High Mannose-type Structures Research Articles

Phosphorylcholine-containing N-glycans of Trichinella spiralis: identification of multiantennary lacdiNAc structures.

Although the presence of phosphorylcholine (PC) in Trichinella spiralis is well established, the precise structure of the PC-bearing molecules is not known. In this paper, we report structural studies of N-glycans released from T.spiralis affinity-purified antigens by peptide N-glycosidase F. Three classes of N-glycan structures were observed: high mannose type structures; those which had been fully trimmed to the trimannosyl core and were sub-stoichiometrically fucosylated; and those with a trimannosyl core, with and without core fucosylation, carrying between one and eight N-acetylhexosamine residues. Of the three classes of glycans, only the last was found to be substituted with detectable levels of phosphorylcholine.

Mass spectrometry study of ecto-5'-nucleotidase from bull seminal plasma.

The structure of ecto-5'-nucleotidase from bull seminal plasma, containing a glycosyl-phosphatidylinositol anchor, was studied using mass spectrometry. MALDI-MS analysis of intact protein indicated a mass of 65 568.2 Da for the monomeric form, and it also showed a heterogeneous population of glycoforms with the glycosidic moiety accounting for approximately 6000 Da. MALDI-MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites of N-glycosylation. GC-MS analysis provided information on the glycosidic structures linked to the four asparagines. Asn53, Asn311 and Asn333 were linked to high-mannose saccharide chains, whereas the glycan chains linked to Asn403 contained a heterogeneous mixture of oligosaccharides, the high-mannose type structure being the most abundant and hybrid or complex type glycans being minor components. By combining enzymatic and/or chemical hydrolysis with GC-MS analysis, detailed characterization of the glycosyl-phpsphatidylinositol anchor was obtained. MALDI spectral analysis indicated that the glycosyl-phosphatidylinositol core contained EtN(P)Man3GlcNH2-myo-inositol(P)-glycerol, principally modified by stearoyl and palmitoyl residues or by stearoyl and myristoyl residues to a minor extent. Moreover, 1-palmitoylglycerol and 1-stearoylglycerol outweighed 2-palmitoylglycerol and 2-stearoylglycerol. The combination of chemical and enzymatic digestions of the protein with the mass spectral analysis yielded a complete pattern of S-S bridges. The protein does not contain free thiols and its eight cysteines are linked by intramolecular disulfide bonds, the pairs being: Cys51-Cys57, Cys353-Cys358, Cys365-Cys387 and Cys476-Cys479. This work resolves details of the structure of ecto-5'-nucleotidase, with particular regard to the localization and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the glycosyl-phosphatidylinositol anchor.

Free N-glycans already occur at an early stage of seed development.

As a part of our studies to elucidate the physiological significance of free N-glycans in differentiating or growing plant cells, we first demonstrate that two kinds of free N-glycans already occur at an early stage of seed development. In this report, we used the developing Ginkgo biloba seeds as a model plant, since we have already revealed a functional feature of the Ginkgo endo-beta-N-acetylglucosaminidase and structural features of N-glycans linked to storage glycoproteins in the developing seeds [Kimura, Y. et al. (1998) Biosci. Biotechnol. Biochem. 62, 253-261; Kimura, Y. and Matsuo, S. (2000) Biosci. Biotechnol. Biochem. 64, 562-568]. The structures of free N-glycans, which were determined by a combination of ESI-MS, sequential a-mannosidase digestions, partial acetolysis, and two dimensional sugar chain map, fell into two categories. One dominant species is a high-mannose type structure having one GlcNAc residue at the reducing end (Man(9-5)GlcNAc(1)). The concentration of this type of free glycan (as the pyridylaminated derivatives) is about 2.2 nmol in 1 g fresh weight. The detailed structural analysis revealed that the high-mannose type structures have a common core unit; Manalpha1-6(Man1-3)Manalpha1-6(Manalpha1-3)Ma nbeta1-4GlcNAc. The other minor species of free N-glycans is the plant complex type structure having an N-acetylchitobiose unit at the reducing end (Man(3)Xyl(1)Fuc(1)GlcNAc(2)). The concentration of this type of free glycan (as the pyridylaminated derivative) was about 75 pmol in 1 g fresh weight.

