N-linked Oligosaccharide Side Chains Research Articles

UPLC-MS/MS Analysis of Urinary Free Oligosaccharides for Lysosomal Storage Diseases: Diagnosis and Potential Treatment Monitoring.

The glycoproteinoses are a subgroup of lysosomal storage diseases (LSDs) resulting from impaired degradation of N-linked oligosaccharide side chains of glycoproteins, which are commonly screened by detecting the accumulated free oligosaccharides (FOSs) in urine via thin layer chromatography (TLC). The traditional TLC method suffers from limited analytical sensitivity and specificity and lacks quantification capability. Therefore, we developed an analytically sensitive and relatively specific assay using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for urinary FOS analysis and validated its use for urine screening of glycoproteinoses and other LSDs. Urine volumes equivalent to 30 μg of creatinine were derivatized with butyl-4-aminobenzoate and then purified through a solid-phase extraction cartridge. A 7-min UPLC-MS/MS analysis was performed on a triple quadrupole mass spectrometer using an amide column for separation of derivatized FOS. Urine samples from >100 unaffected controls and 37 patients with various LSDs were studied. Relative quantification was conducted on 7 selected FOSs using a single internal standard, which allowed the identification of patients with 1 of 8 different LSDs: aspartylglucosaminuria, α-fucosidosis, α-mannosidosis, β-mannosidosis, β-galactosidase deficiency, Sandhoff disease, sialidosis, and galactosialidosis. Patients treated with hematopoietic stem cell transplant show decreased FOS responses compared with untreated patients. This UPLC-MS/MS assay offers a valuable tool for screening of glycoproteinoses and other LSDs, with potential use for future treatment monitoring.

Expression and Characterization of HA1 Protein of Highly Pathogenic H5N1 Avian Influenza Virus for Use in a Serodiagnostic Assay.

The hemagglutinin ectodomain (HA1 subunit) from highly pathogenic avian influenza (HPAI) isolate (A/chicken/Vietnam/14/2005) was cloned and expressed using a baculovirus expression vector. Biosynthesis, glycosylation and secretion of the HA1 proteins, with natural or a melittin signal peptide at the N-terminus anda six-histidine (6xHis) tag at the C-terminus, were examined in insect cells. A 40-kDa unglycosylated precursor and a fully processed, mature form of the HA1 protein migrated around 52kDa were detected by SDS-PAGE and confirmed by Western blot using H5N1-specific antibody. Treatment of tunicamycin and peptide-N-glycosidase F (PNGase F) further revealed that the recombinant HA1 proteins produced in insect cells were indeed glycosylated with N-linked oligosaccharide side chains. Time-course experiments showed that substitution of the HA natural sequence with the signal sequence from honeybee melittin promoted a high level of expression and efficient secretion of the HA1. A high yield, 37μg/ml, of HA1 protein was obtained from recombinant baculovirus-infected cell culture supernatant. In addition, the cell surface expression of rHA1 was detected by indirect immunofluorescent staining and showed biological activity on hemadsorption assays. Recombinant HA1 protein-based ELISA was evaluated and appeared to be sensitive and specific for the rapid detection of H5 subtype-specific antibodies in serum samples. No cross-reactivity to antibodies from 15 other influenza A subtypes was detected. Taken together, the newly developed recombinant HA1-based ELISA could offer an alternative to other diagnostic approaches for the specific detection of H5 avian influenza virus infection.

The dimeric crystal structure of the human fertility lipocalin glycodelin reveals a protein scaffold for the presentation of complex glycans.

