Removal Of Oligosaccharides Research Articles

Enzymatic degradation of oligosaccharides in soybean flours

The oligosaccharide content was determined in the flour of six soybean cultivars grown in India. The effect of cooking and soaking on the removal of oligosaccharides from the whole dry seeds was also studied. Crude α-galactosidase treatment on soybean flour reduced the raffinose and stachyose contents by 90.4% and 91.9%, respectively. This information could stimulate interest in the large-scale production of oligosaccharide-free soybean flour, by modulating its flatulence-causing properties.

Properties of an extracellular glycosidase of Aspergillus niger suitable for removal of oligosaccharides from cowpea meal

The properties of crude and purified α-galactosidase formed by a strain of Aspergillus niger on wheat and rice bran-based media were examined. Cell-free extracts from both media had optimal α-galactosidase activities at 50°C and pH 5.0. The crude preparation from wheat bran had the highest activity (0.13 U ml −1), whereas that from rice bran had a lower activity (0.08 U ml −1) but was more stable over a wider range of temperature and pH. Three peaks of activity occurred after dialysis and chromatography on Sephadex G-100, indicating the presence of multimolecular forms. The specific activity of the pure enzyme solution is 1567 U μg protein −1, and the protein responsible for the highest activity peak consists of two subunits with relative molecular masses of 78 and 69 KDa, respectively. The crude preparation simultaneously hydrolyzed stachyose and raffinose in two cowpea meal fractions (particle size <300 and <850 μm). Raffinose was hydrolyzed 1.7 times faster than stachyose.

A general method for the complete deglycosylation of a wide variety of serum glycoproteins using peptide-N-glycosidase-F

Many indirect serum studies show changes in protein glycosylation in disease, but further progress will require direct investigation of oligosaccharide composition. Current methods of deglycosylation using PNGase-F often result in incomplete removal of oligosaccharides. This is an unsatisfactory situation because only small quantities of material are often available in clinical studies, glycosylation changes may occur in only a small proportion of the molecules and quantification of the released oligosaccharides may be unreliable. The ability of PNGase-F to deglycosylate haptoglobin (Hp) under different conditions has been investigated. Oligosaccharides were completely removed from 50μg of Hp by treatment for 24 h with PNGase-F in 50 mmol/l ammonium formate buffer, pH 8.6, in combination with sodium dodecyl sulphate, mercaptoethanol and Nonidet P40. This modified procedure was equally effective at removing oligosaccharides from other serum glycoproteins (α1 glycoprotein, α1, transferrin) and fetuin, and its efficiency was independent of the polymeric structure of the molecule or the amount of glycosylation. The method has the additional advantage of using 20% less enzyme than previous methods, which substantially reduces costs.

Intestinal mucins inhibit rotavirus replication in an oligosaccharide-dependent manner.

Rotaviruses are important causes of infant morbidity and mortality worldwide. It has been previously shown that mucinous glycoproteins can inhibit rotavirus replication. However, the structure-function relationships of this inhibition have not been completely elucidated. Mucins were purified from epithelial scrapings of rat and human intestine by CsCl density-gradient ultracentrifugation and tested for the inhibition of rotavirus replication in MA-104 cells. Native human and rat intestinal mucins inhibited the replication of human and animal rotaviruses at low concentrations. Antiviral activity was most prominent in the densely glycosylated part of the rat and human mucins. Activity was retained after thiol reduction and alkylation, chloroform methanol partition, and partial removal of oligosaccharides. However, total deglycosylation of the mucins destroyed antiviral activity. Intestinal mucins from humans and other animals are potent inhibitors of rotavirus replication, and this inhibition is dependent on specific mucin-viral interactions.

