Man8GlcNAc2糖基化的酿酒酵母菌株的构建

酿酒酵母糖蛋白的N-糖基化经过高尔基体的修饰后形成聚合度约150-200的甘露寡糖,高尔基体N-糖基化的糖基转移酶Mnn1p和Och1p在甘露寡糖的形成过程中起关键作用。通过同源重组置换敲除了酵母中的MNN1和OCH1基因阻断高尔基体N-糖基化修饰,分离纯化了mnn1 och1突变株中的N-糖蛋白,糖酰胺酶PNGaseF酶解释放的N-糖链经过2-氨基吡啶衍生后,利用HPLC和MALDITOF/MS结合的方法分析了突变株糖蛋白上的N-糖链。结果显示mnn1 och1突变株中的糖蛋白的N-糖链为结构单一的糖链,分子量为1794.66,推测为Man₈GlcNAc₂。     

Structure, position, and biosynthesis of the high mannose and the complex oligosaccharide side chains of the bean storage protein phaseolin

Phaseolin, the major storage protein of the common bean (Phaseolus vulgaris), is a glycoprotein which is synthesized during seed development and accumulates in protein storage vacuoles or protein bodies. The protein has three different N-linked oligosaccharide side chains: Man9(GlcNAc)2, Man7(GlcNAc)2, and Xyl-Man3(GlcNAc)2 (where Xyl represents xylose). The structures of these glycans were determined by 1H NMR spectroscopy. The Man9(GlcNAc)2 glycan has the typical structure found in plant and animal glycoproteins. The structures of the two other glycans are shown below. (Formula; see text) Phaseolin was separated by electrophoresis on denaturing gels into four size classes of polypeptides. The two abundant ones have two oligosaccharides each, whereas the less abundant ones have only one oligosaccharide each. Polypeptides with two glycans have Man7(GlcNAc)2 attached to Asn252 and Man9(GlcNAc)2 attached to Asn341. Polypeptides with only one glycan have Xyl-Man3(GlcNAc)2 attached to Asn252. Both these asparagine residues are in canonical glycosylation sites; the numbering starts with the N-terminal methionine of the signal peptide of phaseolin. The presence of the Man7(GlcNAc)2 and of Xyl-Man3(GlcNAc)2 at the same asparagine residue (position 252) of different polypeptides seems to be controlled by the glycosylation status of Asn341. When Asp341 is unoccupied, the glycan at Asn252 is complex. When Asn341 is occupied, the glycan at Asn252 is only modified to the extent that 2 mannosyl residues are removed. The processing of the glycans, after the removal of the glucose residues, involves enzymes in the Golgi apparatus as well as in the protein bodies. Formation of the Xyl-Man3(GlcNAc)2 glycan is a multistep process that involves the Golgi apparatus-mediated removal of 6 mannose residues and the addition of 2 N-acetylglucosamine residues and 1 xylose. The terminal N-acetylglucosamine residues are later removed in the protein bodies. The conversion of Man9(GlcNAc)2 to Man7(GlcNAc)2 is a late processing event which occurs in the protein bodies. Experiments in which [3H]glucosamine-labeled phaseolin obtained from the endoplasmic reticulum (i.e. precursor phaseolin) is incubated with jack bean alpha-mannosidase show that the high mannose glycan on Asn252, but not the one on Asn341, is susceptible to enzyme degradation. Incubation of [3H] glucosamine-labeled phaseolin obtained from the Golgi apparatus with jack bean beta-N-acetylglucosaminidase results in the removal of the terminal N-acetylglucosamine residues from the complex chain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assessment of N-glycan heterogeneity of cactus glycoproteins by one-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Artificial environmental conditions in tissue culture, such as elevated relative humidity and rich nutrient medium, can influence and modify tissue growth and induce spontaneous changes from characteristic organization pattern to unorganized callus. As succulent plants with crassulacean acid metabolism, cacti are particularly susceptible to this altered growth environment. Glycosylated proteins of Mammillaria gracillis tissues cultivated in vitro, separated by SDS-PAGE, were detected with Con A after the transfer of proteins onto the nitrocellulose membrane. The glycan components were further characterized by affinity blotting with different lectins (GNA, DSA, PNA, and RCA(120)). The results revealed significant differences in glycoprotein pattern among the investigated cactus tissues (shoot, callus, hyperhydric regenerant, and tumor). To test whether the N-glycosylation of the same protein can vary in different developmental stages of cactus tissue, the N-glycans were analyzed by MALDI-TOF MS after in-gel deglycosylation of the excised 38-kDa protein band. Paucimannosidic-type N-glycans were detected in oligosaccharide mixtures from shoot and callus, while the hyperhydric regenerant and tumor shared glycans of complex type. The hybrid oligosaccharide structures were found only in tumor tissue. These results indicate that the adaptation of plant cells to artificial environment in tissue culture is reflected in N-glycosylation, and structures of N-linked glycans vary with different developmental stages of Mammillaria gracillis tissues.     

Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig.

Competitive inhibition of sperm to explants of the oviductal epithelium was used to study the complementary receptor system that may be involved in the establishment of the oviductal sperm reservoir in the pig. Sperm binding to the oviductal explants is expressed as Binding Index (BI = sperm cells/0.01 mm(2)). From a set of glycoproteins with known oligosaccharide structures, only asialofetuin and ovalbumin showed inhibitory activity, indicating that ovalbumin may block high affinity binding sites (IC(50) congruent with 1.3 microM) and asialofetuin low affinity sites (IC(50) congruent with 18 microM) of the complementary receptor systems, whereas fetuin carrying terminal sialic acid has no effect. Ovalbumin glycopeptides were isolated by Con A affinity chromatography and reverse-phase HPLC following tryptic digestion. Glycopeptides and enzymatically released glycans were analyzed by MS, and were shown to represent preferentially the two high mannose type glycans (Man)(5)(GlcNAc)(2) and (Man)(6)(GlcNAc)(2), and as a minor component the hybrid type glycan (Hex)(4)(GlcNAc)(5). Glycopeptides (84% inhibition) and glycans (81% inhibition) significantly reduced sperm-oviduct binding at a concentration of 3 microM, whereas the deglycosylated peptides showed no inhibitory activity. Mannopentaose (IC(50) congruent with 0.8 microM) representing the oligomannose residue of the high mannose glycans of ovalbumin was as effective as ovalbumin. These data indicate that the carbohydrate-based mechanisms underlying the formation of the oviductal sperm reservoir in the pig is the result of the concerted action of at least the high-affinity binding sites for oligomannose or nonreducing terminal mannose residues and low-affinity binding of galactose.     

The expanding horizons of asparagine-linked glycosylation

Asparagine-linked glycosylation involves the sequential assembly of an oligosaccharide onto a polyisoprenyl donor, followed by the en bloc transfer of the glycan to particular asparagine residues within acceptor proteins. These N-linked glycans play a critical role in a wide variety of biological processes, such as protein folding, cellular targeting and motility, and the immune response. In the past decade, research in the field of N-linked glycosylation has achieved major advances, including the discovery of new carbohydrate modifications, the biochemical characterization of the enzymes involved in glycan assembly, and the determination of the biological impact of these glycans on target proteins. It is now firmly established that this enzyme-catalyzed modification occurs in all three domains of life. However, despite similarities in the overall logic of N-linked glycoprotein biosynthesis among the three kingdoms, the structures of the appended glycans are markedly different and thus influence the functions of elaborated proteins in various ways. Though nearly all eukaryotes produce the same nascent tetradecasaccharide (Glc(3)Man(9)GlcNAc(2)), heterogeneity is introduced into this glycan structure after it is transferred to the protein through a complex series of glycosyl trimming and addition steps. In contrast, bacteria and archaea display diversity within their N-linked glycan structures through the use of unique monosaccharide building blocks during the assembly process. In this review, recent progress toward gaining a deeper biochemical understanding of this modification across all three kingdoms will be summarized. In addition, a brief overview of the role of N-linked glycosylation in viruses will also be presented.     

