A specific screen for oligosaccharyltransferase mutations identifies the 9 kDa OST5 protein required for optimal activity in vivo and in vitro.

The central reaction in the process of N-linked protein glycosylation in eukaryotic cells, the transfer of the oligosaccharide Glc(3)Man(9)GlcNAc(2) from the lipid dolicholpyrophosphate to selected asparagine residues, is catalyzed by the oligosaccharyltransferase (OTase). This enzyme consists of multiple subunits; however, purification of the complex has revealed different results with respect to its protein composition. To determine how many different loci are required for OTase activity in vivo, we performed a novel, specific screen for mutants with altered OTase activity. Based on the synthetic lethal phenotype of OTase mutants in combination with a deficiency of dolicholphosphoglucose biosynthesis which results in non-glucosylated lipid-linked oligosaccharide, we identified seven complementation groups with decreased OTase activity. Beside the known OTase loci, STT3, OST1, WBP1, OST3, SWP1 and OST2, a novel locus, OST5, was identified. OST5 is an intron-containing gene encoding a putative membrane protein of 9.5 kDa present in highly purified OTase preparations. OST5 protein is not essential for growth but its depletion results in a reduced OTase activity. Suppression of an ost1 mutation by overexpression of OST5 indicates that this small membrane protein directly interacts with other OTase components, most likely with Ost1p. A strong genetic interaction with a stt3 mutation implies a role in complex assembly.     

The STT3 protein is a component of the yeast oligosaccharyltransferase complex

N-linked protein glycosylation is an essential process in eukaryotic cells. In the central reaction, the oligosaccharyltransferase (OTase) catalyzes the transfer of the oligosaccharide Glc₃Man₉GlcNAc₂ from dolicholpyrophosphate onto asparagine residues of nascent polypeptide chains in the lumen of the endoplasmic reticulum. The product of the essential gene STT3 is required for OTase activity in vivo, but is not present in highly purified OTase preparations. Using affinity purification of a tagged Stt3 protein, we now demonstrate that other components of the OTase complex, namely Ost1p, Wbp1p and Swp1p, specifically co-purify with the Stt3 protein. In addition, different conditional stt3 alleles can be suppressed by overexpression of either OST3 and OST4, which encode small components of the OTase complex. These genetic and biochemical data show that the highly conserved Stt3p is a component of the oligosaccharyltransferase complex. Received: 3 June 1997 / Accepted: 29 July 1997     

The 3.4-kDa Ost4 protein is required for the assembly of two distinct oligosaccharyltransferase complexes in yeast

In the central reaction of N-linked glycosylation, the oligosaccharyltransferase (OTase) complex catalyzes the transfer of a lipid-linked core oligosaccharide onto asparagine residues of nascent polypeptide chains in the lumen of the endoplasmic reticulum (ER). The Saccharomyces cerevisiae OTase has been shown to consist of at least eight subunits. We analyzed this enzyme complex, applying the technique of blue native gel electrophoresis. Using available antibodies, six different subunits were detected in the wild-type (wt) complex, including Stt3p, Ost1p, Wbp1p, Swp1p, Ost3p, and Ost6p. We demonstrate that the small 3.4-kDa subunit Ost4p is required for the incorporation of either Ost3p or Ost6p into the complex, resulting in two, functionally distinct OTase complexes in vivo. Ost3p and Ost6p are not absolutely required for OTase activity, but modulate the affinity of the enzyme toward different protein substrates.     

