Complex-type Carbohydrate Chains Research Articles

The patterns of the complex- and oligomannose-type glycans of uromodulin (Tamm-Horsfall glycoprotein) in the course of pregnancy.

Uromodulin was isolated from urine of three pregnant women. Urine of each donor was collected at subsequent stages of their pregnancy and at one month after gestation. Each batch of uromodulin was enzymatically N-deglycosylated and the released N-glycans were isolated, quantified and profiled by high-pH anion-exchange chromatography. In the course of pregnancy no significant changes were detected in the negative charge distribution stemming from sialic acid and sulfate residues on the complex-type carbohydrate chains of uromodulin. Furthermore, no significant changes in the molar ratio between Man6GlcNAc2 and Man7GlcNAc2 were found in the course of pregnancy, only uromodulin from non-pregnant periods showed small differences.

Regioselective Synthesis of β-D-Gal-(1→3)-D-Glcnac Using β-Galactosidase from Xanthomonas Manihotis

The carbohydrate chains of glycoconjugates are involved in a variety of molecular recognition events. For example, the tetrasaccharide sialyl Lewis a (sLea) plays a pivotal role in the metastasis of cancer cells.1 In order to elucidate the biological tunction of carbohydrate chains, many researchers have synthesized components of carbohydrate chains. 2-Acetamido-2-deoxy-3-O-(β-D-galactopyranosyl)-D-glucopyranose (1) is an important constituent of sLea in complex type carbohydrate chains. This disaccharide has been synthesized using the transglycosylation activity of bovine testes β-galactosidase.2 In this case the product mixtures contained unwanted isomers and had to be treated with Escherichia coli β-galactosidase in order to hydrolyze the undesired isomers. Eventually, 1 was obtained in 12 % yield. This methods is far from ideal as it requies two steps to obtain 1 and bovine testes are expensive and not easily available.

Hamster antithrombin III: Purification, characterization and acute phase response

Antithrombin III was purified to homogeneity from hamster plasma by affinity chromatography on heparin-agrose, ion-exchange chromatography on Mono Q and size-exclusion chromatography on TSK G3000SWG column with 50% yield. The molecular mass of hamster antithrombin III was estimated at 62.5 kDa and the absorption coefficient (A 280nm 1%,1cm) at 6.48 (in 0.1 M sodium phosphate pH 7.0). Several isoforms of the inhibitor were detected with the pI in range of 4.95-5.25. The protein contains all residues characteristic for complex-type carbohydrate chains. The N-terminal amino acid sequence shows 84% of identity to mouse and 76% to human analogue. The hamster antithrombin III gives low immunological cross-reactivity with antibodies to human antithrombin III. Initiation of the acute phase response only slightly affected the plasma concentration of inhibitor (±10% within 72-h period). The kinetic data suggest high efficiency in bovine and human thrombin inhibition. In summary, the study shows only similarities between hamster and other mammal antithrombins.

Biological activity and metabolic clearance of recombinant human follicle stimulating hormone produced in Sp2/0 myeloma cells

Human follicle stimulating hormone is a pituitary glycoprotein that is essential for the maintenance of ovarian follicle development and testicular spermatogenesis. Like other members of the glycoprotein hormone family, it contains a common a subunit and a hormone specificβ subunit. Each subunit contains two glycosylation sites. The specific structures of the oligosaccharides of human follicle stimulating hormone have been shown to influence both thein vitro andin vivo bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the isoform profiles,in vitro bioactivity and metabolic clearance of a preparation of purified recombinant human follicle stimulating hormone derived from a stable, transfected Sp2/0 myeloma cell line, and pituitary human follicle stimulating hormone. Isoelectric focussing and chromatofocussing studies of human follicle stimulating hormone preparations both showed a more basic isoform profile for the recombinant human follicle stimulating hormone compared to that of pituitary human follicle stimulating hormone. The recombinant human follicle stimulating hormone had a significantly higher radioreceptor activity compared to that of pituitary human follicle stimulating hormone, consistent with a greaterin vitro potency. Pharmacokinetic studies in rats indicated a similar terminal half life (124 min) to that of the pituitary human follicle stimulating hormone (119 min). Preliminary carbohydrate analysis showed recombinant human follicle stimulating hormone to contain high mannose and/or hybrid type, in addition to complex type carbohydrate chains, terminating with bothα2,3 andα2,6 linked sialic acids. These results demonstrate that recombinant human follicle stimulating hormone made in the Sp2/0 myeloma cells is sialylated, has a more basic isoform profile, and has a greaterin vitro biological potency compared to those of the pituitary human follicle stimulating hormone.

