Isolation and characterization of two individual glycoprotein components from human milk-fat-globule membranes.

Two individual glycoprotein components from human milk-fat-globule membranes (MFGM) has been purified by selectively extracting the membrane glycoproteins followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-disaggregating agents. The purified glycoprotein components, termed 'epithelial-membrane glycoprotein' (EMGP-155 and EMGP-39) have estimated molecular weights of 155 000 and 39 000 respectively, and yield a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide gel. EMGP-155 and EMGP-39 contain 21.0% and 7.0% carbohydrate by weight, with fucose (13.5%, 12.4%), mannose (3.7%, 6.2%), galactose (28.5%, 22.6%), N-acetylglucosamine (17.8%, 7.4%) and sialic acid (36.4%, 51.4%) of the carbohydrate moiety respectively. For both the glycoprotein components, aspartic and glutamic acid and serine are the major amino acid residues.     

Isolation and characterization of a major glycoprotein from milk-fat-globule membrane of human breast milk.

A major periodate--Schiff-positive component from milk-fat-globule membrane of human breast milk has been purified by selectively extracting the membrane glycoproteins, followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-dissociating agents. The purified glycoprotein, termed epithelial membrane glycoprotein (EMGP-70), has an estimated mol.wt. of 70 000 and yields a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The glycoprotein contains 13.5% carbohydrate by weight, with fucose, mannose, galactose, N-acetylglucosamine and sialic acid 17.2, 17.0, 21.1, 7.9 and 36.6% respectively of the carbohydrate moiety. Aspartic and glutamic acid and serine are the major amino acid residues.     

Delta 6-desaturase activity in liver microsomes of rats fed diets enriched with cholesterol and/or omega 3 fatty acids.

A glycoprotein antigen was purified from human brain by immunoaffinity chromatography using the 44D10-monoclonal IgG, and its chemical nature was investigated. The yield of antigen was estimated at 91% and a 4340-fold purification was obtained relative to the white-matter homogenate. The antigen preparation from brain was further purified by preparative SDS/polyacrylamide-gel electrophoresis (PAGE) to obtain a glycoprotein with an Mr of 80,000 consisting of a single polypeptide. Amino acid analyses revealed a composition which was high in acidic and neutral amino acids, and low in basic residues. The presence of both glucosamine and galactosamine suggested that the glycoprotein contained both N- and O-linked glycans. Neutral sugar analyses showed that fucose, galactose and mannose were present. An assay for sialic acid determined that there were approximately 20 mol of sialic acid per mol of glycoprotein. Chemical cleavage of oligosaccharides by trifluoromethanesulphonic acid followed by SDS/PAGE showed that carbohydrate accounted for 25,000 of the 80,000-Mr glycoprotein.     

Isolation and partial characterization of rat duodenal-gland (Brunner''s-gland) mucus glycoprotein.

A mucus glycoprotein was isolated from the duodenal glands of the rat and purified by repeated density-gradient centrifugation. The characterized glycoprotein is unique to the mucous cells of the duodenal glands and is not present in parts of the small intestine devoid of these glands. The chemical composition of the purified glycoprotein is characteristic for glycoproteins of the mucin-type. Its protein content is relatively high and amount to 35% by weight. No neuraminic acid and little sulphate (2%) is present. Evidence is presented that the native glycoprotein is built up from subunits held together via disulphide bridges in a non-glycosylated region of the protein core.     

Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep.

Crude soluble mucus from sheep small intestine was freed of nearly all the nucleic acid contaminants by precipitation with protamine sulphate and treatment with nucleases. After removal of non-covalently bound proteins by equilibrium density-gradient centrifugation in CsCl, a high-Mr glycoprotein was isolated by repeated h.p.l.c. from the partially purified mucin. The high degree of purity of the high-Mr mucin was borne out by (a) the observation of a single boundary on analytical ultracentrifugation in the presence of 5M-guanidinium chloride and (b) the observation of apparent monodispersity on sedimentation-equilibrium analysis. The Mr of the highly purified mucin, determined by sedimentation equilibrium, was 5.0 (+/- 0.1) X 10(6) and was concentration-independent. Finally, only goblet cells and the mucus blanket lining the intestinal epithelial cells were immunofluorescent when guinea-pig anti-(highly purified mucin) serum was used in an indirect immunofluorescence assay. The above antiserum reacted with apparently equal strength with goblet cells and with free mucin in abomasum, caecum and colon. The chemical composition of the glycoprotein was 66% carbohydrate and 34% protein, 45% of the latter being composed of valine and threonine. The glycoprotein migrated anodally on immunoelectrophoresis and contained 7.1% (w/w) sulphate. Neutral hexoses accounted for nearly half of the total carbohydrate content, followed by galactosamine and glucosamine. Whereas fucose and sialic acid were present in only small amounts, uronic acid was not detectable in the highly purified mucus glycoprotein.     

