Reactive oxygen species modify oligosaccharides of glycoproteins in vivo: a study of a spontaneous acute hepatitis model rat (LEC rat)

The Long-Evans Cinnamon (LEC) rat, an animal model of Wilson's disease, spontaneously develops hepatitis as the result of abnormal copper accumulation in liver. The findings of this study show that copper, hydrogen peroxide, and lipid peroxides accumulate to drastically high levels in LEC rat serum in acute hepatitis but not chronic hepatitis. The effect of these reactive oxygen species (ROS) on oligosaccharides of glycoproteins in the LEC rat serum was examined. Lectin blot and lectin ELISA analyses showed that sialic acid and galactose residues of serum glycoproteins including transferrin were decreased in acute hepatitis. Further analyses of oligosaccharide structures of transferrin demonstrated that di-sialylated and asialo-agalacto biantennary sugar chains, but not tri-sialylated sugar chain, exist on transferrin in the acute hepatitis rats. In addition, treatment of non-hepatitis rat serum with copper ions and hydrogen peroxide decreased tri-sialylated sugar chain of the normal transferrin and increased di-sialylated and asialo-agalacto biantennary sugar chains. This is the first evidence to show that ROS result in the cleavage of oligosaccharides of glycoproteins in vivo, and indicate this cleavage of oligosaccharides may contribute the development of acute hepatitis.     

Detection of aggressive prostate cancer associated glycoproteins in urine using glycoproteomics and mass spectrometry

Clinical management of prostate cancer remains a significant challenge due to the lack of available tests for guiding treatment decisions. The blood prostate‐specific antigen test has facilitated early detection and intervention of prostate cancer. However, blood prostate‐specific antigen levels are less effective in distinguishing aggressive from indolent prostate cancers and other benign prostatic diseases. Thus, the development of novel approaches specific for prostate cancer that can differentiate aggressive from indolent disease remains an urgent medical need. In the current study, we evaluated urine specimens from prostate cancer patients using LC‐MS/MS, with the aim of identifying effective urinary prostate cancer biomarkers. Glycoproteins from urine samples of prostate cancer patients with different Gleason scores were characterized via solid phase extraction of N‐linked glycosite‐containing peptides and LC‐MS/MS. A total of 2923 unique glycosite‐containing peptides were identified. Glycoproteomic comparison on urine and tissues from aggressive and non‐aggressive prostate cancers as well as sera from prostate cancer patients revealed that the majority of AG prostate cancer associated glycoproteins were more readily detected in patient's urine than serum samples. Our data collectively indicate that urine provides a potential source for biomarker testing in patients with AG prostate cancer.     

Swainsonine对胰岛素受体功能影响的研究

以Ｓｗａｉｓｏｎｉｎｅ（Ｓｗ）作为高尔基体付糖链加工酶系中α－甘露糖苷酶Ⅱ的特异抑制剂,研究Ｎ－糖链结构和胰岛素受体（Ｉｎｓ－Ｒ）功能的关系．发现Ｓｗ不影响细胞生长和３Ｈ－亮氨酸参入ＳＭＭＣ７７２１细胞,但明显促进３Ｈ－甘露糖参入细胞总糖蛋白和表面糖蛋白,并使后者的ＣｏｎＡ强结合组分显著增加,提示Ｓｗ使Ｉｎｓ－Ｒ的Ｎ－糖链变成杂合型及高甘露糖型。胰岛素结合试验后作Ｓｃａｔｃｈａｒｄ分析：发现Ｓｗ不改变Ｉｎｓ－Ｒ的结合容量和每个细胞表面的结合位点数,也不改变结合动力学。再用部分纯化的Ｉｎｓ－Ｒ研究自身磷酸化和对外源底物的酪氨酸蛋白激酶活力,也未发现Ｓｗ处理和对照细胞间的明显区别,表示Ｓｗ也不影响Ｉｓｎ－Ｒ的跨膜信息传递,结合已报道的衣霉素使细胞表面Ｉｎｓ－Ｒ减少的结果,提示Ｉｎｓ－Ｒ运送至细胞膜需要Ｎ－糖链存在,但糖链的类型对ＩＮＳ－Ｒ的代谢和结合动力学并不重要     