Isolation of a mutant Arabidopsis plant that lacks N-acetyl glucosaminyl transferase I and is unable to synthesize Golgi-modified complex N-linked glycans.

The complex asparagine-linked glycans of plant glycoproteins, characterized by the presence of beta 1-->2 xylose and alpha 1-->3 fucose residues, are derived from typical mannose9(N-acetylglucosamine)2 (Man9GlcNAc2) N-linked glycans through the activity of a series of glycosidases and glycosyl transferases in the Golgi apparatus. By screening leaf extracts with an antiserum against complex glycans, we isolated a mutant of Arabidopsis thaliana that is blocked in the conversion of high-manne to complex glycans. In callus tissues derived from the mutant plants, all glycans bind to concanavalin A. These glycans can be released by treatment with endoglycosidase H, and the majority has the same size as Man5GlcNAc1 glycans. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, the mutant cells synthesize Man9GlcNAc2 and Man8GlcNAc2 glycans, suggesting that the biochemical lesion in the mutant is not in the biosynthesis of high-mannose glycans in the endoplasmic reticulum but in their modification in the Golgi. Direct enzyme assays of cell extracts show that the mutant cells lack N-acetyl glucosaminyl transferase I, the first enzyme in the pathway of complex glycan biosynthesis. The mutant plants are able to complete their development normally under several environmental conditions, suggesting that complex glycans are not essential for normal developmental processes. By crossing the complex-glycan-deficient strain of A. thaliana with a transgenic strain that expresses the glycoprotein phytohemagglutinin, we obtained a unique strain that synthesizes phytohemagglutinin with two high-mannose glycans, instead of one high-mannose and one complex glycan.     

Analysis of folate binding protein N-linked glycans by mass spectrometry

The folate binding protein (FBP), also known as the folate receptor (FR), is a glycoprotein which binds the vitamin folic acid and its analogues. FBP contains multiple N-glycosilation sites, is selectively expressed in tissues and body fluids, and mediates targeted therapies in cancer and inflammatory diseases. Much remains to be understood about the structure, composition, and the tissue specificities of N-glycans bound to FBP. Here, we performed structural characterization of N-linked glycans originating from bovine and human milk FBPs. The N-linked glycans were enzymatically released from FBPs, purified, and permethylated. Native and permethylated glycans were further analyzed by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS), while tandem MS (MS/MS) was used for their structural characterization. The assignment of putative glycan structures from MS and MS/MS data was achieved using Functional Glycomics glycan database and SimGlycan software, respectively. It was found that FBP from human milk contains putative structures that have composition consistent with high-mannose (Hex(5-6)HexNAc(2)) as well as hybrid and complex N-linked glycans (NeuAc(0-1)Fuc(0-3)Hex(3-6)HexNAc(3-5)). The FBP from bovine milk contains putative structures corresponding to high-mannose (Hex(4-9)HexNAc(2)) as well as hybrid and complex N-linked glycans (Hex(3-6)HexNAc(3-6)), but these glycans mostly do not contain fucose and sialic acid. Glycomic characterization of FBP provides valuable insight into the structure of this pharmacologically important glycoprotein and may have utility in tissue-selective drug targeting and as a biomarker.     

Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing.

