Enhanced anti-rotavirus action of human cystatin C by site-specific glycosylation in yeast

The cDNA encoding human cystatin C (HCC) was subjected to site-specific substitution of alanine for serine at the position 37, to obtain the Asn(35)-Lys(36)-Ser(37) sequence that is a signal for asparagine-linked (N-linked) glycosylation of protein in eukaryotes, and was transformed into Pichia pastoris X33. As a result, 1.2 mg/L oligomannosyl HCC with a carbohydrate chain of Man(10)GlcNAc(2) was produced by the Pichia transformant. The oligomannosyl HCC was more stable at the low ionic strength condition of 50 mM potassium phosphate buffer, pH 7.0, than the wild-type. In addition, the oligomannosylation substantially improved the molecular stability of cystatin against an aspartic proteinase, cathepsin D, in which the susceptibility decreased to less than 50% of nonglycosylated one. The anti-rotavirus activity of HCC was substantially enhanced by the site-directed glycosylation using the yeast expression system. A MA-104 cell line was used as a host cell for human rotavirus type-2 Wa strain in this study, to which both the wild-type and oligomannosyl HCCs did not show cytotoxicity at a concentration of 100 mug/mL. More than 80% viability of the host cell infected with 1.0 x 10(5) PFU/mL of rotavirus was conserved under the condition coexisting with 75 mug/mL of the oligomannosyl HCC, which was 15.2% higher than that of wild-type HCC. Thus, the in vitro anti-rotavirus assay indicated that the supplement of a proper amount of the oligomannosyl HCC could be used as an anti-rotavirus agent.     

Effective reduction of antigenicity of hen egg lysozyme by site-specific glycosylation

Various mutant lysozymes were constructed by genetic modification and secreted in yeast expression system to evaluate the changes in the antigenicity of hen egg lysozyme (HEL). Although Arg68, the most critical residue to antigenicity of HEL, was substituted with Gln, the binding of monoclonal antibodies (mAbs) with the mutant lysozyme did not critically reduce, remaining 60% of the binding with mAb. In contrast, glycosylated mutant lysozyme G49N whose glycine was substituted with asparagine dramatically reduced the binding with mAb. The oligomannosyl type of G49N lysozyme reduced binding with mAb to one-fifth, while the polymannosyl type of G49N lysozyme completely diminished the binding with mAb. This suggests that the site-specific glycosylation of lysozyme in the interfacial region of lysozyme-antibody complex is more effective to reduce the antigenicity than the mutation of single amino acid substitution in the interfacial region.     

Characterisation of the asparagine-linked oligosaccharides from Trypanosoma brucei type-I variant surface glycoproteins

The complete primary structures of the Asn-linked oligosaccharides from the conserved glycosylation site of the type-I variant surface glycoproteins of Trypanosoma brucei MITat 1.4 and MITat 1.6 were determined using a combination of exoglycosidase digestions, permethylation analysis, acetolysis and 1H NMR. Both variants contained almost exclusively oligomannose-type oligosaccharides, identical in structure to those of mammalian glycoproteins. The oligosaccharides ranged in size from (Man)9(GlcNAc)2 to (Man)5(GlcNAc)2. The relative abundance of each component was similar in both variants. The major components were (Man)8(GlcNAc)2 and (Man)7(GlcNAc)2 with slightly less (Man)9(GlcNAc)2 and (Man)6(GlcNAc)2 and much less (Man)5(GlcNAc)2. Both variants also contained the same structural isomers. The close similarity of the oligomannose series indicates identical processing at the conserved site in both variants.     

The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin

Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn(354) are inaccessible because the complex glycans at Asn(445) block access to the site. On IgE, the additional C(H)2 hinge domain blocks access to the oligomannose glycans at Asn(394) on one H chain by adopting an asymmetrically bent conformation. IgE contains 8.3% Man(5)GlcNAc(2) glycans, which are the trimmed products of the Glc(3)Man(9)GlcNAc(2) oligomannose precursor. The presence of these structures suggests that the C(H)2 domain flips between two bent quaternary conformations so that the oligomannose glycans on each chain become accessible for limited trimming to Man(5)GlcNAc(2) during glycan biosynthesis. This is the first study of the glycosylation of human serum IgD and IgE from nonmyeloma proteins.     

A novel glycosylation phenotype expressed by Lec23, a Chinese hamster ovary mutant deficient in alpha-glucosidase I.

