER membrane that mediates trafficking between the ER and post‐ER compartments. It has a unique 33 amino acid luminal tail whose function is not known. Here we have investigated the structural requirements for localization of syntaxin 17 to the ERGIC and its role in trafficking. Results. Deletion analysis showed that syntaxin 17 required its cytoplasmic domain to exit the ER and localize to the ERGIC. Mutation of a conserved tyrosine residue in the cytoplasmic domain resulted in reduced localization of syntaxin 17 in the ERGIC and ER‐exit sites, suggesting the presence of a tyrosine‐based ER export motif. Syntaxin 17 also required its C‐terminal tail to localize to the ERES (ER exit sites) and ERGIC. Knockdown of syntaxin 17 destabilized the ERGIC organization and also caused fragmentation of the Golgi complex. Syntaxin 17 showed direct interaction with transmembrane proteins p23 and p25 (cargo receptors that cycle between the ER and Golgi) with the help of its C‐terminal tail. Overexpression of syntaxin 17 redistributed β‐COP (β‐coatomer protein) which required its C‐terminal tail. Overexpression of syntaxin 17 also blocked the anterograde transport of VSVG (vesicular stomatitis virus G‐protein) in the ERGIC. Conclusions. We show that syntaxin 17 has a tyrosine‐based motif which is required for its incorporation into COPII (coatomer protein II) vesicles, exit from the ER and localization to the ERGIC. Our results suggest that syntaxin 17 cycles between the ER and ERGIC through classical trafficking pathways involving COPII and COPI (coatomer protein I) vesicles, which requires its unique C‐terminal tail. We also show that syntaxin 17 is essential for maintaining the architecture of ERGIC and Golgi.     

Interaction of Golgin‐84 with the COG Complex Mediates the Intra‐Golgi Retrograde Transport

The coiled‐coil Golgi membrane protein golgin‐84 functions as a tethering factor for coat protein I (COPI) vesicles. Protein interaction analyses have revealed that golgin‐84 interacts with another tether, the conserved oligomeric Golgi (COG) complex, through its subunit Cog7. Therefore, we explored the function of golgin‐84 as the tether for COPI vesicles of intra‐Golgi retrograde traffic. First, glycosylic maturation of both plasma membrane (CD44) and lysosomal (lamp1) glycoproteins was distorted in golgin‐84 knockdown (KD) cells. The depletion of golgin‐84 caused fragmentation of the Golgi with the mislocalization of Golgi resident proteins, resulting in the accumulation of vesicles carrying intra‐Golgi soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) and cis‐Golgi membrane protein GPP130. Similar observations were obtained by diminution of the COG complex, suggesting a strong correlation between the two tethers. Indeed, COG complex‐dependent (CCD) vesicles that accumulate in Cog3 or Cog7 KD cells carried golgin‐84. Surprisingly, the interaction between golgin‐84 and another candidate tethering partner CASP (CDP/cut alternatively spliced product) decreased in Cog3 KD cells. These results indicate that golgin‐84 on COPI vesicles interact with the COG complex before SNARE assembly, suggesting that the interaction of golgin‐84 with COG plays an important role in the tethering process of intra‐Golgi retrograde vesicle traffic.     

More than just sugars: Conserved oligomeric Golgi complex deficiency causes glycosylation‐independent cellular defects

The conserved oligomeric Golgi (COG) complex controls membrane trafficking and ensures Golgi homeostasis by orchestrating retrograde vesicle trafficking within the Golgi. Human COG defects lead to severe multisystemic diseases known as COG‐congenital disorders of glycosylation (COG‐CDG). To gain better understanding of COG‐CDGs, we compared COG knockout cells with cells deficient to 2 key enzymes, Alpha‐1,3‐mannosyl‐glycoprotein 2‐beta‐N‐acetylglucosaminyltransferase and uridine diphosphate‐glucose 4‐epimerase (GALE), which contribute to proper N‐ and O‐glycosylation. While all knockout cells share similar defects in glycosylation, these defects only account for a small fraction of observed COG knockout phenotypes. Glycosylation deficiencies were not associated with the fragmented Golgi, abnormal endolysosomes, defective sorting and secretion or delayed retrograde trafficking, indicating that these phenotypes are probably not due to hypoglycosylation, but to other specific interactions or roles of the COG complex. Importantly, these COG deficiency specific phenotypes were also apparent in COG7‐CDG patient fibroblasts, proving the human disease relevance of our CRISPR knockout findings. The knowledge gained from this study has important implications, both for understanding the physiological role of COG complex in Golgi homeostasis in eukaryotic cells, and for better understanding human diseases associated with COG/Golgi impairment.      

pH-dependent cargo sorting from the Golgi

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.     

