Effect Of N-linked Glycosylation Research Articles

Effect of N-linked glycosylation and disulfide bonds on yeast ER proteome refoldability

Effect of N-linked glycosylation and disulfide bonds on yeast ER proteome refoldability

The Effects of N-Linked Glycosylation on SLC6 Transporters.

Membrane transporters of the solute carrier 6 (SLC6) family mediate various physiological processes by facilitating the translocation of amino acids, neurotransmitters, and other metabolites. In the body, the activity of these transporters is tightly controlled through various post-translational modifications with implications on protein expression, stability, membrane trafficking, and dynamics. While N-linked glycosylation is a universal regulatory mechanism among eukaryotes, a consistent mechanism of how glycosylation affects the SLC6 transporter family remains elusive. It is generally believed that glycans influence transporter stability and membrane trafficking; however, the role of glycosylation on transporter dynamics remains disputable, with differing conclusions among individual transporters across the SLC6 family. In this study, we collected over 1 ms of aggregated all-atom molecular dynamics (MD) simulation data to systematically identify the impact of N-glycans on SLC6 transporter dynamics. We modeled four human SLC6 transporters, the serotonin, dopamine, glycine, and B0AT1 transporters, by first simulating all possible combinations of a glycan attached to each glycosylation site followed by investigating the effect of larger, oligo-N-linked glycans to each transporter. The simulations reveal that glycosylation does not significantly affect the transporter structure but alters the dynamics of the glycosylated extracellular loop and surrounding regions. The structural consequences of glycosylation on the loop dynamics are further emphasized with larger glycan molecules attached. However, no apparent differences in ligand stability or movement of the gating helices were observed, and as such, the simulations suggest that glycosylation does not have a profound effect on conformational dynamics associated with substrate transport.

Effect of N-linked glycosylation at position 162 of hemagglutinin in influenza A virus A(H1N1)pdm09

Subtype H1N1 influenza A virus (IAV) that caused a pandemic in 2009, called A(H1N1)pdm09, has been circulating as a seasonal IAV among humans, escaping from herd immunity through amino acid substitutions in hemagglutinin (HA). Additionally, position 162 of HA in A(H1N1)pdm09 evolved to be N-linked glycosylated by 2017. Here the effect of N-linked glycosylation at position 162 of HA in A(H1N1)pdm09 was examined by comparing the ratio of nonsynonymous diversity (πN) to synonymous diversity (πS) (πN/πS) at structurally defined categories of amino acid sites between 1785 (162+) and 7562 (162−) sequences of HA that were and were not considered to be N-linked glycosylated at position 162, respectively. In 162− sequences, πN/πS for the sites located inside 15 Å from position 162 was not significantly smaller than one and was greater than that for the sites located outside 15 Å. In 162+ sequences, πN/πS for the sites located inside and outside 15 Å from position 162 were smaller than the corresponding values in 162− sequences, and were not significantly different from each other. These results suggest that N-linked glycosylation at position 162 of HA may shield epitopes against antibodies in A(H1N1)pdm09.

Effect of N-linked glycosylation on the activity and stability of a β-glucosidase from Putranjiva roxburghii

In this study, the effect of N-glycosylation on the conformational and functional stability of Putranjiva roxburghii family 1 β-glucosidase (PRGH1) enzyme was investigated. The deglycosylation of PRGH1 was carried out by using PNGase F enzyme and confirmed by SDS-PAGE and carbohydrate estimation. Comparative analysis with respect to enzyme activity, stability and aggregation behaviour was carried out for the glycosylated and deglycosylated PRGH1. The deglycosylation of PRGH1 affected enzyme activity to a certain extent only where Km was not affected but a slight reduction in Vmax for various substrates was observed. Circular dichroism, fluorescence studies and differential scanning calorimetry (DSC) analysis demonstrated the possible effect of glycosylation on local and/or global conformational dynamics of protein and its effect on the thermostability of PRGH1. DSC results showed deglycosylated form had lower Tm as compared to the glycosylated form of PRGH1. The PRGH1 was found to be more sensitive to proteolysis after deglycosylation suggesting that the glycosylated PRGH1 was quite compact and rigid. Mutagenesis studies revealed that out of seven potential N-linked glycosylation sites, only three were glycosylated. The results demonstrated that N-linked glycosylation played an important role in conformational stability of PRGH1; however, it did not affect the enzyme function drastically.