Glycosylation in lepidopteran insect cells: identification of a beta 1-->4-N-acetylgalactosaminyltransferase involved in the synthesis of complex-type oligosaccharide chains.

The choice for a heterologous expression system to produce glycoprotein therapeutics highly depends on its potential to perform mammalian-like posttranslational modifications such as glycosylation. To gain more insight into the glycosylation potential of the baculovirus mediated insect cell expression system, we have studied the expression of glycosyltransferases involved in complex-type N-glycosylation. Lepidopteran insect cell lines derived from Trichoplusia ni, Spodoptera frugiperda, and Mamestra brassicae were found to express a beta 1-->4- N-acetylgalactosaminyltransferase (beta 4-GalNAcT) that catalyzes the transfer of GalNAc from UDP-GalNAc to oligosaccharides and glycoproteins carrying a terminal beta-linked GlcNAc residue. These results suggest that Lepidopteran insect cells are capable of synthesizing complex-type carbohydrate chains containing GalNAc beta 1-->4GlcNAc (LacdiNAc) units. Baculovirus infection of the cells, however, resulted in a decrease in the activity of beta 4-GalNAcT from 80 to <1 pmol.min-1 mg-1 protein within 48 h post infection. Furthermore, considerable beta-N-acetylgalactosaminidase and beta-N-acetylglucosaminidase activity was observed in insect cells, whether or not infected with baculovirus, as well as in the culture medium. These enzyme activities could be responsible for degradation of complex-type oligosaccharide chains containing LacdiNAc units. Our findings provide an enzymatic basis for the observation that most recombinant glycoproteins produced by baculovirus infected insect cells carry oligomannosidic-type N-linked glycans, in spite of the fact that uninfected insect cells have the potential for the synthesis of mammalian-like complex-type glycans.

Targeting of neuronal subsets mediated by their sequentially expressed carbohydrate markers

The targeting of sensory afferent neurons in the leech CNS occurs in two discrete steps that are mediated via different carbohydrate recognitions, as shown by molecular perturbations of cultured embryos. A constitutive carbohydrate marker that is generic to all of these neurons mediates their initial defasciculation and arborization across the entire target region via mannose-specific recognition. Subsequently, two subsets of these same neurons can be differentiated by their expression of other markers that are located on hybrid or complex type carbohydrate chains. These developmentally regulated carbohydrate markers then mediate the target assembly of their respective neuronal subsets into discrete subregions. Thus, by performing opposing functions in a temporal sequence, constitutive and developmentally regulated carbohydrate markers collaborate in the targeting of neuronal subsets in the CNS.

HL 60 leukaemia cells chemically induced to differentiate retain some surface glycan features of undifferentiated cells not found in normal leukocytes.

Human HL 60 myeloid leukaemia cells have the potential to differentiate into either macrophage-like cells or granulocyte-like cells under the stimulus of chemical treatments. Using glycotechnology procedures, the glycosylation patterns of differentiated and undifferentiated HL 60 cells were analysed and compared with those of normal human peripheral monocytes. Both in vitro differentiations result in significant morphologic and functional changes, but we observed that the glycosylation patterns of undifferentiated and differentiated HL 60 cells exhibit several common glycosidic features that are absent in normal peripheral monocytes: the presence of (i) bisecting beta-N-acetylglucosamine attached at the C-4 position of the beta-mannose of polyantennary complex-type carbohydrate chains and (ii) complex-type carbohydrate chains enriched with non-reducing terminal beta-N-acetylglucosamine residues. Moreover, the three populations of HL 60 cells express small amounts of biantennary complex-type structures (< 6%), whereas normal peripheral monocytes expressed > 20% of such structures. Thus, the cell glycosylation pattern could reflect the pathological state of the HL 60 cells.

Carbohydrate heterogeneity of human myeloma proteins of the IgA1 and IgA2 subclasses

Comparative studies of the N-linked carbohydrate chains of human myeloma proteins of the IgA1 and IgA2 subclasses were performed. The N-linked carbohydrate chains were released by hydrazinolysis from the polypeptide backbone, converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation and separated into one neutral and two acidic fractions by paper electrophoresis. The acidic oligosaccharides were completely converted to neutral oligosaccharides by sialidase treatment, indicating that they were sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that human myeloma IgA proteins contained significant amounts of biantennary complex-type carbohydrate chains in addition to a small amount of the high mannose-type. The results indicated that the oligosaccharide structures of human IgA1 and IgA2 display a high degree of heterogeneity not only in the number of carbohydrate chains, but also in their composition.