Lec23 Chinese hamster ovary (CHO) cells have been shown to possess a unique lectin resistance phenotype and genotype compared with previously isolated CHO glycosylation mutants (Stanley, P., Sallustio, S., Krag, S. S., and Dunn, B. (1990) Somatic Cell Mol. Genet. 16, 211-223). In this paper, a biochemical basis for the lec23 mutation is identified. The carbohydrates associated with the G glycoprotein of vesicular stomatitis virus (VSV) grown in Lec23 cells (Lec23/VSV) were found to possess predominantly oligomannosyl carbohydrates that bound strongly to concanavalin A-Sepharose, eluted 3 sugar eq beyond a Man9GlcNAc marker oligosaccharide on ion suppression high pressure liquid chromatography, and were susceptible to digestion with jack bean alpha-mannosidase. Monosaccharide analyses revealed that the oligomannosyl carbohydrates contained glucose, indicating a defect in alpha-glucosidase activity. This was confirmed by further structural characterization of the Lec23/VSV oligomannosyl carbohydrates using purified rat mammary gland alpha-glucosidase I, jack bean alpha-mannosidase, and 1H NMR spectroscopy at 500 MHz. [3H]Glucose-labeled Glc3Man9GlcNAc was prepared from CHO/VSV labeled with [3H]galactose in the presence of the processing inhibitors castanospermine and deoxymannojirimycin. Subsequently, [3H]Glc2Man9GlcNAc was prepared by purified alpha-glucosidase I digestion of [3H]Glc3Man9GlcNAc. When these oligosaccharides were used as alpha-glucosidase substrates it was revealed that Lec23 cells are specifically defective in alpha-glucosidase I, a deficiency not previously identified among mammalian cell glycosylation mutants.     

Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway

A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.     

Structures of the N-linked oligosaccharides of Gp63, the major surface glycoprotein, from Leishmania mexicana amazonensis

The extent of protein N-glycosylation in Leishmania mexicana amazonensis has been proposed to be a factor in the virulence of the parasite. The N-linked oligosaccharides of gp63, the major surface glycoprotein of L. mexicana amazonensis, were characterized after their release by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. High voltage paper electrophoresis of the reduced oligosaccharides revealed only neutral species. Gel-permeation chromatography on Bio-Gel P-4 yielded four fractions, and the oligosaccharides present were structurally characterized by sequential exoglycosidase digestion, fragmentation by acetolysis, and methylation analysis. Four major structures were found and were biantennary oligomannose type with compositions of Glc1Man6GlcNAc2 (La), Man6GlcNAc2 (Lb), Man5GlcNAc2 (Lc), and Man4GlcNAc2 (Ld). The largest oligosaccharide (La) was shown to contain a terminal glucopyranosyl residue on the alpha (1----3) arm. The biantennary oligomannose structures (Lb and Lc) and the glucosylated structure Glc1Man6GlcNAc2 (La) have not previously been reported as a component of a mature glycoprotein from any source.     

Enzymatic properties of rhea lysozyme

Rhea lysozyme was analyzed for its enzymatic properties both lytic and oligomer activities to reveal the structural and functional relationships of goose type lysozyme. Rhea lysozyme had the highest lytic activity at pH 6, followed by ostrich and goose at pH 5.5-6, whereas the optimum of cassowary was at pH 5. pH profile was correlated to the net charge of each molecule surface. On the other hand, the pH optimum for oligomer substrate was found to be pH 4, indicating the mechanism of rhea catalysis as a general acid. The time-course of the reaction was studied using beta-1,4-linked oligosaccharide of N-acetylglucosamine (GlcNAc) with a polymerization degree of n ((GlcNAc)n) (n=4, 5, and 6) as the substrate. This enzyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3+(GlcNAc)3 predominating over that to (GlcNAc)2+ (GlcNAc)4. This indicates that the lysozyme hydrolyzed preferentially the third glycosidic linkage from the nonreducing end. Theoretical analysis has shown the highest rate constant value at 1.5 s-1 with (GlcNAc)6. This confirmed six substrate binding subsites as goose lysozyme (Honda, Y., and Fukamizo, T., Biochim. Biophys. Acta, 1388, 53-65 (1998)). The different binding free energy values for subsites B, C, F, and G from goose lysozyme might responsible for the amino acid substitutions, Asn122Ser and Phe123Met, located at the subsite B.     