Cell-Free Synthesis of Vitellin in the Shrimp Penaeus semisulcatus (de Haan)

The vitellogenic ovary of Penaeus semisulcatus contains mRNA specific for vitellin (Vt), low levels of which are found in nonvitellogenic ovaries, and is absent from testes. lmmunoisolation from cell-free translation of poly(A) + RNA using antiserum against purified Vt produced a 35-kDa polypeptide. No differences were found between Vt precipitated from the translation products of the rabbit reticulocyte system and that from the wheat germ extract. The specificity of the immunoprecipitation reaction was demonstrated by the absence of precipitation with nonimmunized rabbit serum and with antibodies prepared against purified hemocyanin or lipoprotein I (LPI), which are crustacean hemolymph proteins. In addition, competition with the radioactively labeled translation product occurred only in the presence of purified Vt, but not BSA or LPI. Vt synthesized in the translation system had a significantly lower molecular weight than that of the purified Vt or that synthesized by ovaries incubated in vitro. The possibility that this difference may be because of the nonglycosylated nature of a cell-free translation product was tested. The removal of oligosaccharides from purified Vt by enzymatic digestion with N-glycosidase F resulted in the appearance of a smaller polypeptide of 36 kDa, which reacted immunologically with Vt antiserum in a Western blot. The size of this fragment is very close to the molecular weight of the translation product.

Oligosaccharides in Canola Meal and Their Effect on Nonstarch Polysaccharide Digestibility and True Metabolizable Energy in Poultry

Balance studies utilizing laying hens and adult cockerels were conducted to determine the influence of oligosaccharides (raffinose and stachyose) present in canola meal (CM) on the digestibility of nonstarch polysaccharides (NSP) and on the TMEn of the meal. Ethanol extraction was used to produce oligosaccharide-free meal, and exogenous dietary enzymes (α-galactosidase and invertase) were employed to bring about oligosaccharide hydrolysis in the intestinal tract of the birds. In each of two balance trials, six hens individually housed were randomly allotted to each of the experimental diets in completely randomized design. Experiment 1 consisted of a factorial arrangement of treatments (two sources of protein with or without enzyme supplementation), whereas Experiment 2 consisted of five diets: semipurified CM control, semipurified ethanol-extracted CM; semipurified ethanol-extracted CM plus raffinose; conventional CM; and conventional ethanol-extracted CM. Elimination of oligosaccharides by the use of exogenous dietary enzymes had no effect on NSP digestion. Removal of oligosaccharides by ethanol extraction increased NSP digestibility from 4 to 8%. A more pronounced effect (17% NSP digestion) was noted in hens fed a wheat-based diet containing 30% oligosaccharide-free CM. This latter effect may have been due to the relatively high content of water-soluble polysaccharides contributed by the wheat portion of the diet. The TMEn content of ethanol-extracted CM was 2,302 kcal/kg as compared with 2,426 kcal/kg for untreated CM. The data indicate no advantage of oligosaccharide removal with regard to the nutritive worth of canola meal.

Significance of the carbohydrate moiety of the rat ovarian luteinizing-hormone/chorionic-gonadotropin receptor for ligand-binding specificity and signal transduction.

The contribution of the carbohydrate moiety of the rat ovarian luteinizing-hormone (LH)/chorionic-gonadotropin (CG) receptor to ligand-binding specificity and signal transduction was investigated by using glycosidases. Purified membranes from pseudo-pregnant rat ovaries were treated with neuraminidase or peptide N-glycosidase F, to remove terminal sialic acids and N-linked oligosaccharides of the receptor, respectively. Ligand blotting and densitometric scanning of the autoradiograms showed that 90-95% of the receptors were deglycosylated, and that desialylation was virtually complete. Neither the desialylated nor the deglycosylated receptors were able to bind human follicle-stimulating hormone or bovine thyroid-stimulating hormone, as revealed by competition binding experiments. The 50% effective dose of hCG for adenylate cyclase activation, as determined by measuring the formation of cyclic [32P]AMP from [alpha-32P]ATP for 15 min at 30 degrees C, was similar in the control and deglycosylated membranes: 10.2 +/- 3.3 nM and 12.2 +/- 3.8 nM respectively. The same was true for the time course of the basal, hCG- and forskolin-stimulated enzyme activity. In addition, removal of oligosaccharides from the receptor did not restore the ability of desialylated hCG, nor of the deglycosylated hormone, to stimulate adenylate cyclase. In conclusion, the carbohydrate moiety of the native membrane-inserted rat ovarian LH/CG receptor does not contribute to the ligand-binding specificity, and it is not required for the functional coupling of the occupied receptor and the adenylate cyclase system. These functions are associated with the polypeptide portion of the receptor.