Changes in N-linked oligosaccharides during seed development of Ginkgo biloba.

Structural changes in N-linked oligosaccharides of glycoproteins during seed development of Ginkgo biloba have been explored to discover possible endogenous substrate(s) for the Ginko endo-beta-N-acetylglucosaminidase (endo-GB; Kimura, Y., et al. (1998) Biosci. Biotechnol. Biochem., 62, 253-261), which should be involved in the production of high-mannose type free N-glycans. The structural analysis of the pyridylaminated oligosaccharides with a 2D sugar chain map, by ESI-MS/MS spectroscopy, showed that all N-glycans expressed on glycoproteins through the developmental stage of the Ginkgo seeds have the xylose-containing type (GlcNAc2 approximately 0Man3Xyl1Fuc1 approximately 0GlcNAc2) but no high-mannose type structure. Man3Xyl1Fuc1GlcNAc2, a typical plant complex type structure especially found in vacuolar glycoproteins, was a dominant structure through the seed development, while the amount of expression of GlcNAc2Man3Xyl1Fuc1GlcNAc2 and GlcNAc1Man3Xyl1Fuc1GlcNAc2 decreased as the seeds developed. The dominantly occurrence of xylose-containing type structures and the absence of the high-mannose type structures on Ginkgo glycoproteins were also shown by lectin-blotting and immunoblotting of SDS-soluble glycoproteins extracted from the developing seeds at various developmental stages. Concerning the endogenous substrates for plant endo-beta-N-acetylglucosaminidase, these results suggested that the endogenous substrates might be the dolicol-oligosaccharide intermediates or some glycopeptides with the high-mannose type N-glycan(s) derived from misfolded glycoproteins in the quality control system for newly synthesized glycoproteins.

Structural analysis of free N-glycans occurring in soybean seedlings and dry seeds.

The structures of unconjugated or free N-glycans in stems of soybean seedlings and dry seeds have been identified. The free N-glycans were extracted from the stems of seedlings or defatted dry seeds. After desalting by two kinds of ion-exchange chromatography and a gel filtration, the free N-glycans were coupled with 2-aminopyridine. The resulting fluorescence-labeled (PA-) N-glycans were purified by gel filtration, Con A affinity chromatography, reverse-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains purified were analyzed by the combination of two-dimensional sugar chain mapping, jack bean alpha-mannosidase digestion, alpha-1,2-mannosidase digestions, partial acetolysis, and ESI-MS/MS. The free N-glycan structures found showed that two categories of free N-glycans occur in the stems of soybean seedlings. One is a high-mannose type structure having one GlcNAc residue at the reducing end (Man 9 approximately 5 GlcNAc1, 93%), that would be derived by endo-GM (Kimura, Y. et al., Biochim. Biophys. Acta, 1381, 27-36 (1998)). The other small component is a xylose-containing type one having two GlcNAc residues at the reducing end (Man3Xyl1GlcNAc2, 7%), which would be derived by PNGase-GM (Kimura, Y. and Ohno, A., Biosci. Biotechnol. Biochem., 62, 412-418 (1998)). The detailed structural analysis of free glycans showed that high-mannose type free N-glycans (Man 9 approximately 5 GlcNAc1) in the soybean seedlings have a common core structural unit; Manalpha1-6(Man1-3)Manalpha1-6(Manalpha1-3)Ma nbeta1-4GlcNAc. Comparing the amount of free N-glycans in the seedling stems and dry seeds, the amount in the stems of seedlings was much higher than that in the dry seeds; approximately 700 pmol per one stem, 8 pmol in one dry seed. This fact suggested that free N-glycans in soybean seedlings could be produced by two kinds of N-glycan releasing enzymes during germination or seedling-development.

Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans.