Deletion of the glucosidase II gene in Trypanosoma brucei reveals novel N-glycosylation mechanisms in the biosynthesis of variant surface glycoprotein

The trypanosomatids are generally aberrant in their protein N-glycosylation pathways. However, protein N-glycosylation in the African trypanosome Trypanosoma brucei, etiological agent of human African sleeping sickness, is not well understood. Here, we describe the creation of a bloodstream-form T. brucei mutant that is deficient in the endoplasmic reticulum enzyme glucosidase II. Characterization of the variant surface glycoprotein, the main glycoprotein synthesized by the parasite with two N-glycosylation sites, revealed unexpected changes in the N-glycosylation of this molecule. Structural characterization by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical and enzymatic treatments revealed that one of the two glycosylation sites was occupied by conventional oligomannose structures, whereas the other accumulated unusual structures in the form of Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, and Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Galbeta1-4GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc. The possibility that these structures might arise from Glc1Man9GlcNAc2 by unusually rapid alpha-mannosidase processing was ruled out using a mixture of alpha-mannosidase inhibitors. The results suggest that bloodstream-form T. brucei can transfer both Man9GlcNAc2 and Man5GlcNAc2 to the variant surface glycoprotein in a site-specific manner and that, unlike organisms that transfer exclusively Glc3Man9GlcNAc2, the T. brucei UDP-Glc: glycoprotein glucosyltransferase and glucosidase II enzymes can use Man5GlcNAc2 and Glc1Man5GlcNAc2, respectively, as their substrates. The ability to transfer Man5GlcNAc2 structures to N-glycosylation sites destined to become Man(4-3)GlcNAc2 or complex structures may have evolved as a mechanism to conserve dolichol-phosphate-mannose donors for glycosylphosphatidylinositol anchor biosynthesis and points to fundamental differences in the specificities of host and parasite glycosyltransferases that initiate the synthesis of complex N-glycans.     

N-glycans of recombinant human interferon-gamma change during batch culture of chinese hamster ovary cells

A recombinant Chinese hamster ovary (CHO) cell line making human interfron-gamma (IFN-gamma) was grown in 12-L stirred tank fermentors in three batch fermentations under conditions of constant temperature, pH, and dissolved oxygen tension. In addition to cell growth, metabolite, and productivity data, a detailed analysis of the carbohydrate structures attached to each glycosylation site of IFN-gamma was achieved using matrix-assisted laser desorption mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. Complex biantennary oligosaccharides (particularly Gal(2)GlcNAc(4)Man(3) which was core alephl-6 fucosylated at Asn(25) but not at Asng(97)) were most prevalent at both glycosylation sites. However, considerable microheterogeneity arising from the presence of triantennary and truncated glycan structures was also observed. The proportion of the dominant core glycan structure (Gal(2)GlcNAc(4)Man(3) +/- Fuc(1)) decreased by 15-26% during batch culture, with increases in the proportion of oligomannose and truncated glycans over the same time period. Prolonged culture resulting from an extended lag phase led to further accumulation of oligomannose and truncated structures, reaching up to 52% of total glycans attached to Asng(97) by 240 h of culture. The implications of these glycosylation changes for optimizing the time for harvesting cell cultures, and for the clearance of recombinant therapeutic products in vivo are discussed. (c) 1995 John Wiley & Sons, Inc.     

Dextranase (alpha-1,6 glucan-6-glucanohydrolase) from Penicillium minioluteum expressed in Pichia pastoris: two host cells with minor differences in N-glycosylation

Differences in glycosylation between the natural alpha-1,6 glucan-6-glucanohydrolase from Penicillium minioluteum and the heterologous protein expressed in the yeast Pichia pastoris were analyzed. Glycosylation profiling was carried out using fluorophore-assisted carbohydrate electrophoresis and amine absorption high-performance liquid chromatography (NH(2)-HPLC) in combination with matrix-assisted laser desorption-time of flight-mass spectrometry. Both microorganisms produce only oligomannosidic type structures, but the oligosaccharide population differs in both enzymes. The native enzyme has mainly short oligosaccharide chains ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), of which Man(8)GlcNAc(2) was the most represented oligosaccharide. The oligosaccharides linked to the protein produced in P. pastoris range from Man(7)GlcNAc(2) up to Man(14)GlcNAc(2), with Man(8)GlcNAc(2) and Man(9)GlcNAc(2) being the most abundant structures. In both enzymes the first glycosylation site (Asn(5)) is always glycosylated. However, Asn(537) and Asn(540) are only partially glycosylated in an alternate manner.     

