Absence Of N-glycosylation Research Articles

Mechanistic study of SCOOPs recognition by MIK2-BAK1 complex reveals the role of N-glycans in plant ligand-receptor-coreceptor complex formation.

Ligand-induced receptor and co-receptor heterodimerization is a common mechanism in receptor kinase (RK) signalling activation. SERINE-RICH ENDOGENOUS PEPTIDEs (SCOOPs) mediate the complex formation of Arabidopsis RK MIK2 and co-receptor BAK1, triggering immune responses. Through structural, biochemical and genetic analyses, we demonstrate that SCOOPs use their SxS motif and adjacent residues to bind MIK2 and the carboxy-terminal GGR residues to link MIK2 to BAK1. While N-glycosylation of plant RKs is typically associated with protein maturation, plasma membrane targeting and conformation maintenance, a surprising revelation emerges from our crystal structural analysis of MIK2-SCOOP-BAK1 complexes. Specific N-glycans on MIK2 directly interact with BAK1 upon SCOOP sensing. The absence of N-glycosylation at the specific site in MIK2 neither affects its subcellular localization and protein accumulation in plant cells nor alters its structural conformation, but markedly reduces its affinity for BAK1, abolishing SCOOP-triggered immune responses. This N-glycan-mediated receptor and co-receptor heterodimerization occurs in both Arabidopsis and Brassica napus. Our findings elucidate the molecular basis of SCOOP perception by the MIK2-BAK1 immune complex and underscore the crucial role of N-glycans in plant receptor-coreceptor interactions and signalling activation, shaping immune responses.

An N-Glycosylated Form of SERINC5 Is Specifically Incorporated into HIV-1 Virions.

SERINC5 is an inhibitor of retroviral infectivity that is counteracted by viral proteins, including HIV-1 Nef. Inhibition of infectivity by SERINC5 is associated with its incorporation into virions. Nef counteracts this inhibition, presumably by removing SERINC5 from sites of virion assembly at the plasma membrane. While evaluating the virion incorporation of SERINC5, we observed that a relatively high molecular weight form was preferentially present in virions. We used various glycosidases to establish that virion-associated SERINC5 is modified by N-linked, complex glycans, whereas the majority of SERINC5 in cells is of relatively low molecular weight and is modified by high-mannose glycans. Sequence alignment of SERINC family proteins led us to identify a conserved N-glycosylation site, N294, in SERINC5. We mutated this site to evaluate its effect on glycosylation, the restrictive activity of SERINC5, and the sensitivity of SERINC5 to antagonism by Nef. Our results demonstrate that N294 is the major site of N-glycosylation in SERINC5. Although N-glycosylation was required neither for restrictive activity nor for sensitivity to Nef per se, we observed a decrease in the steady-state expression of glycosylation-deficient SERINC5 (the N294A mutant) compared to the wild-type protein. Expression of this mutant was partly restored by treatment of cells with MG132 (a proteasome inhibitor) but not with bafilomycin A1 (a lysosomal inhibitor). We conclude that although not required for restrictive activity or Nef sensitivity, N-linked glycosylation is important for maintaining the steady-state expression of SERINC5 and that nonglycosylated SERINC5 is likely subjected to a quality control mechanism that induces its proteasomal degradation.IMPORTANCE SERINC5 is a member of a family of multipass transmembrane proteins that inhibit the infectivity of retroviruses, including HIV-1. These proteins are incorporated into virions and inhibit infection of target cells unless counteracted by viral antagonists such as HIV-1 Nef. The only other biological function with which these proteins have been associated is the formation of serine-containing membrane lipids. Here we show that SERINC5 is a glycosylated protein and that N-glycosylation is important for its steady-state expression. In the absence of N-glycosylation, SERINC5 is prone to proteasomal degradation. Nonetheless, N-glycosylation per se is required neither for the ability of SERINC5 to inhibit HIV-1 infectivity nor for its sensitivity to antagonism by Nef.

