Acid polysaccharides from invertebrate connective tissue: phylogenetic aspects.

Polyfucose sulfate and a chondroitin sulfate were isolated from echinoderm connective tissue. Coelenterate and poriferan connective tissues were devoid of these acid polysaccharides.

Quantitative immunological determination of 12 plasma proteins excreted in human urine collected before and after exercise.

Urine was collected from 6 healthy male adults at rest and from 20 male adults after a marathon race (25 miles). The concentrated urines were quantitatively analyzed, by single radial immunodiffusion, for their content in 12 different plasma proteins: tryptophan-rich prealbumin, albumin, alpha(1)-acid glycoprotein, alpha(1)-antitrypsin, ceruloplasmin, haptoglobin, Gc-globulin, transferrin, hemopexin, beta(2)-glycoprotein I, gammaA-globulin, and gammaG-globulin.Albumin, gammaA-globulin, and gammaG-globulin represent the major part of the plasma proteins detected in normal urine excreted by humans at rest (12, 0.5, and 2.5 mg respectively, out of a total excretion of 17.5 mg of plasma proteins per 24 hr). The other plasma proteins were excreted at a lower rate (< 0.4 mg/24 hr). The relative content of tryptophan-rich prealbumin, alpha(1)-antitrypsin, Gc-globulin, transferrin, and gammaG-globulin was lower in normal urine than in normal serum, whereas that of alpha(1)-acid glycoprotein, beta(2)-glycoprotein I, and gammaA-globulin was higher. The ratio of gammaG-globulin to gammaA-globulin was 4.9:1. When plotted on a logarithmic scale, no direct relationship between the molecular weight of a protein and the value of its renal clearance could be observed.Strenuous exercise increased (up to 50-fold) the excretion of plasma proteins which represent 82% of the total proteins found in urine, instead of 57% in urine collected from humans at rest. There was particularly a significant rise of tryptophan-rich albumin, albumin, alpha(1)-acid glycoprotein, transferrin, gammaA-globulin, and gammaG-globulin (0.26, 127, 11.8, 3.3, 1.2, and 2.0 mug respectively, out of a total excretion of 167 mug of plasma proteins per min). The ratio of gammaG-globulin to gammaA-globulin was 16:1. After exercise, the renal clearance of proteins increased from 2 to 40 times, but, as for the urine of normal subjects at rest, no direct relationship between molecular weight and renal clearance could be observed.

Epiglycanin-immunoreactive glycoproteins in mouse fetal tissues and fetal cells in culture.

Fetal tissues from time-pregnant female A/J mice of 16- and 19-day pregnancies and from neonates 1 day after birth, as well as from fetal cells in culture, absorbed significant amounts of anti-epiglycanin antibody. Detergent-solubilized glycoproteins, with epiglycanin activity, from fetal tissues and cells were separated by polyacrylamide gel electrophoresis, and the protein bands electroblotted onto a nitrocellulose gel. After the antigens were labeled with rabbit anti-epiglycanin antiserum and [125I]epiglycanin, autoradiography revealed two major bands containing the antigenic determinant at Mr 90,000 and 82,000. Bands of similar molecular weights, but with no demonstrated immunologic cross-reactivity, were observed by fluorography, if intact cells prior to solubilization were labeled by galactose oxidase followed by sodium borotritiide. Immunoreactive epiglycanin activity could be destroyed by Pronase, endo-N-acetyl-alpha-D-galactosaminidase (Diplococcus pneumoniae), or periodate oxidation. Activity was enhanced with neuraminidase. The spleen, liver, or erythrocytes from adult A/J mice did not possess the antigen, but incubation of adult spleen or liver with neuraminidase (Vibrio cholerae) exposed the epitope.

Reversible loss in suspension culture of a major cell-surface glycoprotein of the TA3-Ha mouse tumor.

A major cell-surface glycoprotein of the TA3-Ha ascites mammary adenocarcinoma diminished during transfer from ascites growth to cell growth in suspension culture. A sensitive, hemagglutination-inhibition assay that used a lectin from Vicia graminea seeds indicated approximately a 50% loss after 7-10 days of culture and a 90% loss after 2 months. These findings were corroborated by carbohydrate and amino acid analysis with gas-liquid chromatography of trypsin glycopeptides released from the cell surface. Repassage of the cultured cells in vivo caused the reappearance of the surface glycoprotein.