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry. The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing beta1-2 xylosyl and/or alpha1-3 fucosyl residue(s) and occurrence of beta1-4GlcNAc residue in the insect glycoproteins. The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man(9 to approximately 4)GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3 GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1 Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3 Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of beta-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a beta1-4 linkage N-acetylglucosaminyl residue.

Glycosylation and proteolytic processing of 70 kDa C-terminal recombinant polypeptides of Plasmodium falciparum merozoite surface protein 1 expressed in mammalian cells.

The cDNAs that encode the 70 kDa C-terminal portion of Plasmodium falciparum merozoite surface protein 1 (MSP-1), with or without an N-terminal signal peptide sequence and C-terminal glycosylphosphatidylinositol (GPI) signal sequence of MSP-1, were expressed in mammalian cell lines via recombinant vaccinia virus. The polypeptides were studied with respect to the nature of glycosylation, localization, and proteolytic processing. The polypeptides derived from the cDNAs that contained the N-terminal signal peptide were modified with N -linked high mannose type structures and low levels of O -linked oligosaccharides, whereas the polypeptides from the cDNAs that lacked the signal peptide were not glycosylated. The GPI anchor moiety is either absent or present at a very low level in the polypeptide expressed from the cDNA that contained both the signal peptide and GPI signal sequences. Together, these data establish that whereas the signal peptide of MSP-1 is functional, the GPI anchor signal is either nonfunctional or poorly functional in mammalian cells. The polypeptides expressed from the cDNAs that contained the signal peptide were proteolytically cleaved at their C-termini, whereas the polypeptides expressed from the cDNAs that lacked the signal peptide were uncleaved. While the polypeptide expressed from the cDNA containing both the signal peptide and GPI anchor signal was truncated by approximately 14 kDa at the C-terminus, the polypeptide derived from the cDNA with only the signal peptide was processed to remove approximately 6 kDa, also from the C-terminus. Furthermore, the polypeptides derived from cDNAs that lacked the signal peptide were exclusively localized intra-cellularly, the polypeptides from cDNAs that contained the signal peptide were predominantly intracellular, with low levels on the cell surface; none of the polypeptides was secreted into the culture medium to a detectable level. These results suggest that N -glycosylation alone is not sufficient for the efficient extracellular transport of the recombinant MSP-1 polypeptides through the secretory pathway in mammalian cells.

Simplified procedure for fractionation and structural characterisation of complex mixtures of N-linked glycans, released from HIV-1 gp120 and other highly glycosylated viral proteins.

HIV-1 gp120 is heavily glycosylated containing 24 N-glycosylation sites, and this makes elucidation of the significance of glycans at individual glycosylation sites a difficult task. A procedure is described where a complex mixture of biologically radiolabelled glycans of gp120, derived from a relatively small number of virus-infected cells may be characterized by a combination of N-glycanase release, single lectin separation, and normal phase HPLC (NP-HPLC). The method was applied in analysis of three N-linked glycosylation sites essential for the in vivo priming of T-cells, specific for an epitope in their vicinity (Sjölander, S., Bolmstedt, A., Åkerblom, 1996. Virology 215, 124–133.). The carbohydrate compositions of wild type gp120 and of mutant variants gp120 lacking one, two, or all of these three active N-linked glycans were analysed. Cells were infected with r-vaccinia virus expressing wild-type gp120 or mutated gp120, or were infected with HIV-1 BRU (wild type) or mutant virus variants. HIV-1 glycoproteins were purified by immunosorbent affinity chromatography and released glycans were separated on lectins, then analysed with NP-HPLC. Our data showed that the structural composition of glycans occupying two of the three glycosylation sites was heterogeneous but the site located adjacent to the T-cell epitope was equipped with one large, high mannose-type structure (>11 units) with the capacity to cover a substantial part of the gp120 surface.

