Sulfated Glycopeptide Research Articles

A method for the identification of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane.

Two-dimensional electrophoresis on cellulose acetate membrane permits the clean separation of sulfated glycopeptide in a mixture of acidic glycans (glycosaminoglycans and acidic glycopeptides). Two systems were used. In system 1, 0.1 M pyridine-0.47 M formic acid buffer (pH 3.0) was used in the first and 0.1 M barium acetate (pH 8.0) in the second dimension. In system 2, 0.1 M pyridine-0.47 M formic acid buffer (pH 3.0) was used in the first and 0.1 M HCl in the second dimension. All of the acidic glycans on electrophoretogram were stained with alcian blue in 70% ethanol. On the other hand, sulfated glycans alone were made visible with alcian blue in 0.1 M HCl. Alcian blue in 70% ethanol or 0.1 M HCl, when combined with periodic acid-Schiff's reagent identified sulfated glycopeptides on cellulose acetate membrane.

Sulfated glycopeptides, containing desmosine and isodesmosine, isolated from porcine aorta.

Intima-media of porcine thoracic aorta was digested with pronase, after extraction of saline-soluble matters and fat. A crude sulfated glycopeptide fraction (CSGP) was precipitated with 90% (v/v) ethanol from the 80% ethanol-soluble fraction of the trichloroacetic acid (7%)-soluble fraction of the pronase digest. CSGP was fractionated by DEAE-Sephadex A-25 (Cl- form) column chromatography. Of the resulting 9 fractions, 4 major fractions were further purified by gel filtration on Sephadex G-50, followed by affinity chromatography on concanavalin (Con) A-Sepharose 4B to a homogeneous state in electrophoresis on cellulose acetate membrane. All the purified fractions contained glucosamine, galactose, mannose, and sialic acid as the major sugars, and desmosine and isodesmosine as the unique constituents. The fractions with affinity for Con A (S1 and S2) contained much more mannose than those without affinity for this lectin (S3 and S4). The latter contained sulfate. The predominant amino acids in the former were glycine, aspartic acid (and/or asparagine), and serine, while those in the latter were glycine, proline, and alanine.

Separation and characterization of sulfated glycopeptides from ovomucin, chalazae and yolk membrane in chicken eggs.

The sulfated glycopeptides in ovomucin, chalazae and yolk membrane were isolated from the proteolytic digests by gel filtration on a Bio-Gel P-100 column and DEAE-Sephadex A-25 column chromatography. These sulfated glycopeptides contained N-acetylhexosamine (23.3-26.8%), hexose (23.6-24.4%), sialic acid (11.2-18.0%), sulfate (5-12.1%) and peptide (17.5-18.1%). The sulfate contents of glycopeptides in chalazae and yolk membrane were much higher than those in ovomucin, about two times in a molar ratio to hexosamine. The sedimentation patterns of each sulfated glycopeptide were single and the sedimentation constants were around 3 S, suggesting that these sulfated glycopeptides were macromolecular components. Thus, the presence of highly sulfated glycoproteins was confirmed in chalazae and yolk membrane, which were different from those in ovomucin.

Characterization of the sulfated glycopeptide of chicken pepsinogen

S-sulfonated chicken pepsinogen was digested with TPCK-trypsin; large tryptic peptides, separated on Sephadex G-25 fine, were subjected to additional cleavage with α-chymotrypsin. The hold-up fraction of the chymotryptic digest from the Sephadex G-25 column, was resolved by high voltage electrophoresis. The three most acidic zones contained glycopeptides of identical amino acid sequence Val-Ser-Thr-Asn-Glu-Thr-Val-Tyr, yet differed in the composition of the sugar moiety. These glycopeptides, moreover, bear different numbers of sulfate groups which enabled the resolution of the peptides. The most acidic glycopeptide contains 7 glucosamine residues, 3 mannose residues and 5 sulfate groups, the second one 6 glucosamine residues, 3 mannose residues and 4 sulfate groups and the slowest, minority glycopeptide, 5 glucosamine residues, 2 mannose residues and 2 sulfate groups. The entire sugar moiety is attached to one of the chain viaasparagine. In other experiments the glycopeptides were also isolated from the thermolytic digest of chicken pepsin; their C-terminal sequence was shorter by two amino acid residues. The tentative assignment of the glycopeptides to the amino acid sequence of pepsinogen resulted from the analysis of the limited tryptic digest of the whole protein molecule. Chicken pepsinogen is glycosylated at the site of the chain occupied by a phosphoserine residue in hog pepsinogen A.

