N-linked glycoproteins associated with flagellar scales in a flagellate green alga: characterization of interactions.

Glycoproteins associated with one type of flagellar scale (p-scale) isolated from the flagellate green alga Tetraselmis striata (Prasinophyceae) were shown to bind the mannose-specific lectin GNA (Galanthus nivalis agglutinin). Enzymatic deglycosylation of the glycoproteins with N-glycosidase F led to an electrophoretic mobility shift to lower molecular masses in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and abolished GNA-binding strongly indicating that most of the scale-associated glycoproteins contain asparagine-linked oligosaccharide side chains presumably of the high mannose type. To evaluate the significance of N-linked glycoproteins for scale structure and integrity, p-scales were digested with various proteases or extracted with 8 M urea and their ultrastructure and protein composition determined. The results show that while scale-associated N-linked glycoproteins do not determine the overall structure of the scale subunits (which consist of complex polysaccharides), they are apparently involved in mediating linkages between scale subunits; we have tentatively identified one glycoprotein of Mr 280,000 which may link outer scale subunits to one another. In addition, some scale-associated N-linked glycoproteins may provide connections between the layer of p-scales and the underlying flagellar membrane.     

Unraveling unique structure and biosynthesis pathway of N-linked glycans in human fungal pathogen Cryptococcus neoformans by glycomics analysis

The encapsulated fungal pathogen Cryptococcus neoformans causes cryptococcosis in immunocompromised individuals. Although cell surface mannoproteins have been implicated in C. neoformans pathogenicity, the structure of N-linked glycans assembled on mannoproteins has not yet been elucidated. By analyzing oligosaccharide profiles combined with exoglycosidase treatment, we report here that C. neoformans has serotype-specific high mannose-type N-glycans with or without a β1,2-xylose residue, which is attached to the trimannosyl core of N-glycans. Interestingly, the neutral N-glycans of serotypes A and D were shown to contain a xylose residue, whereas those of serotype B appeared to be much shorter and devoid of a xylose residue. Moreover, analysis of the C. neoformans uxs1Δ mutant demonstrated that UDP-xylose is utilized as a donor sugar in N-glycan biosynthesis. We also constructed and analyzed a set of C. neoformans mutant strains lacking genes putatively assigned to the reconstructed N-glycan biosynthesis pathway. It was shown that the outer chain of N-glycan is initiated by CnOch1p with addition of an α1,6-mannose residue and then subsequently extended by CnMnn2p with multiple additions of α1,2-mannose residues. Finally, comparative analysis of acidic N-glycans from wild-type, Cnoch1Δ, Cnmnn2Δ, and Cnuxs1Δ strains strongly indicated the presence of xylose phosphate attached to mannose residues in the core and outer region of N-glycans. Our data present the first report on the unique structure and biosynthesis pathway of N-glycans in C. neoformans.     

Complete genome sequence of the dog commensal and human pathogen Capnocytophaga canimorsus strain 5

Capnocytophaga canimorsus is a commensal Gram-negative bacterium, originally isolated from a dog's mouth, that causes septicemia in humans. C. canimorsus has the unusual ability to feed on host cells, including phagocytes. This capacity depends on surface-exposed glycan-foraging systems. Here we present the first complete genome sequence of a C. canimorsus strain (Cc5).     

Capnocytophaga canimorsus: a human pathogen feeding at the surface of epithelial cells and phagocytes

Capnocytophaga canimorsus, a commensal bacterium of the canine oral flora, has been repeatedly isolated since 1976 from severe human infections transmitted by dog bites. Here, we show that C. canimorsus exhibits robust growth when it is in direct contact with mammalian cells, including phagocytes. This property was found to be dependent on a surface-exposed sialidase allowing C. canimorsus to utilize internal aminosugars of glycan chains from host cell glycoproteins. Although sialidase probably evolved to sustain commensalism, by releasing carbohydrates from mucosal surfaces, it also contributed to bacterial persistence in a murine infection model: the wild type, but not the sialidase-deficient mutant, grew and persisted, both when infected singly or in competition. This study reveals an example of pathogenic bacteria feeding on mammalian cells, including phagocytes by deglycosylation of host glycans, and it illustrates how the adaptation of a commensal to its ecological niche in the host, here the dog's oral cavity, contributes to being a potential pathogen.     

