High-mannose Oligosaccharides Research Articles

The extracellular polysaccharide of Pichia (Hansenula) holstii NRRL Y-2448: The structure of the phosphomannan backbone

The phosphomannan core of the exopolysaccharide of Pichia (Hansenula) holstii NRRL Y-2448 was isolated after hydrolytic removal of the oligosaccharide phosphate side-chains. The core polysaccharide and its dephosphorylated derivative were subjected to extensive 1D and 2D NMR spectroscopy which yielded information on the linkage sites and on the sequence of the mannosyl residues in the major oligosaccharide repeating unit. The most probable structure for the repeating unit was -[6-O-PO 3H 2-α- D-Man-(1→3)-α- D-Man-(1→2)-α- D-Man-(1→2)]-α- D-Man-(1→6)-[-α- D-Man-(1→2)]-α- D-Man-(1→6)- A semiquantitative conformational analysis was performed by Monte Carlo simulations and the result was confirmed by comparison with the experimentally determined NMR data. The distance distribution for the phosphate groups was determined from the modelling and was found to cover the expected range of distances for phosphorylated high-mannose oligosaccharides.

Structural and Electron Microscopic Analysis of Neurocan and Recombinant Neurocan Fragments

Neurocan, a nervous tissue-specific chondroitin sulfate proteoglycan of the aggrecan family which has been shown to interact with neural cell adhesion molecules and tenascin, could be visualized by rotary shadowing electron microscopy as two globular domains interconnected by an extended flexible filament of 60-90 nm. Several recombinant neurocan fragments generated in the human embryonic kidney cell line 293 represent as observed by electron microscopy the expected parts of this structure, which indicates a correct folding of these molecules. Biological activity of the recombinant N-terminal globular domain could be demonstrated by its coelution with hyaluronan in gel permeation chromatography. In addition, the modification of the recombinant fragments with certain carbohydrate structures was analyzed. High mannose oligosaccharides could be mapped to the N-terminal globular domain of the brain-derived molecule. Only recombinant fragments containing parts of the central region of the molecule were modified with chondroitin sulfate chains and with the HNK-1 epitope, and could be considerably altered in their migratory behavior on SDS-polyacrylamide gel electrophoresis by neuraminidase treatment. These findings and the electron microscopic shape indicate a mucin-like character for the central neurocan region.

Processing of human cytomegalovirus glycoprotein B in recombinant adenovirus-infected cells.

Intracellular processing of human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) expressed by a recombinant adenovirus (Ad-gB) was studied in human A549 cells as processing events could affect immunogenicity when such viruses are used as live-recombinant vaccines. Cleavage of [35S]methionine-labelled gp13O into gp93 and gp55 reached a maximum after a 3 h chase. Cleavage was completely inhibited by brefeldin A, suggesting that processing normally occurs as a late Golgi or post-Golgi event. Uncleaved gp 130 remained completely sensitive to endo-beta-N-acetylglucosaminidase H (Endo-H) in untreated cells following long chase periods, indicating high-mannose oligosaccharides at all of the 18 N-linked glycosylation sites (Asn-X-Ser/Thr) and retention in the endoplasmic reticulum. Endo-H analysis of gp55 from swainsonine-treated and untreated cells was consistent with glycosylation at all three potential sites, with two oligosaccharides remaining sensitive to Endo-H and one being processed to Endo-H resistance. The heavily glycosylated N-terminal gp93 subunit was not detected by [35S]methionine-labelling but was easily detected along with gp55 after labelling with [3H]mannose. No cleavage of gp 130 was observed in analogous pulse-chase radiolabelling of Ad-gB-infected human fibroblasts, even though these cells are permissive for HCMV replication and can process the native gB molecule. Processing of gB in recombinant adenovirus-infected A549 cells was generally similar to that previously reported for native gB in HCMV-infected fibroblasts.

