Formation of insoluble oligomers correlates with ST6Gal I stable localization in the golgi

The ST6Gal I is a sialyltransferase that functions in the late Golgi to modify the N-linked oligosaccharides of glycoproteins. The ST6Gal I is expressed as two isoforms with a single amino acid difference in their catalytic domains. The STcys isoform is stably retained in the cell and is predominantly found in the Golgi, whereas the STtyr isoform is only transiently localized in the Golgi and is cleaved and secreted from a post-Golgi compartment. These two ST6Gal I isoforms were used to explore the role of the bilayer thickness mechanism and oligomerization in Golgi localization. Analysis of STcys and STtyr proteins with longer transmembrane regions suggested that the bilayer thickness mechanism is not the predominant mechanism used for ST6Gal I Golgi localization. In contrast, the formation and quantity of Triton X-100-insoluble oligomers was correlated with the stable or transient localization of the ST6Gal I isoforms in the Golgi. Nearly 100% of the STcys and only 13% of the STtyr were found as Triton-insoluble oligomers when Golgi membranes of COS-1 cells expressing these proteins were solubilized at pH 6.3, the pH of the late Golgi. In contrast, both proteins were found in the soluble fraction when these membranes were solubilized at pH 8.0. Analysis of other mutants suggested that a conformational change in the catalytic domain rather than increased disulfide bond-based cross-linking is the basis for the increased ability of STcys protein to form oligomers and the stable localization of STcys protein in the Golgi.     

The two rat alpha 2,6-sialyltransferase (ST6Gal I) isoforms: evaluation of catalytic activity and intra-Golgi localization

alpha2,6-Sialyltransferase (ST6Gal I) functions in the Golgi to terminally sialylate the N-linked oligosaccharides of glycoproteins. Interestingly, rat ST6Gal I is expressed as two isoforms, STtyr and STcys, that differ by a single amino acid in their catalytic domains. In this article, our goal was to evaluate more carefully possible differences in the catalytic activity and intra-Golgi localization of the two isoforms that had been suggested by earlier work. Using soluble recombinant STtyr and STcys enzymes and three asialoglycoprotein substrates for in vitro analysis, we found that the STcys isoform was somewhat more active than the STtyr isoform. However, we found no differences in isoform substrate choice when these proteins were expressed in Chinese hamster ovary cells, and sialylated substrates were detected by lectin blotting. Immuno-fluorescence and immunoelectron microscopy revealed differences in the relative levels of the isoforms found in the endoplasmic reticulum (ER) and Golgi of transiently expressing cells but similar intra-Golgi localization. STtyr was restricted to the Golgi in most cells, and STcys was found in both the ER and Golgi. The ER localization of STcys was especially pronounced with a C-terminal V5 epitope tag. Ultrastructural and deconvolution studies of immunostained HeLa cells expressing STtyr or STcys showed that within the Golgi both isoforms are found in medial-trans regions. The similar catalytic activities and intra-Golgi localization of the two ST6Gal I isoforms suggest that the particular isoform expressed in specific cells and tissues is not likely to have significant functional consequences.     

Multiple signals are required for alpha2,6-sialyltransferase (ST6Gal I) oligomerization and Golgi localization

A single amino acid difference in the catalytic domain of two isoforms of the alpha2,6-sialyltransferase (ST6Gal I) leads to differences in their trafficking, processing, and oligomerization. The STtyr isoform is transiently localized in the Golgi and is ultimately cleaved and secreted, whereas the STcys isoform is stably localized in the Golgi and is not cleaved and secreted. The stable localization of STcys is correlated with its enhanced ability to oligomerize. To test the hypothesis that multiple signals can mediate Golgi localization and further evaluate the role of oligomerization in the localization process, we evaluated the effects of individually and simultaneously altering the cytosolic tail and transmembrane region of the STcys isoform. We found that the localization, processing, and oligomerization of STcys were not substantially changed when either the core amino acids of the cytosolic tail were deleted or the sequence and length of the transmembrane region were altered. In contrast, when these changes were made simultaneously, the STcys isoform was converted into a form that was processed, secreted, and weakly oligomerized like STtyr. We propose that STcys oligomerization is a secondary event resulting from its concentration in the Golgi via mechanisms independently mediated by its cytosolic tail and transmembrane region.     

