Bovine Fetuin Research Articles

Analysis of the mitogenic effect of fetuin preparations on arterial smooth muscle cells: The role of contaminant platelet‐derived growth factor

Fetuin, a major protein of fetal calf serum, partially purified by the method of Pedersen, stimulated growth of aortic smooth muscle cells. More highly purified fetuin preparations stimulated growth less than Pedersen fetuin, as previously described for other cell types, suggesting that this activity is due to a contaminant. Recently bovine alpha 2-macroglobulin or "Embryonin" has been proposed as the mitogenic component of crude fetuin preparations. We found that active fetuin preparations did contain alpha 2-macroglobulin that stimulated smooth muscle cell growth. However, alpha 2-macroglobulin purified directly from platelet-poor bovine plasma or fetuin purified from Pedersen fetuin by gel filtration lacked appreciable mitogenic effect on smooth muscle cells. Since alpha 2-macroglobulin can bind platelet-derived growth factor (PDGF), and since highly acidic fetuin might bind the very basic PDGF molecule non-specifically, we measured the PDGF content of various fetuin preparations and found a good correlation between the PDGF content and mitogenic activity. Gel filtration experiments demonstrated that in Pedersen fetuin PDGF occurred both free, and in association with alpha 2-macroglobulin. We conclude that the principal mitogenic component for smooth muscle cells in crude fetuin preparations is PDGF, since purified bovine alpha 2-macroglobulin or fetuin do not appreciably affect growth of these cells. These results help to resolve a long-standing controversy regarding the nutrition of cultured cells. In addition, we suggest that before alpha 2-macroglobulin or "Embryonin" is accepted as a bona fide growth factor for a given cell type, the role of contamination with PDGF should be assessed.

Monoclonal antibodies to the HN glycoprotein of newcastle disease virus. Biological characterization and use for strain comparisons

We have isolated nine hybridomas secreting monoclonal antibodies directed against the HN glycoprotein of D 26 strain of Newcastle disease virus (NDV) and classified them into three groups by their biological reactivities. The group I antibodies from six clones had high hemagglutination inhibition (HI) and neutralization titers, and exhibited neuraminidase inhibition (NI) activity with substrates, both bovine fetuin and N-acetyl neuraminlactose, although the levels of maximum inhibition were greater with the former substrate. The group II antibodies from two clones also had high HI activity but showed a strongly substrate-dependent NI, with efficient inhibition against fetuin and a total lack of inhibition against neuraminlactose. These antibodies had neutralization capacity but the titers were much lower than those of group I antibodies. One clone (group III secreted antibody which exhibited HI and NI with either substrate but no detectable neutralization activity. Frequency of isolation of antigenic variants and competitive binding assays have shown that group I and group II antibodies may recognize distinct regions not overlapping each other whereas antibodies placed within each of the two groups are to the respective same overlapping regions. The site of group III seemed to be close to or overlap the group I determinant but distinct from group II region. Using the isolated monoclonal antibodies we compared seven heterologous strains by HI and neutralization tests as well as by enzyme-linked immunosorbent assay. The results indicated that the regions recognized by group I antibodies would be conserved among the strains whereas those by the other two antibody groups might undergo considerable changes.

Cell adhesion mediated by a purified fucosyltransferase.

Human embryonic skin fibroblasts attach and spread on surfaces on which a fucosyltransferase purified from human milk has been immobilized. The adhesion-enhancing effect of the transferase involves specific interactions of the enzyme surface with the cell surface carbohydrate acceptors, as suggested by the following findings. About 80% of human embryonic skin fibroblasts attach and spread in 1 hr on fucosyltransferase surfaces; in contrast, bovine serum albumin, fetuin, asialofetuin, and asialotransferrin surfaces fail to enhance adhesion. The adhesion-mediating activity of the transferase is destroyed by alkylation of the sulfhydryl groups or by heating. The adhesion on fucosyltransferase surfaces is inhibited by glycoprotein, glycolipid, and oligosaccharide acceptors containing the sugar sequence galactosyl-(beta 1 leads to 4)-N-acetylglucosamine, in agreement with the substrate specificity of the enzyme. The results suggest that glycosyltransferases are able to stimulate cell adhesion in a manner similar to that proposed for lectins.