Further characterization of structural determinants of rabbit hemopexin function

To further identify structural features of the hemopexin molecule important for its heme transport function, a fragment of the heme-binding domain (residues 1–213, Mr 35 kD, domain I) of rabbit hemopexin was obtained after digestion with subtilisin. Both apo- and heme-domain I were cleaved by subtilisin, and the subtilisin-digested form of domain I (called SD-DI) was shown by microsequencing to have been cleaved at Asp 22 forming a 30 kD subfragment lacking the conserved histidine residue at position 7 and the N-linked oligosaccharide at Asn 9. The 5 kD peptide cleaved from domain I is not disulfide linked to domain I and can be removed by membrane ultrafiltration. SD-DI retains the ability of domain I to bind heme, to associate with the other functional domain of hemopexin (domain II), and to interact with the hemopexin receptor on mouse Hepa cells. Moreover, although the heme complex of SD-DI is less themostable than native heme-domain I, like heme-domain I, heme-SD-DI is stabilized to a large extent when associated with domain II. These results show that the conserved His 7 residue is not involved in heme binding by hemopexin and that residues 1–22 of hemopexin and the N-linked oligosaccharide at Asn 9 are not essential for either receptor binding or interdomain interactions. Nevertheless, these N-terminal residues of hemopexin do contribute significantly to the overall stability of the hemopexin molecule and the interdomain interactions necessary for receptor recognition.     

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.      

Post-translational protein modification and expression of ankyrin-binding site(s) in GP85 (Pgp-1/CD44) and its biosynthetic precursors during T-lymphoma membrane biosynthesis.

In this study, we have investigated the biosynthesis and processing of GP85 (Pgp-1/CD44), a lymphoma transmembrane glycoprotein known to contain ankyrin-binding site(s). Using a standard pulse-chase protocol, we have detected a 52-kDa polypeptide precursor (p52) within the first 5 min of pulse labeling which contains a high mannose-type N-linked oligosaccharide chains. The conversion of p52 to GP85 requires further glycosylation (both complex type N-linked and O-linked) which takes place in the Golgi complex within 10-20 min after p52 is synthesized. GP85 is then incorporated into the plasma membrane where its turnover rate is relatively slow, a t1/2 of approximately 8 h. Following tunicamycin treatment, we have detected two other precursor proteins: p42 which is unglycosylated and p58 which is O-glycosylated. p42 appears to be an immediate precursor of p52 because p52 is converted to p42 upon deglycosylation. Therefore, the biosynthesis of GP85 appears to occur in the following sequence: p42 in equilibrium to p52 in equilibrium to GP85. Further analysis reveals that all of the GP85 precursors (i.e. p42, p52, and p58) contain ankyrin-binding site(s). Chemical composition analysis of GP85 indicates that this molecule contains approximately 3 N-linked and 4-5 O-linked oligosaccharide chains. Although neither N-glycosylation nor O-glycosylation appears to play an important role in the formation of ankyrin-binding site(s), O-glycosylation (and to a lesser extent N-glycosylation) of GP85 is required for T-lymphoma cell surface interaction with both collagen and hyaluronic acid. These findings suggest that GP85 (Pgp-1/CD44) and its biosynthetic precursors play a pivotal role in regulating adhesion functions such as lymphocyte homing and binding to the extracellular matrix.     

Evidence that WapB is a 1,2-glucosyltransferase of Pseudomonas aeruginosa involved in Lipopolysaccharide outer core biosynthesis