Disulphide linkage in mouse ST6Gal-I: determination of linkage positions and mutant analysis

All cloned sialyltransferases from vertebrates are classified into four subfamilies and are characterized as having type II transmembrane topology. The catalytic domain has highly conserved motifs known as sialylmotifs. Besides sialylmotifs, each family has several unique conserved cysteine (Cys) residues mainly in the catalytic domain. The number and loci of conserved amino acids, however, differ with each subfamily, suggesting that the conserved Cys-residues and/or disulphide linkages they make may contribute to linkage specificity. Using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF)-mass spectrometry, the present study performed disulphide linkage analysis on soluble mouse ST6Gal-I, which has six Cys-residues. Results confirmed that there were no free Cys-residues, and all six residues contributed to disulphide linkage formation, C(139)-C(403), C(181)-C(332) and C(350)-C(361). Study of single amino acid-substituted mutants revealed that the disulphide linkage C(181)-C(332) was necessary for molecular expression of the enzyme, and that the disulphide linkage C(350)-C(361) was necessary for enzyme activity. The remaining disulphide linkage C(139)-C(403) was not necessary for enzyme expression or for activity, including substrate specificity. Crystallographic study of pig ST3Gal I has recently been reported. Interestingly, the loci of disulphide linkages in ST6Gal-I differ from those in ST3Gal I, suggesting that the linkage specificity of sialyltransferase may results from significant structural differences, including the loci of disulphide linkages.     

Monte carlo simulations of tau proteins: effect of phosphorylation

We perform Monte Carlo simulations of tau proteins bound to a cylinder that mimics a microtubule (MT), and then study them in solution. Tau protein binds to a highly anionic MT surface to stabilize the cylindrical structure of MT. The negatively charged tail domain floats away from the anionic MT surface while positively charged tau segments localize near the MT surface. Monte Carlo simulations demonstrate that, in 3RS tau isoform (which has three imperfect repeats (R) short (S) isoform), amino acids are more condensed near a highly charged interface compared to 4RL isoform (which has four imperfect repeats (R) long (L) isoform). In 4RL isoform, amino acids in tail domain stay mostly apart from the MT surface. In the bulk solution, dephosphorylated taus are separated due to Coulomb repulsion between similarly charged isoforms. Moderate phosphorylation of 3RS isoform decreases average intermolecular distance between dephosphorylated and phosphorylated taus and lead to their overlap. Further phosphorylation does not change noticeably the intermolecular distances.     

Genetically altered mice with different sialyltransferase deficiencies show tissue-specific alterations in sialylation and sialic acid 9-O-acetylation

Glycan chains on glycoconjugates traversing the Golgi apparatus are often terminated by sialic acid residues, which can also be 9-O-acetylated. This process involves competition between multiple Golgi enzymes. Expression levels of Golgi enzyme mRNAs do not always correlate with enzyme activity, which in turn cannot accurately predict glycan sequences found on cell surfaces. Here we examine the cell type-specific expression of terminal glycans in tissues of normal mice in comparison with animals deficient in ST6Gal-I (transfers alpha2-6-linked sialic acid to Galbeta1-4GlcNAc) or ST3Gal-I (transfers alpha2-3-linked sialic acid to Galbeta1-3GalNAc). Tissues of ST6Gal-I null mice showed minimal binding of an alpha2-6-sialic acid-specific lectin, indicating that no other enzyme generates Siaalpha2-6Galbeta1-4GlcNAc and that Siaalpha2-6GalNAc (sialyl-Tn) is rare in mice. However, exposed Galbeta1-4GlcNAc termini were only moderately increased, indicating that these can be partially capped by other enzymes. Indeed, Galalpha1-3Galbeta1-4GlcNAc and Fucalpha1-2Galbeta1-4GlcNAc termini were enhanced in some tissues. Many tissues of ST3Gal-I null animals showed increases in Galbeta1-3GalNAc termini, and some increases in poly-N-acetyllactosamines. However, overall expression of alpha2-3-linked sialic acid was selectively reduced only in a few instances, indicating that other ST3Gal enzymes can generate this linkage in most tissues. Highly selective losses of 9-O-acetylation of sialic acid residues were also observed, with ST6Gal-I deficiency causing loss on endothelium and ST3Gal-I deficiency giving a marked decrease on CD4(+) lymphocytes. These data demonstrate selective regulation of sialylation and 9-O-acetylation, point to cell types with potential physiological defects in null animals, and show in vivo evidence for competition between Golgi enzymes.     