In vitro hydrolysis of oligomannosyl oligosaccharides by the lysosomal alpha-D-mannosidases

In vitro incubation of the oligomannosyl oligosaccharides Man9GlcNAc and Man5GlcNAc with isolated disrupted lysosomes yields different oligosaccharide isomers resulting from mannosidase hydrolysis. These isomers were isolated by HPLC and characterized by 1H-NMR spectroscopy. The first steps of the degradation involve an (alpha 1-2)mannosidase activity and lead to the formation of one Man8GlcNAc, one Man7GlcNAc, two Man6GlcNAc and two Man5GlcNAc isomers. These reactions do not require Zn2+ as activator. On the other hand, the following steps, which lead to the formation of Man3GlcNAc and Man2GlcNAc, are Zn2(+)-dependent. This process is characterized by the preferential action of an (alpha 1-3)mannosidase activity, and the formation of Man(alpha 1-6)Man(alpha 1-6)Man(beta 1-4)GlcNAc and Man(alpha 1-6)Man(beta 1-4)GlcNAc. Therefore, the digestion of Man9GlcNAc inside the lysosome appears to follow a very specific pathway, since only nine intermediate compounds can be identified instead of the 38 possible isomers. Our results are consistent both with the existence of several specific enzymes for alpha 1-2, alpha 1-3 and alpha 1-6 linkages, and with the presence of a unique enzyme whose specificity would be dependent either on Zn2+ or on the spatial conformation of the glycan. Nevertheless, previous work on the structural analysis of oligosaccharides excreted in the urine of patients suffering from mannosidosis, demonstrates the absence of the core alpha 1-6-linked mannosyl residue in the major storage product derived from oligomannosyl oligosaccharides. This observation indicates the presence of a specific (alpha 1-6)mannosidase form, unaffected in mannosidosis.     

Functional properties of glycosylated lysozyme secreted in Pichia pastoris

Various mutant lysozymes having the N-glycosylation signal sequence, R21T (Asn(19)-Tyr(20)-Thr(21)), G49N (Asn(49)- Ser(50)-Thr(51)), R21T/G49N (Asn(19)-Tyr(20)-Thr(21)/Asn(49)-Ser(50)-Thr(51)), were secreted in the Pichia pastoris expression system. The secreted amounts of these mutant glycosylated lysozymes were almost the same as those of wild-type lysozyme (about 30 mg/liter). Glycosylation of the mutant lysozymes was confirmed by SDS-PAGE patterns, Endo-H treatment, TOF-MS analysis and chemical analysis. The composition of the carbohydrate chain attached to the single glycosylated lysozymes, R21T and G49N, was GlcNAc(2)Man(9-11), while that of the double glycosylated lysozyme, R21T/G49N, was GlcNAc(4)Man(27-32). The results of a CD analysis and lytic activity suggested that the conformation of the single glycosylated lysozymes had been conserved, while that of the double glycosylated lysozyme was less stable. The emulsifying properties of the lysozyme when glycosylated were greatly improved, being especially noteworthy in the double glycosylated lysozyme.     

IL-2, a lectin with specificity for high mannose glycopeptides

Utilizing a solid phase binding assay, we have demonstrated that rIL-2 binds with high affinity to the human urinary glycoprotein uromodulin. This binding is specifically inhibited by the saccharides diacetylchitobiose and Man(alpha 1-3)(Man(alpha 1-6]Man-O-methyl and by the high mannose glycopeptides Man5GlcNAc2-R and Man6GlcNAc2-R, but not by Man9GlcNAc2-R. rIL-2 also binds OVA, a glycoprotein which contains approximately 50% high mannose chains at a single glycosylation site, and to yeast mannan. This binding is inhibited by the same battery of saccharides which inhibit the binding to uromodulin. The conclusion that rIL-2 is a lectin is further supported by the observation that the sequence of IL-2 shares 27% homology with a 33-residue sequence of the carbohydrate-binding domain of human mannose-binding protein. The potential physiologic relevance of the carbohydrate binding activity is further elucidated by studies which show that 1) binding of soluble rIL-2 to immobilized uromodulin is enhanced at a pH of 4 to5 in the presence of divalent cations, and 2) neither uromodulin nor the high mannose glycopeptide Man5GlcNAc2Asn blocks the binding of rIL-2 to the IL-2R. Thus the carbohydrate-binding site of rIL-2 is distinct from the cell surface receptor-binding site, and might function preferentially in acidic microenvironments.     