Pseudomonas aeruginosa is an important opportunistic pathogen infecting debilitated individuals. One of the major virulence factors expressed by P. aeruginosa is lipopolysaccharide (LPS), which is composed of lipid A, core oligosaccharide (OS), and O-antigen polysaccharide. The core OS is divided into inner and outer regions. Although the structure of the outer core OS has been elucidated, the functions and mechanisms of the glycosyltransferases involved in core OS biogenesis are currently unknown. Here, we show that a previously uncharacterized gene, pa1014, is involved in outer core biosynthesis, and we propose to rename this gene wapB. We constructed a chromosomal mutant, wapB::Gm, in a PAO1 (O5 serotype) strain background. Characterization of the LPS from the mutant by Western immunoblotting showed a lack of reactivity to PAO1 outer core-specific monoclonal antibody (MAb) 5c-101. The chemical structure of the core OS of the wapB mutant was elucidated using nuclear magnetic resonance spectroscopy and mass spectrometry techniques and revealed that the core OS of the wapB mutant lacked the terminal β-1,2-linked-d-glucose residue. Complementation of the mutant with wapB in trans restored the core structure to one that is identical to that of the wild type. Eleven of the 20 P. aeruginosa International Antigenic Typing Scheme (IATS) serotypes produce LPSs that lack the terminal d-glucose residue (Glc(IV)). Interestingly, expressing wapB in each of these 11 serotypes modifies each of their outer core OS structures, which became reactive to MAb 5c-101 in Western immunoblotting, suggesting the presence of a terminal d-glucose in these core OS structures. Our results strongly suggested that wapB encodes a 1,2-glucosyltransferase.     

Contribution of the N-linked carbohydrate of erythrocyte antigen CD59 to its complement-inhibitory activity.

The contribution of N-linked carbohydrate to the complement-inhibitory function of the human erythrocyte membrane glycoprotein, CD59, was investigated. Amino acid sequence analysis of tryptic peptides labeled with [3H]borohydride revealed an N-linked carbohydrate moiety at the Asn18 residue. No O-linked carbohydrate was detected, as judged by the failure of asialo-CD59 to bind peanut agglutinin and by its resistance to digestion by O-glycanase. The apparent molecular mass of CD59 was reduced from 18-20 to 14 kDa upon complete digestion with N-glycanase, with no detectable proteolysis. N-glycanase digestion of CD59 was associated with an 88 +/- 4% loss of the complement-inhibitory activity of the protein, as assessed by its capacity to protect chicken erythrocytes from lysis by the human C5b-9 proteins. By contrast, no change in function was observed after digestion of CD59 with neuraminidase, under conditions that removed greater than 60% of [3H]sialic acid residues. Despite loss of functional activity after N-glycanase digestion, we detected no change in the capacity of the deglycosylated CD59 to incorporate into erythrocyte membranes or to bind specifically and with species selectivity to the C8 and C9 components of the membrane attack complex. In order to alter the branched-chain structure of the N-linked carbohydrate of CD59 without enzymatic digestion, Chinese hamster ovary (CHO) cells transfected with cDNA for human CD59 were grown in the alpha-mannosidase inhibitor, 1-deoxymannojirimycin, resulting in conversion of approximately 70% of the membrane glycoprotein to a high mannose. When grown in the presence of 1-deoxymannojirimycin, the C5b-9-inhibitory activity of CD59 expressed on the surface of the transfected CHO cells was reduced by an amount comparable to that observed for the N-glycanase digested protein. Taken together, these data suggest that normal glycosylation of Asn18 in CD59 is required for the normal expression of its complement-inhibitory activity on membrane surfaces, although these N-linked sugar residues do not contribute to CD59's affinity for the C8 and C9 components of the C5b-9 complex.     

Cytochemical characterization of glycoproteins in the developing acrosome of rats. An ultrastructural study using lectin histochemistry, enzymes and chemical deglycosylation.

The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with beta-elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane.     

Enzymatic deglycosylation of thyroid-stimulating hormone with peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase F

We investigated the ability of two enzymes, peptide N-glycosidase F (PNGase F) and endo-beta-N-acetylglucosaminidase F (Endo F), to deglycosylate microgram quantities of bovine TSH and its subunits under nondenaturing conditions. One oligosaccharide chain could be selectively removed from the alpha subunit by PNGase F, and all the oligosaccharide chains from both subunits could be removed by Endo F. These methods of enzymatic deglycosylation should permit study of the functional role of each N-linked carbohydrate chain of various glycoprotein hormones.     