Tumor cell migration and invasion are regulated by expression of variant integrin glycoforms

The ST6Gal-I glycosyltransferase, which adds α2-6-linked sialic acids to glycoproteins, is overexpressed in colon adenocarcinoma, and enzyme activity is correlated with tumor cell invasiveness. Previously we reported that forced expression of oncogenic ras in HD3 colonocytes causes upregulation of ST6Gal-I, leading to increased α2-6 sialylation of β1 integrins. To determine whether ras-induced sialylation is involved in promoting the tumor cell phenotype, we used shRNA to downregulate ST6Gal-I in ras-expressors, and then monitored integrin-dependent responses. Here we show that forced ST6Gal-I downregulation, leading to diminished α2-6 sialylation of integrins, inhibits cell adhesion to collagen I, a β1 ligand. Correspondingly, collagen binding is reduced by enzymatic removal of cell surface sialic acids from ras-expressors with high ST6Gal-I levels (i.e., no shRNA). Cells with forced ST6Gal-I downregulation also exhibit decreased migration on collagen I and diminished invasion through Matrigel. Importantly, GD25 cells, which lack β1 integrins (and ST6Gal-I), do not demonstrate differential invasiveness when forced to express ST6Gal-I, suggesting that the effects of variant sialylation are mediated specifically by β1 integrins. The observation that cell migration and invasion can be blocked in oncogenic ras-expressing cells by forcing ST6Gal-I downregulation implicates differential sialylation as an important ras effector, and also suggests that ST6Gal-I is a promising therapeutic target.     

Epstein-Barr virus latent membrane protein 2 associates with and is a substrate for mitogen-activated protein kinase.

The latent membrane protein 2 (LMP2) of Epstein-Barr virus interferes with B-lymphocyte signal transduction through the immunoglobulin (Ig) receptor. Two isoforms of LMP2 exist and differ only in that one isoform (LMP2a) contains an N-terminal cytoplasmic domain that the other isoform does not. LMP2a is a phosphoprotein that is phosphorylated on tyrosines and serines in the cytoplasmic domain. GST1-119, a glutathione S-transferase (GST) fusion protein containing the 119 amino acids of the cytoplasmic domain, affinity precipitated serine kinase activity from BJAB cell extracts. The affinity-precipitated kinase phosphorylated LMP2a sequences, and kinase activity was increased following induction. Probing of Western immunoblots of affinity-precipitated proteins showed that the Erk1 form of mitogen-activated protein kinase (MAPK) was present. Purified MAPK phosphorylated GST fusion proteins containing the cytoplasmic domain of LMP2a and mutational analyses were used to identify S15 and S102 as the sites of in vitro phosphorylation. A polyclonal rabbit antiserum was prepared against a maltose binding protein-LMP2a cytoplasmic domain fusion protein (MBP1-119) and used to immunoprecipitate LMP2a from the in vitro-immortalized lymphoblastoid B-cell line B95-8CR. LMP2a immunoprecipitates from B95-8CR contained MAPK as a coprecipitated protein. Cross-linking surface Ig on B95-8CR cells failed to induce MAPK activity within the cells. Treatment of B95-8CR with phorbol myristate acetate (PMA) was able to bypass the Ig receptor block and activate MAPK activity. Phosphorylation of LMP2a on serine residues increased after PMA induction. The possible role for LMP2a serine phosphorylation by MAPK in the control of latency is discussed.     