Isolation and partial characterization of surface components of cell line MDA-MB-231 derived from a human metastatic breast carcinoma.

Neuraminidase (Vibrio cholerae) treatment of human metastatic mammary carcinoma MDA-MB-231 cells grown in culture released 0.60-0.63 mg of N-acetylneuraminic acid from 10(9) cells. Incubation of intact cells with a modified trypsin and fractionation by gel filtration gave mainly O-glycopeptides. The presence of O-glycosyl-linked chains having one or two carbohydrate residues was confirmed by treatment of the glycopeptide fractions with galactose oxidase, followed by reduction with alkaline sodium borotritide and fractionation. The major glycopeptide fraction, which consisted of 53% carbohydrate and 47% protein, and a minor glycopeptide fraction each inhibited hemagglutination by peanut lectin. These results suggest the presence of O-beta-D-galactopyranosyl-(1 leads to 3)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1 leads to 3)-L-serine (threonine) residues. The absorptive capacities for anti-HLA-A2 and anti-HLA-B8 antisera were slightly greater for intact than for lyophilized cells, which suggested that masking of these major histocompatibility antigens did not occur in intact cells.

Further studies on the relationship between large glycoprotein molecules and allotransplantability in the TA3 tumor of the mouse: studies on segregating TA3-HA hybrids.

In a series of six TA3-HA/A.CA hybrid cell lines formed by the fusion of the TA3-HA mammary carcinoma of a strain A mouse with a normal embryonic fibroblast of an A.CA mouse and then converted to the ascites form in parental strain A, the capacity to grow in foreign strains was inversely related to the ability to absorb anti-H-2a antibody. The absorptive capacities of the hybrid cell lines were intermediate between the low absorptive capacity of the non-strain-specific parent TA3-HA ascites cell line and the much higher absorptive capacity of the strain-specific ascites line TA3-St of the same tumor. Each hybrid cell line possessed an abundance of large endogenous cell-surface glycoprotein molecules similar to epiglycanin, a glycoprotein detected at the surface of the parent TA3-HA cell. The results suggested that the amount of epiglycanin-like material at the hybrid cell surfaces, determined by chemical and immunochemical methods, may have been directly related to the capacities of the cells to grow in foreign mouse strains and inversely related to their capacities to absorb anti-H-2a antibody.

Isolation and partial characterization of an epiglycanin-like glycoprotein from a new non-strain-specific subline of TA3 murine mammary adenocarcinoma.

A new non-strain-specific ascites subline of the TA3 mammary adenocarcinoma TA3-MM, which arose in vivo from the strain-specific TA3-St subline during an acute respiratory illness of the syngeneic mouse strain A/HeHa hosts, possessed at its surface a glycoprotein not found on the parent TA3-St cell. This glycoprotein, termed TA3-MM epiglycanin, was characterized by a high molecular weight (500,000), by potent inhibition of hemagglutination by the Vicia gramines lectin, and by carbohydrate and amino acid compositions nearly identical to those of the glycoprotein epiglycanin present at the surface of the allotransplantable TA3-Ha ascites cell. By electron microscopic examination, TA3-MM epiglycanin appeared as long extended rods with widths (2.5 nm) and lengths (450--500 nm) similar to those of TA3-Ha epiglycanin. Incubation of each of two sublines of the TA3-MM ascites cell, TA3-MM/1 and TA3-MM/2, with a modified trypsin followed by column chromatography produced approximately 1.0- and 0.2-fold as much epiglycanin-like material, respectively, as was obtained from the TA3--a ascites cell. Continuous growth of the TA3-MM cell in suspension culture resulted in an almost complete disappearance of epiglycanin in a manner demonstrated earlier for the TA3-Ha cell grown under similar conditions. Allotransplantability in the TA3-MM cell may be due, at least in part, to masking a histocompatibility antigens by epiglycanin-like molecules.

Antibody to epiglycanin and radioimmunoassay to detect epiglycanin-related glycoproteins in body fluids of cancer patients.