Effect of domestic processing on oligosaccharide content of some dry legume seeds

Raffinose family oligosaccharides (raffinose, stachyose and verbascose) present in legume seeds have been reported to cause flatulence. The object of this work was to study the effect of methods of domestic processing, including soaking in tap water and 0.5% sodium bicarbonate solution, cooking and autoclaving of unsoaked and soaked seeds, on oligosaccharide contents of four commonly consumed legumes in Egypt, namely: faba bean ( Vicia faba L.), lentil ( Lens culinaris), common bean ( Phaseolus vulgaris) and cowpea ( Vigna sinensis). The identification and quantitative determination of oligosaccharides using gas-liquid chromatography (GLC) indicated that the four raw legume seeds contained sucrose, raffinose, stachyose and verbascose. The raffinose family oligosaccharides account for 67.3, 63.2, 53.0 and 51.0% of the total soluble sugars in cowpea, faba bean, lentil and common bean, respectively. The major oligosaccharide was verbascose in faba bean and stachyose in the other three legumes. The soaking of legume seeds in tap water or sodium bicarbonate solution resulted in a decreased concentration of sucrose and the raffinose family of sugars in each case. The extent of losses was enhanced as the time of soaking was increased. The removal of oligosaccharides from legume seeds was higher in alkaline medium than in water. In addition, the loss of an individual sugar was not related to its solubility or molecular weight. Cooking of unsoaked seeds led to a large decrease in oligosaccharide content of all studied legumes compared with soaking for 12 h. The loss in the raffinose family oligosaccharides after cooking of soaked seeds was highest in common bean and lowest in faba bean. Autoclaving had a greater effect on removal of oligosaccharides than did cooking. In all cases, autoclaving of soaked seeds resulted in complete disappearance of raffinose.

The influence of N-linked glycosylation on the antigenicity and immunogenicity of rubella virus E1 glycoprotein

Rubella virus E1 glycoprotein contains three functional Winked glycosylation sites. The role of Winked glycosylation on the antigenicity and immunogenicity of E1 glycoprotein was studied using vaccinia recombinants expressing E1 glycosylation mutants. Expressed E1 glycosylation mutant proteins were recognized by a panel of E1-specific monoclonal antibodies in radioimmunoprecipitation, immunofluorescence, and immunoblotting, indicating that carbohydrate side chains on E1 are not involved in the constitution of epitopes recognized by these monoclonal antibodies. This observation was further supported by the fact that removal of oligosaccharides on E1 by glycosidase digestion did not significantly change the antigenicity of E1. All the glycosylation mutants were capable of eliciting anti-RV E1 antibodies. The single glycosylation mutants (G1, G2, and G3), but not the double mutant (G23) or the triple mutant (G123), were found to be capable of inducing virus neutralizing antibodies. Among the single glycosylation mutants, only G2 and G3 were active in producing hemagglutination inhibition antibodies in mice. Our findings suggest that although carbohydrate on E1 is not directly involved in the antigenic structures of E1, it is important in maintaining proper protein folding and stable conformation for expression of immunological epitopes on E1.

Effect of Ethanol Extract on Nitrogen-Corrected True Metabolizable Energy for Soybean Meal with Broilers and Roosters

Forty-day-old Arbor Acre × Ross male broilers were precision-fed 44% soybean meal (SBM) or 80%-ethanol extracted SBM (ESBM) to determine differences in TMEn values due to the removal of oligosaccharides. The SBM had a dry matter crude protein content of 47.2% and the TMEn value of 2,546 kcal/kg. The ESBM had a dry matter crude protein content of 64.4% and TMEn value of 3,117 kcal/kg. In a second experiment, Leghorn roosters were precision-fed SBM, ESBM, ESBM containing 5.3% raffinose (RAF), or ESBM plus the alcohol extract (EXT). The TMEn of ESBM with Leghorn roosters was approximately 420 kcal/kg higher than that of SBM. The TMEn values for RAF and EXT were similar to that of SBM. In a third experiment, SBM and freeze-dried ESBM (FDE) were precision-fed to Leghorn roosters. Excreta were collected for 48, 72, and 96 h. The TMEn values did not differ significantly with the longer collection times. Significantly higher TMEn values were observed for FDE than for SBM for all three collection periods.Alcohol extraction increased the TMEn of SBM as determined with both roosters and broilers. The recombination of the alcohol extract or an equivalent amount of raffinose to the ESBM resulted in TMEn values that were not significantly different from that of SBM.