Identification of tyrosines 154 and 307 in the extracellular domain and 653 and 766 in the intracellular domain as phosphorylation sites in the heparin-binding fibroblast growth factor receptor tyrosine kinase (flg).

Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.     

Location and mechanism of alpha 2,6-sialyltransferase dimer formation. Role of cysteine residues in enzyme dimerization, localization, activity, and processing

A significant proportion of the alpha2,6-sialyltransferase of protein Asn-linked glycosylation (ST6Gal I) forms disulfide-bonded dimers that exhibit decreased activity, but retain the ability to bind asialoglycoprotein substrates. Here, we have investigated the subcellular location and mechanism of ST6Gal I dimer formation, as well as the role of Cys residues in the enzyme's trafficking, localization, and catalytic activity. Pulse-chase analysis demonstrated that the ST6Gal I disulfide-bonded dimer forms in the endoplasmic reticulum. Mutagenesis experiments showed that Cys-24 in the transmembrane region is required for dimerization, while catalytic domain Cys residues are required for trafficking and catalytic activity. Replacement of Cys-181 and Cys-332 generated proteins that are largely retained in the endoplasmic reticulum and minimally active or inactive, respectively. Replacement of Cys-350 or Cys-361 inactivated the enzyme without compromising its localization or processing, suggesting that these amino acids are part of the enzyme's active site. Replacement of Cys-139 or Cys-403 generated proteins that are catalytically active and appear to be more stably localized in the Golgi, since they exhibited decreased cleavage and secretion. The Cys-139 mutant also exhibited increased dimer formation suggesting that ST6Gal I dimers may be critical in the oligomerization process involved in stable ST6Gal I Golgi localization.     

The relationship between ST6Gal I Golgi retention and its cleavage-secretion

The ST6Gal I is a sialyltransferase that modifies N-linked oligosaccharides of glycoproteins. Previous results suggested a role for luminal stem and active domain sequences in the efficiency of ST6Gal I Golgi retention. Characterization of a series of STtyr isoform deletion mutants demonstrated that the stem is sensitive to proteases and that preventing cleavage in this region leads to increased cell surface expression. A mutant lacking amino acids 32-104 (STDelta4) is not active or cleaved and secreted like the wild type STtyr, but does exhibit increased cell surface expression. It is probable that the STDelta4 mutant lacks the stem region and some amino acids of the active domain because the STDelta5 mutant lacking amino acids 86-104 is also not active but is cleaved and secreted. In contrast, deletion of stem amino acids between residues 32 and 86 in the STDelta1, STDelta2, and STDelta3 mutants does not inactive these enzyme forms, eliminate their cleavage and secretion, or increase their cell surface expression. Surprisingly, cleavage occurs even though the previously identified Asn63-Ser 64 cleavage site is missing. Further evaluation demonstrated that a cleavage site between Lys 40 and Glu 41 is used in COS cells. Mutagenesis of Lys 40 significantly decreased, but did not eliminate cleavage, suggesting that there are additional secondary sites of cleavage in the ST6Gal I stem.     