The biosynthesis of the various types of N-linked oligosaccharide structures involves two series of reactions: 1) the formation of the lipid-linked saccharide precursor, Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol, by the stepwise addition of GlcNAc, mannose and glucose to dolichyl-P, and 2) the removal of glucose and mannose by membrane-bound glycosidases and the addition of GlcNAc, galactose, sialic acid, and fucose by Golgi-localized glycosyltransferases to produce different complex oligosaccharide structures. For most glycoproteins, the precise role of the carbohydrate is still not known, but specific N-linked oligosaccharide structures are key players in targeting of lysosomal hydrolases to the lysosomes, in the clearance of asialoglycoproteins from the serum, and in some cases of cell:cell adhesion. Furthermore, many glycoproteins have more than one N-linked oligosaccharide, and these oligosaccharides on the same protein frequently have different structures. Thus, one oligosaccharide may be of the high-mannose type whereas another may be a complex chain. One approach to determining the role of specific structures in glycoprotein function is to use inhibitors that block the modification reactions at different steps, causing the cell to produce glycoproteins with altered carbohydrate structures. The function of these glycoproteins can then be assessed. A number of alkaloid-like compounds have been identified that are specific inhibitors of the glucosidases and mannosidases involved in glycoprotein processing. These compounds cause the formation of glycoproteins with glucose-containing high mannose structures, or various high-mannose or hybrid chains, depending on the site of inhibition. These inhibitors have also been useful for studying the processing pathway and for comparing processing enzymes from different organisms.     

An engineered Saccharomyces cerevisiae strain binds the broadly neutralizing human immunodeficiency virus type 1 antibody 2G12 and elicits mannose-specific gp120-binding antibodies

The glycan shield of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein serves as a barrier to antibody-mediated neutralization and plays a critical role in transmission and infection. One of the few broadly neutralizing HIV-1 antibodies, 2G12, binds to a carbohydrate epitope consisting of an array of high-mannose glycans exposed on the surface of the gp120 subunit of the Env protein. To produce proteins with exclusively high-mannose carbohydrates, we generated a mutant strain of Saccharomyces cerevisiae by deleting three genes in the N-glycosylation pathway, Och1, Mnn1, and Mnn4. Glycan profiling revealed that N-glycans produced by this mutant were almost exclusively Man(8)GlcNAc(2), and four endogenous glycoproteins that were efficiently recognized by the 2G12 antibody were identified. These yeast proteins, like HIV-1 gp120, contain a large number and high density of N-linked glycans, with glycosidase digestion abrogating 2G12 cross-reactivity. Immunization of rabbits with whole Delta och1 Delta mnn1 Delta mnn4 yeast cells produced sera that recognized a broad range of HIV-1 and simian immunodeficiency virus (SIV) Env glycoproteins, despite no HIV/SIV-related proteins being used in the immunization procedure. Analyses of one of these sera on a glycan array showed strong binding to glycans with terminal Man alpha1,2Man residues, and binding to gp120 was abrogated by glycosidase removal of high-mannose glycans and terminal Man alpha1,2Man residues, similar to 2G12. Since S. cerevisiae is genetically pliable and can be grown easily and inexpensively, it will be possible to produce new immunogens that recapitulate the 2G12 epitope and may make the glycan shield of HIV Env a practical target for vaccine development.     

Differential release of high mannose structural isoforms by fungal and bacterial endo-β-N-acetylglucosaminidases

Endo-β-N-acetylglucosaminidases (ENGases) are widely used to remove N-linked oligosaccharides from glycoproteins for glycomic and proteomic studies and biopharmaceutical processes. Although several ENGases are widely available and their main oligosaccharide structural preferences are generally known (i.e. high mannose, hybrid or complex), the preferences of ENGases from different kingdoms for individual structural isoforms within the major classes of N-linked oligosaccharides have previously not been compared. In this work, a fungal ENGase (Endo Tv) was purified for the first time from a commercial Trichoderma viride chitinase mixture by sequential anion exchange and size exclusion chromatography, a commonly used strategy for purification of chitinases and endo enzymes. Oligosaccharides released from substrate glycoproteins by Endo Tv were identified and quantified by high pH anion exchange chromatography with pulsed amperometric detection and verified by mass spectrometric analysis. Unlike the widely-used bacterial ENGases, Endo H and Endo F1, Endo Tv released exclusively high mannose N-linked oligosaccharides from RNase B, ovalbumin, and yeast invertase. Endo Tv did not hydrolyze fucosylated, hybrid, complex type or bisecting N-acetylglucosamine-containing structures from bovine fetuin, ovalbumin and IgG. When compared to the bacterial ENGase, Endo H, the relative ratio of high-mannose oligosaccharide structural isoforms released from RNase B by Endo Tv was found to differ, with Endo Tv releasing more Man?GlcNAc and Man?GlcNAc isoform I and less Man(9)GlcNAc from RNase B. Based on these data, it is suggested that use of ENGases from multiple sources may serve to balance an introduced bias in quantitative analysis of released structural isoforms and may further prove valuable in biochemical structure-function studies.     