Intracellular Synthesis, Processing, and Transport of Proteins Encoded by ORFs 5 to 7 of Porcine Reproductive and Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a small enveloped virus containing a positive-strand RNA genome, possesses at least three major structural proteins designated N, M, and E. The N protein is considered as the major component of the nucleocapsid, whereas M and E are membrane-associated. Previous studies using peptide-specific antibodies assigned these proteins to ORFs 7, 6, and 5, respectively. In the present report, monospecific antisera raised againstEscherichia coli-expressed ORFs 5, 6, and 7 products were used to study the synthesis and processing of PRRSV structural proteins in the highly permissive MARC-145 cell line. Treatment of viral proteins with various glycosidases showed that only E was modified by N-linked glycans. Pulse–chase experiments revealed that intracellular transport of the major envelope glycoprotein was delayed in the premedial Golgi compartment. During the first 30 min of chase, E undergoes a gradual downward shift of its apparent molecular weight, thought to result from trimming of the mannose-rich glycan structures. Once E is transported to the medial Golgi or proximal elements, some molecules undergo complete processing of all their high-mannose N-linked oligosaccharides to complex type, while in other molecules only a fraction of N-linked glycans are terminally glycosylated. These two differentially glycosylated forms of E were found to be incorporated into extracellular virions. In cells and virions, both M and E were shown to occur in heterodimeric complexes linked by disulfide bonds. The oligomerization process, as analyzed from pulse–chase experiments, showed that M and E are incorporated into M-E complexes with different kinetics and efficiencies, in a fashion similar to their counterparts in equine arteritis virus. Apparently, all steps of E protein N-glycans processing proceed after its association with M which occurs in the endoplasmic reticulum (ER). In the infected cells, E and M appear highly membrane-associated, while N is predominantly cytosolic.

Glycoprotein H of human cytomegalovirus is a major antigen for the neutralizing humoral immune response.

A recombinant baculovirus expressing glycoprotein H (gpUL75) of human cytomegalovirus was used to examine the humoral immune response in naturally infected individuals. Recombinant baculovirus infected insect cells produced two forms of gH with molecular masses of 78-82 kDa and 94 kDa. The 94 kDa polypeptide was modified by high mannose oligosaccharide side-chains as shown by reduction in molecular mass after treatment with endoglycosidases H and F. The 78-82 kDa protein represented the non-glycosylated precursor which was resistant to the enzymes. In contrast to gH expressed in mammalian cells, the recombinant baculovirus expressed gH was transported to the cell surface. Glycoprotein H produced in insect cells was reactive with human convalescent sera and all tested neutralizing monoclonal antibodies recognizing either linear or conformational epitopes. Antibodies reacting with insect cell derived gH were detected in 96 percent of HCMV seropositive human sera. Using insect cells infected with the gH expressing recombinant baculovirus as immunoabsorbent, between 0 percent and 58 percent of the total virus neutralizing activity was removed from sera of individuals with a past HCMV infection. gH must therefore be considered a major antigen for the induction of neutralizing antibodies during natural infection.

Biochemistry, molecular biology, and genetics of the oligosaccharyltransferase

Asparagine-linked glycosylation is a highly conserved protein modification reaction that occurs in all eukaryotes. The initial stage in the biosynthesis of N-linked glycoproteins, catalyzed by the enzyme oligosaccharyltransferase (OST), involves the transfer of a preassembled high-mannose oligosaccharide from a dolichol-linked oligosaccharide donor onto asparagine acceptor sites in nascent proteins in the lumen of the rough endoplasmic reticulum. Biochemical, molecular biological, and genetic studies conducted during the past 5 years have resulted in an explosive growth in our knowledge concerning the OST. Although the basic biochemical properties of the enzyme were determined more than a decade ago using intact microsomal membranes, recent studies provide novel insight into the catalytic mechanism of the enzyme. The OST was recently purified as a large heteroligomeric membrane protein complex; the sequences of many of the subunits have been determined from both fungal and vertebrate sources. Consistent with the evolutionary conservation of N-linked glycosylation, protein sequence comparisons reveal significant homologies between vertebrate, invertebrate, plant, and fungal OST subunits. Yeast molecular genetic methods have been instrumental in the functional characterization of the OST subunits, and have proven to be powerful tools for the identification of novel gene products that influence oligosaccharide transfer in vivo.

Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan.