Characterization of a high-molecular-weight glycoprotein isolated from the pulmonary secretions of patients with alveolar proteinosis

A high-molecular-weight glycoprotein was isolated, purified and partially characterized from the insoluble pulmonary secretions accumulating in lungs of patients suffering from pulmonary alveolar proteinosis. On electrophoresis in 5% polyacrylamide gel in the presence of sodium dodecyl sulphate and 2-mercaptoethanol, the purified protein gave one major band as detected by Coomassie Blue as well as with periodic acid/Schiff staining. An apparent mol.wt. of 250000 was estimated for this glycoprotein. Amino acid analysis showed that it contains hydroxyproline, and relatively high amounts of glycine, glutamic acid, aspartic acid and leucine. It contains approx. 6% hexose, 3% sialic acid and 2% glucosamine. The neutral sugars are galactose, mannose and fucose. An antiserum prepared in rabbits against this high-molecular-weight glycoprotein cross-reacted with two smaller glycoproteins (mol.wts. 62000 and 36000) isolated from the same pulmonary secretions of these patients. A complementary observation was also made when this large alveolar glycoprotein cross-reacted with an antiserum prepared in rabbits against the smaller glycoprotein (mol.wt. 36000). It appears that this high-molecular-weight glycoprotein may be the precursor of the two smaller glycoproteins present in the same diseased pulmonary secretions.     

Characterization of pepsin-resistant peptides present in a glycoprotein isolated from lung lavage of normal rabbit

A glycoprotein of Mr 36 000 has been isolated from lung lavage of normal rabbit and purified to homogeneity by gel chromatography. Three peptides containing hydroxyproline and nearly 30% glycine have been isolated and purified from pepsin-digested native glycoprotein. Partial NH2-terminal amino acid sequence analysis on one of the peptides indicated the presence of -Gly-Pro-Hyp-Gly- sequence in the peptide chain, suggesting that collagen-like region(s) may be present in this glycoprotein.     

Further Studies of Pollen Wall Glycoproteins in Cucurbita pepo L.

Samples of pollen wall protein of Cucurbita pepo were prepared as reported in previous paper. Gas chromoatographic analyses snowed that the carbohydrate fraction of the pollen wall glycoprotcin contained 20.4% rhamnose, 15.3% fucose, 11% mannose, 11% galactose, 31% glucose, 4% arabinose and traces of xylose. The glycoproteins were further purified by Con. A affinity chromatography, Isoelectric focussing electrophoresis of the purified sample showed 3 PAS-positive bands, with respective PI 5.2, 6.0 and 6.3. The glycoprotein samples were subjected to hydrolysis with 6N HC1. After hydrolysis, the mixture was analyzed for amino acid composition with Backman 121-MB automatic amino acid analyzer, Results show the amino acid composition of the 3 glycoprotein was very similar, They all have glycine, glutamic acid and serine as their major component (these three amino acids constitute 50–60% of the total amino acids); and they all contain very small amount of methionine, phenylalanine, isoleucine and tyrosine. The lysine content of each glycoprotein is consistent with its respective PI, the glycoprotein which contains more lysine has higher PI.     

In vitro acylation of rat gastric mucus glycoprotein with [3H]palmitic acid

The incorporation of fatty acids into gastric mucus glycoproteins was studied by incubating rat gastric mucosal cell suspensions with [9,10-3H]palmitic acid and [3H]proline. The mucus glycoprotein polymer, secreted into the growth medium (extracellular) and that contained within the cells (intracellular), was purified from the other components of the secretion, thoroughly delipidated, and then analyzed for the radiolabeled tracers. Both pools of mucus glycoprotein, incubated in the presence of [3H]palmitic acid, contained radioactive label which could not be removed by gel filtration, CsCl density gradient centrifugation, sodium dodecyl sulfate-gel electrophoresis, or lipid extraction. Treatment of the purified mucus glycoprotein with 1 M hydroxylamine or 0.3 M methanolic KOH released the radioactivity, thus indicating that [3H]palmitic acid was covalently bound by ester linkage to the glycoprotein. The released radioactivity was associated mainly (87%) with palmitic acid. The incorporation ratio of [3H]proline to [3H]palmitic acid was 0.12:1.0 in the extracellular glycoprotein and 1.38:1.0 in the intracellular glycoprotein, which suggested that acylation of mucus glycoprotein occurs in the intracellular compartment after completion of its polypeptide core. The fact that incorporation of [3H]palmitic acid was greater in the glycoprotein subunits than in the glycoprotein polymer indicates that acylation takes place near the end of subunit processing but before their assembly into the high molecular weight mucus glycoprotein polymer.     