A Screen for Swainsonine in Select North American Astragalus Species

Swainsonine is found in several plant species worldwide, and causes severe toxicosis in livestock grazing these plants, leading to a chronic condition characterized by weight loss, altered behavior, depression, decreased libido, infertility, and death. Swainsonine has been detected in 13 North American Astragalus species of which eight belong to taxa in four taxonomic sections, the Densifolii, Diphysi, Inflati, and Trichopodi. These sections belong to two larger groups representing several morphologically related species, the Pacific Piptolobi and the small‐flowered Piptolobi. The objective of this study was to screen the other 31 species for swainsonine in sections Densifolii, Diphysi, Inflati, and Trichopodi previously not known to contain swainsonine. Furthermore, to broaden the scope further, 21 species within the 8 sections of the Pacific Piptolobi and the small flowered Piptolobi were screened for swainsonine. Swainsonine was detected for the first time in 36 Astragalus taxa representing 29 species using liquid and gas chromatography coupled with mass spectrometry. Several taxonomic sections were highly enriched in species that contain swainsonine while others were not. A systematic examination for swainsonine in these species will provide important information on the toxic risk of these species and may be a valuable reference for diagnosticians and land managers.     

Synthesis and secretion of colonic glycoproteins: Evidence for shedding in vivo of low molecular weight membrane components

In vivo glycoprotein synthesis and secretion was studied in rat colonic epithelial cells using precursor labelling with radiolabelled glucosamine. Sepharose 4B gel filtration of radiolabelled glycoproteins obtained from isolated colonic epithelial cells revealed two major fractions: (1) high molecular weight mucus in the excluded fraction and (2) lower molecular weight glycoproteins in the included volume. These glycoproteins were further fractionated by affinity chromatography on concanavalin A-Sepharose. The low molecular weight [³H]glucosamine-labelled glycoproteins contained a major subfraction which specifically adhered to concanavalin A, and could be eluted with 0.2 M α-methylmannoside. Fractionation of the concanavalin A-reactive glycoproteins on Sephadex G-100 revealed a major peak with a molecular weight of 15 000. In contrast, high molecular weight mucus glycoprotein did not adhere appreciably to concanavalin A-Sepharose. Perfusion experiments indicated that colonic secretions contained both mucus and concanavalin A-reactive glycoproteins. The major concanavalin A-reactive glycoprotein in the colonic perfusate was not derived from serum, but was released directly from the colonic membrane into the lumen.     

Trypanosoma brucei: radioimmunoassay of variant surface glycoproteins from organisms grown in vitro and in vivo

Bloodstream forms of Trypanosoma brucei that were infective for mammals, when grown in vitro at 37 C for 29 days or 25 months had amounts of variant surface glycoprotein similar to the amounts from bloodstream forms isolated from infected rat blood. The amounts were measured by competition radioimmunoassays for both unique and cross-reacting determinants and the results were the same, providing evidence that a single type of variant surface glycoprotein was measured. Neither radioimmunoassay detected determinants of variant surface glycoproteins in trypanosomes transformed by culturing at 27 C to insect forms not infective for mammals.     

Oligosaccharide heterogeneity of glycoproteins sulfated during the vegetative growth of Dictyostelium discoideum

Macromolecules are sulfated during the vegetative growth of Dictyostelium discoideum. A characterisation of the structures of sulfated oligosaccharides associated with these macromolecules indicates that the oligosaccharides are heterogeneous. Endoglycosidase and pronase digestion were used with gel-filtration chromatography to obtain two different oligosaccharide fractions and a glycopeptide fraction; these were further characterised by ion-exchange and lectin-affinity chromatography and by acid hydrolysis. The data indicate that up to 43% of the sulfate is associated with typical N-linked oligosaccharides, that up to 5% is associated with N-linked oligosaccharides that are either very large or extremely highly charged, and that the remaining sulfate is associated with oligosaccharides non-N-linked to protein. Each fraction was also shown to be heterogeneous at most other structural levels. Electrophoretic analyses following the endoglycosidase and pronase treatments indicated that all of the macromolecules are glycoproteins and suggested further that at least two of the oligosaccharide fractions are located on different groups of glycoproteins.     

Biodegradation of Swainsonine by Acinetobacter calcoaceticus strain YLZZ-1 and its isolation and identification

Eight swainsonine (SW)-degrading bacteria were isolated from the soil where locoweed was buried for 6 months and one of the strains (YLZZ-1) was selected for further study. Based on morphology, physiologic tests, 16S rRNA gene sequence, and phylogenetic characteristics, the strain showed the greatest similarity to members of the order Acinetobacters and within the order to members of the Acinetobacter calcoaceticus group. The ability of the strain for degrading SW, as sole carbon source, was investigated under different culture conditions. The preferential temperature and initial pH for the strain were 25–35°C and 6–9, respectively. The optimal temperature for the strain was 30°C and the optimal pH was 7.0. There was a positive correlation between degradation rate and inoculation amount. The concentration of SW affected the degradation ability. When the concentration of SW was lower than 100 mg/l, SW decreased immediately after incubation, and when the concentration of SW was 200 mg/l, there was an inhibiting effect for bacteria growth and SW degradation. The strain could degrade SW completely within 14 h when the concentration of SW was 50 mg/l. These results highlight the potential of this bacterium to be used in detoxifying of SW in livestock consuming locoweed.     