Human glycodelin (Gd) is an abundant glycoprotein from the lipocalin family and is involved in crucial biological processes such as reproduction and immune reaction. In females and males, Gd is found in four distinct glycoforms-A, C, F and S-that arise from different N-linked oligosaccharide side chains at amino acid residues Asn28 and Asn63. We have expressed Gd (carrying two amino acid substitutions to improve solubility) as a non-glycosylated protein in Escherichia coli via periplasmic secretion and determined its X-ray structure at 2.45 Å resolution. Gd reveals a classical lipocalin fold including two disulfide bridges, which is however unusually compact and lacks a pronounced central pocket inside the β-barrel, in line with its low affinity for hydrophobic ligands. Instead, this lipocalin exhibits a unique homodimeric quaternary structure that appears ideally suited as a scaffold for the presentation of specific glycans. In fact, the four oligosaccharides are presented in close proximity on the same side of the dimer surface, which increases avidity for cellular receptors, e.g. during sperm-egg recognition. A bioinformatic analysis indicated that Gd orthologues exclusively occur in certain suborders of primates that have a menstrual cycle, suggesting that this lipocalin with its role in fertility only recently emerged during evolution.

Site-specific analysis of N-linked oligosaccharides of recombinant lysosomal arylsulfatase A produced in different cell lines

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a deficiency of the lysosomal enzyme arylsulfatase A (ASA). Enzyme replacement therapy (ERT) is a therapeutic option for MLD and other lysosomal disorders. This therapy depends on N-linked oligosaccharide-mediated delivery of intravenously injected recombinant enzyme to the lysosomes of patient cells. Because of the importance of N-linked oligosaccharide side chains in ERT, we examined the composition of the three N-linked glycans of four different recombinant ASAs in a site-specific manner. Depending on the culture conditions and the cell line expressing the enzyme, we detected a high variability of the high-mannose-type N-glycans which prevail at all glycosylation sites. Our data show that the composition of the glycans is largely determined by substantial trimming in the medium. The susceptibility for trimming is different for the glycans at the three N-glycosylation sites. Interestingly, which of the glycans is most susceptible to trimming also depends on production conditions. CHO cells cultured under bioreactor conditions yielded recombinant ASA with the most preserved N-glycan structures, the highest mannose-6-phosphate content and the highest similarity to non-recombinant enzyme. Notably, roughly one-third of the N-glycans released from the three glycosylation sites were fucosylated. In the last years, numerous recombinant lysosomal enzymes were used for preclinical ERT trials. Our data show that the oligosaccharide structures were very different in these trials making it difficult to draw common conclusions from the various investigations.

Essential Role of Dengue Virus Envelope Protein N Glycosylation at Asparagine-67 during Viral Propagation

Dengue virus envelope protein (E) contains two N-linked glycosylation sites, at Asn-67 and Asn-153. The glycosylation site at position 153 is conserved in most flaviviruses, while the site at position 67 is thought to be unique for dengue viruses. N-linked oligosaccharide side chains on flavivirus E proteins have been associated with viral morphogenesis, infectivity, and tropism. Here, we examined the relevance of each N-linked glycan on dengue virus E protein by removing each site in the context of infectious viral particles. Dengue viruses lacking Asn-67 were able to infect mammalian cells and translate and replicate the viral genome, but production of new infectious particles was abolished. In addition, dengue viruses lacking Asn-153 in the E showed reduced infectivity. In contrast, ablation of one or both glycosylation sites yielded viruses that replicate and propagate in mosquito cells. Furthermore, we found a differential requirement of N-linked glycans for E secretion in mammalian and mosquito cells. While secretion of E lacking Asn-67 was efficient in mosquito cells, secretion of the same protein expressed in mammalian cells was dramatically impaired. Finally, we found that viruses lacking the carbohydrate at position 67 showed reduced infection of immature dendritic cells, suggesting interaction between this glycan and the lectin DC-SIGN. Overall, our data defined different roles for the two glycans present at the E protein during dengue virus infection, highlighting the involvement of distinct host functions from mammalian and mosquito cells during dengue virus propagation.

Oligosaccharide side chains on human secretory IgA serve as receptors for ricin.