By means of a radioimmunoassay, which utilized [125I]-epiglycanin and anti-epiglycanin antiserum induced in rabbits by injections of viable TA3-Ha ascites cells with Freund's complete adjuvant, picogram quantities of epiglycanin could be detected. Anti-epiglycanin antiserum was similarly produced in allogeneic mice. Unlabeled epiglycanin lost the capacity to compete with [125I]epiglycanin in the radioimmunoassay as a result of periodate oxidation or incubation with endo-alpha-N-acetyl-D-galactosaminidase (Diplococcus pneumoniae), an enzyme found to cleave only the disaccharide beta-D-galactopyranosyl-(1----3)-2-acetamido-2-deoxy-D-galactose chain from serine or threonine residues in epiglycanin. Glycosylhydrolases known to cleave alpha-D-mannose, beta-D-galactose (1,4-linked), beta-N-acetyl-D-glucosamine, and alpha-N-acetyl-D-galactosamine did not reduce the activity of epiglycanin. Neuraminidase enhanced the activity twofold to fivefold. The finding that little or no activity was demonstrated by the disaccharide, the reduced disaccharide, or other glycoproteins containing the same disaccharide chain suggested that the antigenic determinant probably involved the disaccharide and a unique amino acid sequence at the site of its attachment. By means of the radioimmunoassay epiglycanin cross-reactive antigens were detected in the peritoneal or pleural fluid and in the sera of patients with metastatic cancer. Lower concentrations of epiglycanin-like antigen(s) were found in the peritoneal fluid of patients with hepatitis or liver cirrhosis but not in normal serum.

Effect of heterogeneity of carcinoembryonic antigen on liver cell membrane binding and its kinetics of removal from circulation.

Carcinoembryonic antigen (CEA) is a glycoprotein metabolized primarily by the liver. Subcellular fractions of rat liver were examined for CEA binding activity. Hepatocyte plasma membrane and microsome fractions bound CEA, and this binding shared the calcium requirement, neuraminidase sensitivity, and carbohydrate specificity of the hepatocyte asialoglycoprotein receptor. CEA had previously been shown to react with this galactose-specific receptor, in vivo, only following neuraminidase treatment. Galactose receptor binding of CEA was measured in three different purified CEA preparations. The fraction of CEA capable of binding to excess levels of galactose receptor on membranes varied (46.5%, 40.2%, and 4.7% for CEA-1, -2, and -3, respectively). These CEAs were shown to be 2.3%, 7.9%, and 0.7% as effective, respectively, as asialo-alpha 1-acid glycoprotein in inhibiting the binding of radiolabeled asialo-alpha 1-acid glycoprotein to liver cell membranes. Each of the three CEA preparations showed different clearance kinetics from the circulation of mice. Coinjection of asialo-alpha 1-acid glycoprotein with the CEAs revealed differing inhibition of the clearances. These results show that differences in the carbohydrate components of purified CEA preparations affect their rate of removal from circulation and thus possibly the relationship between CEA production and observed plasma levels in patients. The possible origin of these CEA differences is discussed with their clinical implications.

Physicochemical and morphological characterization of a new allotransplantable mammary carcinoma ascites subline, TA3-St/ticol/-A.

The TA3-St/ticol ascites cell (I), immunoselected from the strain-specific TA3-St mammary carcinoma ascites cell of the strain A mouse for decreased H-2a antibody-binding capacity, underwent a spontaneous transition in vivo to a new cell line, TA3-St/ticol/-A (II). Line II was more allotransplantable than was the parental line (line I), and its absorptive capacity for anti-H-2a antibody was manyfold less than line I. Disruption of line II cells by lyophilization did not increase the absorptive capacity, in contrast to its marked enhancement in the TA3-Ha ascites cell under similar conditions. An explanation for the enhanced allotransplantability of line II may be related to either a loss of H-2a antigens or altered macromolecular structures at the cell surface: sialic acid, consisting of 93% N-glycolylneuraminic acid for line II and 9% for line I; altered chemical structures of cell surface glycoproteins, particularly a high-molecular-weight glycoprotein present in much greater proportion in line II than in line I; a macromolecular complex released by a protease from line I, but not line II; and I being agglutinable by concanavalin A, but not line II. Electron microscopy showed line II to be more pleomorphic and less rounded than was line I. Under high-resolution electron microscopy, the cell surfaces of both allotransplantable cells, lines II and I, exhibited thin filamentous material, material not observed at the surface of the nonallotransplantable TA3-St ascites cell.

Cell surface characteristics of an allotransplantable TA3 ascites subline resulting from a process of immunoselection.