Effect of Phosphorylation of CM2 Protein on Influenza C Virus Replication.

CM2 is the second membrane protein of the influenza C virus and has been demonstrated to play a role in the uncoating and genome packaging processes in influenza C virus replication. Although the effects of N-linked glycosylation, disulfide-linked oligomerization, and palmitoylation of CM2 on virus replication have been analyzed, the effect of the phosphorylation of CM2 on virus replication remains to be determined. In this study, a phosphorylation site(s) at residue 78 and/or 103 of CM2 was replaced with an alanine residue(s), and the effects of the loss of phosphorylation on influenza C virus replication were analyzed. No significant differences were observed in the packaging of the reporter gene between influenza C virus-like particles (VLPs) produced from 293T cells expressing wild-type CM2 and those from the cells expressing the CM2 mutants lacking the phosphorylation site(s). Reporter gene expression in HMV-II cells infected with VLPs containing the CM2 mutants was inhibited in comparison with that in cells infected with wild-type VLPs. The virus production of the recombinant influenza C virus possessing CM2 mutants containing a serine-to-alanine change at residue 78 was significantly lower than that of wild-type recombinant influenza C virus. Furthermore, the virus growth of the recombinant viruses possessing CM2 with a serine-to-aspartic acid change at position 78, to mimic constitutive phosphorylation, was virtually identical to that of the wild-type virus. These results suggest that phosphorylation of CM2 plays a role in efficient virus replication, probably through the addition of a negative charge to the Ser78 phosphorylation site.IMPORTANCE It is well-known that many host and viral proteins are posttranslationally modified by phosphorylation, which plays a role in the functions of these proteins. In influenza A and B viruses, phosphorylation of viral proteins NP, M1, NS1, and the nuclear export protein (NEP), which are not integrated into the membranes, affects the functions of these proteins, thereby affecting virus replication. However, it was reported that phosphorylation of the influenza A virus M2 ion channel protein, which is integrated into the membrane, has no effect on virus replication in vitro or in vivo We previously demonstrated that the influenza C virus CM2 ion channel protein is modified by N-glycosylation, oligomerization, palmitoylation, and phosphorylation and have analyzed the effects of these modifications, except phosphorylation, on virus replication. This is the first report demonstrating that phosphorylation of the influenza C virus CM2 ion channel protein, unlike that of the influenza A virus M2 protein, plays a role in virus replication.

ADAMTS‐13 glycans and conformation‐dependent activity

Background ADAMTS-13 activity can be regulated by its conformation, whereby interactions between the C-terminal CUB domains and the spacer domain maintain ADAMTS-13 in a closed conformation. ADAMTS-13 contains 10 N-linked glycans, with four sites present in theTSP2 through to CUB domains that may contribute to its conformation. Objectives/Methods We hypothesized that glycosylation contributes to ADAMTS-13 conformation and function. The proteolytic activity of glycan-modified ADAMTS-13 was assessed under static and shear stress conditions. Results Enzymatic removal of terminal silaic acid or entire N-linked glycan chains decreased activity against FRETS-VWF73 at pH 7.4 and against full-length von Willebrand factor (VWF) under shear stress. Using truncated ADAMTS-13, we demonstrated that this was attributable to loss of sialic acid from the glycans in the metalloprotease domain and an effect of N-linked glycosylation in the TSP2 through to CUB domains. Mutation of the N-linked glycan sites in the MDTCS domains reduced or abolished protein expression. However, the N707Q, N828Q, N1235Q and N1354Q (TSP2, TSP4, CUB1, and CUB2 domains, respectively) variants were expressed normally. Interestingly, the N707Q and N828Q variants showed reduced activity against FRETS-VWF73, but normal activity under flow conditions. In contrast, the N1235Q and N1354Q variants had enhanced activity against FRETS-VWF73 and VWF under shear stress. Immunoprecipitation experiments confirmed that loss of N-linked glycans in the CUB domains significantly reduced the interaction with the spacer domain and enhanced binding to the 6A6 anti-ADAMTS-13 antibody, which recognizes a cryptic epitope in the metalloprotease domain. Conclusions Together, these data demonstrate that the N-linked glycans of ADAMTS-13 play a crucial role in regulating ADAMTS-13 activity.