Secretory IgA (sIgA) Abs are polymeric Igs comprised of two or more IgA monomers joined together at their C termini and covalently associated with a 70-kDa glycoprotein called secretory component. As the predominant Ig type in gastrointestinal sections, sIgA Abs are centrally important in adaptive immunity to enteropathogenic bacteria, viruses, and toxins. In this study, we demonstrate that sIgA Abs may also function in innate defense against ricin, a naturally occurring, galactose-specific plant lectin with extremely potent shiga toxin-like enzymatic activity. In lectin blot overlay assays, we found that ricin bound to secretory component and the H chain of human IgA, and this binding was inhibited by the addition of excess galactose. The toxin also recognized IgM (albeit with less affinity than to IgA), but not IgG. Ricin bound to both human IgA1 and IgA2, primarily via N-linked oligosaccharide side chains. At 100-fold molar excess concentration, sIgA (but not IgG) Abs inhibited ricin attachment to the apical surfaces of polarized intestinal epithelial cells grown in culture. sIgA Abs also visibly reduced toxin binding to the luminal surfaces of human duodenum in tissue section overlay assays. We conclude that sIgA Abs in mucosal secretions may serve as receptor analogues for ricin, thereby reducing the effective dose of toxin capable of gaining access to glycolipid and glycoprotein receptors on epithelial cell surfaces.

Characterization of the molecular chaperone calnexin in the channel catfish, Ictalurus punctatus, and its association with MHC class II molecules

Folding and assembly of MHC molecules in mammals occurs in the endoplasmic reticulum (ER), but has not been studied in teleosts. Calnexin (CNX) is an ER chaperone that associates with glycoproteins bearing a monoglucosylated N-linked oligosaccharide side chain. Here we report the first identification and characterization of a full-length CNX cDNA clone in a teleost, and the association of the CNX chaperone with MHC class II in a channel catfish T cell line. The 1.8 kb CNX clone encodes a protein of 607 amino acids that is 72% identical to the consensus sequence of mammalian CNXs. The association of CNX with class II is of particular interest because the native MHC class II α chain of Ictalurus punctatus does not bear any N-linked oligosaccharide consensus glycosylation sequences. Thus the assembly of class II molecules in the catfish probably proceeds via different steps than occurs in mammals.

Adenoviral-expressed GP5 of porcine respiratory and reproductive syndrome virus differs in its cellular maturation from the authentic viral protein but maintains known biological functions.

Summary. The ORFs 5, 6 and 7, encoding for the three major structural proteins, GP5, M and N, of the IAF-Klop strain of PRRSV were cloned and expressed in 293 cells using replication-defective human type 5 adenoviral vectors (hAdVs). Although the M protein gene could be cloned into hAdVs and expressed constituvely in 293 cells under the control of the hCMV immediate early promotor/enhancer, hAdVs expressing N and GP5 proteins, which appeared to be toxic or interfered with adenovirus replication, could only be generated by inclusion of a tetracycline-regulatable promotor in the transfer vector pAdTR5. The recombinant (rec) proteins appeared similar to the authentic viral proteins in regards to their M rs and antigenicities. However, the recGP5 apparently possesses different N-linked oligosaccharides residues. Its sensitivity to endo-β-galactosidase digestion indicates that poly-N-acetyllactosamine is present on the individually-expressed protein, but not on the authentic GP5 anchored into the virion envelope. The recGP5 apparently accumulates within the ER compartment as a glycoprotein that possesses high-mannose N-linked oligosaccharide side chains sensitive to endo-β-N-acetylglucosaminidase H treatment, by contrast to its viral counterpart for which N-linked oligosaccharide side chains are of both high-mannose and complex types. Coinfection of 293 cells with hAdVs expressing the M and GP5 did not lead to M-GP5 heterodimer formation, as demonstrated in PRRSV-infected cells. Moreover, cells infected with inducible hAdV/ORF5 showed that GP5 of the North American strain is proapoptotic. Indeed, when the expression cassette was turned-on, caspase 3 activity in hAdV/ORF5 infected cells was enhanced and DNA fragmentation could be detected by TUNEL assays. Pigs intradermally injected twice with hAdV/ORF5 developed antibody titers to the authentic viral GP5 as soon as 10 days following challenge with the homologous virulent PRRSV strain, as revealed by Western blot and virus neutralization tests, suggesting the establishment of a specific immune memory.