Efficient production of active industrial enzymes in plants

An industrial bulk enzyme, Bacillus licheniformis α-amylase was produced in transgenic tobacco at a maximum level of about 0.3% of total soluble protein. The molecular weight of the enzyme produced in tobacco differed from the bacterial enzyme due to complex-type carbohydrate chains attached to the protein. Milled transgenic seeds containing α-amylase were successfully applied in the liquefaction of starch. The resulting hydrolysis products were virtually identical to those obtained from degradation with B. licheniformis α-amylase. No effect was observed of the presence of the enzyme on endogenous starch. Homogenization of transgenic leaves, followed by incubation at 95°C, resulted in degradation of the endogenous starch. Different approaches to the concept of production and application of industrial enzymes in plants are presented that show the efficacy of transgenic plants as a new source of active industrial enzymes.

Production of active Bacillus licheniformis alpha-amylase in tobacco and its application in starch liquefaction.

As a first example of the feasibility of producing industrial bulk enzymes in plants, we have expressed Bacillus licheniformis alpha-amylase in transgenic tobacco, and applied the seeds directly in starch liquification. The enzyme was properly secreted into the intercellular space, and maximum expression levels of about 0.3% of total soluble protein were obtained. No apparent effect of the presence of the enzyme on plant phenotype was observed. The molecular weight of the enzyme produced in tobacco was around 64 kD. The difference, compared to 55.2 kD for the bacterial enzyme, was found to result from complex-type carbohydrate chains attached to the protein. Application studies on the liquefaction of starch were done with transgenic seeds containing the recombinant alpha-amylase. The resulting hydrolysis products were virtually identical with those obtained from degradation with alpha-amylase from Bacillus licheniformis.

Modulation of the carbohydrate moiety of thyroglobulin by thyrotropin and calcium in Fisher rat thyroid line-5 cells.

Thyroglobulin secreted in the medium by Fisher rat thyroid line-5 (FRTL-5) cells cultured in the presence of thyroid stimulating hormone (TSH) shows a slower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a higher density position in a CsCl gradient than thyroglobulin secreted by FRTL-5 cells cultured in the absence of TSH for 5-7 days. Such a TSH effect is much less or not evident when secreted thyroglobulin is digested with peptide N-glycohydrolase F or when intracellular thyroglobulin is compared. Intracellular thyroglobulin migrates faster than thyroglobulin secreted either in the presence or in the absence of TSH. Evaluation of the mannose and galactose content of thyroglobulin demonstrates that intracellular thyroglobulin has more mannose and less galactose than extracellular thyroglobulin; it also shows that TSH decreases the mannose content of thyroglobulin while increasing its galactose content. Bio-Gel P6 chromatography shows that TSH increases the complex type carbohydrate chains while decreasing the high mannose chains in the secreted thyroglobulin. High mannose type oligosaccharides were characterized by fast atom bombardment-mass spectrometry analysis. Treatment with the calcium ionophore A23187 (5 microM) of FRTL-5 cells cultured with or without TSH causes the appearance of a "fast" migrating form of thyroglobulinin in the culture medium. Bio-Gel P6 chromatography shows that A23187 causes a dramatic decrease of the complex carbohydrate chains of the secreted thyroglobulin.

Biosynthesis and intracellular movement of the melanosomal membrane glycoprotein gp75, the human b ( brown) locus product

A 75-kDa melanosomal glycoprotein (gp75) is the product of a gene that maps to the b ( brown) locus, a genetic locus that determines coat color in the mouse. The b locus is conserved (88% identity) between mouse and human. The mouse monoclonal antibody TA99 was used to study the biosynthesis and processing of gp75. gp75 was synthesized as a 55-kDa polypeptide, glycosylated by addition and processing of five or more Asnlinked carbohydrate chains through the cis and trans Golgi, and transported to melanosomes as a mature 75kDa form. Synthesis and processing of gp75 was rapid ( T 1 2 < 30 min ), and early steps in processing were required for efficient export of gp75 to melanosomes. Fully processed mature gp75 was quite stable ( T 1 2 = 22–24 h ) in the melanosome. Digestion of high-mannose carbohydrate chains with endo-β- N-acetylglucosaminidase H revealed two alternative processed forms of gp75 that differed in the number or composition of complex-type carbohydrate chains. The rate of synthesis and movement through intracellular membrane compartments was the same for both glycosylated forms. Studies with inhibitors of steps in oligosaccharide processing showed that alternative forms of gp75 were generated during trimming reactions by mannosidase IA/IB and that further maturation resulted in the two mature forms of gp75. We propose that the kinetics of biosynthesis and processing reflect events in the biogenesis and maturation of melanosomes.