α(1,3)-Fucosyltransferases FUT4 and FUT7 Control Murine Susceptibility to Thrombosis

The α(1,3)-fucosyltransferases, types IV and VII (FUT4 and FUT7, respectively), are required for the synthesis of functional selectin-type leukocyte adhesion molecule ligands. The selectins and their ligands modulate leukocyte trafficking, and P-selectin and its ligand, P-selectin glycoprotein ligand-1, can modulate hemostasis and thrombosis. Regulation of thrombosis by FUT4 and/or FUT7 activity was examined in mouse models of carotid artery thrombosis and collagen/epinephrine-induced thromboembolism. Mice lacking both FUT4 and FUT7 (Fut(-/-) mice) had a shorter time to occlusive thrombus formation in the injured carotid artery and a higher mortality due to collagen/epinephrine-induced pulmonary thromboemboli. Mice lacking P-selectin or P-selectin glycoprotein ligand-1 did not have a prothrombotic phenotype. Whole blood platelet aggregation was enhanced, and plasma fibrinogen content, clot weight, and clot strength were increased in Fut(-/-) mice, and invitro clot lysis was reduced compared with wild type. Fut4(-/-), but not Fut7(-/-), mice had increased pulmonary thromboembolism-induced mortality and decreased thromboemboli dissolution invivo. These data show that FUT4 and FUT7 activity regulates thrombosis in a P-selectin- and P-selectin glycoprotein ligand-1-independent manner and suggest that FUT4 activity is important for thrombolysis.

Effects of N-linked glycosylation of the fusion protein on replication of human metapneumovirus in vitro and in mouse lungs

The fusion (F) protein is an important membrane glycoprotein necessary for cellular entry and replication of human metapneumovirus (hMPV). Selective prevention of N-linked glycosylation may compromise the catalytic and fusion functions of the F protein. By using site-directed mutagenesis and reverse genetics, recombinant mutant viruses lacking one or two N-linked glycosylation sites in the F protein were constructed. M1, which lacked glycosylation at position 57 of the F protein, had slightly compromised replication, whereas M2 and M4, which lacked glycosylation at position(s) 172 or 57 and 172, respectively, showed profound impairment of replication when compared with wild-type (WT) NL/1/00-GFP virus in both Vero E6 cells and mouse lungs. M2 was less fit than WT virus in vitro and in immunocompromised mouse lungs. The F proteins of WT and mutant viruses were similarly expressed on the infected cell membrane, while the activated fusion protein subunits, F1 of M2 and M4, were produced in lower quantities compared with those of WT and M1 virus. The mutated viruses lacking N-linked glycosylation at position 353, either individually or together with other sites, could not be recovered. Thus, N-linked glycosylation may be involved in the catalysis of the fusion protein from F0 to F1 and F2, which is critical for fusion function. Strategies targeting N-linked glycosylation may be helpful for developing attenuated live vaccines or antiviral drugs for hMPV.