How Golgi glycosylation meets and needs trafficking: the case of the COG complex

Protein glycosylation is one of the major biosynthetic functions occurring in the endoplasmic reticulum and Golgi compartments. It requires an amazing number of enzymes, chaperones, lectins and transporters whose actions delicately secure the fidelity of glycan structures. Over the past 30 years, glycobiologists hammered that glycan structures are not mere decorative elements but serve crucial cellular functions. This becomes dramatically illustrated by a group of mostly severe, inherited human disorders named congenital disorders of glycosylation (CDG). To date, many types of CDG have been defined genetically and most of the time the defects impair the biosynthesis, transfer and remodeling of N-glycans. Recently, the identification of the several types of CDG caused by deficiencies in the conserved oligomeric Golgi (COG) complex, a complex involved in vesicular Golgi trafficking, expanded the field of CDG but also brought novel insights in glycosylation. The molecular mechanisms underlying the complex pathway of N-glycosylation in the Golgi are far from understood. The availability of COG-deficient CDG patients and patients' cells offered a new way to study how COG, and its different subunits, could influence the Golgi N-glycosylation machinery and localization. This review summarizes the recent findings on the implication of COG in Golgi glycosylation. It highlights the need for a dynamic, finely tuned balance between anterograde and retrograde trafficking for the correct localization of Golgi enzymes to assure the stepwise maturation of N-glycan chains.     

Reversible sialylation: synthesis of cytidine 5'-monophospho-N-acetylneuraminic acid from cytidine 5'-monophosphate with alpha2,3-sialyl O-glycan-, glycolipid-, and macromolecule-based donors yields diverse sialylated products

Sialyltransferases transfer sialic acid from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to an acceptor molecule. Trans-sialidases of parasites transfer alpha2,3-linked sialic acid from one molecule to another without the involvement of CMP-NeuAc. Here we report another type of sialylation, termed reverse sialylation, catalyzed by mammalian sialyltransferase ST3Gal-II. This enzyme synthesizes CMP-NeuAc by transferring NeuAc from the NeuAcalpha2,3Galbeta1,3GalNAcalpha unit of O-glycans, 3-sialyl globo unit of glycolipids, and sialylated macromolecules to 5'-CMP. CMP-NeuAc produced in situ is utilized by the same enzyme to sialylate other O-glycans and by other sialyltransferases such as ST6Gal-I and ST6GalNAc-I, forming alpha2,6-sialylated compounds. ST3Gal-II also catalyzed the conversion of 5'-uridine monophosphate (UMP) to UMP-NeuAc, which was found to be an inactive sialyl donor. Reverse sialylation proceeded without the need for free sialic acid, divalent metal ions, or energy. Direct sialylation with CMP-NeuAc as well as the formation of CMP-NeuAc from 5'-CMP had a wide optimum range (pH 5.2-7.2 and 4.8-6.4, respectively), whereas the entire reaction comprising in situ production of CMP-NeuAc and sialylation of acceptor had a sharp optimum at pH 5.6 (activity level 50% at pH 5.2 and 6.8, 25% at pH 4.8 and 7.2). Several properties distinguish forward/conventional versus reverse sialylation: (i) sodium citrate inhibited forward sialylation but not reverse sialylation; (ii) 5'-CDP, a potent forward sialyltransferase inhibitor, did not inhibit the conversion of 5'-CMP to CMP-NeuAc; and (iii) the mucin core 2 compound 3-O-sulfoGalbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-benzyl, an efficient acceptor for ST3Gal-II, inhibited the conversion of 5'-CMP to CMP-NeuAc. A significant level of reverse sialylation activity is noted in human prostate cancer cell lines LNCaP and PC3. Overall, the study demonstrates that the sialyltransferase reaction is readily reversible in the case of ST3Gal-II and can be exploited for the enzymatic synthesis of diverse sialyl products.