Naturally occurring hemagglutinins in the hemolymph of the scorpionParuroctonus mesaensis Stahnke

Lectins which bind sialic acid-containing receptors are present in the hemolymph of the scorpionParuroctonus mesaensis Stahnke. Glycoproteins like bovine submaxillary mucin, fetuin and human orosomucoid behave as strong inhibitors forParuroctonus lectins; desialylation of glycoproteins results in a drastic reduction of their inhibitory capabilities confirming that sialic acids are the terminal monosaccharides ofParuroctonus lectin receptors.

Model for specific rescue of normal hepatocytes during methotrexate treatment of hepatic malignancy.

We utilized the presence of hepatic receptors for galactose-terminal glycoproteins to achieve specific rescue of differentiated hepatocytes from methotrexate toxicity. This was accomplished by administration of a conjugate formed by covalent coupling of the methotrexate antagonist, folinic acid (leucovorin), to galactose-terminating (desialylated) bovine fetuin. Two cultured hepatocyte-derived cell lines were chosen to test rescue from methotrexate toxicity: Hep G2 cells are capable of receptor-mediated endocytosis of galactose-terminating glycoproteins; PLC/PRF/5 cells are not. In the presence of methotrexate alone, both cells lines were reduced in number to 20% of control populations by day 5. Growth rates of both lines returned to normal when free folinic acid was added to the medium containing methotrexate. When asialofetuin-folinic acid conjugate was given to both cell lines in the presence of methotrexate, Hep G2 cells doubled their numbers by day 4 and reached control (without methotrexate) populations by day 8. However, PLC/PRF/5 cells failed to respond to administered asialofetuin-folinic acid conjugate under identical conditions, and the growth curve was the same as that for cells which received methotrexate alone. Conjugates of asialofetuin bound to folic acid (folate derivative requiring dihydrofolate reductase activity for conversion to an active metabolite) and fetuin (non-galactose-terminal glycoprotein) linked to folinic acid also were made for control studies. These conjugates failed to rescue either cell line.

Carbohydrate-chain analysis by lectin binding to mixtures of glycoproteins, separated by polyacrylamide slab-gel electrophoresis, with in situ chemical modifications

The interactions of five 125I-lectins of differing binding-specificities with seven glycoproteins containing known carbohydrate chains were investigated on polyacrylamide gels after electrophoretic separation. The glycoproteins were also chemically modified in the gels in order to gather additional information. The glycoproteins used were: porcine thyroglobulin (PTG); bovine-serum fibronectin (BFN); human-serum transferrin (HTF), bovine fetuin (BFE); subfractionated, hen ovalbumins having galactosylated, hybrid-type, carbohydrate chains (OVG); agalactosyl, hybrid-type-carbohydrate chains (OVH); and high-mannose-type-carbohydrate chains (OVM). Wheat-germ agglutinin (WGA) stained BFE, OVG, and OVH, but removal of sialic acid from BFE resulted in loss of WGA binding. After desialosylation and Smith degradation in situ, BFE and HTF reacted strongly, and PTG slightly with WGA. Ricinus communis agglutinin I stained PTG, BFN, HTF, and BFE after removal of sialic acid. Arachis hypogaea (peanut) agglutinin reacted only with BFN after removal of sialic acid. Lens culinaris hemagglutinin bound only to PTG, and concanavalin A bound to PTG, BFE, HTF, OVH, and OVM. The results were consistent with previously proposed lectin-carbohydrate-binding specificities, indicating that lectins may be used to determine the carbohydrate-chain structures of particular glycoproteins, even in complex mixtures.