Human Thyroperoxidase in Its Alternatively Spliced Form (TPO2) Is Enzymatically Inactive and Exhibits Changes in Intracellular Processing and Trafficking

Thyroid peroxidase (TPO1) is a membrane-bound heme-containing glycoprotein that catalyzes the synthesis of thyroid hormones. We generated stable cell lines expressing TPO1 and the alternatively spliced isoform TPO2. Pulse-chase studies showed that TPO2 half-life was dramatically decreased as compared with TPO1. The sensitivity of TPO2 to endo-beta-N-acetylglucosaminidase H indicated that the protein is processed through the endoplasmic reticulum and bears high mannose-type structures. Cell surface biotinylation experiments showed that the two isoforms also differ in their intracellular trafficking. TPO2 was totally retained in the cell, whereas 15% of TPO1 reached the cell surface. The inability of TPO2 to come out of the intracellular compartments was related to structural changes in the molecule. Evidence of these changes was obtained through the lack of recognition of TPO2 by half of the 13 TPO monoclonal antibodies tested in immunoprecipitation experiments. Our data suggest that because of an improper folding, TPO2 is trapped in the endoplasmic reticulum and rapidly degraded. The failure of incorporation of [14C]aminolevulinic acid in the cultured cells showed that TPO2 did not bind to heme, whereas TPO1 did. This result was confirmed through a guaiacol assay showing that TPO2 is enzymatically inactive.

Characterisation of the phosphorylcholine-containing N-linked oligosaccharides in the excretory-secretory 62 kDa glycoprotein of Acanthocheilonemaviteae

The major excretory-secretory product of the rodent filarial nematode Acanthocheilonema viteae is a 62 kDa glycoprotein (ES-62), which has phosphorylcholine, attached to the N-linked carbohydrates. In this paper, we describe structural studies of N-glycans released from ES-62 by peptide N-glycosidase F. Three major classes of N-glycan structures were observed: high mannose type structures; those which had been fully trimmed to the trimannosyl core and were sub-stoichiometrically fucosylated; and those with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine residues. Of the three classes of glycans, only the last was found to be substituted with detectable levels of phosphorylcholine. The implications of these results with respect to the probable glycosylation pathways operating in A. viteae are discussed.

Dityrosine bridge formation and thyroid hormone synthesis are tightly linked and are both dependent on N-glycans

Formation of dityrosine bridges is a ubiquitous process mainly attributed to oxidative stress leading to protein degradation and cellular damages. Here we show that dityrosine formation is involved in a physiological process, thyroid hormone synthesis, and is strictly dependent on structural characteristics, namely N-glycans, presented by the protein acting as the prothyroid hormone. We used two isoforms of the N-terminal thyroid hormone forming domain (NTD) of human thyroglobulin: one without N-glycan (19 kDa isoform) and the other with high mannose type structures (25 kDa isoform). Both isoforms were able to form iodotyrosines after in vitro iodination. However, iodotyrosine coupling to form thyroxine did not occur with the unglycosylated 19 kDa NTD. In contrast, the 25 kDa isoform formed thyroxine. Strikingly, thyroxine synthesis was accompanied by dimerization of the 25 kDa isoform and formation of a dityrosine bridge; none of this was observed with the 19 kDa isoform. Taken as a whole, our results indicate that dimerization through dityrosine bridging accompanies and could have a role in thyroid hormone synthesis.

Analysis of glycosylation of prostate-specific membrane antigen derived from LNCaP cells, prostatic carcinoma tumors, and serum from prostate cancer patients