The effects of oligosaccharide trimming inhibitors on glycoprotein expression and infectivity of Junin virus

Abstract The effects of specific inhibitors of glycoprotein trimming reactions on Junin virus (JV) replication were investigated. Bromoconduritol, an inhibitor of glucosidase II, significantly reduced infective virus production (DE50: 1.1 mM) and viral protein expression. Neither 1-deoxynojirimycin, an inhibitor of both glucosidases I and II, nor 1-deoxymannojirimycin and swainsonine, inhibitors of mannosidase I and II, respectively, showed any activity against JV multiplication. These results are the first evidence that the acquisition of a complex form of the envelope glycoprotein oligosaccharide chains is not essential for JV infectivity. The effect of bromoconduritol was reversible and probably due to the formation of an unstable intermediate oligosaccharide structure which may be more sensitive to degradative proteolysis.     

Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method

We have developed a new method for the large scale preparation of pyridylaminated (PA-) oligosaccharides from glycoproteins. Phenol/chloroform extration was adapted for the removal of protein and excess 2-aminopyridine, improving the efficiency of preparation. From a 2.5 g sample of human apo-transferrin, 25–30 mol of agalacto biantennary PA-oligosaccharide could be obtained. By increasing the concentration of PA-oligosaccharide substrate, we were able to detect a very low level ofN-acetylglucosaminlytransferase IV activity in CHO cell extracts.Abbreviations PA 2-aminopyridine - SDS sodium dodecyl sulfate - GlcNAc N-acetylglucosamine - GnT N-acetylglucosaminyltransferase - Gn,Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-tri-PA GlcNAc1-2(GlcNAc1-4)Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-trí-PA GlcNAc1-2Man1-3({GlcNAc1-2(GlcNAc1-6)Man1-6})Man1-4GlcNac1-4GlcNAc-2-aminopyridine - Gn,(Gn),Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-4)(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine     

Similarity in the bound carbohydrate groups of glycoproteins from cells of several vertebrate classes

The carbohydrate groups of the glycoproteins of human, hamster, chick, reptile and fish cells growing in culture have been fractionated in succession according to size (Sephadex G-50), affinity for concanavalin A, charge (DEAE-Sephadex) and by thin-layer chromatography. It was found that despite the complexity of the array of separable glycopeptides in each type of cell, most of these structures seemed to be common to all of the cells. This suggests that they have existed in a relatively stable state for several hundreds of millions of years throughout the evolution of the vertebrates.     

Swainsonine treatment accelerates intracellular transport and secretion of glycoproteins in human hepatoma cells

We are interested in determining whether carbohydrates are important regulatory determinants in the intracellular transport and secretion of glycoproteins. In the present study, we have used swainsonine, an indolizidine alkaloid, to modify the structure of N-glycosidically linked complex oligosaccharides. By inhibiting Golgi mannosidase II, swainsonine prevents the trimming of GlcNAc(Man)5(GlcNAc)2 to GlcNAc-(Man)3(GlcNAc)2, resulting in the formation of hybrid-type oligosaccharides. We find, from pulse-chase experiments using [35S]methionine and immunoprecipitation of individual proteins from culture media, that swainsonine treatment (1 microgram/ml) accelerated the secretion of glycoproteins (transferrin, ceruloplasmin, alpha 2-macroglobulin, and alpha 1-antitrypsin) by decreasing the lag period by 10-15 min relative to untreated cultures. The enhanced secretion was specific for glycoproteins since the secretion of albumin, a nonglycoprotein, was unaffected. When alpha 1-antitrypsin was immunoprecipitated from the cell lysates, sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorographic analysis demonstrated that the conversion of the high-mannose precursor to the hybrid form in swainsonine-treated cells occurred more rapidly (by about 10 min) than the conversion to the complex form in control cells. Since both the hybrid and complex forms of alpha 1-antitrypsin are terminally sialylated by sialyltransferase in the trans-Golgi, these results suggest that swainsonine-modified glycoproteins traverse the Golgi more rapidly than their normal counterparts. Therefore, accelerated transport within this organelle may account for the decreased lag period of glycoprotein secretion in the swainsonine-treated cultures.     