Intracellular Assembly and Secretion of Recombinant Subviral Particles from Tick-Borne Encephalitis Virus

It is believed that flavivirus assembly occurs by intracellular budding of the nucleocapsid into the lumen of the endoplasmic reticulum (ER). Recombinant expression of tick-borne encephalitis (TBE) virus envelope proteins prM and E in mammalian cells leads to their incorporation into enveloped recombinant subviral particles (RSPs), which have been used as a model system for studying assembly and entry processes and are also promising vaccine candidates. In this study, we analyzed the formation and secretion of TBE virus RSPs and of a membrane anchor-free E homodimer in mammalian cells. Immunofluorescence microscopy showed that E was accumulated in the lumen of the ER. RSPs were observed by electron microscopy in the rough and smooth ER and in downstream compartments of the secretory pathway. About 75% of the particles appeared to be of the size expected for RSPs (about 30 nm in diameter), but a number of larger particles and tubular structures were also observed in these compartments. Secretion of membrane anchor-free E dimers was detected 30 min after synthesis of prM and E, and secretion of RSPs was detected 1 h after synthesis of prM and E. We also found that the presence of the single N-linked oligosaccharide side chain on the E protein and its trimming by glucosidases was necessary for secretion of RSPs and truncated E dimers. Our results suggest that incorporation of prM and E into RSPs occurs at the ER membrane without other viral elements being required, followed by rapid transport along the compartments of the secretory pathway and secretion. Moreover, the carbohydrate side chain of E is involved in at least one assembly or transport step.

N-Glycans attached to the stem domain of haemagglutinin efficiently regulate influenza A virus replication.

The haemagglutinin (HA) protein of fowl plague virus A/FPV/Rostock/34 (H7N1) contains three N-linked oligosaccharide side chains in its stem domain. These stem glycans, which are attached to the Asn residues at positions 12, 28 and 478, are highly conserved throughout all HA protein sequences analysed to date. In a previous study, in which mutant HA proteins lacking individual stem glycosylation sites had been expressed from an SV-40 vector, it was shown that these glycans maintain the HA protein in the metastable form required for fusion activity. In the present study, the functional role of the stem N-glycans for virus replication was investigated using recombinant influenza viruses generated by an RNA polymerase I-based system. Studies in Madin-Darby canine kidney cells and embryonated chickens' eggs revealed that the N-glycan at Asn(12) is crucial for virus replication. In both culture systems, growth of virus lacking this glycan (mutant cg1) was completely blocked at 37 degrees C and inhibited at 33 degrees C. Loss of the glycan from Asn(478) (mutant cg3) caused less striking, but still measurable, effects. Interestingly, it was not possible to generate mutant viruses containing the HA protein lacking the N-glycan at Asn(28). It is concluded from this that the N-glycan at Asn(28) is indispensable for the formation of replication-competent influenza viruses. When compared to viruses containing wild-type HA protein, mutants cg1 and cg3 showed a significantly decreased pH stability. Taken together, these data show that the HA stem glycans are potent regulators of influenza virus replication.