All in one: Leishmania major STT3 proteins substitute for the whole oligosaccharyltransferase complex in Saccharomyces cerevisiae

The transfer of lipid-linked oligosaccharide to asparagine residues of polypeptide chains is catalyzed by oligosaccharyltransferase (OTase). In most eukaryotes, OTase is a hetero-oligomeric complex composed of eight different proteins, in which the STT3 component is believed to be the catalytic subunit. In the parasitic protozoa Leishmania major, four STT3 paralogues, but no homologues to the other OTase components seem to be encoded in the genome. We expressed each of the four L. major STT3 proteins individually in Saccharomyces cerevisiae and found that three of them, LmSTT3A, LmSTT3B, and LmSTT3D, were able to complement a deletion of the yeast STT3 locus. Furthermore, LmSTT3D expression suppressed the lethal phenotype of single and double deletions in genes encoding other essential OTase subunits. LmSTT3 proteins did not incorporate into the yeast OTase complex but formed a homodimeric enzyme, capable of replacing the endogenous, multimeric enzyme of the yeast cell. Therefore, these protozoan OTases resemble the prokaryotic enzymes with respect to their architecture, but they used substrates typical for eukaryotic cells: N-X-S/T sequons in proteins and dolicholpyrophosphate-linked high mannose oligosaccharides.     

Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases.

The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the membrane of the ER. We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the alpha-1,6 mannose to dolichol-linked Man7GlcNAc2. The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. Using the combination of mutant strains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branching. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum.     

Cell-free N-glycosylation in Dictyostelium discoideum: analysis of wild-type and mutants defective in lipid-linked oligosaccharide biosynthesis

N-glycosylation was measured in wild-type cell lysates of Dictyostelium discoideum and in two mutant strains that synthesize a truncated lipid-linked oligosaccharide, Man6GlcNAc2 lacking terminal mannose and glucose residues. Endogenous lipid-linked oligosaccharide (LLO) was transferred to octanoyl-Asn-[125I]Tyr-ThrNH2 by membrane fractions. About 50% of the glycopeptide product remained associated with membranes. Taurocholate and saponin promoted and preserved glycosylation, but NP-40 and Triton X-100 did not. Using this artificial assay, the rate and extent of transfer of the truncated lipid-linked oligosaccharide in extracts of the two mutant strains, HL241 and HL243, was reduced 5-10-fold relative to that of wild-type. The low activity found in the mutant strains appears to result from either reduced affinity of the truncated LLO for the transferase or from its improper topological localization in the membrane. When protein N-glycosylation is measured in living cells it is nearly normal in HL241, but it is 3-4-fold decreased in HL243. Although the results of the in vitro and in vivo assays differ, they are not in conflict. Rather, they suggest that the static in vitro assay may be capable of revealing subtleties in the productive positioning of LLO and the oligosaccharyl transferase. The decrease in glycosylation seen in intact HL243 cells may be a consequence of the pleiotropic effects of the primary mutation rather than a direct result of the altered LLO structure. Genetic analysis showed that the mutation in HL241 is recessive, while the mutation in HL243 is dominant and prevents normal development. Thus, the two mutants share a lesion in lipid-linked oligosaccharide biosynthesis and in cell-free glycosylation, but differ in their in vivo glycosylation. Their primary defects are probably different.     

Quality control in biosynthetic pathways of N-linked glycoproteins in the yeast endoplasmic reticulum

Summary The initial steps in N-glycosylation involve the synthesis of dolichol-linked Glc₃Man₉GlcNAc₂ oligosaccharides and the transfer of these oligosaccharides to nascent polypeptides. These processes take place at the membrane of the endoplasmic reticulum (ER) and are conserved among eukaryotes. Once transferred to the protein the N-linked oligosaccharides are immediately trimmed by glycosidases located in the ER. This review focuses on the N-linked glycosylation pathway in the ER ofSaccharomyces cerevisiae andSchizosaccharomyces pombe. In particular, we outline how yeast cells ensure that only completely assembled lipid-linked oligosaccharides are transferred to nascent polypeptides. We will discuss the oligosaccharide trimming of glycoproteins with respect to glycoprotein quality control and degradation, focusing on the two different quality control mechanisms ofS. cerevisiae andS. pombe.Abbreviations CPY carboxypeptidase Y - ER endoplasmic reticulum - LLO lipid-linked oligosaccharide - NLO protein-linked oligosaccharide - OTase oligosaccharyltransferase     