Optimized deglycosylation of glycoproteins by peptide-N4-(N-acetyl-β-glucosaminyl)-asparagine amidase fromFlavobacterium meningosepticum

Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F(PNGase F) from Flavobacterium meningosepticum is a highly useful enzyme for the structural analysis of N (asparagine)-linked carbohydrate chains derived from glycoproteins. The enzyme was enriched using a published procedure [Tarentino AL, Gomez CM, Plummer TH, Jr (1984) Biochemistry 1985:4665-71; Tarentino AL, Plummer TH, Jr (1987) Methods Enzymol 138:770-78] and further purified by hydrophobic interaction HPLC on a weak hydrophobic TSK-Ether column from which it was eluted by a decreasing gradient of 1.7 M ammonium sulphate in 100 mM sodium phosphate, pH 7.0, containing 5 mM EDTA. To determine the optimal conditions for a complete deglycosylation of glycoproteins by PNGase F, experiments were performed with human alpha 1-acid glycoprotein, because the five complex type carbohydrate chains are quite resistant to enzymic hydrolysis. The influence of different detergents on the enzyme reaction was studied. Complete deglycosylation of human alpha 1-acid glycoprotein was achieved by the use of 60 mU/ml PNGase F in 0.25 M sodium phosphate buffer, pH 8.6, containing 0.2% (w/v) SDS, 20 mM mercaptoethanol and 0.5% Mega-10.

Microheterogeneity of the carbohydrate moiety of the human erythrocyte glucose transporter

The carbohydrate moiety of the human erythrocyte glucose transporter was isolated using two independent methods: hydrazinolysis andN-glycanase treatment. The major structure observed was constituted of complex-type carbohydrate chains carrying repetitive units ofN-acetyllactosamine. This structure exhibited microheterogeneity: a broad variability in the number of repetitive units, presence of branched structures and substitution by fucosyl residues. Moreover, significant amounts of bi-antennary and hybrid structures were present.

Isolation and sequencing of a cDNA clone encoding 96 kDa sialoglycoprotein in rat liver lysosomal membranes

We isolated and sequenced LGP 96, a cDNA clone corresponding to the entire coding sequence of the rat liver lysosomal membrane sialoglycoprotein with an apparent Mr of 96 K, LGP 96. The deduced amino acid sequence indicates that LGP 96 consists of 411 amino acid residues (Mr 45,163) and the 26 NH 2-terminal residues presumably constitute a cleavable signal peptide. The major portion of LGP 96 resides on the luminal side of the lysosome and bears a large number of N-linked heavily sialylated complex type carbohydrate chains, giving the mature molecule of 96 kDa. The protein has 17 potential N-glycosylation sites and 32.1 and 65.3% sequence similarities in amino acid to LGP 107 and human lamp-2,respectively. The glycosylation sites are clustered into two domains separated by a hinge-like structure enriched with proline and threonine. LGP 96 possesses one putative transmembrane domain consisting of 24 hydrophobic amino acids near the COOH-terminus and contains a short cytoplasmic segment constituting 12 amino acid residues at the COOH-terminal end. Comparison of LGP 96 and recently cloned lysosomal membrane glycoprotein sequences reveals strong similarity in the putative transmembrane domain and cytoplasmic tail. It is very likely that these portions are important for the targeting of molecules to lysosomes. A comparison of LGP 96 and LGP 107 showed numberous structural similarities.

Insulin-like growth factor I receptors on mouse neuroblastoma cells. Two beta subunits are derived from differences in glycosylation.

We have characterized receptors for the insulin-like growth factor (IGF-I) on the mouse neuroblastoma cell line N18 as well as NG108, the hybrid cell line of N18 and rat glioma (C6). In this cell-free system, IGF-I and insulin stimulated the phosphorylation of 95-kDa and 105-kDa proteins. Using appropriate antibodies we were able to demonstrate that the IGF-I receptor beta subunit has two subtypes of 95 kDa and 105 kDa. On the other hand, insulin receptor beta subunit is a separate single 95-kDa protein. Enzymatic digestion of IGF-I receptor beta subunit subtypes by glycopeptidase F resulted in similar molecular masses (84 kDa and 86 kDa) on SDS-PAGE, which suggests that the difference in molecular masses between two subtypes is attributable to the differences in N-linked complex-type carbohydrate chains on the extracellular domain of beta subunits. This conclusion is further supported by peptides of similar molecular mass following staphylococcal V8 protease digestion. Analysis of IGF-I receptor beta subunit subtypes in these cells may provide insights into the mechanism of action of IGF-I on neural tissues.