Purification, physicochemical characterization, and immunohistochemical localization of a major 11.7 S glycoprotein from the jelly coats of the anuran Lepidobatrachus laevis

Embryos of the frog Lepidobatrachus laevis are encased by a fertilization envelope and two jelly layers, termed J1 (innermost) and J2 (outermost). From preparations of total jelly solubilized from cleavage-stage embryos by a solution of alkaline beta-mercaptoethanol we have purified one jelly coat glycoprotein to homogeneity via FPLC gel permeation chromatography on Superose 6H. The purified glycoprotein was 94% protein and 6% carbohydrate, had an s0(20),w of 11.7 S, with a molecular weight of 245,000 measured by sedimentation equilibrium and 263,000 by gel permeation chromatography. SDS-PAGE revealed that the glycoprotein is composed of a single subunit near 29,700 molecular weight; thus we propose that eight of these subunits comprise the native molecule. Amino acid analysis of the glycoprotein indicated a high content of Glx + Asx (32.4 mole%), a low content of basic amino acids (Arg + Lys = 12.2 mole%), and a single cysteine residue per subunit. The N-terminal amino acid was threonine and the sequence of the first twenty amino acids was determined. Monospecific antisera to the glycoprotein were prepared in rabbits and were used to immunohistochemically localize the glycoprotein throughout the matrix of both jelly layers. Antiserum against the glycoprotein had virtually no effect on the fertilizability of jellied eggs in vitro; thus we hypothesize that the glycoprotein fulfills a structural role in both jelly layers.     

Isolation and partial characterization of the major glycoprotein from the plasma membranes of AH-66 hepatoma cells.

A major glycoprotein of the plasma membranes of AH-66 hepatoma ascites cells was isolated in essentially pure form and in milligram amounts. The plasma membranes were solubilized with a solution containing both 0.3 M lithium diiodosalycylate and 0.2% cetylpyridinium chloride, and further extracted with 50% phenol, followed by gel filtration on Sepharose 6B in the presence of 0.1% Ammonyx-LO at pH 8.0. The apparent molecular weight of the purified glycoprotein was estimated to be 165 000 in 5.6% polyacrylamide gels, of which 54% was carbohydrate and 46% was protein. The chemical composition of the glycoprotein resembles glycophorin A from human erythrocyte membranes in that it has a high content of N-acetylgalactosamine, N-acetylglucosamine, galactose and sialic acid and a particularly large proportion of serine, threonine, aspartic acid and glutamic acid.     

One-step purification of the serotonin transporter located at the human platelet plasma membrane.

A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.     

Purification and characterization of proline/hydroxyproline-rich glycoprotein from pearl millet coleoptiles infected with downy mildew pathogen Sclerospora graminicola

Hydroxyproline-rich glycoproteins (HRGPs) are important plant cell wall structural components, which are also involved in response to pathogen attack. In pearl millet, deposition and cross-linking of HRGPs in plant cell walls was shown to contribute to the formation of resistance barriers against the phytopathogenic oomycete Sclerospora graminicola. In the present study, the purification and characterization of HRGPs that accumulated in coleoptiles of pearl millet seedlings in response to S. graminicola inoculation has been carried out. Periodic acid Schiff's staining revealed that the purified protein was a glycoprotein. The protein to carbohydrate ratio was determined to be 95.5%:4.5% (w/w). Proline amounted for 20 mol% of the total amino acids as indicated by amino acid composition analysis. The isolated protein had a pI of 9.8 and was shown to be composed of subunits of 27, 17, and 14 kDa. Cross reactivity with the monoclonal antibody MAC 265 and the presence of the signature amino acid sequence, PVYK, strongly suggested to classify the purified glycoprotein as a member of the P/HRGPs class. In the presence of horseradish peroxidase and H2O2 the purified glycoprotein served as a substrate for oxidative cross-linking processes.     

Purification and chemical characterization of the major glycoprotein of avian myeloblastosis virus.