Controlling N-linked glycan site occupancy

N-linked glycosylation, a common co-translational modification in eukaryotic cells, involves the transfer of a lipid-linked oligosaccharide onto asparagine residues in a tripeptide sequon on a nascent protein in the lumen of the endoplasmic reticulum. The attachment of an oligosaccharide unit to the polypeptide at the site of occupancy can enhance solubility, improve folding, facilitate secretion, modulate antigenicity, and increase in vivo half-life of the glycoprotein. A number of proteins exhibit variable site occupancy. The efficiency of protein N-glycosylation is dependent on the kinetics of the individual steps in the biosynthesis of the dolichol-linked oligosaccharide and the transfer of the oligosaccharide from the lipid donor substrate to the nascent polypeptide. In this review, we will discuss the role of N-linked glycan site occupancy and give an overview of the possible limitations associated with variable site occupancy. The characterization of the dolichol pyrophosphate biosynthetic pathway and the recent identification of potential rate limiting enzymes in yeast and mammalian cells has made it possible to investigate their role in site occupancy. Genetic and biochemical characterization of oligosaccharide transferase (OST) complex in yeast and mammalian cells have demonstrated the importance of specific OST subunits in protein N-glycosylation. In addition, insights into the location and residues in and around the acceptor tripeptide sequon suggest an influence on N-glycan site occupancy. Insights from these characterizations are being used to elucidate methodologies to control N-glycosylation site heterogeneity.     

ALG9 mannosyltransferase is involved in two different steps of lipid-linked oligosaccharide biosynthesis

N-linked protein glycosylation follows a conserved pathway in eukaryotic cells. The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for the oligosaccharyltransferase (OST), is catalyzed by different glycosyltransferases located at the membrane of the endoplasmic reticulum (ER). The substrate specificity of the different glycosyltransferase guarantees the ordered assembly of the branched oligosaccharide and ensures that only completely assembled oligosaccharide is transferred to protein. The glycosyltransferases involved in this pathway are highly specific, catalyzing the addition of one single hexose unit to the lipid-linked oligosaccharide (LLO). Here, we show that the dolichylphosphomannose-dependent ALG9 mannosyltransferase is the exception from this rule and is required for the addition of two different alpha-1,2-linked mannose residues to the LLO. This report completes the list of lumen-oriented glycosyltransferases required for the assembly of the LLO.     

The alpha subunit of the Saccharomyces cerevisiae oligosaccharyltransferase complex is essential for vegetative growth of yeast and is homologous to mammalian ribophorin I

Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae Ost1 protein and ribophorin I, a previously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OST1 locus was not tolerated in haploid yeast showing that expression of the Ost1 protein is essential for vegetative growth of yeast. An analysis of a series of conditional ost1 mutants demonstrated that defects in the Ost1 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins at both the permissive and restrictive growth temperatures. Microsomal membranes isolated from ost1 mutant yeast showed marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Microsomal membranes isolated from the ost1 mutants contained elevated amounts of the Kar2 stress-response protein.     

Identification of genes involved in the biosynthesis and attachment of Methanococcus voltae N-linked glycans: insight into N-linked glycosylation pathways in Archaea

N-linked glycosylation is recognized as an important post-translational modification across all three domains of life. However, the understanding of the genetic pathways for the assembly and attachment of N-linked glycans in eukaryotic and bacterial systems far outweighs the knowledge of comparable processes in Archaea. The recent characterization of a novel trisaccharide [beta-ManpNAcA6Thr-(1-4)-beta-GlcpNAc3NAcA-(1-3)-beta-GlcpNAc]N-linked to asparagine residues in Methanococcus voltae flagellin and S-layer proteins affords new opportunities to investigate N-linked glycosylation pathways in Archaea. In this contribution, the insertional inactivation of several candidate genes within the M. voltae genome and their resulting effects on flagellin and S-layer glycosylation are reported. Two of the candidate genes were shown to have effects on flagellin and S-layer protein molecular mass and N-linked glycan structure. Further examination revealed inactivation of either of these two genes also had effects on flagella assembly. These genes, designated agl (archaeal glycosylation) genes, include a glycosyl transferase (aglA) involved in the attachment of the terminal sugar to the glycan and an STT3 oligosaccharyl transferase homologue (aglB) involved in the transfer of the complete glycan to the flagellin and S-layer proteins. These findings document the first experimental evidence for genes involved in any glycosylation process within the domain Archaea.     

Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway

N-Linked protein glycosylation in most eukaryotic cells initiateswith the transfer of the oligosaccharide Glc₃Man₉GlcNAc₂ fromthe lipid carrier dolichyl pyrophosphate to selected asparagineresidues. In the yeast Saccharomyces cerevisiae, alg mutationswhich affect the assembly of the lipid-linked oligosaccharideat the membrane of the endoplasmic reticulum result in the accumulationof lipid-linked oligosaccharide intermediates and a hypoglycosylationof proteins. Exploiting the synthetic growth defect of alg mutationsin combination with mutations affecting oligosaccharyl transferaseactivity (Stagljar et al., 1994), we have isolated the ALG6locus. alg6 mutants accumulate lipid-linked Man₉GlcNAc₂, suggestingthat this locus encodes an endoplasmic glucosyltransferase.Alg6p has sequence similarity to Alg8p, a protein required forglucosylation of Glc₁Man⁹GlcNAc². Saccharomyces cerevisiae endoplasmic reticulum glycosyltransferase dolichol     

The essential OST2 gene encodes the 16-kD subunit of the yeast oligosaccharyltransferase, a highly conserved protein expressed in diverse eukaryotic organisms

Oligosaccharyltransferase catalyzes the transfer of a preassembled high mannose oligosaccharide from a dolichol-oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six non-identical subunits (alpha-zeta). The alpha, beta, gamma, and delta subunits of the oligosaccharyltransferase are encoded by the OST1, WBP1, OST3, and SWP1 genes, respectively. Here we describe the functional characterization of the OST2 gene that encodes the epsilon- subunit of the oligosaccharyltransferase. Genomic disruption of the OST2 locus was lethal in haploid yeast showing that expression of the Ost2 protein is essential for viability. Overexpression of the Ost2 protein suppresses the temperature-sensitive phenotype of the wbp1-2 allele and increases in vivo and in vitro oligosaccharyltransferase activity in a wbp1-2 strain. An analysis of a series of conditional ost2 mutants demonstrated that defects in the Ost2 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins. Microsomal membranes isolated from ost2 mutant yeast show marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Surprisingly, the Ost2 protein was found to be 40% identical to the DAD1 protein (defender against apoptotic cell death), a highly conserved protein initially identified in vertebrate organisms. The protein sequence of ost2 mutant alleles revealed mutations at highly conserved residues in the Ost2p/DAD1 protein sequence.     

Identification and functional analysis of a defect in the human ALG9 gene: definition of congenital disorder of glycosylation type IL

Defects of lipid-linked oligosaccharide assembly lead to alterations of N-linked glycosylation known as "type I congenital disorders of glycosylation" (CDG). Dysfunctions along this stepwise assembly pathway are characterized by intracellular accumulation of intermediate lipid-linked oligosaccharides, the detection of which contributes to the identification of underlying enzymatic defects. Using this approach, we have found, in a patient with CDG, a deficiency of the ALG9 alpha 1,2 mannosyltransferase enzyme, which causes an accumulation of lipid-linked-GlcNAc(2)Man(6) and -GlcNAc(2)Man(8) structures, which was paralleled by the transfer of incomplete oligosaccharides precursors to protein. A homozygous point-mutation 1567G-->A (amino acid substitution E523K) was detected in the ALG9 gene. The functional homology between the human ALG9 and Saccharomyces cerevisiae ALG9, as well as the deleterious effect of the E523K mutation detected in the patient with CDG, were confirmed by a yeast complementation assay lacking the ALG9 gene. The ALG9 defect found in the patient with CDG--who presented with developmental delay, hypotonia, seizures, and hepatomegaly--shows that efficient lipid-linked oligosaccharide synthesis is required for proper human development and physiology. The ALG9 defect presented here defines a novel form of CDG named "CDG-IL."     

Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway

We describe in this report the characterization of the recently discovered N-linked glycosylation locus of the human bacterial pathogen Campylobacter jejuni, the first such system found in a species from the domain Bacteria. We exploited the ability of this locus to function in Escherichia coli to demonstrate through mutational and structural analyses that variant glycan structures can be transferred onto protein indicating the relaxed specificity of the putative oligosaccharyltransferase PglB. Structural data derived from these variant glycans allowed us to infer the role of five individual glycosyltransferases in the biosynthesis of the N-linked heptasaccharide. Furthermore, we show that C. jejuni- and E. coli-derived pathways can interact in the biosynthesis of N-linked glycoproteins. In particular, the E. coli encoded WecA protein, a UDP-GlcNAc: undecaprenylphosphate GlcNAc-1-phosphate transferase involved in glycolipid biosynthesis, provides for an alternative N-linked heptasaccharide biosynthetic pathway bypassing the requirement for the C. jejuni-derived glycosyltransferase PglC. This is the first experimental evidence that biosynthesis of the N-linked glycan occurs on a lipid-linked precursor prior to transfer onto protein. These findings provide a framework for understanding the process of N-linked protein glycosylation in Bacteria and for devising strategies to exploit this system for glycoengineering.     

Two yeast mutations in glucosylation steps of the asparagine glycosylation pathway

Two complementing mutations in lipid-linked oligosaccharide biosynthesis have been isolated following a [3H]mannose suicide enrichment. Rather than making the wild type precursor oligosaccharide, Glc3man9Glc-NA2-P-P-dolichol, the mutants, alg5-1 and alg6-1, accumulate Man9GlcNAc2-P-P-dolichol as their largest lipid-linked oligosaccharide in vivo and in vitro. When UDP-[3H]Glc was added to microsomal membranes of each mutant, neither could elongate Man9GlcNAc2-P-P-dolichol and only alg6-1 could synthesize dolichol-phosphoglucose. When dolicholphospho[3H]glucose was added to microsomes from alg5-1, alg6-1, or the parental strain, only alg5-1 and the parental strain made glucosylated lipid-linked oligosaccharides. These results indicate that alg5-1 cells are unable to synthesize dolichol phosphoglucose while alg6-1 cells are unable to transfer glucose from dolichol phosphoglucose to the unglucosylated lipid-linked oligosaccharide. We also present evidence that both mutants transfer Man9GlcNAc2 to protein.     

Biochemical characterization of the O-linked glycosylation pathway in Neisseria gonorrhoeae responsible for biosynthesis of protein glycans containing N,N'-diacetylbacillosamine

The O-linked protein glycosylation pathway in Neisseria gonorrhoeae is responsible for the synthesis of a complex oligosaccharide on undecaprenyl diphosphate and subsequent en bloc transfer of the glycan to serine residues of select periplasmic proteins. Protein glycosylation (pgl) genes have been annotated on the basis of bioinformatics and top-down mass spectrometry analysis of protein modifications in pgl-null strains [Aas, F. E., et al. (2007) Mol. Microbiol. 65, 607-624; Vik, A., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 4447-4452], but relatively little biochemical analysis has been performed to date. In this report, we present the expression, purification, and functional characterization of seven Pgl enzymes. Specifically, the enzymes studied are responsible for synthesis of an uncommon uridine diphosphate (UDP)-sugar (PglD, PglC, and PglB-acetyltransferase domain), glycan assembly (PglB-phospho-glycosyltransferase domain, PglA, PglE, and PglH), and final oligosaccharide transfer (PglO). UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-hexose (DATDH), which is the first sugar in glycan biosynthesis, was produced enzymatically, and the stereochemistry was assigned as uridine diphosphate N'-diacetylbacillosamine (UDP-diNAcBac) by nuclear magnetic resonance characterization. In addition, the substrate specificities of the phospho-glycosyltransferase, glycosyltransferases, and oligosaccharyltransferase (OTase) were analyzed in vitro, and in most cases, these enzymes exhibited strong preferences for the native substrates relative to closely related glycans. In particular, PglO, the O-linked OTase, and PglB(Cj), the N-linked OTase from Campylobacter jejuni, preferred the native N. gonorrhoeae and C. jejuni substrates, respectively. This study represents the first comprehensive biochemical characterization of this important O-linked glycosylation pathway and provides the basis for further investigations of these enzymes as antibacterial targets.     

Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step.

Asparagine-linked glycosylation is a form of covalent modification that distinguishes proteins that are either membrane bound or are in cellular compartments topologically outside of the cell from those proteins that remain soluble in the cytoplasm. This type of glycosylation occurs stepwise, with core oligosaccharide added in the endoplasmic reticulum and subsequent modifications occurring in the golgi. We used tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharide, to select for mutants that are resistant to this antibiotic. Genetic, biochemical, and physiological experiments led to the following conclusions. The synthesis of N-linked oligosaccharide is an essential function in cells. In contrast to mammalian cells, yeast cells do not transport tunicamycin by a glucosamine transport function. We identified a gene, ALG7, that is probably the structural gene for UDP-N-acetylglucosamine-1-P transferase, the enzyme inhibited by tunicamycin. Dominant mutations in this gene result in increased activity of the transferase and loss of the ability of the cell to sporulate. In addition, we identified another gene, TUN1, in which recessive mutations result in resistance to tunicamycin. The ALG7 and TUN1 genes both map on chromosome VII.     

Physical interactions between the Alg1, Alg2, and Alg11 mannosyltransferases of the endoplasmic reticulum

The early steps of N-linked glycosylation involve the synthesis of a lipid-linked oligosaccharide, Glc(3)Man(9)GlcNAc(2)-PP-dolichol, on the endoplasmic reticulum (ER) membrane. Prior to its lumenal translocation and transfer to nascent glycoproteins, mannosylation of Man(5)GlcNAc(2)-PP-dolichol is catalyzed by the Alg1, Alg2, and Alg11 mannosyltransferases. We provide evidence for a physical interaction between these proteins. Using a combination of biochemical and genetic assays, two distinct complexes that contain multiple copies of Alg1 were identified. The two Alg1-containing complexes differ from one another in that one complex contains Alg2 and the other contains Alg11. Alg1 self-assembles through a C-terminal domain that is distinct from the region required for its association with Alg2 or Alg11. Missense mutations affecting catalysis but not Alg1 protein stability or assembly with Alg2 or Alg11 were also identified. Overexpression of these catalytically inactive alleles resulted in dominant negative phenotypes, providing genetic evidence for functional Alg1-containing complexes in vivo. These data suggest that an additional level of regulation that ensures the fidelity of complex oligosaccharide structures involves the physical association of the related catalytic enzymes in the ER membrane.     

Distinctive roles of endoplasmic reticulum and golgi glycosylation in functional surface expression of mammalian E-NTPDase1, CD39

CD39 is a membrane-bound ecto-nucleoside triphosphate diphosphohydrolase that is involved in the regulation of purinergic signaling. It has been previously reported that N-linked glycosylation is essential for the surface localization of CD39 and for its cellular activity. Here we have addressed the roles of different stages of N-linked glycosylation on CD39's activity and surface expression by using various glycosylation inhibitors, glycosylation deficient CHO cells, and oligosaccharide removal enzymes. The results demonstrate that endoplasmic reticulum glycosylation is required for protein folding and essential for functional surface expression of CD39, while Golgi glycosylation is less important. The study has also shown that N-linked glycosylation of CD39 is dispensable for the activity after the protein is properly folded and targeted.