Minimal structural and glycosylation requirements for ST6Gal I activity and trafficking

The influence of N-linked glycosylation on the activity and trafficking of membrane associated and soluble forms of the STtyr isoform of the ST6Gal I has been evaluated. We have demonstrated that the enzyme is glycosylated on Asn 146 and Asn 158 and that glycosylation is not required for the endoplasmic reticulum to Golgi transport of the membrane-associated form of the STtyr isoform. In addition, N-linked glycosylation may stabilize the protein but is not absolutely required for catalytic activity in vivo. In contrast, soluble forms of the protein consisting of amino acids 64-403, 89-403, and 97-403 are efficiently secreted and active in their fully glycosylated forms, but retained in the endoplasmic reticulum and inactive in their unglycosylated forms. These results suggest that membrane associated and soluble forms of the STtyr protein have different requirements for N-linked glycosylation. Elimination of the oligosaccharide attached to Asn 158 in the full length STtyr single and double glycosylation mutants generates proteins that are not cleaved and secreted but stably localized in the Golgi, like the STcys isoform of the ST6Gal I. This stable Golgi localization is correlated with the observation that these two mutants are active in in vivo assays but inactive in in vitro assays of membrane lysates. We predict that removal of N-linked oligosaccharides leads to an increased ability of the STtyr protein to self-associate or oligomerize which subsequently allows more stable retention in the Golgi and increased aggregation and inactivity when membranes are lysed in the in vitro activity assays.     

The signal anchor and stem regions of the beta-galactoside alpha 2,6-sialyltransferase may each act to localize the enzyme to the Golgi apparatus.

The beta-galactoside alpha 2,6-sialyltransferase has been localized to the trans cisternae of the Golgi apparatus and the trans Golgi network where it transfers sialic acid residues to terminal positions on N-linked oligosaccharides. It is a type II transmembrane protein possessing a 9-amino acid amino-terminal cytoplasmic tail, a 17-amino acid signal anchor domain, and a 35-amino acid stem region which tethers the large luminal catalytic domain to the membrane anchor. Previous work has demonstrated that the soluble sialytransferase catalytic domain is rapidly secreted from Chinese hamster ovary cells. These results suggest that the signals for Golgi apparatus localization do not reside in the catalytic domain of the enzyme but must reside in the cytoplasmic tail, signal anchor domain, and/or stem region. To determine which amino-terminal regions are required for Golgi apparatus localization, mutant sialyltransferase proteins were constructed by in vitro oligonucleotide-directed mutagenesis, expressed in Cos-1 cells, and localized by indirect immunofluorescence microscopy. Signal cleavage-sialyltransferase mutants which consist of only the stem and catalytic domain of the enzyme are not rapidly secreted but are retained intracellularly and predominantly localized to the Golgi apparatus. However, deletion of either the stem region or the cytoplasmic tail of the membrane-bound sialyltransferase does not alter its Golgi apparatus localization. In addition, sequential replacement of the amino acids of the sialyltransferase signal anchor domain with amino acids from the signal anchor domain of a plasma membrane protein, the influenza virus neuraminidase does not alter the Golgi apparatus localization of the sialyltransferase. These observations suggest that sequences in the signal anchor region and stem region allow the Golgi apparatus localization of the membrane-bound and soluble forms of the sialytransferase, respectively, and that both regions may contain Golgi apparatus localization signals.     

Different glycosyltransferases are differentially processed for secretion, dimerization, and autoglycosylation

Modification of Golgi glycosyltransferases, such as formation of disulfide-bonded dimers and proteolytical release from cells as a soluble form, are important processes to regulate the activity of glycosyltransferases. To better understand these processes, six glycosyltransferases were selected on the basis of the donor sugars, including two N-acetylglucosaminyltransferases, core 1 beta1,3-N-acetylglucosaminyltransferase (C1-beta3GnT) and core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT-I); two fucosyltransferases, alpha1,2-fucosyltransferase-I (FucT-I) and alpha1,3-fucosyltransferase-VII (FucT-VII); and two sialyltransferases, alpha2,3-sialyltransferase-I (ST3Gal-I) and alpha2,6-sialyltransferase-I (ST6Gal-I). These enzymes were fused with enhanced green fluorescence protein and stably expressed in Chinese hamster ovary cells. Spectrofluorimetric detection and immunoblotting analyses showed that all of these glycosyltransferases except FucT-VII were secreted in the medium. By examining dimers formed in cells and culture media, we found that all of the enzymes, except ST3Gal-I, form a combination of monomers and dimers in cells, whereas the molecules released in the media are either exclusively monomers (C2GnT-I and ST6Gal-I), dimers (FucT-I) or a mixture of both (C1-beta3GnT). These results indicate that dimerization does not always lead to Golgi retention. Analysis of the N-glycosylation status of the enzymes revealed that the secreted proteins are generally more heavily N-glycosylated and sialylated than their membrane-associated counterparts, suggesting that the proteolytic cleavage occurs before the glycosylation is completed. Using FucT-I and ST6Gal-I as a model, we also show that these glycosyltransferases are able to perform autoglycosylation in the dimeric forms. These results indicate that different glycosyltranferases differ significantly in dimerization, proteolytic digestion and secretion, and autoglycosylation. These results strongly suggest that disulfide-bonded dimerization and secretion differentially plays a role in the processing and function of different glycosyltransferases in the Golgi apparatus.     