Prostate-specific membrane antigen (PSMA) has been detected in human prostatic cancer tissues, serum, and seminal fluid based on Western blot data with the monoclonal antibody 7E11.C5. The reactive protein is very similar in size to that from human prostatic carcinoma LNCaP cells and corresponds to a protein with a molecular size of about 110,000 daltons. Given that PSMA is known to be a 750 amino acid protein of about 84,000 daltons, a substantial portion, perhaps 20-25% of the native molecular weight, is composed of carbohydrates. In this study, we have begun initial analyses of the glycosylation of the PSMA protein from multiple sources using a variety of exo- and endoglycosidase treatments. The results indicate that the carbohydrate is primarily N-linked and in each case the deglycosylated protein has an apparent molecular weight of about 86,000 daltons. The glycan present on in vivo-derived PSMA from tumor tissue or serum was found to be primarily N-linked complex type. A small amount of O-linked glycan also appears to be present. In contrast, only high mannose-type N-linked glycans are present on the PSMA from LNCaP cells. Oligosaccharides present on PSMA derived from both tissue culture LNCaP cells and in vivo specimens are primarily N-linked and comprise about 20-25% of the native molecular weight. N-linked glycans of PSMA derived from in vivo sources were found to be complex type, lacking polylactosamine structures. In contrast, LNCaP cells express only high mannose-type structures. These results will be useful in our ongoing efforts to develop monoclonal antibodies which are specific for protein epitopes present in the extracellular domain of the protein.

N-linked sugar chain of 55-kDa royal jelly glycoprotein.

An N-linked sugar chain from 55-kDa royal jelly glycoprotein (RJGP), which maintains the high viability of rat liver primary cultured cell and is a different molecular species from 350-kDa RJGP [Kimura et al., Biosci. Biotech. Biochem., 59, 507-509 (1995)], has been identified. The sugar chains were released by hydrazinolysis followed by N-acetylation and pyridylamination. The structural analysis of the pyridylaminated sugar chain was done by a combination of sequential exo-mannosidase digestions, MALDI-TOF MS, and 500 MHz 1H-NMR. For the carbohydrate moiety of 55-kDa RJGP, only one N-linked sugar chain has been detected. The structure has been found to be Man alpha 1-->2Man alpha 1-->6(Man alpha 1--> 2Man alpha 1-->3)Man alpha 1-->6(Man alpha 1-->2Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc, which is a non-processed high mannose type structure.

N-Glycans Modulate in Vivo and in Vitro Thyroid Hormone Synthesis: STUDY AT THE N-TERMINAL DOMAIN OF THYROGLOBULIN

Thyroglobulin (Tg) is the substrate for thyroid hormone biosynthesis, which requires tyrosine iodination and iodotyrosine coupling and occurs at the apical membrane of the thyrocytes. Tg glycoconjugates have been shown to play a major role in Tg routing through cellular compartments and recycling after endocytosis. Here we show that glycoconjugates also play a direct role in hormonosynthesis. The N-terminal domain (NTD; Asn1-Met171) of human Tg, which bears the preferential hormonogenic site, brings two N-glycans (Asn57 and Asn91). NTD preparations were purified from Tg with low and mild iodine content in vivo and from poorly iodinated Tg after in vitro iodination and coupling. NTD separated from poorly iodinated Tg was also submitted to iodination and coupling after desialylation and deglycosylation. The various NTD isoforms were analyzed for their N-glycan structures and hormone contents. Our results show that 1) in vivo as well as in vitro unglycosylated isoforms did not synthesize hormones, whereas fully or partially (at Asn91) glycosylated isoforms did; 2) high mannose type structures enhanced the hormone content; and 3) desialylation did not affect in vitro hormone synthesis. Evidence of a direct involvement in hormonosynthesis adds to the role of N-glycans in Tg function and opens the way to new mechanisms for regulation (e.g. TSH modulation of N-glycan) or alteration (e.g. Asn91 mutation) of thyroid hormone synthesis.

Identification and structural characterization of a mannose-6-phosphate containing oligomannosidic N-glycan from human erythropoietin secreted by recombinant BHK-21 cells

A sialidase resistant mono-charged N-glycan was isolated from glycosylation site I (Asn-24) of recombinant human eruthropoietin expressed from baby hamster kidney (BHK-21) cells and constituted approximately 2–4% of the oligosaccharide material at this glycosylation site. Mass spectrometry and both 1- and 2-dimensional NMR techniques revealed a high mannose type structure (Man 6) with a phospho-diesterbridged N-acetylglucosamine as follows: ▪