Deletion of the TbALG3 gene demonstrates site-specific N-glycosylation and N-glycan processing in Trypanosoma brucei

We recently suggested a novel site-specific N-glycosylation mechanism in Trypanosoma brucei whereby some protein N-glycosylation sites selectively receive Man9GlcNAc2 from Man9GlcNAc2-PP-Dol while others receive Man5GlcNA(2 from Man5GlcNAc2-PP-Dol. In this paper, we test this model by creating procyclic and bloodstream form null mutants of TbALG3, the gene that encodes the alpha-mannosyltransferase that converts Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol. The procyclic and bloodstream form TbALG3 null mutants grow with normal kinetics, remain infectious to mice and tsetse flies, respectively, and have normal morphology. However, both forms display aberrant N-glycosylation of their major surface glycoproteins, procylcin, and variant surface glycoprotein, respectively. Specifically, procyclin and variant surface glycoprotein N-glycosylation sites that are modified with Man9GlcNAc2 and processed no further than Man5GlcNAc2 in the wild type are glycosylated less efficiently but processed to complex structures in the mutant. These data confirm our model and refine it by demonstrating that the biantennary glycan transferred from Man5GlcNAc2-PP-Dol is the only route to complex N-glycans in T. brucei and that Man9GlcNAc2-PP-Dol is strictly a precursor for oligomannose structures. The origins of site-specific Man5GlcNAc2 or Man9GlcNAc2 transfer are discussed and an updated model of N-glycosylation in T. brucei is presented.     

Effects of the protein matrix on glycan processing in glycoproteins

In the biosynthesis of glycoproteins containing asparagine-linked glycans, a number of regulatory factors must be involved in converting the single glycan precursor into the variety of different final structures observed in different eukaryotic species. Among these factors are the kind of glycan-processing enzymes available in the Golgi apparatus of different cells, the specificity and regulatory properties of these enzymes, and the unique properties of the protein matrix in which a given glycan resides during the biosynthetic processing. In examining the role of this latter regulatory factor, we have considered a simplified model in which a few key steps are common to all cells, regardless of the nature of the processing enzymes available. The protein-bound oligomannose precursor Man8GlcNAc2-, arriving in the Golgi after the initial trimming in the endoplasmic reticulum (ER), first undergoes a series of preprocessing steps to yield Man5GlcNAc2- in animals and plants or Man13-15GlcNAc2- in yeast. At this stage the key commitment step--to process or not to process--determines whether the above intermediates will remain as unprocessed oligomannose structures or be initiated into a new series of reactions to yield processed structures characteristic of the organisms involved (complex or hybrid for vertebrates, polymannose for yeast, xylosylated glycans for plants and some invertebrates, or Man3GlcNAc2- structures for other invertebrates). It is proposed that this commitment step, along with the obligatory preprocessing steps, is regulated primarily by each glycan's unique exposure on its protein matrix. Subsequent processing steps leading to complex or hybrid structures, fucosylation, extent of branching, and specific structures at the nonreducing terminals are most likely determined primarily by the enzyme makeup of the individual processing machineries, but with the protein matrix still playing a significant role.     

Structural and functional properties of hen egg-white lysozyme deamidated by protein engineering.

The structural and functional properties of lysozymes genetically deamidated at positions 103 (N103D) and 106 (N106D) were studied by a protein engineering technique. The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation-exchange chromatography on CM-Toyopearl. The lytic activity of deamidated lysozymes was almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation. The Gibbs free energy changes of unfolding (delta G) at 20 degrees C for N103D and N106D were almost the same as that of wild-type. On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation. The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme. These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability.     

Large-scale isolation of dolichol-linked oligosaccharides with homogeneous oligosaccharide structures: determination of steady-state dolichol-linked oligosaccharide compositions