Proteolytic shedding of ST6Gal-I by BACE1 regulates the glycosylation and function of alpha4beta1 integrins

Differentiation of monocytes into macrophages is accompanied by increased cell adhesiveness, due in part to the activation of alpha4beta1 integrins. Here we report that the sustained alpha4beta1 activation associated with macrophage differentiation results from expression of beta1 integrin subunits that lack alpha2-6-linked sialic acids, a carbohydrate modification added by the ST6Gal-I sialyltransferase. During differentiation of U937 monocytic cells and primary human CD14(+) monocytes, ST6Gal-I is down-regulated, leading to beta1 hyposialylation and enhanced alpha4beta1-dependent VCAM-1 binding. Importantly, ST6Gal-I down-regulation results from cleavage by the BACE1 secretase, which we show is dramatically up-regulated during macrophage differentiation. BACE1 up-regulation, ST6Gal-I shedding, beta1 hyposialylation, and alpha4beta1-dependent VCAM-1 binding are all temporally correlated and share the same signaling mechanism (protein kinase C/Ras/ERK). Preventing ST6Gal-I down-regulation (and therefore integrin hyposialylation), through BACE1 inhibition or ST6Gal-I constitutive overexpression, eliminates VCAM-1 binding. Similarly, preventing integrin hyposialylation inhibits a differentiation-induced increase in the expression of an activation-dependent conformational epitope on the beta1 subunit. Collectively, these results describe a novel mechanism for alpha4beta1 regulation and further suggest an unanticipated role for BACE1 in macrophage function.     

ST6Gal-I restrains CD22-dependent antigen receptor endocytosis and Shp-1 recruitment in normal and pathogenic immune signaling

The ST6Gal-I sialyltransferase produces Siglec ligands for the B-cell-specific CD22 lectin and sustains humoral immune responses. Using multiple experimental approaches to elucidate the mechanisms involved, we report that ST6Gal-I deficiency induces immunoglobulin M (IgM) antigen receptor endocytosis in the absence of immune stimulation. This coincides with increased antigen receptor colocalization with CD22 in both clathrin-deficient and clathrin-enriched membrane microdomains concurrent with diminished tyrosine phosphorylation of Igalpha/beta, Syk, and phospholipase C-gamma2 upon immune activation. Codeficiency with CD22 restores IgM antigen receptor half-life at the cell surface in addition to reversing alterations in membrane trafficking and immune signaling. Diminished immune responses due to ST6Gal-I deficiency further correlate with constitutive recruitment of Shp-1 to CD22 in unstimulated B cells independent of Lyn tyrosine kinase activity and prevent autoimmune disease pathogenesis in the Lyn-deficient model of systemic lupus erythematosus, resulting in a significant extension of life span. Protein glycosylation by ST6Gal-I restricts access of antigen receptors and Shp-1 to CD22 and operates by a CD22-dependent mechanism that decreases the basal rate of IgM antigen receptor endocytosis in altering the threshold of B-cell immune activation.