The asparagine-linked carbohydrate of honeybee venom hyaluronidase

Hyaluronidase from the venom of the honeybee (Apis mellifera) has been purified by gelpermeation and cation exchange chromatography. Its asparagine-linked carbohydrate chains were released from tryptic glycopeptides with N-glycosidase A and reductively aminated with 2-aminopyridine. Separation of the fluorescent derivatives by size-fractionation and reversed-phase HPLC afforded eighteen fractions which were analysed by two-dimensional HPLC mapping combined with exoglycosidase digestions. The bulk of the N-linked glycans of hyaluronidase consisted of small oligosaccharides (Man1-3GlcNAc2), most of which were either alpha 1,3-monofucosylated or alpha 1,3-(alpha 1,6-)difucosylated at the innermost GlcNAc residue. High-mannose type structures constituted the minor fractions, together making up about 5% of the oligosaccharide pool from hyaluronidase. Four fractions, making up 8% of the N-linked glycans, contained the terminal trisaccharide GalNAc beta 1-4[Fuc alpha 1-3]GlcNAc beta 1- in beta 1,2-linkage to the core alpha 1,3-mannosyl residue. No evidence for the presence of O-glycans or sialic acids could be found.

Factor X-activating glycoprotein of Russell's viper venom. Polypeptide composition and characterization of the carbohydrate moieties.

There is contradictory information regarding the molecular weight and polypeptide chain composition of RVV-X, a glycoprotein in Russell's viper venom that is capable of activating factor X to Xa. We show that RVV-X is a 92,880-Da glycoprotein. It consists of three disulfide-linked polypeptide chains, one heavy chain (alpha-chain, M(r) 57,600) and two light chains (beta- and gamma-chains, M(r) 19,400 and 16,400, respectively). The two light chains seen on SDS-polyacrylamide gel electrophoresis under reducing conditions are two distinct components of the molecule, rather than a heterogeneous mixture of a single chain as previously reported (Takeya, H., Nishida, S., Miyata, T., Kawada, S., Saisaka, Y., Morita, T., and Iwanaga, S. (1992) J. Biol. Chem. 267, 14109-14117). The following evidence supports this conclusion. (i) The two light chains of RVV-X are present in equal proportion. (ii) The estimated molecular weight of an alpha 1 beta 1 gamma 1-structure closely matches the molecular weight determined by matrix-assisted laser desorption mass spectrometry. (iii) The amino acid compositions and NH2-terminal sequences of the beta- and gamma-chains are different. (iv) Although both the beta- and gamma-chains contain one N-linked oligosaccharide chain each, they are glycosylated differentially. RVV-X contains six N-linked oligosaccharides, four in the alpha-chain and one in each of the beta- and gamma-chains. The carbohydrate structures are different from those known for other snake venom glycoproteins, and they resemble closely those in various mammalian glycoproteins. The majority of the oligosaccharides are complex bi-, tri-, and tetraantennary structures, with a small proportion of multiantennary and high mannose-type structures. Two notable structural features of RVV-X oligosaccharides are as follows. (i) Sialic acid residues are linked to beta-galactosyl residues solely by alpha 2,3-linkages, and (ii) bisecting N-acetylglucosamine residues are present in the majority of the oligosaccharides.

The number of oligosaccharides borne by porcine thyroglobulin is variable.

Purified porcine thyroglobulin (Tg) was fractionated on a concanavalin A-Sepharose 4B column by a step-wise elution with increasing concentrations of methyl alpha-mannoside (fraction A, 50 mM; B, 100 mM; C, 200 mM; D, 500 mM, and E, 1 M), and its fractional ratio was 12.8:28.6:26.4:19.7:12.4. These five fractions showed the same profile in polyacrylamide gel electrophoresis. The subfractions were analyzed for their relative contents in oligosaccharides of each structure type and for their monosaccharide contents. In fractions B, C, D, and E the former varied between 15-22% for triantennary complex-type, 47-60% for biantennary complex-type, and 22-30% for high mannose-type oligosaccharide. Fraction A showed a higher percentage of triantennary complex-type structures (36%) and a lower percentage of biantennary complex-type structures (17%). The monosaccharide numbers increased from fraction A to E: 85 to 135 mannose residues, 60 to 82 galactose residues, 84 to 115 N-acetyl glucosamine residues, and 22 to 28 sialic acid residues. After analysis of the number of mannose residues contained in the high mannose-type structures, it was possible to calculate the number of oligosaccharides borne by each Tg subfraction. This number was approximately the same for fractions A and B (22.4 and 21.7), then it increased from B to E (21.8 to 32.9). These results account for the separation obtained on the concanavalin A-Sepharose 4B column. Separation of the two first subfractions bearing the same number of oligosaccharides is certainly due to the higher number of high mannose-type structures in B. In conclusion, the studies reported here show that porcine Tg is heterogeneous, and mainly so in terms of total number of N-glycan structures.