The effect of inhibitors of glycoprotein synthesis and processing on the phagocytosis of rod outer segments by cultured retinal pigment epithelial cells

Retinal pigment epithelial cells selectively phagocytize rod outer segments by a process that may be mediated by specific cell surface receptors. Since many receptors are glycoproteins, we have studied the effect of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, and of castanospermine and swainsonine, which are inhibitors of oligosaccharide processing, on the ability of cultured retinal pigment epithelial cells to phagocytize rod outer segment. Tunicamycin inhibits the glycosylation of newly synthesized glycoproteins by 85-90%; concomitantly, the phagocytosis of rod outer segments is inhibited by 70-80%. The effect of tunicamycin is to initially reduce rod outer segments binding, and therefore the subsequent ingestion of rod outer segments. SDS-PAGE analysis and autoradiography of [35S]methionine labelled extracts of tunicamycin-treated cells, demonstrates the disappearance of a number of glycoprotein bands, and the appearance of a number of protein bands of lower Mr. Kinetic analysis of the disappearance and reappearance of specific glycoproteins suggests that the lower Mr bands are the non-glycosylated forms of the higher Mr bands. By contrast, castanospermine and swainsonine have no effect on the ability of retinal pigment epithelial cells to phagocytize rod outer segments, or on the SDS-PAGE pattern of treated cells, although they were shown to inhibit oligosaccharide processing as expected. These results support the hypothesis that rod outer segment phagocytosis by retinal pigment epithelial cells is mediated by specific glycoprotein receptors. N-Glycosylation of these receptors is required for their function, or for their insertion into the plasma membrane, whereas processing of the N-linked oligosaccharide chains of these receptors is not crucial for rod outer segment phagocytosis by retinal pigment epithelial cells.     

Murine Ia-associated invariant chain's processing to complex oligosaccharide forms and its dissociation from the I-Ak complex

The processing of murine invariant chain (Ii) to a cell surface form bearing complex N-linked oligosaccharides has been demonstrated in the B cell lymphoma, AKTB-1b. In addition, the rate of processing of pulse-labeled Ii has been determined relative to its rate of dissociation from the alpha/beta complex of I-Ak. Ii, alpha-, and beta-chains were immunoprecipitated with anti-I-Ak or anti-Ii monoclonal antibodies. The heretofore uncharacterized complex oligosaccharide form of Ii (Ii-c) was identified in gel-purified immunoprecipitates by peptide mapping with reverse-phase HPLC. Ii-c is resistant to deglycosylation by Endo H, which is specific for high-mannose N-linkages, but can be digested with Endo F, a glycosidase capable of cleaving both complex and high-mannose N-linked oligosaccharides. Immunoprecipitation of surface iodinated cells indicates that Ii-c is expressed on the plasma membrane. Pulse-chase metabolic labeling data show that the processing of Ii to Ii-c occurs with a t1/2 of about 120 min. In contrast, the processing of both alpha- and beta-chains of I-Ak to complex forms occurs with a t1/2 of 15 to 20 min. Our data show that Ii-hm begins to dissociate rapidly from the I-Ak complex after 100 to 120 min of chase. Only a small amount (less than 5% on a per mole basis) of Ii-c was found associated with the I-Ak complexes after 300 min of continuous metabolic labeling. These results are consistent with Ii serving as a carrier for Ia antigens as they are transported to the cell surface. In addition, they suggest that the processing of Ii to Ii-c, or a late processing event of the alpha- and beta-chains, such as their sialylation, may be a possible mechanism for inducing the dissociation of Ii from the I-Ak complex.     

The carbohydrate moiety of the activator protein for glucosylceramide beta-glucosidase

SAP-2 is a family of heat-stable, acidic glycoproteins which stimulate enzymatic hydrolysis of glucosylceramide. We studied the carbohydrate moieties of a ConA-binding form of SAP-2. The protein contained glucosamine, galactose, mannose, and fucose; galactosamine and sialic acid were not detectable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed three bands of 6.5, 8.5, and 10 kDa. After deglycosylation with peptide N-glycosidase, SAP-2 eluted more slowly from the C4 column and showed a single band of 4 kDa. From carbohydrate analysis it was evident that deglycosylation had removed more than 90% of the sugars. These data indicate that SAP-2 possesses N-linked complex or hybrid type oligosaccharide chains. The specific activity of the deglycosylated protein in the glucosidase stimulation assay was unaffected.     