The major glycoprotein of avian myeloblastosis virus (AMV) has been purified to an apparent state of homogeneity by gel filtration on a Sepharose 4B column in the presence of 6 m guanidine hydrochloride followed by dialysis against distilled water and then extraction with chloroform-methanol. The AMV glycoprotein remains soluble in the aqueous phase whereas contaminating proteins precipitate, either upon dialysis against distilled water or after treatment with chloroform-methanol.Carbohydrate, represented by glucosamine, mannose, galactose, fucose, and sialic acid, constitutes 40% of the weight of AMV glycoprotein. Glucosamine is the major carbohydrate component whereas fucose and sialic acid are present in relatively low amount. Amino acid analysis indicates a relatively high content of aspartic and glutamic acid, serine, threonine, and glycine. Based on SDS-polyacrylamide gel electrophoresis, a molecular weight value of 77,500 ± 500 was determined for AMV glycoprotein.     

A collagenous glycoprotein found in dissociative extracts of foetal bovine nuchal ligament. Evidence for a relationship with type VI collagen.

A collagenous glycoprotein (Mr 140000) was isolated from dissociative extracts of foetal bovine nuchal ligament and purified by a combination of ion-exchange and gel-filtration chromatography. This glycoprotein (designated MFPI) exists as a large-Mr disulphide-bonded aggregate in the absence of a reducing agent. The purified glycoprotein was shown to contain about 6% (w/w) carbohydrate, mostly as galactose, glucose and mannose. Amino acid analysis showed the presence of hydroxyproline and hydroxylysine, indicative of its collagenous nature. The collagenous nature of this glycoprotein was further investigated by enzyme digestion. Pepsin digestion produced three major fragments, which were identical with peptides of type VI collagen. Bacterial-collagenase digestion of the unreduced glycoprotein also produced several discrete peptides. However, reduction of the glycoprotein before bacterial-collagenase digestion resulted in the degradation of these discrete peptides. Glycoprotein MFPI extracted in dissociative conditions appears to be a larger-Mr form of type VI collagen, believed to originate from microfibrillar components in the intact tissue.     

Extracellular hydroxyproline-rich glycoprotein of suspension-cultured tobacco cells

Hydroxyproline-rich glycoprotein was isolated from the used media of tobacco XD-6 cells cultured in suspension and purified by repeated ion exchange- and gel filtration-chromatography. The preparation was judged to be homogeneous from ultracentrifugation and isoelectric focusing. The dry wt of the glycoprotein was composed of 94% polysaccharide and 6% protein. Hydroxyproline accounted for 23% of the amino acids in the protein moiety. The polysaccharide moiety consisted of 44% galactose, 30% arabinose, 5% rhamnose, and 21% uronic acid. About 10% of the uronic acid residues were present as methyl esters.     

Purification and characterization of a major glycoprotein in rat hepatoma plasma membranes. One of the membrane proteins released by phosphatidylinositol-specific phospholipase C.

A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen.     

Identification of the milk fat globule membrane proteins: I. Isolation and partial characterization of glycoprotein B

The salt soluble proteins from the fat globule membrane of cow's milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.     

Purification and some chemical properties of 30 kDa Ginkgo biloba glycoprotein, which reacts with antiserum against beta 1-->2 xylose-containing N-glycans

From the seeds of Ginkgo biloba, a glycoprotein, which is a major component that reacts with an antiserum against beta 1-->2 xylose-containing N-glycans, has been purified and characterized. The N-terminal amino acid sequence of the purified glycoprotein was H-K-A-N-X-V-T-V-A-F-V-M-T-Q-H-L-L-F-G-Q-. The molecular mass was estimated to be 17 kDa and 16 kDa by SDS-PAGE under reducing conditions, however, the molecular mass of this glycoprotein in the native state was 30,762 by MALDI-TOF MS, suggesting that this glycoprotein consists of two subunits; one is glycosylated and the other is not. The structure of N-glycan linked to this glycoprotein (designated 30 kDa GBGP) was identified as Man3Fuc1Xyl1GlcNAc2, which is the predominant N-glycan linked to the storage glycoproteins in the same seeds (Kimura, Y et al. (1998) Biosci. Biotechnol. Biochem. 62, 253-261). From the peptic digest of the carboxymethylated glycosylated subunit, one glycopeptide was purified by RP-HPLC and the amino acid sequence was identified as H-K-A-N-N(Man3Fuc1Xyl1Glc-NAc2)-V-T-V-A-F, which corresponded to the N-terminal amino acid sequence.     

Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin

The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.