CD44-mediated cell adhesion to hyaluronate is controlled by mechanisms which are poorly understood. In the present work we examine the role of N-linked glycosylation and Ser-Gly motifs in regulating CD44-hyaluronate interaction. Our results show that treatment of a panel of human cell lines which constitutively express CD44 with the inhibitor of N-linked glycosylation tunicamycin results in the loss of attachment of these cells to hyaluronate-coated substrate. In contrast, treatment of the same cells with deoxymannojirimycin, which inhibits the conversion of high mannose oligosaccharides to complex N-linked carbohydrates, results in either no change or an increase in CD44-mediated adhesion to hyaluronate, suggesting that complex N-linked oligosaccharides may not be required for and may even inhibit CD44-HA interaction. Using human melanoma cells stably transfected with CD44 N-linked glycosylation site-specific mutants, we show that integrity of five potential N-linked glycosylation sites within the hyaluronate recognition domain of CD44 is critical for hyaluronate binding. Mutation of any one of these potential N-linked glycosylation sites abrogates CD44-mediated melanoma cell attachment to hyaluronate-coated surfaces, suggesting that all five sites are necessary to maintain the HA-recognition domain in the appropriate conformation. We also demonstrate that mutation of serine residues which constitute the four Ser-Gly motifs in the membrane proximal domain, and provide potential sites for glycosaminoglycan side chain attachment, impairs hyaluronate binding. Taken together, these observations indicate that changes in glycosylation of CD44 can have profound effects on its interaction with hyaluronic acid and suggest that glycosylation may provide an important regulatory mechanism of CD44 function.

Identification of the Lysosomal Membrane Glycoprotein Lamp-1 as a Receptor for Type-1-Fimbriated (Mannose-Specific)Escherichia coli

The presence of several glycosylated sites with high-mannose oligosaccharides on the lysosome-associated membrane glycoproteins (Lamps) combined with the fact that neutrophil Lamps are present in mobilizable organelles inspired us to investigate their ability to bind type-1 fimbriated (mannose-binding)Escherichia coliand subsequently define a potential function for the Lamps in human neutrophils. Bacterial binding to Lamps purified from chronic myeloic leukemia cells was investigated by separation of the proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transfer to a blotting membrane and overlay with type-1-fimbriated bacteria. The overlays were developed by growth. The bacteria bound readily to Lamp-1 while there was almost no binding to Lamp-2. Hence, we state that a possible function for neutrophil Lamp-1 is bacterial binding.

FSH isoforms: bio and immuno-activities in post-menopausal and normal menstruating women.

The full expression of gonadotrophin biological activity depends on the gonadotrophin carbohydrate component. Our aim was to study serum FSH isoforms present in the follicular phase (FPS) and in the menopause (PMS) since the endocrine status may influence the structure of incorporated oligosaccharides. Ten healthy post-menopausal women (age range 53-68) not receiving any hormonal treatment and 10 healthy women (age range 20-28) in the follicular phase of their menstrual cycle were studied. Bio and immuno FSH-activities (Sertoli cell aromatase induction assay and RIA, respectively) were determined in separated isoforms after concanavalin A chromatography. Isolated isoforms were: UB, unbound; WB, weakly bound and FB, firmly bound to the lectin. PMS showed two groups of immuno and bio-active FSH isoforms: WB, bearing biantennary galactosylated type and FB, bearing high mannose or hybrid type oligosaccharides. Immuno and bio-active FSH were not detected in the UB fractions. WB isoforms represented 82 +/- 6% of the total bioactivity recovered in samples analysed individually; their B/I ratio was 0.85 +/- 0.20. FB isoforms were 18 +/- 6%; their B/I ratio was 3.27 +/- 0.60. Whole serum B/I ratio was 1.20 +/- 0.30. Similar results were obtained when pooled sera was analysed: WB: 77%, B/I: 0.82; FB: 23%, B/I: 3.75. Whole serum B/I in pooled samples was 1.10. FPS showed a different pattern. UB isoforms, bearing triantennary or bisecting oligosaccharides, were 41 +/- 3% of the total bioactivity recovered in samples analysed individually. Their B/I ratio was 0.61 +/- 0.23. WB isoforms were 59 +/- 3% and their B/I 0.76 +/- 0.14. FB FSH isoforms were not detected. The whole serum B/I ratio was 0.60 +/- 0.30. Similar results were obtained when pooled sera was analysed: WB 43%, B/I 0.42; FB 57%, B/I 0.62. Whole serum B/I in pooled samples was 0.70. These results show that, in normal women, circulating FSH bioactivity is associated with isoforms with different oligosaccharide [correction of oligosacharide] structures according to hormonal status. FSH in the follicular phase has a higher degree of branching and a more complete carbohydrate chain than the FSH secreted during the menopause.