The Reduced Susceptibility to Develop CNS-Autoimmunity in the Absence of N-Glycosylation Is Associated to Impaired Differentiation and Function of Induced Regulatory T Cells (P5.330)

The Reduced Susceptibility to Develop CNS-Autoimmunity in the Absence of N-Glycosylation Is Associated to Impaired Differentiation and Function of Induced Regulatory T Cells (P5.330)

Pilin Processing Follows a Different Temporal Route than That of Archaellins in Methanococcus maripaludis.

Methanococcus maripaludis has two different surface appendages: type IV-like pili and archaella. Both structures are believed to be assembled using a bacterial type IV pilus mechanism. Each structure is composed of multiple subunits, either pilins or archaellins. Both pilins and archaellins are made initially as preproteins with type IV pilin-like signal peptides, which must be removed by a prepilin peptidase-like enzyme. This enzyme is FlaK for archaellins and EppA for pilins. In addition, both pilins and archaellins are modified with N-linked glycans. The archaellins possess an N-linked tetrasaccharide while the pilins have a pentasaccharide which consists of the archaellin tetrasaccharide but with an additional sugar, an unidentified hexose, attached to the linking sugar. In this report, we show that archaellins can be processed by FlaK in the absence of N-glycosylation and N-glycosylation can occur on archaellins that still retain their signal peptides. In contrast, pilins are not glycosylated unless they have been acted on by EppA to have the signal peptide removed. However, EppA can still remove signal peptides from non-glycosylated pilins. These findings indicate that there is a difference in the order of the posttranslational modifications of pilins and archaellins even though both are type IV pilin-like proteins.

Increased N-glycosylation of Asn⁸⁸ in serum pancreatic ribonuclease 1 is a novel diagnostic marker for pancreatic cancer.

Alterations of carbohydrate structures in cancer cells are the most promising targets for developing clinical diagnostic reagents. Pancreatic cancer is one of the most difficult cancers to diagnose because it lacks definitive symptoms. Two antibodies were raised against human pancreatic ribonuclease 1 that bind to the enzyme containing unglycosylated Asn88, but not when its Asn88 is N-glycosylated. Differential studies using these antibodies in immunoassays and Western blot analyses showed a significant increase in the serum levels of pancreatic ribonuclease 1 containing N-glycosylated Asn88 in pancreatic cancer patients compared with normal human subjects. Focusing on the increase in an N-glycosylated Asn residue of serum pancreatic ribonuclease 1, specifically Asn88, affords a new diagnostic marker for pancreatic cancer. This is the first report of a diagnostic cancer marker that takes advantage of the presence or absence of N-glycosylation at a specific Asn residue of a glycoprotein.

Endoplasmic reticulum microenvironment and conserved histidines govern ELOVL4 fatty acid elongase activity

Autosomal dominant Stargardt-like macular dystrophy (STGD3) in humans results from mutations in elongation of very long chain FAs-like 4 (ELOVL4), which leads to vision loss in young adults. ELOVL4 is an integral endoplasmic reticulum (ER) protein that mediates the elongation of very long chain (VLC) FAs. Mutations in ELOVL4 lead to truncation and mislocalization of the translated protein from the ER, the site of FA elongation. Little is known about the enzymatic elongation of VLC-FAs by ELOVL4. We over-expressed full-length mouse ELOVL4, an N-glycosylation-deficient mutant, an ER-retention mutant, and mutants of active site histidines to parse their individual roles in VLC-FA elongation. ELOVL4 elongated appropriate precursors to the corresponding VLC-FA species ≥28 carbons. Active site histidine mutants of ELOVL4 did not elongate appropriate precursors, establishing ELOVL4 as the elongase. Displacing ELOVL4 from the ER was sufficient to cause loss of condensation activity, while absence of N-glycosylation was irrelevant for enzyme function. This study shows that ELOVL4 enzymatic activity is governed by individual histidines in its active site and the ER microenvironment, both of which are essential for elongation of VLC-FAs.