Human Vascular Adhesion Protein-1 in Smooth Muscle Cells

Human vascular adhesion protein-1 (VAP-1) is a dual-function molecule with adhesive and enzymatic properties. In addition to synthesis in endothelial cells, where it mediates lymphocyte binding, VAP-1 is expressed in smooth muscle cells. Here we studied the expression, biochemical structure, and function of VAP-1 in muscle cells and compared it to those in endothelial cells. VAP-1 is expressed on the plasma membrane of all types of smooth muscle cells, but it is completely absent from cardiac and skeletal muscle cells. In tumors, VAP-1 is retained on all leiomyoma cells, whereas it is lost in half of leiomyosarcoma samples. In smooth muscle VAP-1 predominantly exists as a approximately 165-kd homodimeric glycoprotein, but a trimeric (approximately 250 kd) form of VAP-1 is also found. It contains N-linked oligosaccharide side chains and abundant sialic acid decorations. In comparison, in endothelial cells dimeric VAP-1 is larger, no trimeric forms are found, and VAP-1 does not have N-glycanase-sensitive oligosaccharides. Unlike endothelial VAP-1, VAP-1 localized on smooth muscle cells does not support binding of lymphocytes. Instead, it deaminates exogenous and endogenous primary amines. In conclusion, VAP-1 in smooth muscle cells is structurally and functionally distinct from VAP-1 present on endothelial cells.

Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum

Human hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylation and oligosaccharide processing. Secretion of HL was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), monensin, brefeldin A (BFA), tunicamycin, castanospermine and N-methyldeoxynojirimycin, but not by 1-deoxymannojirimycin. Secretion of alpha1-antitrypsin, an unrelated N-glycoprotein, was also inhibited by monensin, BFA and tunicamycin, but not by CCCP, castanospermine or N-methyldeoxynojirimycin. Intracellular HL activity decreased with CCCP, tunicamycin, castanospermine and N-methyldeoxynojirimycin, but increased with monensin and BFA. In the absence of protein synthesis de novo, HL activity secreted into the medium was 7.8+/-2.1-fold higher (mean+/-S.D., n=7) than the simultaneous fall in intracellular HL activity. In cells pretreated with monensin or BFA, this factor decreased to 1.3+/-0.5, indicating that the apparent increase in HL activity had already occurred within these cells. After chromatography on Sepharose-heparin, the specific triacylglycerol hydrolase activity of secreted HL was only 1.7+/-0. 3-fold higher than that of intracellular HL, indicating that the secretion-coupled increase in HL activity is only partly explained by true activation. We conclude that oligosaccharide processing by glucosidases in the endoplasmic reticulum is necessary for the transport of newly synthesized human HL, but not alpha1-antitrypsin, to the Golgi, where the catalytic activity of HL is unmasked.

Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum

Human hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylation and oligosaccharide processing. Secretion of HL was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), monensin, brefeldin A (BFA), tunicamycin, castanospermine and N-methyldeoxynojirimycin, but not by 1-deoxymannojirimycin. Secretion of α1-antitrypsin, an unrelated N-glycoprotein, was also inhibited by monensin, BFA and tunicamycin, but not by CCCP, castanospermine or N-methyldeoxynojirimycin. Intracellular HL activity decreased with CCCP, tunicamycin, castanospermine and N-methyldeoxynojirimycin, but increased with monensin and BFA. In the absence of protein synthesis de novo, HL activity secreted into the medium was 7.8±2.1-fold higher (mean±S.D., n = 7) than the simultaneous fall in intracellular HL activity. In cells pretreated with monensin or BFA, this factor decreased to 1.3±0.5, indicating that the apparent increase in HL activity had already occurred within these cells. After chromatography on Sepharose–heparin, the specific triacylglycerol hydrolase activity of secreted HL was only 1.7±0.3-fold higher than that of intracellular HL, indicating that the secretion-coupled increase in HL activity is only partly explained by true activation. We conclude that oligosaccharide processing by glucosidases in the endoplasmic reticulum is necessary for the transport of newly synthesized human HL, but not α1-antitrypsin, to the Golgi, where the catalytic activity of HL is unmasked.