Novel beta-D-galactofuranose-containing high-mannose type oligosaccharides in ascorbate oxidase from Acremonium sp. HI-25.

Ascorbate oxidase from the fungus Acremonium sp. HI-25 is a copper-containing glycoprotein that catalyzes the oxidation of ascorbic acid to dehydroascorbic acid. Monosaccharide composition analysis showed that the enzyme contains exclusively N-linked oligosaccharide chains. Following liberation by hydrazinolysis/re-N-acetylation, and fractionation by HPLC on anion exchange. Amide-80 and/or octadecyl silica columns after derivatization with p-aminobenzoic ethyl ester, the structures of the twelve major neutral oligosaccharides were identified by FAB-MS, 400 MHz 1H-NMR, methylation analysis, mild acid hydrolysis, and/or sequential exoglycosidase digestions. Acremonium sp. ascorbate oxidase was found to consist of high-mannose type oligosaccharides (76.3%) having 4 to 9 mannose residues and a series of novel D-galactofuranose-containing high-mannose type oligosaccharides (18.6%) with the following structure.

Free Energy Simulation Studies of the Binding Specificity of Mannose-Binding Protein

Free energy simulations and standard molecular mechanics calculations have been used to examine the binding specificity of the C-type lectin rat mannose-binding protein. This protein, which is important in inflammation and immunoglobin-independent immune response, recognizes and binds to mannose and high-mannose oligosaccharides but not to galactose. Molecular dynamics simulations were used to estimate the free energy difference between these two sugars in aqueous solution and in the binding site of the protein. While the simulations found mannose to be favored over galactose, interactions with the solvent tended to favor galactose. The calculated binding free energy difference for these two sugars to MBP was 3.1 kcal/mol favoring mannose, approximately twice the experimental value. An estimate of the resolution into components found that enthalpy favored mannose but that entropy favored galactose, a reversal of the situation in solution. From standard MD simulations, the galactose was found to rotate in ...

IgM polymerization inhibits the Golgi-mediated processing of the μ-chain carboxy-terminal glycans

Secreted glycoproteins generally contain oligosaccharides of the complex type. However, several molecules have been described in which individual glycans are processed differently from one another. Folding, assembly and oligomerization could affect the maturation of certain glycans by hindering them to the Golgi processing machinery. We have tested this possibility by analysing a panel of engineered murine μ chains secreted as μ2L2 monomers or as polymers, and having or not the carboxy-terminal glycan (Asn563). In secreted IgM polymers, Asn563 bears high-mannose oligosaccharides, typical of endoplasmic reticulum resident proteins, while complex sugars are found at the other four sites (Brenckle and Kornfeld, 1980 Arch. Biochem. Biophys. 243, 605–618). Polymeric and monomeric IgM contain μ chains whose glycans are processed differently. We show here that this is mainly due to the differential processing at the Asn563 glycan, which undergoes Golgi-mediated processing when IgM are secreted in the monomeric form. These results indicate that the oligomerization-dependent accessibility to the sugar modifying enzymes can be one of the key features that dictate the extent of oligosaccharide processing in multimeric glycoproteins. The presence of high mannose glycans at Asn563 implies that IgM polymerization takes place before encountering mannosidase II, likely in a pre-Golgi compartment.

Immunological and biological activities of pituitary FSH isoforms in prepubertal male rats: effect of antiandrogens.

In male rats androgens are involved in the regulation of follicle-stimulating hormone (FSH) synthesis and secretion. Two nonsteroidal antiandrogens, flutamide and Casodex, were used to study the influence of androgens on the carbohydrate structure of FSH isoforms and the relationship with their bioactivity in prepubertal male rats. Different doses of flutamide or Casodex (vehicle, 1, 5, or 10 mg/rat/day) were administered subcutaneously for 10 days to 23-day-old rats. Immunological FSH was determined by radioimmunoassay and the bioactivity by in vitro Sertoli cell bioassay. Concanavalin A affinity chromatography was used to study the distribution of immunoactive and bioactive pituitary FSH isoforms. A significant depletion of immunological and biological pituitary FSH contents was observed even at the lowest dose of flutamide or Casodex used. The bioactive/immunoactive ratio of pituitary FSH was reduced at the highest dose of flutamide; however, no change was observed in Casodex-treated rats, suggesting a differential effect of the antiandrogens on the FSH bioactivity. Flutamide treatment provoked a significant decrease in proportion and bioactivity of FSH isoforms bearing biantennary and truncated hybrid oligosaccharide side chains and an increase in the proportion but a decrease in bioactivity of FSH isoforms bearing high-mannose oligosaccharides. Conversely, Casodex administration did not modify the proportions of FSH isoforms, although those bearing biantennary and truncated hybrid structures were less bioactive, while those bearing high-mannose oligosaccharides were more bioactive. The highest dose of flutamide decreased the bioactive/immunoactive ratio of FSH isoforms with a high degree of branching in their carbohydrate chains. Our results suggest that androgens, acting directly and indirectly at the pituitary, regulate the selective incorporation of sugar residues to the FSH molecule, thus modulating its biological activity.