FCER2 (CD23) Asthma-Related Single Nucleotide Polymorphisms Yields Increased IgE Binding and Egr-1 Expression in Human B Cells

CD23 is the low-affinity Fc receptor for IgE. When expressed on B cells, CD23 appears to play a role in regulation of IgE synthesis. Polymorphisms within FCER2, the gene encoding CD23, have been associated with atopy, increased risk of exacerbations in patients with asthma, and high serum IgE levels. A single-nucleotide polymorphism (rs2228137) present in exon 4 of FCER2 encodes a nonsynonymous amino acid change (R62W) and is the subject of the present analysis. Human B cell stable transfectants were established to characterize the functional relevance of the R62W SNP. We demonstrate that CD23b-R62W-expressing human B cells bind IgE with greater affinity than wild-type cells and display differences in kinetics of CD23-mediated ERK1/2 activation that may be responsible for the increased levels of Egr-1 mRNA observed after stimulation through CD23. Finally, the R62W SNP seems to alter the tertiary or quaternary structure of CD23 because in the absence of N-glycosylation the CD23b-R62W-expressing cells appear to be less sensitive to endogenous proteases. These observations may have implications in mechanisms responsible for the atopic phenotypes observed in patients with asthma who possess this genotype.

Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca2 + in adult cardiac myocytes

Endothelin receptors are present on the nuclear membranes in adult cardiac ventricular myocytes. The objectives of the present study were to determine 1) which endothelin receptor subtype is in cardiac nuclear membranes, 2) if the receptor and ligand traffic from the cell surface to the nucleus, and 3) the effect of increased intracellular ET-1 on nuclear Ca2+ signaling. Confocal microscopy using fluorescently-labeled endothelin analogs confirmed the presence of ETB at the nuclear membrane of rat cardiomyocytes in skinned-cells and isolated nuclei. Furthermore, in both cardiac myocytes and aortic endothelial cells, endocytosed ET:ETB complexes translocated to lysosomes and not the nuclear envelope. Although ETA and ETB can form heterodimers, the presence or absence of ETA did not alter ETB trafficking. Treatment of isolated nuclei with peptide: N-glycosidase F did not alter the electrophoretic mobility of ETB. The absence of N-glycosylation further indicates that these receptors did not originate at the cell surface. Intracellular photolysis of a caged ET-1 analog ([Trp-ODMNB21]ET-1) evoked an increase in nucleoplasmic Ca2+ ([Ca2+]n) that was attenuated by inositol 1,4,5-trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and prevented by pre-treatment with ryanodine. A caged cell-permeable analog of the ETB-selective antagonist IRL-2500 blocked the ability of intracellular cET-1 to increase [Ca2+]n whereas extracellular application of ETA and ETB receptor antagonists did not. These data suggest that 1) the endothelin receptor in the cardiac nuclear membranes is ETB, 2) ETB traffics directly to the nuclear membrane after biosynthesis, 3) exogenous endothelins are not ligands for ETB on nuclear membranes, and 4) ETB associated with the nuclear membranes regulates nuclear Ca2+ signaling.

Identification of the Active Site of DS-epimerase 1 and Requirement of N-Glycosylation for Enzyme Function

Dermatan sulfate is a highly sulfated polysaccharide and has a variety of biological functions in development and disease. Iduronic acid domains in dermatan sulfate, which are formed by the action of two DS-epimerases, have a key role in mediating these functions. We have identified the catalytic site and three putative catalytic residues in DS-epimerase 1, His-205, Tyr-261, and His-450, by tertiary structure modeling and amino acid conservation to heparinase II. These residues were systematically mutated to alanine or more conserved residues, which resulted in complete loss of epimerase activity. Based on these data and the close relationship between lyase and epimerase reactions, we propose a model where His-450 functions as a general base abstracting the C5 proton from glucuronic acid. Subsequent cleavage of the glycosidic linkage by Tyr-261 generates a 4,5-unsaturated hexuronic intermediate, which is protonated at the C5 carbon by His-205 from the side of the sugar plane opposite to the side of previous proton abstraction. Concomitant recreation of the glycosidic linkage ends the reaction, generating iduronic acid. In addition, we show that proper N-glycosylation of DS-epimerase 1 is required for enzyme activity. This study represents the first description of the structural basis for epimerization by a glycosaminoglycan epimerase.