Effects of N-linked glycosylation on the creatine transporter

The CRT (creatine transporter) is a member of the Na+- and Cl--dependent neurotransmitter transporter family and is responsible for the import of creatine into cells, and thus is important for cellular energy metabolism. We established for CRT an expression system in HEK-293 cells that allowed biochemical, immunological and functional analysis of CRT wild-type and glycosylation-deficient mutants. Analysis of HA (haemagglutinin)-tagged CRT-NN (wild-type rat CRT with an HA-tag at the C-terminus) revealed several monomeric immunoreactive species with apparent molecular masses of 58, 48 and 43 kDa. The 58 kDa species was shown to be plasma-membrane-resident by EndoHf (endoglycosidase Hf) and PNGase F (peptide N-glycosidase F) treatments and represents fully glycosylated CRT, whereas the 48 kDa and 43 kDa species were glycosylation intermediates and non-glycosylated CRT respectively. Glycosylation-deficient mutants (Asn192Asp, Asn197Asp and Asn192Asp/Asn197Asp) showed altered electrophoretic mobility, indicating that CRT is indeed N-glycosylated. In addition, a prominent CRT band in the range of 75-91 kDa was also detected. Pharmacological inhibition of N-linked glycosylation by tunicamycin in CRT-NN-expressing cells gave a similar reduction in molecular mass, corroborating the finding that Asn192 and Asn197 are major N-glycosylation sites in CRT. Although the apparent Km was not significantly affected in glycosylation-deficient mutants compared with CRT-NN, we measured reduced Vmax values for all mutants (21-28% residual activity), and 51% residual activity after enzymatic deglycosylation of surface proteins in intact CRT-NN cells by PNGase F. Moreover, immunocytochemical analysis of CRT-NN- and CRT-DD-expressing cells (where CRT-DD represents a non-glycosylated double mutant of CRT, i.e. Asn192Asp/Asn197Asp) showed a lower abundance of CRT-DD in the plasma membrane. Taken together, our results suggest that plasma-membrane CRT is glycosylated and has an apparent monomer molecular mass of 58 kDa. Furthermore, N-linked glycosylation is neither exclusively important for the function of CRT nor for surface trafficking, but affects both processes. These findings may have relevance for closely related neurotransmitter transporter family members.

Effect of N-linked glycosylation on secretion, activity, and stability of alpha-amylase from Aspergillus oryzae.

The effect of N-linked glycosylation on secretion, activity, and stability of alpha-amylase from Aspergillus oryzae grown as dispersed filaments was studied. In the presence of tunicamycin the fungus grew either as dispersed filaments or as one large pellet, whereas growth was as dispersed filaments in all control cultures. The presence of tunicamycin affected neither biomass, level of secreted alpha-amylase, nor total amount of secreted protein in cultures growing as dispersed filaments. In these cultures both glycosylated and nonglycosylated alpha-amylase appeared in the culture medium as well as in the cells, whereas in control cultures only the glycosylated form of alpha-amylase was found in the medium and in the cells. The presence of nonglycosylated alpha-amylase in the medium seemed to result from active secretion rather than from autolysis of the mycelium or extracellular deglycosylation. Deglycosylation with Endo H of crude alpha-amylase in culture filtrate did not affect its stability towards heat, acid pH, or proteolytic degradation.

The Effect of N-linked Glycosylation on Molecular Weight, Thrombin Cleavage, and Functional Activity of Human Protein S

Human protein S (HPS) has three potential N-linked glycosylation sites at Asn458, 468, 489. To study the role of glycosylation at these sites, PCR mutagenesis was used to abolish the consensus sequence of each N-linked glycosylation site (Asn458-->Gln, Ser460-->Gly; Asn468-->Gln, Thr470-->Gly; Asn489-->Gln, Thr491-->Gly) in full-length HPS cDNA. Each resulting construct was expressed in human kidney 293 cells by stable transfection of cDNA/SV40/adeno/pBR322-derived expression vectors, and conditioned medium was collected for recombinant protein purification. SDS-PAGE gels revealed that glycosylation mutants migrate identically and faster than the wild-type rHPS, showing that each of the three potential N-glycosylation sites contain a similar amount of carbohydrate. Mass spectral analysis yielded similar results and a molecular mass of approximately 78,000 for wild-type HPS. To demonstrate that the difference in mobility between wild-type and mutant protein S is due to their carbohydrate content, plasma-derived HPS and recombinant HPS were subjected to N-glycanase digestion and subsequently shown to migrate identically on SDS-PAGE gels. All forms of HPS have similar time courses for cleavage by alpha-thrombin. Functional studies indicate that wild-type rHPS possesses the same cofactor specific activity as plasma-derived HPS, as tested by a standard clotting assay. Asn458 and Ser460 mutant rHPS have only a slightly higher cofactor activity, whereas the other four mutants have similar clotting activities, compared to wild-type rHPS. In a purified component system, glycosylation mutants of protein S showed a slightly enhanced ability to stimulate APC-mediated factor Va inactivation after an initial lag phase. The interaction of rHPS glycosylation mutants with human C4b-binding protein (C4bp) was also studied by solution phase equilibrium binding assay. Two mutants (Asn458, Ser480) have marginally lower dissociated constants (Kd) with C4bp, whereas the others have the same apparent Kd as wild-type rHPS.