Removal of N-linked oligosaccharides of presumptive ectoderm impairs neural induction in Pleurodeles waltl

Studies were carried out on the embryo of the amphibian Pleurodeles waltl to investigate the potential role of the N-linked oligosaccharides of the ectodermal cell membrane in the neural induction process. Glycopeptidase F (GPase F) was used to cleave N-linked oligosaccharides on presumptive ectoderm. Removal of oligosaccharide moieties from ectoderm membrane glycoconjugates completely inhibited natural neural induction in vitro. On the other hand, Swainsonine (Sw) and 1-deoxynojirimycin (dNM), specific inhibitors of enzymes involved in glycosylation, provoked strong and persistent changes in the structure of the N-linked oligosaccharides of presumptive ectoderm but did not prevent neuralisation of treated ectoderm. We conclude that N-linked carbohydrates are implicated in the phenomenon of neural induction. However, the structural integrity of N-linked carbohydrates of target tissue is not itself critical in this process. The existence of specific carbohydrates on presumptive ectoderm was still questioned as receptors of neural signal.

Synthesis and glycosylation of fibronectin in hepatocytes from vitamin A-deficient rats

Recent work from our laboratory (Kim and Wolf, J Biol Chem 262:365-371, 1987) has shown increased uptake of labeled amino acids into fibronectin (FN), increased net synthesis of FN and increased levels of FN-mRNA in primary cultures of hepatocytes from vitamin A-deficient rats compared to controls. We now find, surprisingly, decreased uptake of labeled sugars into the oligosaccharide chains of FN from vitamin A-deficient hepatocytes. This decrease could be reversed by added retinoic acid at physiological concentration. At the same time, FN from deficient hepatocytes (-A.FN) was more susceptible to proteolytic degradation. Decreased uptake of the core sugar mannose into -A.FN was similar to that of glucosamine, yet the percent of label in sialic acid was the same as in + A.FN, suggesting a smaller number of oligosaccharide chains per molecule of -A.FN. Upon enzymatic removal of oligosaccharide and labeling with sodium borotritide, it was found that both -A.FN and +A.FN had biantennary oligosaccharide structures. Selective enzymatic removal of sialic acid showed that +A.FN had both sialic acids in an alpha 2----3 linkage, whereas -A.FN apparently had one alpha 2----3 and one alpha 2----6-linked sialic acid. The borotritide experiments allowed us to calculate that +A.FN appeared to have 5 oligosaccharide chains per FN monomer, whereas the -A.FN showed only 4 chains. These results would account for the decreased glycosylation and increased susceptibility to proteolysis of the -A.FN. We conclude that vitamin A controls both the rate of synthesis of the polypeptide chain of FN via its mRNA, as well as the rate of its glycosylation.

Effect of Oligosaccharide-Free Soybean Meal on True Metabolizable Energy and Fiber Digestion in Adult Roosters

Adult Leghorn roosters were precision-fed 44% protein soybean meal (SBM) or 80% ethanol-extracted soybean meal (ESBM) to determine nutritional differences due to the removal of oligosaccharides. Dry-matter digestibility and TMEn, as well as apparent hemicellulose and cellulose digestion were determined. The transit time, rate of passage, and pH of the cecal contents were also established with roosters that had consumed a .3% chromic-oxide diet that contained SBM or ESBM.The protein content of the SBM and the ESBM was 46.1 and 64.4%, respectively. The true-dry matter digestibility of SBM was 54%. The TMEn of the SBM on a dry-matter basis was 2,794 kcal per kg; and the apparent hemicellulose and cellulose digestibility was 9% and 0%, respectively. The true dry-matter digestibility of the ESBM was 67%. On a dry-matter basis, the TMEn of the ESBM was 3,368 kcal per kg; and the apparent hemicellulose and cellulose digestibility was 62% and 35%, respectively. Oligosaccharide digestion occurred mainly anterior to the ileo-cecal junction. Hemicellulose and cellulose digestion occurred predominantly in the lower gastrointestinal tract.The transit time for the ESBM diet was 115 min, compared to 71 min for the SBM diet. Based on a 16-h collection of excreta, the rate of passage was approximately 50% slower for the ESBM diet than for the SBM diet. The pH of the cecal contents from roosters consuming the ESBM diet was 7.21 versus 6.51 for the SBM diet.