N-Glycosylation of the alpha subunit does not influence trafficking or functional activity of the human organic solute transporter alpha/beta

BackgroundThe organic solute transporter (OSTα-OSTβ) is a heteromeric transporter that is expressed on the basolateral membrane of epithelium in intestine, kidney, liver, testis and adrenal gland and facilitates efflux of bile acids and other steroid solutes. Both subunits are required for plasma membrane localization of the functional transporter but it is unclear how and where the subunits interact and whether glycosylation is required for functional activity. We sought to examine these questions for the human OSTα-OSTβ transporter using the human hepatoma cell line, HepG2, and COS7 cells transfected with constructs of human OSTα-FLAG and OSTβ-Myc.ResultsTunicamycin treatment demonstrated that human OSTα is glycosylated. In COS7 cells Western blotting identified the unglycosylated form (~31 kD), the core precursor form (~35 kD), and the mature, complex glycoprotein (~40 kD). Immunofluorescence of both cells indicated that, in the presence of OSTβ, the alpha subunit could still be expressed on the plasma membrane after tunicamycin treatment. Furthermore, the functional uptake of 3H-estrone sulfate was unchanged in the absence of N-glycosylation. Co-immunoprecipitation indicates that the immature form of OSTα interact with OSTβ. However, immunoprecipitation of OSTβ using an anti-Myc antibody did not co-precipitate the mature, complex glycosylated form of OSTα, suggesting that the primary interaction occurs early in the biosynthetic pathway and may be transient.ConclusionIn conclusion, human OSTα is a glycoprotein that requires interaction with OSTβ to reach the plasma membrane. However, glycosylation of OSTα is not necessary for interaction with the beta subunit or for membrane localization or function of the heteromeric transporter.

Proteasome degradation of protein C and plasmin inhibitor mutants

Protein C (PC) deficiency and plasmin inhibitor (PI) deficiency are inherited thrombotic and haemorrhagic disorders. We investigated the intracellular degradation of mutant proteins, using naturally occurring PC and PI mutants that lead to congenital deficiencies. To examine the necessity of N-linked glycosylation for the proteasomal degradation of PC and PI, PC178 and PC331 mutants treated with tunicamycin and N-glycosylation-lacking mutants, PC92Stop and PI-America were pulse chased. The analysis revealed that the speed of degradation of the tunicamycin-treated PC mutants, PC92Stop and PI-America lacking glycosylation, was slower than that of N-glycosylated mutants. Immunoprecipitation and immunoblot analysis showed that PC178 and PC331 mutants were associated with molecular chaperones, Bip, GRP94, and calreticulin. PI-America was associated with only Bip. Although degradation of mutants was mediated by proteasomes, no association with ubiquitin was detected. Cotransfection of endoplasmic reticulum (ER) degradation enhancing alpha-mannosidase-like protein (EDEM) accelerated the degradation of N-glycosylated PC. In the absence of autophagy using Atg5-deficient cell lines, the degradation of the PC331 mutant was mildly accelerated but that of PC178, PI-America and PI-Okinawa mutants was not influenced. While the degradation of the PC and PI mutants was facilitated by N-glycosylation moieties, they were ubiquitin-independently degraded by proteasomes, irrespective of the presence or absence of N-glycosylation. Molecular chaperone binding was influenced by the presence of N-glycosylation moieties. When the misfolded or truncated mutant proteins are functionally active, proteasome inhibitors such as bortezomib may have therapeutic potential for treatment of protein deficiencies.

The cation-independent mannose 6-phosphate receptor is involved in lysosomal delivery of serglycin

To clarify the sorting mechanism of the lysosomal/granular proteoglycan serglycin, we treated human promonocytic U937 cells with p-nitrophenyl-beta-D-xyloside (PNP-xyl) and cycloheximide. In the absence of protein synthesis, the carbohydrate moiety of serglycin was synthesized as PNP-xyl-chondroitin sulfate (CS), and most of it was delivered to lysosomes and degraded. Further, an augmented lysosomal targeting of serglycin in the presence of tunicamycin suggested that a sorting/lectin receptor with multiple specificity was involved with an increased capacity for serglycin in the absence of N-glycosylation. Correspondingly, the cation-independent mannose 6-phosphate receptor (CI-MPR) and sortilin were observed to bind to immobilized CS. These receptors were eluted in the presence of 200-400 mM and 100-250 mM NaCl, respectively. After treating the cells with a cross-linking reagent, a portion of the sulfated proteoglycan was coimmunoprecipitated with the CI-MPR but not with sortilin. In the presence of phorbol ester, lysosomal targeting of serglycin and to a lesser extent, of cathepsin D was inhibited. We conclude that the CI-MPR participates in lysosomal and granular targeting of serglycin and basic proteins such as lysozyme associated with the proteoglycan in hematopoietic cells.