Structural and functional analysis of the human vasoactive intestinal peptide receptor glycosylation. Alteration of receptor function by wheat germ agglutinin.

The vasoactive intestinal peptide (VIP) receptor from the human melanoma cell line IGR39 has been shown to be a 60-kDa glycoprotein. Using serial lectin affinity chromatography, as well as specific glycosidases, we demonstrate that VIP receptor-linked carbohydrates are predominantly tri- or tetraantennary sialylated N-linked oligosaccharides, 27% of which are fucosylated, and some may have terminal galactose residues. Treatment of 125I-VIP receptor complexes with peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase revealed the presence of at least three N-linked carbohydrate chains/receptor polypeptide. To investigate the functional role of the carbohydrate moiety, 125I-VIP binding to IGR39 cell membranes was tested in the presence of soluble lectins. Among the lectins tested, only wheat germ agglutinin (WGA) was found to markedly inhibit VIP binding in a dose-dependent manner. Binding data indicated that the presence of the lectin led to a 3-fold increase in Kd value, from 0.15 to 0.44 nM, without any change in the number of available binding sites. The potent inhibitor of WGA binding, N,N',N"-triacetylchitotriose, completely reversed the effect of the lectin. On the other hand, VIP binding inhibition persisted even after neuraminidase treatment, suggesting that sialic acids were not directly involved. Furthermore, WGA inhibition was not abolished although most, if not all, VIP receptor oligosaccharides were converted to high mannose type structures by treating IGR39 cells with deoxymannojirimycin. Finally, whereas the pharmacological profile of VIP receptor was virtually identical, the presence of WGA greatly reduced the VIP-stimulated cAMP in IGR39 cells, indicating that the lectin alters the ability of the receptor to interact with the adenylate cyclase system.

Characterization of the oligosaccharide structures on recombinant human prorenin expressed in Chinese hamster ovary cells.

Prorenin was isolated by immunoprecipitation from the culture medium of Chinese hamster ovary cells transfected with a human prorenin cDNA. The N-linked oligosaccharide structures on the in vivo [3H]mannose-labeled, purified protein were characterized using a combination of serial lectin affinity chromatography, high-pressure liquid chromatography, ion-exchange chromatography, and size-exclusion chromatography and treatment with specific glycosidases and methylation analysis. Approximately 61% of the oligosaccharides on the molecule are complex type, in the form of tetraantennary (2%), 2,6-branched triantennary (13%), 2,4-branched triantennary (3%), and biantennary (43%) structures. The majority of all complex type structures are core-fucosylated. Sialic acids are linked at the C-3 position of terminal galactose, and the degree of sialylation of the bi- and triantennary structures varies between nonsialylated and fully sialylated; no tetraatennary structure contains more than three sialic acid residues. Recombinant prorenin contains 4% hybrid-type structures, all of which carry a terminal sialic acid residue. The remaining 35% of the structures on the molecule are high mannose type, composed of 5, 6, or 7 mannose residues. Approximately 6% of the high mannose type structures and 10% of the hybrid structures are phosphorylated, as judged by their susceptibility to treatment with alkaline phosphatase. Compositional analysis of an unlabeled preparation of the protein suggested the presence of approximately 1.4 oligosaccharide units per molecule.