Exocellular (glyco)proteins of Lupinus albus plants and suspension-cultured cells

Plant species from genus Lupinus are among the oldest known legumes, and various aspects of their biology are considerably different from those commonly observed within Leguminosae. To study this issue in more detail, a suspension culture of Lupinus albus cells was developed, and the glycosylation patterns of exocellular proteins analysed. N-linked oligosaccharide side-chains were detected with two lectins: concanavalin A (ConA) and wheat germ agglutinin (WGA) used with respective anti-lectin antibodies, while O-linked arabinosylated side-chains of (hydroxy)proline-rich glycoproteins were identified with anti-(42 kDa French bean chitin-binding protein) antibodies. The obtained data were compared with analogous ones for exocellular (glyco)proteins from suspension-cultured Phaseolus vulgaris cells and from various tissues of L. albus plants. Major species-specific differences between exocellular (glyco)proteins from lupin and bean cells were identified. Similarly, developmentally regulated glycosylation changes following transition from organised plant tissue to dedifferentiated suspension-cultured lupin cells were detected and analysed.

Interaction of the thiol-dependent reductase ERp57 with nascent glycoproteins.

Calnexin and calreticulin interact specifically with newly synthesized glycoproteins in the endoplasmic reticulum (ER) and function as molecular chaperones. The carbohydrate-specific interactions between ER components and glycoproteins synthesized in isolated canine pancreatic microsomes were analyzed using a cross-linking approach. A carbohydrate-dependent interaction between newly synthesized glycoproteins, the thiol-dependent reductase ERp57, and either calnexin or calreticulin was identified. The interaction between ERp57 and the newly synthesized glycoproteins required trimming of the N-linked oligosaccharide side chain. Thus, it is likely that ERp57 functions as part of the glycoprotein-specific quality control machinery operating in the lumen of the ER.

Loss of glycosylation at Asn144 alters the substrate preference of the N8 influenza A virus neuraminidase.

Role of asparagine-linked (N-linked) oligosaccharide side chains in the maturation and the function of influenza virus neuraminidase (NA) subtype N8 was examined by site-directed mutagenesis and vaccinia virus expression system. Mutations in the consensus sequence for N-linked glycosylation at Asn 84 or 398 prevent the proper maturation of mutant NAs. On the contrary, mutation at Asn 144, that is conserved in all except two strains of influenza virus NA ever sequenced, did not affect the proper maturation and the transport of the mutant NA to the cell surface. Furthermore, this mutation led the alternation of substrate preference of this enzyme. These observations indicate that N-glycosylation at Asn 144 of N8 NA may be conserved from the functional requirement, but not from the structural necessity.

Structure and functional significance of the carbohydrates of the LH/CG receptor

The LH/CG appears to contain 1-4 bi- or multi-antennary complex-type N-linked oligosaccharide side chains, which appear to locate apart from the hormone-binding regions. The exact sites to which the N-linked chains are attached remain to be delineated. The carbohydrates of the mature membrane-inserted receptor do not contribute to either specific high-affinity ligand-binding or signal transduction of the receptor. Thus, the polypeptide core of the receptor is responsible for both high affinity binding and dictating the hormone specificity. Moreover, the deglycosylated receptor, once inserted to the plasma membrane in a functionally mature form, retains its functional conformation or permits the conformational change that is required for coupling of the receptor to effector enzymes. Addition of oligosaccharides to the nascent LH/CG receptor but not their subsequent conversion to complex-type ones appears to be required for acquiring the hormone-binding conformation. On the other hand, neither addition of oligosaccharides to the nascent receptor, nor their further maturation are needed for the transport of the receptor to the plasma membrane. Thus, one function of the N-linked oligosaccharides in the LH/CG receptor appears to be to direct the proper folding of the receptor.

Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors.