N-glycan structures of human transferrin produced by Lymantria dispar (gypsy moth) cells using the LdMNPV expression system

N-glycan structures of recombinant human serum transferrin (hTf) expressed by Lymantria dispar (gypsy moth) 652Y cells were determined. The gene encoding hTf was incorporated into a Lymantria dispar nucleopolyhedrovirus (LdMNPV) under the control of the polyhedrin promoter. This virus was then used to infect Ld652Y cells, and the recombinant protein was harvested at 120 h postinfection. N-glycans were released from the purified recombinant human serum transferrin and derivatized with 2-aminopyridine; the glycan structures were analyzed by a two-dimensional HPLC and MALDI-TOF MS. Structures of 11 glycans (88.8% of total N-glycans) were elucidated. The glycan analysis revealed that the most abundant glycans were Man1-3(+/-Fucalpha6)GlcNAc2 (75.5%) and GlcNAcMan3(+/-Fucalpha6)GlcNAc2 (7.4%). There was only approximately 6% of high-mannose type glycans identified. Nearly half (49.8%) of the total N-glycans contained alpha(1,6)-fucosylation on the Asn-linked GlcNAc residue. However alpha(1,3)-fucosylation on the same GlcNAc, often found in N-glycans produced by other insects and insect cells, was not detected. Inclusion of fetal bovine serum in culture media had little effect on the N-glycan structures of the recombinant human serum transferrin obtained.     

Elicitors and suppressors of the defense response in tomato cells. Purification and characterization of glycopeptide elicitors and glycan suppressors generated by enzymatic cleavage of yeast invertase.

Cleavage of yeast invertase by alpha-chymotrypsin produced a number of small glycopeptides that were highly active as elicitors of ethylene biosynthesis and phenylalanine ammonia-lyase in suspension-cultured tomato cells. Five of these elicitors were purified and their amino acid sequence determined. They all had sequences corresponding to known sequences of yeast invertase, and all contained an asparagine known to carry a N-linked small high mannose glycan. The most active glycopeptide elicitor induced ethylene biosynthesis and phenylalanine ammonia-lyase half-maximally at a concentration of 5-10 nM. Structure-activity relationships of the peptide part were analyzed by further cleavage of a defined glycopeptide elicitor with various proteolytic enzymes. Removal of the C-terminal phenylalanine enhanced the elicitor activity, whereas removal of N-terminal arginine impaired it. A glycopeptide with the peptide part trimmed to the dipeptide arginine-asparagine was still fully active as elicitor. Glycopeptides with identical amino acid sequences were further separated into fractions differing in the oligosaccharide side chain. A given peptide had high elicitor activity when carrying a glycan with 10-12 mannosyl residues (Man10-12GlcNAc2), a 3-fold lower activity when carrying Man9GlcNAc2 and a 100-fold lower activity when carrying Man8GlcNAc2. The oligosaccharides, released by endo-beta-N-acetylglucosaminidase H from the pure glycopeptide elicitors, acted as suppressors of elicitor-induced ethylene biosynthesis and phenylalanine ammonia-lyase activity. A series of such oligosaccharides in the size range of Man8-13GlcNAc was purified. The structure and composition of the purified oligosaccharides corresponded to the known small high mannose glycans of yeast invertase as verified by 1H NMR spectroscopy at 600 MHz. The highest suppressor activities were obtained with the oligosaccharides containing 10-12 mannosyl residues (Man10-12GlcNAc). The oligosaccharide Man8 GlcNAc was ineffective as a suppressor. Thus, the structural requirements for the free oligosaccharides to act as efficient suppressors were the same as for the oligosaccharide side chains of the glycopeptides for high elicitor activity. We propose that the glycan suppressors bind to the same recognition site as the glycopeptide elicitors without inducing a response.     