Carbohydrate and glycoprotein inhibitors of naturally occurring and induced agglutinins in the earthworm, Lumbricus terrestris

1. 1. Agglutinins in the coelomic fluid of Lumbricus terrestris are inhibited by a number of glycoproteins and polysaccharides. Naturally occurring agglutinins are inhibited most strongly by bovine submaxillary mucin and fetuin, and induced agglutinins by thyroglobulin, bovine submaxillary mucin, hyaluronic acid and fetuin. 2. 2. The only simple sugar to be specifically inhibitory was 2-keto-3-deoxyoctonate, a monosaccharide found in bacteria. 3. 3. Desialation of erythrocytes resulted in increased agglutination titers, possibly by allowing easier access of agglutinins to underlying carbohydrates. 4. 4. Positively charged substances reduced agglutination in proportion to their strength of charge, probably a non-specific effect. 5. 5. Inhibitor data suggest that the combining site of Lumbricus agglutinins may normally accommodate several sugars.

Partial isolation and characterisation of a hemagglutinating factor from avocado seed.

Extracts of ground avocado seeds (Fuerte and Hass varieties), prepared in different buffer solutions (pH 2.0-12.0), show hemagglutinating activity towards A, B, AB, and H (0) human erythrocytes. The extract showing the highest titer of aggulination was extracted at pH 10.5. The crude extract also causes hemolysis of fresh washed erythrocytes. The hemagglutinating factor is not inhibited by most of the simple sugars tested, e.g., D-glucose, D-mannose, D-galactose, and glucose-amine. The only sugars which show some inhibitory effect are N-Acetyl-neuraminic acid, melibiose, and stachiose. Basic amino acids, e.g., lysine and arginine also inhibit its activity. However the most potent inhibitors of the agglutinin are proteins and glycoproteins such as bovine serum albumin, collagen, thyroglobulin, ovalbumin, mucin, and fetuin. The agglutinin is adsorbed on polymer beads such as Sepharose 4B, Sephadex G100, Agarose, and Chitin, and it reacts with hog erythrocyte membranes. It can be partially eluted from those materials with alkaline buffers (pH 9.0-10.5).

Isolation and characterization of a growth factor (embryonin) from bovine fetuin which resembles alpha 2-macroglobulin.

A serum-free hormone-supplemented medium which was previously formulated for the growth of mouse embryonal carcinoma (EC) cells and rat mammary epithelial (REM) cells required the presence of crude bovine fetuin as a medium supplement for maintaining cell growth. This requirement could not be replaced by purified fetuin preparations. The major growth-promoting activity (embryonin) in the crude fetuin preparation has been purified to near homogeneity by carboxymethylcellulose chromatography and high performance gel filtration chromatography. Purified embryonin, 2 to 4 micrograms/ml, is able to stimulate the growth of mouse EC cells in a serum-free hormone-supplemented medium to a level that is achieved with 0.5 to 1 mg/ml of the crude fetuin preparation. The biological activity resides in a high molecular weight glycoprotein (Mr = 270,000). Three polypeptide chains are observed following reduction, a major polypeptide (Mr = 185,000) and two minor chains (Mr = 116,000 to 120,000 and 68,000). Embryonin differs from pure fetuin in molecular weight, isoelectric point, amino acid composition, and immunological reactivity. However, embryonin is similar to human alpha 2-macroglobulin (alpha 2M) in these physicochemical and immunological properties. Resemblance to alpha 2M is also indicated by the observation that alpha 2M used over the same protein concentration range as embryonin produces a comparable stimulation of EC cell growth in the absence of serum. In addition, embryonin and alpha 2M produce a 2- to 10-fold differential stimulation of collagen production in cultures of normal rat kidney and RME cells, a response which may be linked to the growth-promoting activity of these two proteins.

The structural heterogeneity of the carbohydrate moiety of desialylated human transferrin.