The baculovirus-insect cell expression system is well-suited for recombinant glycoprotein production because baculovirus vectors can provide high levels of expression and insect cells can modify newly synthesized proteins in eucaryotic fashion. However, the N-glycosylation pathway of baculovirus-infected insect cells differs from the pathway found in higher eucaryotes, as indicated by the fact that glycoproteins produced in the baculovirus system typically lack complex biantennary N-linked oligosaccharide side chains containing penultimate galactose and terminal sialic acid residues. We recently developed a new type of baculovirus vector that can express foreign genes immediately after infection under the control of the viral ie1 promoter. These immediate early baculovirus expression vectors can be used to modify the insect cell N-glycosylation pathway and produce a foreign glycoprotein with more extensively processed N-linked oligosaccharides. These vectors can also be used to study the influence of the late steps in N-linked oligosaccharide processing on glycoprotein function. Further development could lead to baculovirus-insect cell expression systems that can produce recombinant glycoproteins with complex biantennary N-linked oligosaccharides structurally identical to those produced by higher eucaryotes.

Analysis of the role of N-glycosylation in cell-surface expression and binding properties of angiotensin II type-2 receptor of rat pheochromocytoma cells

We previously demonstrated that the AT2 receptor is a glycoprotein containing N-linked oligosaccharide side chains and that the marked disparity between the sizes of AT2 receptors from different tissues was related to different degrees of N-glycosylation. In the present study, we used an inhibitor of N-glycosylation, tunicamycin, as well as an endoglycosidase, glycopeptidase-F, to examine the contribution of carbohydrate moieties to the ligand-binding properties, cell-surface expression and apparent molecular mass of AT2 receptors of rat pheochromocytoma cells (PC-12 cells). Photoaffinity labelling of cell-surface AT2 receptors revealed that PC-12 cells grown in the presence of tunicamycin expressed, in addition to the previously described 140 kDa receptor, lower-molecular-mass receptors of 63 kDa, 47 kDa and 32 kDa. Lectin affinity chromatography revealed that the 63 kDa and the 47 kDa receptors are partially glycosylated and that the 32 kDa receptor is completely deglycosylated. Competitive binding experiments were carried out on tunicamycin-treated cells that expressed predominantly the 63 kDa or the 47 kDa receptors. Both receptor forms exhibited a high affinity for angiotensin II, although a slight decrease (of about 2-fold) was consistently observed on tunicamycin-treated cells as compared with control cells. Endoglycosidase digestion of AT2 receptors of PC-12 cells also yielded smaller receptor forms of 47 kDa and 32 kDa. Similarly, angiotensin II showed a high but slightly decreased binding affinity (of about 2-fold) for deglycosylated membranes as compared with control membranes. In conclusion, the stepwise action of tunicamycin suggests the presence of at least three N-linked oligosaccharide side chains on the AT2 receptor of PC-12 cells. These oligosaccharide side chains have a minor contribution to the affinity of the receptor. Interestingly, glycosylation is not an essential requirement for the expression of AT2 receptor at the surface of PC-12 cells.

Carbohydrate analysis of the B2 bradykinin receptor from rat uterus.

The B2 bradykinin receptor purified from rat uterus has an apparent molecular mass of 81 kDa. This is higher than the value of 42 kDa estimated from the sequence data of rat and human B2 receptors. Carbohydrate analysis of the rat B2 bradykinin receptor indicated that it was a sialoglycoprotein with three N-linked complex oligosaccharide side chains. This was consistent with the sequence, which has three potential glycosylation sites. The receptor did not appear to possess O-linked carbohydrate side chains. Removal of the N-linked carbohydrates with endo-beta-N-acetylglucosaminidase yielded a core protein of 42-44 kDa. The presence of these N-linked carbohydrates explains the discrepancy between the molecular size of the purified receptor protein and that estimated from the sequence. The sequence of the rat receptor suggests an isoelectric point of about pH 7.0, but the purified receptor had an isoelectric point of pH 4.5-4.7. Sialic acid residues on the N-linked side chains are likely to be responsible for the acidic nature of the rat receptor. Carbohydrate does not appear to play a role in ligand-receptor interactions, as deglycosylation did not alter the affinity of the B2 bradykinin receptor for bradykinin or the B2-selective antagonist HOE-140.