A Serum Mannose-Binding Lectin Mediates Complement-Dependent Lysis of Influenza Virus-Infected Cells

The mechanism of lysis of influenza virus-infected BHK-21 cells by guinea pig serum (GPS) was investigated. Lysis was shown to involve activation of the classical complement pathway and was dependent on the presence of a mannose-binding lectin in GPS. FAGS analysis demonstrated Ca2+-dependent binding of the lectin to influenza virus-infected, but not uninfected, cells. Cells infected with mutant strains of virus lacking a particular high-mannose oligosaccharide at the tip of the hemagglutinin molecule showed reduced binding of the lectin and were correspondingly less sensitive to lysis by GPS than cells infected with the parent viruses. The degree or pattern of glycosylation of influenza viruses thus influences susceptibility to this mechanism of viral clearance. By interfering with the infectious process, lectin-dependent complement-mediated lysis of infected cells may be an important component of innate immunity to influenza and other enveloped viruses.

Intermediate Forms of Glycoconjugates Are Present in the Envelope of Herpes Simplex Virions during Their Transport along the Exocytic Pathway

In cells infected with herpes simplex virus 1, intracellular virions in transit along the exocytic pathway carry glycoconjugates that react, in fracture–label technique, with helix pomatia lectin. This lectin is specific for unsubstitutedN-acetylgalactosamine, an intermediate sugar added in O-linkage to ser/thr residues incis-Golgi and then substituted with galactose and sialic acid in thetrans-Golgi. Virions in the perinuclear space do not react with helix pomatia lectin. In intracellular transport vesicles and vacuoles, close to the Golgi complex, virions are positively labeled by helix pomatia lectin and variably labeled by wheat germ agglutinin, a lectin specific for fully mature glycoconjugates. Extracellular virions react only with wheat germ agglutinin. The detection of glycoconjugates at intermediate steps of maturation, coupled with previous results that virions in the perinuclear space carry high mannose oligosaccharides (Torrisiet al., J. Virol.66, 554–561, 1992), favor the view that maturation of herpes simplex virion envelope proceeds in a stepwise manner along the exocytic pathway. Should transit of virions involve a deenvelopment of enveloped virions followed by reenvelopment of naked nucleocapsids, our results rule out reenvelopment attrans- or post-Golgi compartments and could be consistent with reenvelopment occurring earlier in the exocytic pathway, most likely at thecis-Golgi.

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

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

Cloning of complementary DNA encoding a 135-kilodalton protein secreted from porcine corpus epididymis and its identification as an epididymis-specific alpha-mannosidase.

In the preceding study (Okamura et al., 1992; Biol Reprod 47:1040-1052) we suggested that a 135-kDa protein secreted by porcine epididymis is involved in the sperm maturation. In this work, we have isolated the cDNA clone coding the 135-kDa protein in an effort to investigate its structure and function. The 135-kDa protein was purified from porcine cauda epididymal fluid. Three oligonucleotide probes were synthesized according to the amino acid sequences of N-termini of the native protein and trypsin-digested peptides. A cDNA clone hybridizing with these three probes was isolated from the cDNA library derived from the porcine proximal corpus epididymis. It encodes a novel protein with 1,006 amino acid residues in an open reading frame. Its overall amino acid sequence was significantly homologous (25.7%) to the alpha-mannosidase precursor of Dictiostelium discoideum (P34098). The 135-kDa protein could digest both p-nitro-phenyl-alpha-D-mannoside and high mannose oligo saccharide (Man8-GlcNAc2), strongly suggesting that it is an alpha-mannosidase homologue. The expression of this protein was specific to porcine and was localized to the very narrow parts of epididymis: the border of the caput and corpus epididymis. This protein may serve as a good marker for the functional differentiation in porcine epididymis. A possible role of this protein in the species-specific sperm-egg interaction is discussed.