Hormonal Effects on the Sulfation of Sulfated Glycoprotein in a Particulate Fraction of the Endometrium of Rabbit Uterus1

A particulate fraction was separated from endometrial scrapings of the uterus of hormone- or sham-treated ovariectomized rabbit. The effects of estrogen and progesterone on the incorporation of [35S]sulfate from 3'-[35S]phosphoadenosine 5'-phosphosulfate (PAPS) into endogenous acceptors in the particulate fraction were investigated. Estrogen increased the incorporation of [35S]sulfate, but progesterone suppressed this effect. The results of DEAE-Sephadex A-25 (Cl-form) column chromatography of the pronase digest of the 35S-labeled substances indicated that the fraction eluted with 0.9 M NaCl (0.9 Fr) was most sensitive to the hormones. The major component in 0.9 Fr was resistant to crude heparinase, whereas the minor component was susceptible to this enzyme. The present observation, together with previous findings, suggested that the former was sulfated glycopeptide and the latter heparan sulfate. The results of the present study indicated that PAPS can serve as the direct "activated" sulfate donor in the enzymatic sulfation of sulfated glycoprotein in the particulate fraction, that the sulfation is greatly stimulated by pre-treatment of the rabbit with estrogen, and that the estrogen effect is suppressed by progesterone.

Pattern of glycosaminoglycans and glycoproteins associated with nuclei of regenerating liver of rat

1. 1. Hyaluronic acid was detected as the largest glycosaminoglycan component in the glycosaminoglycan fraction from purified nuclei of regenerating livers as in the case of normal livers (Furukawa, K. and Tarayama, H. (1977) Biochim. Biophys. Acta 499, 278–289). However, the nuclear content of glycosaminoglycans tended to decrease after partial hepatectomy, reaching one-third of the normal liver level at 24–30 h after partial hepatectomy. On the other hand, two new polyanionic components were detected in the glycosaminoglycan fraction from regenerating liver nuclei. 2. 2. One of these new components seems to be a sulfated glycopeptide. The 35SO 4 incorporation into this component was stimulated biphasically after partial hepatectomy; the first stimulation occurring immediately after partial hepatectomy and the second stimulation occurring almost in parallel to the DNA synthesis. 3. 3. Another polyanionic component which also increases in the nuclear content after partial hepatectomy lacks hexuronic acid, sialic acid and 35SO 4 and yet it is intensely stained by Alcian Blue. Preliminary investigations revealed the presence of hexose, ribose and phosphate as the major components. 4. 4. In contrast to the primary localization of hyaluronic acid in the chromatin fraction and also in the nonhistone chromosomal protein fraction from it, these new polyanionic components were detected mainly in the karyosol fraction.

A method for micro-scale isolation of sulfated glycopeptide from tissue.

Micro-scale isolation of sulfated glycopeptide from tissue was achieved by successive application of pronase digestion (Step 1), cetylpyridinium chloride-fractionation (Step 2), crude heparinase digestion or chondroitinase ABC digestion plus nitrous acid treatment (Step 3) and preparative cellulose acetate membrane-electrophoresis (Step 4). By this method, sulfated glycopeptide was obtained in a high yield from estrogen-treated rabbit uterus.

Isolation of a sulfated glycopeptidic antigen from human gastric tumors: Its localization in normal and cancerous gastrointestinal tissues

A sulfated glycopeptidic antigen (SGA) was purified from papain-digested cancerous human gastric mucosa. The amino acid composition of this antigen was characterized by a high percentage of threonine and proline. Serine was present in small quantities and aromatic amino acids were absent. The amount of sulfate present was evaluated at 7.5%. Fucose, galactose, N-acetyl glucosamine, N-acetyl galactosamine and sialic acid were found to be present in the molar ratio 1:4.6:3.0:6.2:5.0. With immunofluorescence techniques, a rabbit antiserum against the sulfated glycopeptide stained adult gastric mucosa when this tissue had intestinal metaplasia and stained the goblet cells of the intestinal tract (small and large intestines). About 50% of colonic carcinomas and some gastric carcinomas contained SGA. This sulfated antigen was present in well-differentiated tumors and there was a good correlation between tumoral acid mucous secretory activity and the SGA positivity.

Metabolic rate of acidic complex saccharides in rabbit uterus under estrogenic condition.

Uterine slices obtained from estrogen-treated rabbits were incubated in vitro with N-acetyl-D-[1-3H]glucosamine together with D-[U-14C]glucose. The isotope-labelled acidic complex saccharides were then isolated by pronase digestion, Dowex 1 column chromatography and preparative electrophoresis on cellulose acetate membrane, in succession. In this way, individual acidic complex saccharides (hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, sulfated glycopeptide, and sialoglycopeptide) were separated into 2-5 subfractions. The specific radioactivity of hexosamine in the subfractions indicated that the metabolic rate of the uterine complex saccharides as follows: hyaluronic acid greater than sulfated glycopeptide greater than heparan sulfate greater than chondroitin sulfate C greater than dermatan sulfate. In addition, metabolic heterogeneity of heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, and dermatan sulfate was suggested.