Oligosaccharides in several Philippine indigenous food legumes: Determination, localization and removal

The oligosaccharide profile of raw mature seeds of seven different legumes indigenous to the Philippines was measured in 70% ethanol extracts of the seeds by thin layer chromatography using HPTLC plates and quantified by a densitometer. Based on the results, the legumes could be ranked according to decreasing oligosaccharide content or flatulence potential as follows: Sam-samping (Clitoria ternatea) greater than hyacinth bean (Dolichos lablab) greater than sabawel (Mucuna pruriens) greater than lima bean (Phaseolus lunatus) greater than swordbean (Canavalia gladiata) greater than rice bean (Vigna umbellata) greater than jack bean (Canavalia ensiformis). Sam-samping had 4.79% total oligosaccharides and hyacinth bean or batao, 3.66%. A jack bean accession had 1.79% oligosaccharides. Simple processing methods were tested to detoxify the oligosaccharides. Soaking the batao seeds had no effect while boiling even resulted in a net 23-31% increase in the levels of raffinose, stachyose and verbascose. On the other hand, two min of dry roasting resulted in complete removal of oligosaccharides whereas germination resulted in about 30-40% decrease after 1 and 2 days, respectively.

Isolation and characterization of multiple variants of recombinant human interleukin 4 expressed in mammalian cells.

We have purified recombinant human interleukin 4 (huIL-4), formerly named B-cell stimulatory factor-1, from supernatants of COS-7 monkey kidney and L-929 cells transfected with the cDNA for huIL-4. The purified protein exhibited a specific activity of 2.6 X 10(7) units/mg in a T-cell proliferation assay and consisted of multiple components on sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibiting Mr values of 15,000, 18,000, and 19,000. All forms of huIL-4 eluted on gel filtration chromatography with an apparent Mr of 22,000. Gas-phase microsequencing identified 26 and 8 amino acid residues at the N and C termini, respectively, all of which were consistent with the cDNA sequence. The site of processing of the signal sequence was found to occur between Gly-24 and His-25. Incubation with N-glycanase converted the 18- and 19-kDa variants to a 15-kDa form. Treatment with endo-beta-N-acetylglucosaminidase H reduced the molecular mass of the 18-kDa variant to 15 kDa, but did not have any apparent effects on the mass of the 19-kDa species. The removal of oligosaccharide by any of these treatments did not affect bioactivity in the T-cell proliferation assay. Neither O-glycanase nor endo-beta-N-acetylglucosaminidase D affected the molecular weight of any of these species. These data suggest that differences in carbohydrate structure account, at least in part, for the observed microheterogeneity.

Significant steroidogenic activity of luteinizing hormone is maintained after enzymatic removal of oligosaccharides.

Several reports have described the destruction of the N-linked oligosaccharides on glycoprotein hormones by hydrogen fluoride treatment and have noted the accompanying marked reduction, or complete loss, in biological activity. This has led to the concept that the oligosaccharides have an obligatory role in glycoprotein hormone steroidogenic function. Using a less radical and more complete method for removing sugar units, endoglycosidase treatment and ovine LH (oLH) and human LH (hLH) as examples, we examined the role of oligosaccharides in hormone function. Ovine LH and hLH were digested with endoglycosidase F. After treatment cleavage of oligosaccharides was demonstrated by compositional studies, greater than 87% cleavage was demonstrated and only N-acetylglucosamine or N-acetylglucosamine-Fucose shown to remain attached to the peptide, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (appropriate size change) and by chromatofocusing (appearance of a single basic peak). Biological activities and relative potencies of preparations were then assessed in an in vitro assay, in which the ability of samples to promote testosterone production by testicular interstitial cells was measured. Although endoglycosidase F treatment reduced relative potencies 2- to 3-fold in the bioassay, (possibly in part due to subunit dissociation) it did not lessen abilities to induce maximal testosterone response (that of native hLH and oLH). These findings contrast with those obtained from hydrogen fluoride studies and indicate that the oligosaccharides, per se, do not play an obligatory role in the steroidogenic activity of LH.