Comparison of the cell surface characteristics of the parental strain-specific TA3-St ascites cell (I) of the strain A mouse and the more allotransplantable TA3-St/ticol ascites cell (II), immunoselected for reduced absorption of anti-H-2a antibody from Cell I, revealed the following. Cell II, like Cell I, possessed no detectable epiglycanin at its surface, as neither cell absorbed more than 0.5% as much of the antiepiglycanin antibody as was absorbed by the epiglycanin-containing allotransplantable TA3-Ha ascites cell. Tritium-labeled glycoproteins, with polyacrylamide slab gel electrophoresis with sodium dodecyl sulfate and with isoelectric focusing of detergent-treated cells, exhibited marked quantitative differences, but qualitative differences were not established. Glycopeptides cleaved from each cell by proteolysis and fractionated by gel filtration gave similar elution profiles, and the column fractions possessed similar carbohydrate and amino acid compositions. Less sialic acid (170 micrograms/10(9) cells) was removed by neuraminidase from Cell II than from Cell I (270 micrograms/10(9) cells), and the compositions (9% N-glycolylneuraminic acid for Cell II and 20% for Cell I) were different. Transmission and scanning electron microscopy showed rough irregular folds and ridges on the surfaces of each cell, but Cell II appeared more pleomorphic and less rounded than did Cell I. High-resolution transmission electron microscopy showed filamentous material at the surface of Cell II, but not at the surface of Cell I.

Studies on bonnet monkey cervical mucus. The effect of proteases on mucus glycoproteins of Macaca radiata.

The influence of proteinases on monkey cervical glycoproteins was investigated to assess their effect on cervical mucus and, thereby, on sperm penetration. The major component of periovulatory cervical mucus, a high molecular weight glycoprotein, was treated with Pronase, trypsin, chymotrypsin, papain, and bovine seminal peptidase, and the enzyme-resistant glycoprotein was purified by gel filtration on Sepharose 2B. A macromolecular component in high yield was recovered containing carbohydrate and protein moieties. Asialoglycoprotein, on treatment with Pronase, trypsin, and bovine seminal peptidase released more than one glycoprotein fragment. The carbohydrate and amino acid components of the native and degraded glycoproteins were similar in composition with variations in proportions. The structure of the carbohydrate-rich, pronase-resistant glycoprotein, further purified on Sepharose 2B, was examined. Sequential Smith degradation and methylation of the degraded glycoprotein fragment established a structure that shows some differences to that of the native glycoprotein. The influence of proteinases on cervical-mucus glycoproteins and a possible mechanism of sperm penetration through Pronase-treated glycoproteins is discussed.

Solubilization of an alpha-(1 leads to 3)-D-mannosyltransferase from pancreas which utilizes synthetic dolichyl pyrophosphate trisaccharide beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4)GlcNAc as substrate.

Calf pancreas microsomes were treated with 0.5-1% Triton X-100 and the resulting soluble enzyme preparation was incubated with GDP-D-[14C]mannose. The addition of synthetic Dol-PP derivative of the trisaccharide beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4)GlcNAc stimulated the synthesis of labeled lipid-bound tetrasaccharide 50-100-fold. The labeled tetrasaccharide thus formed was identified as alpha-Man-(1 leads to 3)-beta-Man-(1 leads to 4)-beta-GlcNAc- (1 leads to 4)GlcNAc by its chromatographic properties and by its sensitivity to alpha-mannosidase and to endo-beta-N-acetylglucosaminidase D. The solubilized alpha-(1 leads to 3)mannosyltransferase did not require divalent cation and was active in the presence of 10 mM EDTA.

Characterization of oligosaccharides from the urine of loco-intoxicated sheep.

Two major oligosaccharides were isolated by preparative HPLC from the urine of a locoweed-fed sheep. Analysis by gas-liquid chromatography and mass-spectrometry indicated compositions of (Man)4(GlcNAc)2 and (Man)5(GlcNAc)2, respectively. Structures were determined by digestion with alpha-D-mannosidase and endo-beta-N-acetylglucosaminidases D and H, and comparison of the products by HPLC with synthetic standards, and oligosaccharides isolated from human mannosidosis urine. Incubation with an exo-beta-N-acetylglucosaminidase was without effect.

Induced mannosidosis-excretion of oligosaccharides by locoweed-intoxicated sheep.

Daily urine samples were collected from a locoweed-fed sheep, and the oligosaccharide content examined by thin-layer and liquid chromatography. An unusual pattern of urine oligosaccharides was observed, which appears to be characteristic of loco intoxication. Changes in the pattern could be correlated with the onset of visible disease, which occurred approximately 5 weeks after the typical urine sugars were first detected. HPLC showed that these sugars consisted of two homologous series of oligosaccharides containing one and two residues of 2-acetamido-2-deoxy-D-glucose, respectively.