Structural determination of the N-glycans of a lepidopteran arylphorin reveals the presence of a monoglucosylated oligosaccharide in the storage protein

The structures of the oligosaccharides attached to arylphorin from Chinese oak silkworm, Antheraea pernyi, have been determined. Arylphorin, a storage protein present in fifth larval hemolymph, contained 4.8% (w/w) of carbohydrate that was composed of Fuc:GlcNAc:Glc:Man=0.2:4.0:1.4:13.6 moles per mole protein. Four moles of GlcNAc in oligomannose-type oligosaccharides strongly suggest that the protein contains two N-glycosylation sites. Normal-phase HPLC and mass spectrometry oligosaccharide profiles confirmed that arylphorin contained mainly oligomannose-type glycans as well as truncated mannose-type structures with or without fucosylation. Interestingly, the most abundant oligosaccharide was monoglucosylated Man9-GlcNAc2, which was characterized by normal-phase HPLC, mass spectrometry, Aspergillus saitoi alpha-mannosidase digestion, and 1H 600 MHz NMR spectrometry. This glycan structure is not normally present in secreted mammalian glycoproteins; however, it has been identified in avian species. The Glc1Man9GlcNAc2 structure was present only in arylphorin, whereas other hemolymph proteins contained only oligomannose and truncated oligosaccharides. The oligosaccharide was also detected in the arylphorin of another silkworm, Bombyx mori, suggesting a specific function for the Glc1Man9GlcNAc2 glycan. There were no processed glucosylated oligosaccharides such as Glc1Man5-8GlcNAc2. Furthermore, Glc1Man9GlcNAc2 was not released from arylophorin by PNGase F under nondenaturing conditions, suggesting that the N-glycosidic linkage to Asn is protected by the protein. Glc1Man9GlcNAc2 may play a role in the folding of arylphorin or in the assembly of hexamers.     

Structural analysis of the N-glycans of the major cysteine proteinase of Trypanosoma cruzi. Identification of sulfated high-mannose type oligosaccharides

Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine proteinase, cruzipain. This lysosomal enzyme bears an unusual C-terminal extension that contains a number of post-translational modifications, and most antibodies in natural and experimental infections are directed against it. In this report we took advantage of UV-MALDI-TOF mass spectrometry in conjunction with peptide N-glycosidase F deglycosylation and high performance anion exchange chromatography analysis to address the structure of the N-linked oligosaccharides present in this domain. The UV-MALDI-TOF MS analysis in the negative-ion mode, using nor-harmane as matrix, allowed us to determine a new striking feature in cruzipain: sulfated high-mannose type oligosaccharides. Sulfated GlcNAc2Man3 to GlcNAc2Man9 species were identified. In accordance, after chemical or enzymatic desulfation, the corresponding signals disappeared. In addition, by UV-MALDI-TOF MS analysis (a) a main population of high-mannose type oligosaccharides was shown in the positive-ion mode, (b) lactosaminic glycans were also identified, among them, structures corresponding to monosialylated species were detected, and (c) as an interesting fact a fucosylated oligosaccharide was also detected. The presence of the deoxy sugar was further confirmed by high performance anion exchange chromatography. In conclusion, the total number of oligosaccharides occurring in cruzipain was shown to be much higher than previous estimates. This constitutes the first report on the presence of sulfated glycoproteins in Trypanosomatids.     