The effect of N-linked glycosylation on activity of the Na(+)- and Cl(-)-dependent serotonin transporter expressed using recombinant baculovirus in insect cells

The rat Na(+)- and Cl(-)-dependent serotonin transporter was expressed in Sf9 insect cells using the baculovirus system. Expression of the serotonin transporter caused the Sf9 cells to accumulate [3H]serotonin (Km 78 nM) and to bind the specific transport inhibitor [125I]RT155 (2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane) (Kd 0.22 nM). Ligand binding assays on isolated membranes showed 500,000 copies of the serotonin transporter/cell (9 pmol/mg of membrane protein). Immunoreactive bands of apparent M(r) 54,000 (unglycosylated) and 60,000 (glycosylated) were observed in Western blots of membrane proteins from infected cells. The 54-kDa band was significantly smaller than the expected M(r) of 72,500 predicted from the cDNA sequence. The 54-kDa band was shown to represent the intact serotonin transporter by expressing a recombinant serotonin transporter that contained c-Myc and FLAG epitope tags engineered at the N and C termini, respectively. Both tags were present on a membrane protein that migrated slightly slower than the previously observed 54-kDa band, consistent with the extra mass added by the tags. The tags did not affect the Kd for [125I]RT155 binding. The effect of N-linked glycosylation on ligand binding and the level of expression were studied. The expression of the serotonin transporter in tunicamycin-treated Sf9 cells resulted in low levels of ligand binding activity (0.2 pmol/mg) but unchanged Kd. Similarly, mutated serotonin transporters that contained reduced numbers of N-linked glycosylation sites had unchanged Kd for [125I]RT155 binding whether there were 2, 1, or 0 N-linked glycosylation sites present on the serotonin transporter. In contrast, Bmax was dramatically reduced; levels of expression of the unglycosylated serotonin transporter (0.4 pmol/mg) were 20-fold lower compared with levels of the fully glycosylated serotonin transporter. The Km for [3H]serotonin uptake was also unchanged. These data indicate that glycosylation is required for optimal stability of the serotonin transporter in the membrane but not for serotonin transport or ligand binding per se.

The conformational effects of N-linked glycosylation

The conformational effects of N-linked glycosylation

Secretion of N-glycosylated interleukin-1 beta in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N-linked glycosylation on biological activity

Human interleukin-1 beta (IL-1 beta) is expressed in activated monocytes as a 31-kDa precursor protein which is processed and secreted as a mature, unglycosylated 17-kDa carboxyl-terminal fragment, despite the fact that it contains a potential N-linked glycosylation site near the NH2 terminus (-Asn7-Cys8-Thr9-). cDNA coding for authentic mature IL-1 beta was fused to the signal sequence from the Candida albicans glucoamylase gene, two amino acids downstream from the signal processing site. Upon expression in Saccharomyces cerevisiae, approximately equimolar amounts of N-glycosylated (22 kDa) and unglycosylated (17 kDa) IL-1 beta protein were secreted. The N-glycosylated yeast recombinant IL-1 beta exhibited a 5-7-fold lower specific activity compared to the unglycosylated species. The mechanism responsible for inefficient glycosylation was also studied. We found no differences in secretion kinetics or processing between the two extracellular forms of IL-1 beta. The 17-kDa protein, which was found to lack core sugars, does not result from deglycosylation of the 22-kDa protein in vivo and does not result from saturation of the glycosylation enzymatic machinery through overexpression. Alteration of the uncommon Cys8 residue in the -Asn-X-Ser/Thr-glycosylation site to Ser also had no effect. However, increasing the distance between Asn7 and the signal processing site increased the extent of core N-linked glycosylation, suggesting a reduction in glycosylation efficiency near the NH2 terminus.