Surface glycoproteins determine the feature of the 2009 pandemic H1N1 virus

After the outbreak of the swine-origin influenza A H1N1 virus in April 2009, World Health Organization declared this novel H1N1 virus as the first pandemic influenza virus (2009 pH1N1) of the 21^st century. To elucidate the characteristics of 2009 pH1N1, the growth properties of A/Korea/01/09 (K/09) was analyzed in cells. Interestingly, the maximal titer of K/09 was higher than that of a seasonal H1N1 virus isolated in Korea 2008 (S/08) though the RNP complex of K/09 was less competent than that of S/08. In addition, the NS1 protein of K/09 was determined as a weak interferon antagonist as compared to that of S/08. Thus, in order to confine genetic determinants of K/09, activities of two major surface glycoproteins were analyzed. Interestingly, K/09 possesses highly reactive NA proteins and weak HA cell-binding avidity. These findings suggest that the surface glycoproteins might be a key factor in the features of 2009 pH1N1. [BMB Reports 2012; 45(11): 653-658]     

Transfer of glucose in the biosynthesis of thyroid glycoproperties I. Inhibition of glucose transfer to oligosaccharide lipids by GDP-mannose

Thyroid rough microsomes catalyzed the synthesis of glucose-containing oligosaccharide lipids which were compared to those extracted from labeled thyroid cells and were found to be largely similar.Glucose transfer to these oligosaccharide lipids in the microsomal system was shown to be markedly depressed by an addition of GDPmannose. This sugar nucleotide, already at 1μM, blocked dolichol-P-glucose synthesis, thus restraining further glucosylation of oligosaccharide lipids. Using this concentration of radioactive GDPmannose in the incubation medium lead to the detection of three glucose containing mannose-labeled oligosaccharide lipids. Double labeling experiments suggested a precursor-product relationship between them.Previously labeled oligosaccharide lipids, containing glucose or not were compared in their efficiency to acr as donors of their oligosaccharide chain to an exogenous synthetic Asn-X-Thr containing peptide. It was foun that the presence of glucose did not signifantly influence the transfer. Free glucose was released during the reaction when using the glucose-labeled oligosaccharide lipid.     

Steps in the biosynthesis of mosquito cell membrane glycoproteins and the effects of tunicamycin

A cultured cell line of the mosquito, Aedes aegypti, is sensitive to tunicamycin as expected from the ability of crude membrane preparations to catalyse the formation of N-acetylglucosamine-linked dolichyl pyrophosphate. Formation of dolichylphosphomannose was also detected and this reaction was totally insensitive to tunicamycin. Incorporation of radioactive mannose into total acid-precipitable glycoproteins was inhibited greater than 90% in whole cells by tunicamycin, while the incorporation of leucine and glucosamine was less affected. Separation of the radioactive hexosamines from acid hydrolysates of cells incubated with [¹⁴C]glucosamine and tunicamycin showed predominant labelling of galactosamine, whereas in control cells not treated with the drug both glucosamine and galactosamine were labelled equally. Evidently, mosquito cells synthesise N-glycosidically linked carbohydrate chains assembled through tunicamycin-sensitive steps involving dolichyl pyrophospho-oligosaccharides, and O-glycosidically linked chains rich in N-acetylgalactosamine, the assembly of which is unaffected by tunicamycin. These results support structural evidence (Butters, T.D. and Hughes, R.C. (1981) Biochim. Biophys. Acta 640, 655–671) for the presence of high mannose N-glycans and N-acetylgalactosamine-richO-glycans in mosquito cell glycoproteins. The absence of complex N-glycans was confirmed by the demonstration of negligible activities of N-acetylglucosaminyl-, galactosyl- and sialyltransferases responsible for assembly of the terminal sequences of N-glycans of mature mammalian glycoproteins.     

Prestaining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by dansylhydrazine

A new fluorescent prestaining method for gel‐separated glycoproteins in 1D and 2D SDS‐PAGE was developed by using dansylhydrazine in this study. The prestained gels could be easily imaged after electrophoresis without any time‐consuming steps needed for poststains. As low as 4–8 ng glycoproteins (transferrin, α1‐acid glycoprotein) could be selectively detected, which is comparable to that of Pro‐Q Emerald 488, one of the most commonly used glycoprotein stain. In addition, a subsequent study of deglycosylation, glycoprotein affinity isolation, and LC‐MS/MS analysis was performed to confirm the specificity of the newly developed method.     