Function-dependent Conformational Changes of the ABCG2 Multidrug Transporter Modify Its Interaction with a Monoclonal Antibody on the Cell Surface

The human ABCG2 protein is an important primary active transporter for hydrophobic compounds in several cell types, and its overexpression causes multidrug resistance in tumors. A monoclonal antibody (5D3) recognizes this protein on the cell surface. In ABCG2-expressing cells 5D3 antibody showed a saturable labeling and inhibited ABCG2 transport and ATPase function. However, at low antibody concentrations 5D3 binding to intact cells depended on the actual conformation of the ABCG2 protein. ATP depletion or the addition of the ABCG2 inhibitor Ko143 significantly increased, whereas the vanadate-induced arrest of ABCG2 strongly decreased 5D3 binding. The binding of the 5D3 antibody to a non-functional ABCG2 catalytic center mutant (K86M) in intact cells was not affected by the addition of vanadate but still increased with the addition of Ko143. In isolated membrane fragments the ligand modulation of 5D3 binding to ABCG2 could be analyzed in detail. In this case 5D3 binding was maximum in the presence of ATP, ADP, or Ko143, whereas the non-hydrolysable ATP analog, adenosine 5'-(beta,gamma-imido)triphosphate (AMP-PNP), and nucleotide trapping by vanadate decreased antibody binding. In membranes expressing the ABCG2-K86M mutant, ATP, ADP, and AMP-PNP decreased, whereas Ko143 increased 5D3 binding. Based on these data we suggest that the 5D3 antibody can be used as a sensitive tool to reveal intramolecular changes, reflecting ATP binding, the formation of a catalytic intermediate, or substrate inhibition within the transport cycle of the ABCG2 protein.

The mutation Ser511Asn leads to N-glycosylation and increases the cleavage of high molecular weight kininogen in rats genetically susceptible to inflammation

AbstractCrohn disease is immunologically mediated and characterized by intestinal and systemic chronic inflammation. In a rat model, injection of peptidoglycan-polysaccharide complexes into the intestinal wall induced chronic inflammation in Lewis but neither Fischer nor Buffalo rats, indicating a differential genetic susceptibility. Proteolysis of plasma high molecular weight kininogen (HK) yielding bradykinin and cleaved HK (HKa) was faster in Lewis than in Fischer or Buffalo rat plasma. A single point mutation at nucleotide 1586 was found translating from Ser511 (Buffalo and Fisher) to Asn511 (Lewis). The latter defines an Asn-Xaa-Thr consensus sequence for N-glycosylation. We expressed these domains in Escherichia coli and found no differences in the rate of cleavage by purified kallikrein in the 3 strains in the absence of N-glycosylation. We then expressed these domains in Chinese hamster ovary (CHO) cells, which are capable of glycosylation, and found an increased rate of cleavage of Lewis HK. The Lewis mutation is associated with N-glycosylation as evidenced by a more rapid migration after treatment with N-glycosidase F. When CHO cells were cultured in the presence of tunicamycin, the kallikrein-induced cleavage rate of Lewis HK was not increased. This molecular alteration might be one contributing factor resulting in chronic inflammation in Lewis rats.

Pharmacokinetic and tumor-seeking properties of recombinant and nonrecombinant anti-carcinoembryonic antigen antibody fragments.