Effect of N-linked glycosylation on hepatic lipase activity

Hepatic lipase (HL) is a secretory protein synthesized in hepatocytes and bound to liver endothelium. Previous studies have suggested that HL N-linked glycans are required for catalytic activity. To directly test this hypothesis, Xenopus laevis oocytes were used to express native rat HL or HL lacking one or both N-linked glycosylation sites. The expressed and secreted native HL had an apparent molecular mass of 53 kDa, consistent with purified rat liver HL. The mutant lacking both glycosylation sites, while poorly secreted, had an apparent molecular mass of 48 kDa, the same size observed for HL after enzymatic removal of N-linked oligosaccharides. Mutants lacking one of the two sites were intermediate in size and showed reduced secretion. Each of these expressed and secreted proteins had full catalytic activity that was inhibited by antisera to rat HL. Thus, N-linked glycosylation of rat HL, while important to lipase secretion, is not essential for the expression of lipase activity.

The effect of N-linked glycosylation on the substrate preferences of UDP glucuronosyltransferases

The presence of potential N-linked glycosylation sites (Asn-X-Ser/Thr) in two forms of UDP glucuronosyltransferase, designated UDPGTr-2 and UDPGTr-4, has been deduced from cDNA sequence data. These forms were glycosylated when synthesized from expression vectors transfected into COS cells and were converted to faster migrating species on SDS polyacrylamide gels when treated with endoglycosidase H. The role of glycosylation was investigated by determining the substrate specificities and stabilities of the glycosylated enzymes and their unglycosylated variants which were synthesized in the presence of tunicamycin. Analysis of the activities towards 13 different aglycones showed that the glycosyl moiety was not essential for catalytic activity and had no effect on the substrate preference of each form. The stabilities of the proteins were not adversely affected by the absence of this posttranslational modification. A possible effect of N-linked oligosaccharides on the catalytic properties of these two forms of UDP glucuronosyltransferase is discussed.

Maturation and secretion of lipoprotein lipase in cultured adipose cells. II. Effects of tunicamycin on activation and secretion of the enzyme

The effects of N-linked glycosylation on the activation and secretion of lipoprotein lipase were studied in Ob17 cells. The cells were first depleted of any activity and enzyme content by cycloheximide treatment and of precursors of oligosaccharide chains by tunicamycin. The repletion of lipoprotein lipase content was studied in these cells maintained in the presence of tunicamycin after cycloheximide removal. During the repletion phase, the EC 50 values of inhibition by tunicamycin (approx. 0.2 μg/ml) of the incorporation of labeled glucose, mannose or galactose into trichloroacetic acid-insoluble material were found to be identical. Under these conditions, the rate of protein synthesis was maximally decreased by 30%. The results showed clearly that the recovery in lipoprotein lipase activity was parallel to the recovery in hexose incorporation, no activity being recovered in the absence of glycosylation. An inactive form of lipoprotein lipase from tumicamycin-treated cells was detected by competition experiments with mature active lipoprotein lipase for the binding to immobilized antilipoprotein lipase antibodies, as well as by immunofluorescence staining. SDS-polyacrylamide gel electrophoresis and Western blots of cellular extracts and of extracellular media, obtained after tunicamycin-treated cells were exposed to heparin, revealed a single immunodetectable M r 52000 protein, whereas a single M r 57000 protein was detected in control cells. Therefore, the results indicate that the acquisition by lipoprotein lipase of a catalytically active conformation is linked directly or indirectly to glycosylation. Despite this lack of activation, the lipoprotein lipase molecule was able to migrate intracellularily and to undergo secretion after heparin stimulation of the tunicamycin-treated cells.