The release of oligosaccharides from glycoproteins by endo- β- N-acetylglucosaminidase of Flavobacterium sp.

Endo- β- N-acetylglucosaminidase, purified to homogenicity from the cultural filtrate of Flavobacterium sp., liberated oligosaccharides from various glycoproteins. The enzyme could liberate the carbohydrate chain from native ovalbumin. The release of oligosaccharides from ribonuclease B, yeast carboxypeptidase and a Ricinus lectin was also observed. These glycoproteins contain neutral oligosaccharides that are attached to the protein through glycosyl asparagine bonds. The treatment of glycoprotein with SDS and boiling was more effective for removal of oligosaccharides by the enzyme. The enzyme hydrolyzed all five heterogeneous ovalbumin glycopeptides, although the rate of hydrolysis decreased as the size of the sugar moiety increased. Removal of the neutral oligosaccharides did not appear to effect the enzymatic properties of the hemagglutination ability of these glycoproteins.

Microscale elucidation of an N-linked glycosylation site by comparative high-performance liquid chromatographic peptide mapping

The identification of specific sites of post-translational modifications of polypeptides is an important component in understanding protein structure—function relationships. This report describes the application of specific enzymatic methods of N-linked oligosaccharide removal ( endo H and endo F) to the rapid microscale identification of specific sites of N-linked glycosylation. Following deglycosylation the protein is subjected to proteolytic digestion and comparative high-performance liquid chromatographic peptide mapping with an unmodified protein digest. Peptides from which oligosaccharide(s) have been removed exhibit a measurable increase in retention time on reversed-phase high-performance liquid chromatography and can be clearly identified. Once identified, the peptide can be subjected to direct protein microsequence analysis to elucidate the specific site of glycosylation. As illustrated here, these methods are compatible with microscale protein purification by sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by electroelution and are applicable to the 100-pmol range.

Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-β- N-acetylglucosaminidase H

The ability of endo-β-acetylglucosaminidase H (Endo H) from Streptomyces plicatus to hydrolyze high-mannose oligosaccharides from glycoproteins is influenced by numerous factors, including the tertiary structure of the substrate glycoproteins, the amount of Endo H used, the time of incubation, and the presence or absence of reagents that affect protein configuration. Endo H levels below 10 to 20 milliunits/ml may incompletely hydrolyze oligosaccharides, regardless of the incubation time, because even though the enzyme remains active, it becomes trapped or sequestered and is unavailable. Endo H activity can be potentiated by first denaturing substrate glycoproteins in a 1.2-fold weight excess of sodium dodecyl sulfate prior to hydrolysis. However, low levels of Endo H are sensitive to inactivation by sodium dodecyl sulfate, with considerable activity being lost over 4 h when the unbound detergent concentration exceeds protein by 0.02% (0.2 mg/ml). Other denaturants such as the Tritons, the zwittergents, the Brij series, or octylglucoside do not enhance or inhibit Endo H removal of oligosaccharides, but the chaotropic salt sodium thiocyanate at 0.5 m enhances Endo H action on some glycoproteins, particularly bovine thyroglobulin. Under denaturing conditions, proteolytic contaminants are a potential problem. Addition of 1 m m phenylmethylsulfonyl fluoride to Endo H incubations completely inhibits the residual Endo H-associated protease(s). Furthermore, Endo H is unaffected by a wide range of proteolytic inhibitors that may be used to protect substrate glycoproteins.

REMOVAL OF OLIGOSACCHARIDES FROM SOYBEAN WATER EXTRACTS BY ULTRAFILTRATION

ABSTRACTThe feasibility of using ultrafiltration (UF) for removal of oligosaccharides from aqueous extracts of soybeans was investigated. Soybeans were soaked, blanched, ground, prefiltered, and the resulting full‐fat extract processed in a hollow fiber UF unit. Initial permeate flux of 43‐60 L/m2/hr at 3.5% total solids decreased semi‐logarithmically to 13 L/m2/hr at 14% total solids. Low pH resulted in higher flux. Rates of removal of oligosaccharides closely followed theoretical behavior for a nonrejected solute during ultrafiltration and continuous diafiltration, and up to 96% could be removed by a two‐stage UF process. Final product assayed 60% protein, 35% fat and 0.6% oligosaccharides (dry basis).