A Drosophila metallophosphoesterase mediates deglycosylation of rhodopsin

Oligosaccharide chains of newly synthesized membrane receptors are trimmed and modified to optimize their trafficking and/or signalling before delivery to the cell surface. For most membrane receptors, the functional significance of oligosaccharide chain modification is unknown. During the maturation of Rh1 rhodopsin, a Drosophila light receptor, the oligosaccharide chain is trimmed extensively. Neither the functional significance of this modification nor the enzymes mediating this process are known. Here, we identify a dmppe (Drosophila metallophosphoesterase) mutant with incomplete deglycosylation of Rh1, and show that the retained oligosaccharide chain does not affect Rh1 localization or signalling. The incomplete deglycosylation, however, renders Rh1 more sensitive to endocytic degradation, and causes morphological and functional defects in photoreceptors of aged dmppe flies. We further demonstrate that the dMPPE protein functions as an Mn(2+)/Zn(2+)-dependent phosphoesterase and mediates in vivo dephosphorylation of α-Man-II. Most importantly, the dephosphorylated α-Man-II is required for the removal of the Rh1 oligosaccharide chain. These observations suggest that the glycosylation status of membrane proteins is controlled through phosphorylation/dephosphorylation, and that MPPE acts as the phosphoesterase in this regulation.     

Characterization of the carbohydrate moiety of Clerodendron trichotomum lectins. Its structure and reactivity toward plant lectins

Lectins were isolated from fruits and leaves of Clerodendron trichotomum by affinity chromatography on lactamyl-Sepharose. The purified lectins (C. trichotomum agglutinin: CTA) were homogeneous on SDS/polyacrylamide gel electrophoresis, and the carbohydrate moiety was characterized by physicochemical and immunochemical methods. The asparagine-linked oligosaccharides were released by treatment with N-oligosaccharide glycopeptidase (almond, EC 3.5.1.52) of peptic glycopeptides obtained from fruit CTA, and separated by gel filtration and thin-layer chromatography. The structure of the predominant oligosaccharide was determined as Xyl beta 1----2 (Man alpha 1----6)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc by high-performance liquid chromatography, sugar analysis and 1H-NMR spectroscopy. The reactivity of the carbohydrate moiety of CTA toward various lectins was studied. Fruit and leaf CTAs were applied to polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets and detected with horseradish-peroxidase-conjugated lectins. Concanavalin A, lentil lectin, pea lectin, Vicia faba lectin and Ulex europeus agglutinin I, but not wheat germ lectin, bound to fruit CTA. The results indicate new binding properties of these plant lectins: a beta-xylosyl residue substituted at C-2 of the beta-mannosyl residue of N-linked oligosaccharide does not affect the binding with mannose-specific lectins, lentil, pea and Vicia faba lectins can bind to N-linked oligosaccharides containing an alpha-L-fucosyl residue attached to C-3 of the asparagine-linked N-acetyl-D-glucosamine residue, and Ulex europeus agglutinin I can bind to the (alpha 1----3)-linked fucose residue of the N-linked oligosaccharide.     

Peptide-specific Transfer of N-Acetylgalactosamine to O-Linked Glycans by the Glycosyltransferases β1,4-N-Acetylgalactosaminyl Transferase 3 (β4GalNAc-T3) and β4GalNAc-T4