Production of recombinant antibody fragments in bacterial expression systems results in intentional or fortuitous differences compared to the original products prepared by hybridoma technology. These differences may have significant effects not only on antigen-binding properties but also on pharmacokinetic and tumor-seeking properties. Our major goal was to investigate some of these possible differences. We produced in Escherichia coli an rFab' fragment containing only 1 cysteine residue in the hinge region; the fragment was derived from a mouse MAb (F6) specific for CEA. The rFab' had a slightly lower m.w. and a higher isoelectric point relative to the corresponding nonrecombinant fragment (pFab'). This was explained by the absence of N-glycosylation on the V kappa domain of rFab'. V kappa glycosylation had no significant effect on antibody-binding affinity and kinetics. However, rFab' was eliminated from the circulation much faster than pFab', and the maximal dose accumulated in the tumor was reduced relative to pFab'. Thus, glycosylation appears to modify the targeting efficiency of antibody fragments. rF(ab')(2) fragments were obtained either spontaneously from the culture supernatant of E. coli or by chemical cross-linking [rcF(ab')(2)]. We observed improved tumor targeting with rcF(ab')(2) compared to rF(ab')(2), which could be explained by the greater stability of the thioether compared to the disulfide linkage. These results demonstrate that a single cysteine residue in the hinge region of rFab' is particularly well suited to prepare stable, chemically coupled, bivalent or bispecific antibodies, avoiding intrahinge disulfide bonding and thus achieving higher production yields.

Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the apical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation.

Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic (35)S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.

Characterization of Fibroblast Growth Factor-6 Expressed by Chinese Hamster Ovary Cells as a Glycosylated Mitogen for Human Vascular Endothelial Cells

The gene for fibroblast growth factor (FGF)-6/hst-2 was originally identified by its close homology with the FGF-4/hst-1 gene. Aside from its ability to transform cultured fibroblasts, the characteristics of FGF-6 protein have only been studied using a simple preparation from E. coli. In the present study, we expressed FGF-6 cDNA in CHO cells and characterized the resultant protein. We found that CHO cells secreted several forms of the FGF-6 polypeptide, and that there were multiple N-terminal modifications. The longest form (18-kDa) contained the sequence, SerProAlaGlyAlaArg, as its N-terminus, which was consistent with the signal peptide cleavage site predicted from its primary structure. The core polypeptide was primarily modified by heterogeneous N-glycans that were sialylated to a small degree; among them, biantennary structures were found to predominate. Moreover, possible O-glycosylation was also detected. N-glycosylated FGF-6 potently induced DNA synthesis and proliferation of human vascular endothelial cells, whereas in the absence of N-glycosylation, FGF-6 mitogenicity was substantially diminished. The results clearly indicate that FGF-6 expressed by mammalian cells is a glycosylated mitogen for vascular endothelial cells and further suggests that N-glycosylation plays a key role in determining the mitogenicity of FGF-6.

N-Glycosylation is not a prerequisite for glutamate receptor function but Is essential for lectin modulation.

All ionotropic glutamate receptor (iGluR) subunits analyzed so far are heavily N-glycosylated at multiple sites on their amino-terminal extracellular domains. Although the exact functional significance of this glycosylation remains to be determined, it has been suggested that N-glycosylation may be a precondition for the formation of functional ion channels. In particular, it has been argued that N-glycosylation is required for the formation of functional ligand binding sites. We analyzed heterologously expressed recombinant glutamate receptors (GluRs) of all three pharmacological subclasses of glutamate receptors, N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, and kainate receptors. By expressing the GluR subunits in tunicamycin-treated, nonglycosylating Xenopus laevis oocytes, we determined that in neither case is N-glycosylation required for ion channel function, although for NMDA receptors, functional expression in the absence of N-glycosylation is very low. Furthermore, we analyzed and compared the interaction of the desensitization-inhibiting lectin concanavalin A (ConA) with all functional GluR subunits. We show that although ConA has its most pronounced effects on kainate receptors, it potentiates currents at most other receptor subtypes as well, including certain NMDA receptor subunits, although to a much lesser extent. One notable exception is the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor GluR2, which is not affected by ConA. Furthermore, we show that ConA acts directly via binding to the carbohydrate side chains of the receptor protein.

Absence of N-glycosylation at asparagine 43 in human lipoprotein lipase induces its accumulation in the rough endoplasmic reticulum and alters this cellular compartment.