Structures of N-linked oligosaccharides of glycoproteins from tobacco BY2 suspension cultured cells

The structures of N-linked sugar chains of glycoproteins expressed in tobacco BY2 cultured cells are reported. Five pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the glycoproteins by reversed-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were identified by two-dimensional PA-sugar chain mapping, ion-spray MS/MS analysis, and exoglycosidase digestions. The five structures fell into two categories; the major class (92.5% as molar ratio) was a xylose containing-type (Man3Fuc1 Xyl1GlcNAc2 (41.0%), GlcNAc2Man3Fuc1Xyl1GlcNAc2 (26.5%), GlcNAc1Man3Fuc1Xyl1GlcNAc2 (21.7%), Man3 Xyl1GlcNAc2 (3.3%)), and the minor class was a high-mannose type (Man5GlcNAc2 (7.5%)). This is the first report to show that alpha(1-->3) fucosylation of N-glycans does occur but beta(1-->4) galactosylation of the sugar chains does not in the tobacco cultured cells.     

Characterization of N-linked oligosaccharides in chorion peroxidase of Aedes aegypti mosquito

A peroxidase is present in the chorion of Aedes aegypti eggs and catalyzes chorion protein cross-linking during chorion hardening, which is critical for egg survival in the environment. The unique chorion peroxidase (CPO) is a glycoprotein. This study deals with the N-glycosylation site, structures, and profile of CPO-associated oligosaccharides using mass spectrometric techniques and enzymatic digestion. CPO was isolated from chorion by solubilization and several chromatographic methods. Mono-saccharide composition was analyzed by HPLC with fluorescent detection. Our data revealed that carbohydrate (D-mannose, N-acetyl D-glucosamine, D-arabinose, N-acetyl D-galactosamine, and L-fucose) accounted for 2.24% of the CPO molecular weight. A single N-glycosylation site (Asn328-Cys- Thr) was identified by tryptic peptide mapping and de novo sequencing of native and PNGase A-deglycosylated CPO using matrix-assisted laser/desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The Asn328 was proven to be a major fully glycosylated site. Potential tryptic glycopeptides and profile were first assessed by MALDI/TOF/MS and then by precursor ion scanning during LC/MS/MS. The structures of N-linked oligosaccharides were elucidated from the MS/MS spectra of glycopeptides and exoglycosidase sequencing of PNGase A-released oligosaccharides. These CPO-associated oligosaccharides had dominant Man3GlcNAc2 and Man3 (Fuc) GlcNAc2 and high mannose-type structures (Man(4-8)GlcNAc2). The truncated structures, Man2GlcNAc2 and Man2 (Fuc) GlcNAc2, were also identified. Comparison of CPO activity and Stokes radius between native and deglycosylated CPO suggests that the N-linked oligosaccharides influence the enzyme activity by stabilizing its folded state.     

Unique N-glycan moieties of the 66-kDa cell wall glycoprotein from the red microalga Porphyridium sp

We report here the structural determination of the N-linked glycans in the 66-kDa glycoprotein, part of the unique sulfated complex cell wall polysaccharide of the red microalga Porphyridium sp. Structures were elucidated by a combination of normal phase/reverse phase HPLC, positive ion MALDI-TOF MS, negative ion electrospray ionization, and MS/MS. The sugar moieties of the glycoprotein consisted of at least four fractions of N-linked glycans, each composed of the same four monosaccharides, GlcNAc, Man, 6-O-MeMan, and Xyl, with compositions Man(8-9)Xyl(1-2)Me(3)GlcNAc(2). The present study is the first report of N-glycans with the terminal Xyl attached to the 6-mannose branch of the 6-antenna and to the 3-oxygen of the penultimate (core) GlcNAc. Another novel finding was that all four glycans contain three O-methylmannose residues in positions that have never been reported before. Although it is known that some lower organisms are able to methylate terminal monosaccharides in glycans, the present study on Porphyridium sp. is the first describing an organism that is able to methylate non-terminal mannose residues. This study will thus contribute to understanding of N-glycosylation in algae and might shed light on the evolutionary development from prokaryotes to multicellular organisms. It also may contribute to our understanding of the red algae polysaccharide formation. The additional importance of this research lies in its potential for biotechnological applications, especially in evaluating the use of microalgae as cell factories for the production of therapeutic proteins.     