Neuraminidase Is Essential for Fowl Plague Virus Hemagglutinin to Show Hemagglutinating Activity

When hemagglutinin (HA) of fowl plague virus (FPV) was expressed in CV-1 cells by a simian virus 40 vector, hemadsorption was barely detectable, although HA was exposed at the cell surface. However, treatment of HA-expressing cells with Vibrio cholerae neuraminidase (VCNA) resulted in extensive hemadsorption. VCNA treatment enhanced the electrophoretic mobility of the HA1 subunit of HA, indicating the removal of sialic acid. When two oligosaccharides in the vicinity of the receptor binding site of FPV HA were deleted by site-specific mutagenesis, VCNA treatment was not required for hemadsorption. Mutants which retained one of these oligosaccharides and mutants in which oligosaccharides not adjacent to the receptor binding site were deleted needed VCNA treatment to show hemadsorption. VCNA treatment also enhanced hemadsorption of vector-expressed HA of the WSN strain, which had a complex-type oligosaccharide in the vicinity of the receptor binding site, but had no effect on hemadsorption of Hong Kong type HA, which has a high-mannose type oligosaccharide adjacent to the receptor binding site. These results indicate that sialic acid on oligosaccharides near the receptor binding site interferes with hemadsorption. Thus, the neuraminidase is essential for FPV HA to show hemagglutinating activity.

Synthesis of photolabile mono- and di-valent α- d-mannoside-6-phosphates as chemically modifying probes for mannose-6-phosphate-receptors

Two photoaffinity probes, disodium 3′-azibutyl α- d-mannopyranoside-6-phosphate and tetrasodium 2-azi-1,10-bis(α- d-mannopyranosyloxy-6-phosphate)decane were synthesized for the regioselective chemical modification of mannose-6-phosphate receptors. Disodium 3′-azibutyl α- d-mannopyranoside-6-phosphate was obtained by chemical α-mannosylation of 3-azibutanol and subsequent 6-phosphorylation of the mannosyl residue. The corresponding divalent ligand, mimicking a high mannose oligosaccharide with two mannose-6-phosphate end groups, was obtained by the same procedure but with 2-azi-1,10-decanediol as the aglyconic alcohol. In preliminary experiments, both photolabile ligands had affinity to cation-independent mannose-6-phosphate receptors from bovine testes.

Functional characterization of Ost3p. Loss of the 34-kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased underglycosylation of acceptor substrates.

Within the lumen of the rough endoplasmic reticulum, oligosaccharyltransferase catalyzes the en bloc transfer of a high mannose oligosaccharide moiety from the lipid-linked oligosaccharide donor to asparagine acceptor sites in nascent polypeptides. The Saccharomyces cerevisiae oligosaccharyltransferase was purified as a heteroligomeric complex consisting of six subunits (alpha-zeta) having apparent molecular masses of 64 kD (Ost1p), 45 kD (Wbp1p), 34 kD, 30 kD (Swp1p), 16 kD, and 9 kD. Here we report a structural and functional characterization of Ost3p which corresponds to the 34-kD gamma-subunit of the oligosaccharyltransferase. Unlike Ost1p, Wbp1p, and Swp1p, expression of Ost3p is not essential for viability of yeast. Instead, ost3 null mutant yeast grow at wild-type rates on solid or in liquid media irrespective of culture temperature. Nonetheless, detergent extracts prepared from ost3 null mutant membranes are twofold less active than extracts prepared from wild-type membranes in an in vitro oligosaccharyltransferase assay. Furthermore, loss of Ost3p is accompanied by significant underglycosylation of soluble and membrane-bound glycoproteins in vivo. Compared to the previously characterized ost1-1 mutant in the oligosaccharyltransferase, and the alg5 mutant in the oligosaccharide assembly pathway, ost3 null mutant yeast appear to be selectively impaired in the glycosylation of several membrane glycoproteins. The latter observation suggests that Ost3p may enhance oligosaccharide transfer in vivo to a subset of acceptor substrates.