Lipoprotein lipase (LPL) is the enzyme responsible for the hydrolysis of plasma triglycerides from apolipoprotein C-II-containing lipoproteins at the capillary endothelium and it is synthesized in parenchymal cells of several tissues. Intracellular LPL processing is a major aspect of LPL regulation. The present study aims to determine the intracellular accumulation site of the LPL that is not glycosylated at Asn43. Human LPL (hLPL) cDNA was mutated by site-directed mutagenesis. An Ala residue was substituted for Asn at position 43 of the protein generating N43A hLPL. Wild type hLPL and the mutant hLPL were expressed in COS1 cells. Using immunofluorescence and immunoelectron microscopy we found that wild type hLPL in addition to being secreted into the medium was present in the rough endoplasmic reticulum (ER), Golgi compartments, and vesicles. Neither LPL activity nor protein was found in medium of cells expressing the mutant hLPL and all detectable protein was present exclusively in the ER identified witha specific antibody against the protein disulfide isomerase (PDI), an ER marker. In addition, the intracellular distribution of the ER of the cells that expressed the mutant protein was grossly altered. Treatment of COS1 cells with tunicamycin for 24 h had the same effect on wild type hLPL processing and edoplasmic reticulum distribution. Next, we investigated the influence of the accumulation of mutant hLPL on the intracellular transport of three other proteins that are N-glycosylated before reaching the plasma membrane: the related Bo,+ amino acid transporter (rBAT), the insulin-regulated glucose transporter (GLUT4), and the placental alkaline phosphatase (PLAP) protein. Coexpression of the mutant hLPL (but not wild type) caused the accumulation of rBAT and GLUT4 in the ER while PLAP reached the plasma membrane. Our findings demonstrate that glycosylation of Asn43 of human lipoprotein lipase in the endoplasmic reticulum is essential for its efflux from this compartment and that the retention of the non-glycosylated LPL induces morphological changes in the ER that could also affect its ability to modify the transport of other proteins.

Biosynthesis and Processing of the Autographa californica Nuclear Polyhedrosis Virus gp64 Protein

gp64 is a major virion envelope glycoprotein of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). gp64 plays an important role in AcMNPV infection, probably mediating penetration of one form of the virus into host cells through the endocytic pathway. gp64 also represents an excellent probe for studying the membrane glycoprotein processing capabilities of baculovirus-infected insect cells, which are used widely as a eucaryotic expression system. The goals of this study were to characterize gp64 biosynthesis and processing and determine how N-glycosylation and N-linked oligosaccharide processing influence the fate and function of gp64 in AcMNPV-infected insect cells. We found that gp64 was synthesized in a bipbasic fashion, with peaks at 8 and 24 hr postinfection in both the intracellular and extracellular fractions. Interestingly, the first peak preceded detectable budded virus (BV) production, suggesting that gp64 is shed from infected cells early in infection. Transcriptional regulation accounted for the bipbasic mode of gp64 protein synthesis, as transcription initiated at a consensus early motif during early times of infection, at late motif during late times of infection, and there was a leg between the peak of early and the onset of late transcription. In vitro transcription-translation assays showed that the second ATG in the AcMNPV gp64 long open reading frame is used as the translational initiation codon and that downstream sequences encode a functional signal peptide. Pulse-chase analyses, endoglycosidases, and various inhibitors were used to show that some N-linked oligosaccharides on gp64 are processed by glucosidases and α-mannosidases in AcMNPV-infected insect cells. These experiments also revealed that at least two differentially processed gp64 glycoforms are produced in these cells and that both can reach the cell surface and assemble into progeny BV. However, N-linked oligosaccharide processing was not required for gp64 cell surface expression, its assembly into infectious BV, or its fusogenic activity. This suggested that any gp64 glycoform produced during infection, regardless of its N-linked carbohydrate structure, can have essentially normal biological properties. By contrast, transport of gp64 to the cell surface, production of infectious BV, and fusogenic activity were reduced in the absence of N-glycosylation, indicating that this modification is necessary for optimal gp64 function.