Why two are not enough: degradation of p-toluenesulfonate by a bacterial community from a pristine site in Moorea, French Polynesia

It has been demonstrated previously that Enterococcus faecalis produces secreted endoglycosidases that enable the bacteria to remove N-linked glycans from glycoproteins. One enzyme potentially responsible for this activity is EF0114, comprising a typical GH18 endoglycosidase domain and a GH20 domain. We have analyzed the other candidate, EF2863, and show that this predicted single domain GH18 protein is an endo-β-N-acetylglucosaminidase. EF2863 hydrolyzes the glycosidic bond between two N-acetylglucosamines (GlcNAc) in N-linked glycans of the high-mannose and hybrid type, releasing the glycan and leaving one GlcNAc attached to the protein. The activity of EF2863 is similar to that of the well known deglycosylating enzyme EndoH from Streptomyces plicatus. According to the CAZy nomenclature, the enzyme is designated EfEndo18A.     

Site-specific glycosylation analysis of the bovine lysosomal alpha-mannosidase

Lysosomal alpha-mannosidase is a broad specificity exoglycosidase involved in the ordered degradation of glycoproteins. The bovine enzyme is used as an important model for understanding the inborn lysosomal storage disorder alpha-mannosidosis. This enzyme of about 1,000 amino acids consists of five peptide chains, namely a- to e-peptides and contains eight N-glycosylation sites. The N(497) glycosylation site of the c-peptide chain is evolutionary conserved among LAMANs and is very important for the maintenance of the lysosomal stability of the enzyme. In this work, relying on an approach based on mass spectrometric techniques in combination with exoglycosidase digestions and chemical derivatizations, we will report the detailed structures of the N-glycans and their distribution within six of the eight N-glycosylation sites of the bovine glycoprotein. The analysis of the PNGase F-released glycans from the bovine LAMAN revealed that the major structures fall into three classes, namely high-mannose-type (Fuc(0-1)Glc(0-1)Man(4-9)GlcNAc(2)), hybrid-type (Gal(0-1)Man(4-5)GlcNAc(4)), and complex-type (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(3-5)) N-glycans, with core fucosylation and bisecting GlcNAc. To investigate the exact structure of the N-glycans at each glycosylation site, the peptide chains of the bovine LAMAN were separated using SDS-PAGE and in-gel deglycosylation. These experiments revealed that the N(497) and N(930) sites, from the c- and e-peptides, contain only high-mannose-type glycans Glc(0-1)Man(5-9)GlcNAc(2), including the evolutionary conserved Glc(1)Man(9)GlcNAc(2) glycan, and Fuc(0-1)Man(3-5)GlcNAc(2), respectively. Therefore, to determine the microheterogeneity within the remaining glycosylation sites, the glycoprotein was reduced, carboxymethylated, and digested with trypsin. The tryptic fragments were then subjected to concanavalin A (Con A) affinity chromatography, and the material bound by Con A-Sepharose was purified using reverse-phase high-performance liquid chromatography (HPLC). The tandem mass spectrometry (ESI-MS/MS) and the MALDI analysis of the PNGase F-digested glycopeptides indicated that (1) N(692) and N(766) sites from the d-peptide chain both bear glycans consisting of high-mannose (Fuc(0-1)Man(3-7)GlcNAc(2)), hybrid (Fuc(0-1) Gal(0-1)Man(4-5)GlcNAc(4)), and complex (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(4-5)) structures; and (2) the N(367) site, from the b-peptide chain, is glycosylated only with high-mannose structures (Fuc(0-1)Man(3-5)GlcNAc(2)). Taking into consideration the data obtained from the analysis of either the in-gel-released glycans from the abc- and c-peptides or the tryptic glycopeptide containing the N(367) site, the N(133) site, from the a-peptide, was shown to be glycosylated with truncated and high-mannose-type (Fuc(0-1)Man(4-5)GlcNAc(2)), complex-type (Fuc(0-1)Gal(0-1)Man(3)GlcNAc(5)), and hybrid-type (Fuc(0-1)Gal(0-1)Man(5)GlcNAc(4)) glycans.