Glycosaminoglycans and acidic glycoproteins in rabbit uterus under estrogenic conditions

Uterine slices obtained from the estrogen-treated rabbits were digested with pronase. Glycosaminoglycans and acidic glycopeptides were then isolated by Dowex 1 column chromatography and preparative electrophoresis on celulose acetate membrane (Separax), in succession. Each subfraction thus obtained was identified by the mobility on Separax electrophoresis and the digestibility with mucopolysaccharidases ( Streptomyces hyaluronidase, testicular hyaluronidase, chondroitinase AC, chondroitinase ABC and heparinase). The resulting data showed that each complex saccharide (hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, sulfated glycopeptide and sialoglycopeptide) was separated into 2–5 fractions, indicating charge and/or molecular heterogeneity of each complex saccharide.

Turnover of sulfate in glycopeptides from egg shell membranes and hen oviduct sulfated glycoproteins

Abstract The turnover of sulfated glycoproteins from oviduct and egg shell membranes was studied after in vivo labeling with radioactive sulfate. This study shows that sulfated glycopeptide A from egg shell membranes and from the isthmus and sulfated glycopeptide B from membranes and from the magnum turn over at the same rate. They have half-lives ranging from 1.2 to 1.5 days. The turnover of chondroitin sulfate, only present in the oviduct, is slower, with a half-life of about 4 days. UDP- N -acetylgalactosamine-4-sulfate (UDP-GalNAc-4-SO 3 H), the major sulfated sugar nucleotide of the isthmus also has a half-life of about 1.2 days which is incompatible with an active role in the biosynthesis of sulfated glycoproteins. These metabolic investigations together with structural data are in accordance with an identity between sulfated glycoproteins from egg shell membranes and sulfated glycoproteins secreted into the oviduct.

Antipeptic activity of a sulfate glycopeptide different behavior with purified human pepsin or human gastric juice.

A sulfated glycopeptide with antiulcer properties in animals and man was administered orally to 10 patients with duodenal ulcer, and its effects on peptic activity tested in vivo. A synthetic dipeptide,N-acetyl-l-phenylalanyl-l-diiodotyrosine, was used as substrate. Sulfated glycopeptide, 50 and 100 mg, completely inhibited peptic activity 15 minutes after administration in 2 of 3 patients and 3 of 3 patients examined, respectively; the inhibitory effect lasted 30 minutes. Sulfated glycopeptide, 200 mg, completely inhibited peptic activity in 4 of 4 patients examined 15 minutes after administration. In 2 patients complete enzymatic inhibition lasted 1 hour. In vitro, sulfated glycopeptide inhibited peptic activity of human gastric juice but not of purified human pepsin. These results may indicate that pepsin is present in gastric juice as a component of a mucoprotein complex. Sulfated glycopeptide may inhibit pepsin activity in gastric juice by directly precipitating this glycoproteic complex.

Inhibition of experimentally-induced gastric ulcers in the rat by a new sulfated glycopeptide

A sulfated glycopeptide (GLPS) obtained by sulfonation of a glycopeptide derived from pig duodenum was tested as an inhibitor of experimental gastric ulcers in rat. GLPS, given orally, (15–190 mg/kg) protected the rats against gastric ulceration induced by pyloric ligation or by administration of hypertonic glucose to fasting rats; after oral administration (10–25 mg/kg) it was active against hydrocortisone and restraint-induced ulcers. A protective effect of GLPS against Shay ulcers was also demonstrated after intraperitoneal or intravenous administration. A dose-response relationship was always found except for restraint ulcers. In pyloric-ligated rats GLPS revealed a strong anti-peptic activity and an inhibitory effect on gastric secretion when given orally. The latter effect was also seen after intravenous administration. On the basis of its anti-peptic and anti-secretory activity, an explanation of the protectice effect of GLPS against gastric ulceration is presented.

Sulfated glycopeptides and glycosaminoglycan peptides isolated from intestinal mucosae of rabbit

1. 1. Two sulfated glycopeptides and a highly sulfated uronic acid-containing glycosaminoglycan peptide were isolated in an highly pure state from protease-digested mucosal scrapings from either rabbit small intestine or colon. 2. 2. Small intestinal mucosa yielded much more highly sulfated fractions than did colonic mucosa. 3. 3. All the sulfated glycopeptides were similar in having galactose, equimolar ratios of glucosamine and galactosamine, the major carbohydrate components, with fucose and sialic acid in addition. Sulfate, present in each sulfated glycopeptide, had a molar ratio to hexosamine ranging from 0.11 to 0.41; this ratio increased with increasing molarity of eluting salt. 4. 4. The most acidic fraction, highly sulfated uronic acid-containing glycosamino-glycan peptide, contained a large quantity of sulfate and an appreciable quantity of uronic acid, in addition to galactose, hexosamine (glucosamine/galactosamine = 0.5), fucose and peptide. 5. 5. Threonine, proline and serine were the predominant amino acids in all the purified fractions. The results of the alkali treatment suggested that the linkages were through the hydroxyl group of threonine and serine to carbohydrates. 6. 6. All the purified fractions showed dextrorotation. The optical activity and sedimentation coefficient of the corresponding fractions from the two different tissues were similar.