Synthesis of lactosaminoglycan-containing glycoproteins by vascular endothelial cells

Human vascular endothelial cells synthesize lactosaminoglycan-type glycoproteins which are found both associated with cells and secreted into the culture medium. Pronase-derived glycopeptides prepared from [³H]glucosamine-labeled glycoproteins were found to contain about 10% of the labeled products as a large size (M_r > 5000) ³H-labeled glycopeptide. Digestion of these ³H-labeled glycopeptides with endo-β-galactosidase resulted in the release of smaller size saccharides, which were characterized as having the structure sialic acid → Gal → GlcNAc → Gal. Treatment of [³H]glucosamine-labeled cells with melittin caused ³H-labeled glycoconjugates to be released from the cells. Separation of released glycoproteins from proteoglycans by DEAE-cellulose chromatography indicated that melittin had released 25% of the total ³H-labeled glycoproteins from the cell and 3% of the ³H-labeled proteoglycans. The ³H-labeled glycoproteins were digested with Pronase and the resulting ³H-labeled glycopeptides were fractionated on Sephadex G-50. The large size fraction (M_r > 5000) now comprised about 30% of these released ³H-labeled glycopeptides. These high molecular weight ³H-labeled glycopeptides were degraded with endo-β-galactosidase but not with testicular hyaluronidase. Analysis of the released ³H-labeled glycoproteins indicated a preferential release of glycoproteins of 70–90 kDa enriched in lactosaminoglycan-type oligosaccharides.     

Swainsonine induces the production of hybrid glycoproteins and accumulation of oligosaccharides in male reproductive tissues of the rat

Early studies have shown that spotted locoweed (Astragalus lentiginosus) has an adverse effect on male reproduction. Rams fed locoweed showed a reduced number of primary and secondary spermatocytes and spermatids in the testis, and of spermatozoa in the epididymis and vas deferens. In addition, the Sertoli cells and other epithelial cells were severely vacuolated. Swainsonine, an indolizidine alkaloid, has been identified as the sole or principal toxin in locoweed and perhaps in the plants of genus Swainsona. The toxin is an inhibitor of lysosomal alpha-D-mannosidase, cytosolic alpha-D-mannosidase, and Golgi mannosidase II. The in vitro and in vivo inhibition of Golgi mannosidase II induces the production of abnormal glycoproteins. Since epididymis-mediated modifications of sperm-surface glycoproteins are believed to be important for sperm-egg interactions, we initiated studies to determine effects of swainsonine on processing and catabolism of N-linked glycoproteins in male reproductive tissues. The results presented in this report indicate that feeding of the alkaloid led to accumulation of mannose-rich oligosaccharides (OS) in the testis and epididymis of rats. The major OS was purified from the reproductive tissues of swainsonine-fed rats, and its structure was deduced by comparison of the size of the OS before and after treatment with jack bean alpha-D-mannosidase, and by affinity column chromatography. In addition, the rat epididymal epithelial cells produced abnormal glycoproteins when cultured in the presence of the toxin. This result provides indirect evidence for the presence of a swainsonine-sensitive mannosidase II-like processing enzyme in the epididymal epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural basis for the presence of a monoglucosylated oligosaccharide in mature glycoproteins

Arylphorin is an insect hexameric storage protein. The structures of the oligosaccharides attached to this protein have recently been determined. However, their precise functions remain to be established. Proteolysis and MALDI MS studies disclose that the amino acid residues Asn196 and Asn344 are N-glycosylated with Glc(1)Man(9)GlcNAc(2) and Man(5-6)GlcNAc(2) oligosaccharides, respectively. Interestingly, significant variations in the amounts of glycans involving Glc(1)Man(9)GlcNAc(2) are evident in arylphorins purified from larvae reared at different seasons. The data suggest that the metabolism of larvae and local protein structure contribute to glycan development. Three-dimensional model of the protein speculated that N-glycosidic linkage to Asn196 in the Glc(1)Man(9)GlcNAc(2) structure was buried inside the twofold axis of the hexamer, whereas oligosaccharide linkages to Asn344 were completely exposed to solvent. This finding is in agreement with previous biochemical data showing that limited Glc(1)Man(9)GlcNAc(2) was released by protein-N-glycosidase F under non-denaturing conditions, in contrast to Man(5-6)GlcNAc(2) oligosaccharides.