N- and O-linked oligosaccharides on pro-opiomelanocortin both bear the unique terminal sequence SO(4)-4-GalNAcβ1,4GlcNAcβ. We previously demonstrated that protein-specific transfer of GalNAc to N-linked oligosaccharides on glycoprotein substrates is dependent on the presence of both an oligosaccharide acceptor and a peptide recognition motif consisting of a cluster of basic amino acids. We characterized how two β1,4-N-acetylgalactosaminyltransferases, β4GalNAc-T3 and β4GalNAc-T4, require the presence of both the peptide recognition motif and the N-linked oligosaccharide acceptors to transfer GalNAc in β1,4-linkage to GlcNAc in vivo and in vitro. We now show that β4GalNAc-T3 and β4GalNAc-T4 are able to utilize the same peptide motif to selectively add GalNAc to β1,6-linked GlcNAc in core 2 O-linked oligosaccharide structures to form Galβ1,3(GalNAcβ1,4GlcNAcβ1,6)GalNAcαSer/Thr. The β1,4-linked GalNAc can be further modified with 4-linked sulfate by either GalNAc-4-sulfotransferase 1 (GalNAc-4-ST1) (CHST8) or GalNAc-4-ST2 (CHST9) or with α2,6-linked N-acetylneuraminic acid by α2,6-sialyltransferase 1 (ST6Gal1), thus generating a family of unique GalNAcβ1,4GlcNAcβ (LacdiNAc)-containing structures on specific glycoproteins.     

Structure determination of the glycans of human-serum alpha 1-antichymotrypsin using 1H-NMR spectroscopy and deglycosylation by N-glycanase

alpha 1-Antichymotrypsin purified from normal human serum was separated by affinity chromatography into th ree microheterogeneous forms on a concanavalin-A-Sepharose column: a pass-through (peak 1), a retarded (peak 2) and a bound form (peaks 3 + 4). For each form the asparagine-linked carbohydrate chains were liberated as oligosaccharides by hydrazinolysis, submitted to reduction with NaBH4 after re-N-acetylation and further separated by affinity chromatography on a concanavalin-A-Sepharose column. The complete primary structure of the glycans was determined by high-resolution 1H-NMR spectroscopy. The results indicated the presence of disialyl diantennary and of trisialyl triantennary type glycanic structures, the latter being accompanied by traces of disialylated triantennary oligosaccharide. The N-glycanase was used for the deglycosylation of the unfractionated alpha 1-antichymotrypsin; the successive removal of the N-linked complex-type oligosaccharide side chains of alpha 1-antichymotrypsin was studied in the presence of detergents. From these experiments it is concluded that alpha 1-antichymotrypsin carries four oligosaccharide side chains. Moreover our results show that the peak 1 contains four triantennary glycans, the peak 2 three triantennary and one diantennary glycans while the bound peaks 3 + 4 possess, on average, about one triantennary and three diantennary glycans per molecule. Since we showed that the peak 4 contains mostly diantennary glycans, it can be deduced that in peak 3 there are molecules carrying two triantennary and two diantennary glycans and others carrying one triantennary and three diantennary glycans.     

The effects of inhibiting oligosaccharide trimming by 1-deoxynojirimycin on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor has a subunit stoichiometry of alpha 2 beta gamma delta; all 5 subunits contain N-linked oligosaccharides. We investigated what role trimming of the oligosaccharides played in the post-translational processing of the subunits and assembly of the receptor by examining the receptor synthesized in the presence of an inhibitor of oligosaccharide trimming, 1-deoxynojirimycin. BC3H-1 cells express one-third fewer receptors when grown in the presence of 1-deoxynojirimycin. The receptor subunits that are expressed have decreased mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating an inhibition of oligosaccharide trimming. In control cells, 40% of the translated alpha subunit acquires the capacity to bind alpha-bungarotoxin with a half-time of 40 min before assembly with the other subunits; the rest is rapidly degraded. In 1-deoxynojirimycin-treated cells approximately the same amount of alpha subunit is translated as in control cells, but that alpha subunit is degraded more rapidly, and only 25% acquires the capacity to bind alpha-bungarotoxin. From these results, we conclude that oligosaccharide processing either may aid in protecting the alpha subunit primary translation product from degradation or may be required for the conformational change or other post-translational modification(s) necessary for formation of the alpha-bungarotoxin binding form of the alpha subunit, which is then protected from proteolytic degradation. The cell surface receptor that is expressed in the presence of 1-deoxynojirimycin, however, is not altered in its affinity for cholinergic ligands. Thus, we conclude that differential N-linked oligosaccharide trimming of the 2 alpha subunits does not appear to play a part in the differences in affinities of the 2 alpha subunits for cholinergic ligands.     

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.