Solubilization and characterization of guinea-pig pancreatic somatostatin receptors.

The solubilization of somatostatin receptors from guinea-pig pancreas by different non-denaturing detergents was investigated after stabilization of the receptors by prior binding of 125I-[Tyr11]somatostatin or its analogue 125I-[Leu8,DTrp22,Tyr25]somatostatin 28, to pancreatic plasma membranes. The somatostatin-receptor complexes were solubilized in a high yield by Zwittergent 3-14 (3-[tetradecyldimethylammonio]-1-propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X-100, Chaps (3-[cholamidopropyldimethylammonio]-1-propanesulfonate) and octyl beta-D-glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol. In order to characterize solubilized somatostatin-receptor complexes, membranes receptors were covalently labelled using N-5-azido-2-nitrobenzoyloxysuccinimide as cross-linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent Mr = 93,000 which interacted with the two radioligands. In addition, a similar component of Mr = 88,000 was characterized after analysis by SDS-PAGE of membrane receptors covalently cross-linked with 125I-[Leu8,DTrp22,Tyr25]somatostatin 28 by different heterobifunctional reagents: N-5-azido-2-nitrobenzoyloxysuccinimide, N-hydroxysuccinimidyl 4-azidobenzoate, N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate. Optimal cross-linking results were obtained with N-5-azido-2-nitrobenzoyloxysuccinimide. The solubilized somatostatin-receptor complex was adsorbed to wheat-germ agglutinin-agarose column and eluted by specific sugars. We concluded that the guinea-pig pancreatic somatostatin receptor in the membrane and in the non-denaturing detergent solution behaves as a protein monomer of apparent Mr approximately 85,000-90,000. The somatostatin receptor is a glycoprotein which contains complex-type carbohydrate chains.

The mammalian beta-adrenergic receptor: structural and functional characterization of the carbohydrate moiety.

Mammalian beta-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of Mr approximately 64,000. Treatment of purified [125I]-labeled hamster lung beta-adrenergic receptor with alpha-mannosidase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [125I]-labeled receptor with endoglycosidase F results initially in the formation of a Mr approximately 57,000 peptide which is further converted to Mr approximately 49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [125I]-labeled receptor reveals the presence of two complex type carbohydrate chains (approximately 10% of which are fucosylated) on approximately 45% of the receptors. The remaining approximately 55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of beta-adrenergic agonists and antagonists. Moreover, the ability of control, alpha-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, alpha-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleotide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the beta-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.

Glycosylation of the bovine coronavirus hemagglutinin protein.

The bovine coronavirus hemagglutinin protein gp140 is composed of disulfide-linked subunits of 65 kDa. This protein was further characterized with regard to its glycosylation. The glycosylated subunits of the protein are polypeptides having a molecular mass of 42.5 kDa. Both subunits appear to be processed to the same extent by the addition of N-linked oligosaccharides. Each subunit on the mature virion has 6-7 high mannose and 3-4 complex type carbohydrate chains attached to it.

Photoaffinity cross-linked A1 adenosine receptor-binding subunits. Homologous glycoprotein expression by different tissues.

Mammalian A1 adenosine receptor-binding peptides can be visualized by covalently labeling them with the photoaffinity cross-linking ligand N6-2-(4-amino-3-[125I] iodophenyl)ethyladenosine followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography. The proteins comprising the A1 adenosine receptor-binding subunit of rat brain and fat migrate with Mr 38,000. In this study, the glycoproteins representing the radiolabeled A1 adenosine receptor-binding subunit expressed in each of these tissues (brain and fat) were compared through the use of peptide mapping and exo- and endoglycosidase treatments. Peptide mapping studies with several enzymes demonstrate that the protein component of the radiolabeled A1 adenosine receptor-binding subunit is conserved between different tissues. Both labeled receptor peptides demonstrate a sensitivity to neuraminidase as evidenced by increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the receptors contain complex-type carbohydrate chains. Insensitivity to alpha-mannosidase suggests a lack of high mannose-type carbohydrate chains. Deglycosylation of the labeled receptor-binding subunits with endoglycosidase F results in a single labeled polypeptide of Mr 32,000 for both systems. These data suggest that the A1 adenosine receptor-binding subunits expressed in the rat brain and fat are similar glycoproteins as evidenced by similar overall molecular weights, identical peptide maps, and equivalent responses to endo- and exoglycosidase treatment.