Oligosaccharides from placenta: early diagnosis of feline mannosidosis.

High-pressure liquid chromatography analysis of oligosaccharides from placentas allowed the diagnosis of alpha-mannosidosis in three litters of kittens. The chromatography also afforded a detailed comparison of the oligosaccharide pattern and levels in placenta, liver, brain, urine and ocular fluid of the affected animals. In all cases, two series of compounds were observed, with one or two residues of N-acetylglucosamine at the reducing terminus, respectively, and between two and nine mannose residues. This pattern is unlike that of human mannosidosis, and resembles that of ruminants, except that the major oligosaccharide contains three mannose residues instead of two.

Changes in the sialic acid concentration in the major cervical glycoprotein from the bonnet monkey (Macaca radiata) during a hormonally induced cycle.

The major macromolecular component of monkey cervical mucus has been purified from crude monkey cervical mucus at various times during a hormonally induced estrous cycle. The major component, a high-molecular weight glycoprotein, was secreted in large amounts in midcycle mucus. The glycoprotein exhibited human blood group B activity and contained the same concentration of L-fucose, D-galactose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine during the cycle. There were no changes in the amino acid composition or in the number of carbohydrate side chains. There was a significant increase in the amount of sialic acid in the glycoprotein purified from midcycle mucus. Changes in the specific viscosity and the far ultraviolet circular dichroic spectra were also observed. The possible control of sperm penetration by the glycoprotein is discussed.

Urinary excretion of immunoglobulins and their subunits in human subjects before and after exercise.

Immunoglobulins and their subunits in urines collected before and after exercise from healthy human subjects were studied. Quantitative analysis showed that only gamma A- and gamma G-immunoglobulins were excreted as whole molecules in normal urine, at a concentration of 0.35 and 1.70 micrograms/min., respectively. Exercise enhanced the excretion of both types of molecules. In some cases, gamma D-immunoglobulins were found in "exercise urine." Gel filtration on Sephadex G-150 showed that only gamma G-subunits were present in normal and in "exercise urine." The distribution of gamma G-subunits was not affected by exercise. The authors consider the importance of the present data in relation to renal physiology.

Methyl derivatives of 2-acetamido-2-deoxy-3-O-(beta-D-glucopyranosyluronic acid)-D-glucose (hyalobiouronic acid) from methylated hyaluronic acid.

Methanolysis of methylated hyaluronic acid, followed by acetylation, gave, in 70% yield, crystalline methyl 2-acetamido-2-deoxy-4,6-di-O-methyl-3-O-(methyl 4-O-acetyl-2,3-di-O-methyl-beta-D-glucopyranosyluronate) -alpha-D-glucopyranoside. Removal of the O-acetyl and methyl ester groups gave compounds that are useful in the investigation, by 1H-n.m.r. spectroscopy, of interaction within chains of hyaluronic acid in solution.

Isolation and partial purification of lipid-linked oligosaccharides from calf pancreas.

Lipid-linked oligosaccharides containing at least five mannose residues (0.7 nmol/g of tissue) were isolated from calf pancreas by chloroform-methanol-water extraction and purification on DEAE-cellulose and Sephadex LH-20, and affinity chromatography on concanavalin A-Sepharose in the presence of nonionic detergent. The addition, prior to the chromatographic steps, of 14C-labeled lipid-linked oligosaccharides (synthesized in vitro by calf pancreas microsomes in the presence of GDP-D-[14C]mannose and UDP-D-[14C]glucose, respectively) as internal standards, indicated a final yield ranging from 38 to 50%. Analysis of the oligosaccharide residues by liquid chromatography of the lipid-free preparation, monitored by u.v. absorbance and radioactivity measurement of the tritiated compounds, indicated a heterogeneous mixture of oligosaccharides. Its components, ranging from Man5(GlcNAc)2 to Glc3Man9(GlcNAc)2, cochromatographed with the 14C-labeled derivatives from in vitro synthesis. Calf pancreas contains lipid intermediates bearing at least six mannose residues, such as Man9(GlcNAc)2-P-P-lipid, in almost equal or even higher amounts than Glc3Man9(GlcNAc)2-P-P-dolichol.