The dolichol-linked oligosaccharide donor (Glc(3)Man(9)GlcNAc(2)-PP-Dol) for N-linked glycosylation of proteins is assembled in a series of reactions that initiate on the cytoplasmic face of the rough endoplasmic reticulum and terminate within the lumen. The biochemical analysis of the oligosaccharyltransferase and the glycosyltransferases that mediate assembly of dolichol-linked oligosaccharides (OS-PP-Dol) has been hindered by the lack of structurally homogeneous substrate preparations. We have developed an improved method for the preparative-scale isolation of dolichol-linked oligosaccharides from vertebrate tissues and yeast cells. Preparations that were highly enriched in either Glc(3)Man(9)GlcNAc(2)-PP-Dol or Man(9)GlcNAc(2)-PP-Dol were obtained from porcine pancreas and a Man(5)GlcNAc(2)-PP-Dol preparation was obtained from an alg3 yeast culture. Chromatography of the OS-PP-Dol preparations on an aminopropyl silica column was used to obtain dolichol-linked oligosaccharides with defined structures. A single chromatography step could achieve near-baseline resolution of dolichol-linked oligosaccharides that differed by one sugar residue. A sensitive oligosaccharyltransferase endpoint assay was used to determine the concentration and composition of the OS-PP-Dol preparations. Typical yields of Glc(3)Man(9)GlcNAc(2)-PP-Dol, Man(9)GlcNAc(2)-PP-Dol, and Man(5)GlcNAc(2)-PP-Dol ranged between 5 and 15 nmol per chromatographic run. The homogeneity of these preparations ranged between 85 and 98% with respect to oligosaccharide composition. Purification of dolichol-linked oligosaccharides from cultures of alg mutant yeast strains provides a general method to obtain authentic OS-PP-Dol assembly intermediates of high purity. The analytical methods described here can be used to accurately evaluate the steady-state dolichol-linked oligosaccharide compositions of wild-type and mutant cell lines.     

Deficiency of GDP-Man:GlcNAc2-PP-dolichol mannosyltransferase causes congenital disorder of glycosylation type Ik

The molecular nature of a severe multisystemic disorder with a recurrent nonimmune hydrops fetalis was identified as deficiency of GDP-Man:GlcNAc(2)-PP-dolichol mannosyltransferase, the human orthologue of the yeast ALG1 gene (MIM 605907). The disease belongs to the group of congenital disorders of glycosylation (CDG) and is designated as subtype CDG-Ik. In patient-derived serum, the total amount of the glycoprotein transferrin was reduced. Moreover, a partial loss of N-glycan chains was observed, a characteristic feature of CDG type I forms. Metabolic labeling with [6-(3)H]glucosamine revealed an accumulation of GlcNAc(2)-PP-dolichol and GlcNAc(1)-PP-dolichol in skin fibroblasts of the patient. Incubation of fibroblast extracts with [(14)C]GlcNAc(2)-PP-dolichol and GDP-mannose indicated a severely reduced activity of the beta 1,4-mannosyltransferase, elongating GlcNAc(2)-PP-dolichol to Man(1)GlcNAc(2)-PP-dolichol at the cytosolic side of the endoplasmic reticulum. Genetic analysis of the patient's hALG1 gene identified a homozygous mutation leading to the exchange of a serine residue to leucine at position 258 in the hALG1 protein. The disease-causing nature of the hALG1 mutation for the glycosylation defect was verified by a retroviral complementation approach in patient-derived primary fibroblasts and was confirmed by the expression of wild-type and mutant hALG1 in the Saccharomyces cerevisiae alg1-1 strain.     

Control of carbohydrate processing: the lec1A CHO mutation results in partial loss of N-acetylglucosaminyltransferase I activity.

Lec1 CHO cell glycosylation mutants are defective in N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and therefore cannot convert the oligomannosyl intermediate (Man5GlcNAc2Asn) into complex carbohydrates. Lec1A CHO cell mutants have been shown to belong to the same genetic complementation group but exhibit different phenotypic properties. Evidence is presented that lec1A represents a new mutation at the lec1 locus resulting in partial loss of GlcNAc-TI activity. Structural studies of the carbohydrates associated with vesicular stomatitis virus grown in Lec1A cells (Lec1A/VSV) revealed the presence of biantennary and branched complex carbohydrates as well as the processing intermediate Man5GlcNAc2Asn. By contrast, the glycopeptides from virus grown in CHO cells (CHO/VSV) possessed only fully processed complex carbohydrates, whereas those from Lec1/VSV were almost solely of the Man5GlcNAc2Asn intermediate type. Therefore, the Lec1A glycosylation phenotype appears to result from the partial processing of N-linked carbohydrates because of reduced GlcNAc-TI action on membrane glycoproteins. Genetic experiments provided evidence that lec1A is a single mutation affecting GlcNAc-TI activity. Lec1A mutants could be isolated at frequencies of 10(-5) to 10(-6) from unmutagenized CHO cell populations by single-step selection, a rate inconsistent with two mutations. In addition, segregants selected from Lec1A X parental cell hybrid populations expressed only Lec1A or related lectin-resistant phenotypes and did not include any with a Lec1 phenotype. The Lec1A mutant should be of interest for studies on the mechanisms that control carbohydrate processing in animal cells and the effects of reduced GlcNAc-TI activity on the glycosylation, translocation, and compartmentalization of cellular glycoproteins.