Human transferrin consists of a single chain polypeptide which supports two N-glycosidically linked glycans at sequons a and b. Glycopeptides were released from human transferrin by proteolytic digestion, desialylated by mild acid hydrolysis, and then isolated by chromatographic methods. The structures of the glycans located on each sequon were determined by a combination of analytical techniques including Smith degradation, permethylation, and enzymic degradation. Approximately 79% of the total glycan from sequon a was of the biantennary type as previously described by Dorland and his colleagues (FEBS Lett. 77, 15-20 (1977)). The remaining 21% consisted of a mixture of triantennary and tetraantennary glycans, each amounting to approximately 10% of the total glycan for this sequon. The triantennary structure resembled that described for the N-glycosidic triantennary glycans of bovine fetuin by Nilsson and his colleagues (J. Biol. Chem. 254, 4545-4553 (1979)). Of the tetraantennary glycan, approximately half of the structures were incomplete, i.e., one antenna terminated by N-acetylglucosamine. On sequon b, 81% of the glycan was biantennary, identical to those biantennary glycans of sequon a, and the reminder was triantennary, also of the fetuin type. The glycan structures and their locations on the polypeptide are related to the known subpopulations of human transferrin.

Agglutination of mycelial cell wall fragments and spores of Colletotrichum lindemuthianum by plant extracts, and by various proteins

Mycelial cell wall fragments and spores of Colletotrichum lindemuthianum were agglutinated by seed and hypocotyl extracts from three resistant or susceptible cultivars of the host plant Phaseolus vulgaris and by seed extracts from two non-hosts, Vigna radiata and Ricinus communis. Agglutinating activity in seed extracts was unaffected by the presence of several sugars known to inhibit some classical lectins, and by heating at 100 °C for 15 min, but was greatly reduced by treatment with pronase. Two glycoproteins with high agglutinating activity were purified from seeds of P. vulgaris, cultivar Princor, but results from the purification indicate that a number of other proteins were present in the crude extract which had varying abilities to cause agglutination. Bovine serum albumin, ovalbumin, fetuin, cytochrome c, haemoglobin, thyroglobulin, γ-globulin and protamine sulphate also caused agglutination but the minimum concentration required for agglutination varied. The specific agglutinating activities of the most effective of the commercial proteins were similar to those of the two purified seed glycoproteins. It is concluded that the observed agglutination by plant extracts probably results primarily from non-specific interactions of proteins with the fungal cell wall, and the possibility that such interactions may be involved in attachment or recognition processes during pathogenesis is discussed. These results also demonstrate that agglutination phenomena in general do not necessarily involve group-specific binding such as a lectin-carbohydrate interaction.

A sialic acid-specific lectin from the slug Limax flavus

The slug, Limax flavus, contains a lectin that appears to be highly specific for sialic acid residues of glycoproteins. The carbohydrates which inhibited the hemagglutinating activity of the slug lectin and the concentration of the carbohydrate which gave a 50% inhibition are as follows: N-acetylneuraminic acid, 0.13 m m; N-glycolylneuraminic acid, 0.90 m m; d-glucosamine, 4.9 m m; d-galactosamine, 7.6 m m; N-acetyl- d-glucosamine, 23 m m; and N-acetyl- d-galactosamine, 24 m m. d-Galactose, d-glucose, d-mannose, α-methyl- d-glucoside, α-methyl- d-mannoside, l-arabinose, d-xylose, l-fucose, d-glucuronic acid, lactose, and sucrose were found to be ineffective as inhibitors of the hemagglutinating activity of the slug lectin. Hemagglutination by slug lectin was strongly inhibited by bovine submaxillary mucin and fetuin but not by sialic acid-free bovine submaxillary mucin or fetuin.

Partial purification and characterization of a binding protein for biologically active phorbol and ingenol esters from murine sera.

We have purified a protein (Mr approximately 71,000) from murine sera 104-fold which directly binds biologically active phorbol esters, ingenol esters, and mezerein in a specific, reversible, and saturable manner. The binding of labeled phorbol-12,13-dibutyrate (PDBu) to the purified protein is rapid and dose-dependent. Those phorbol and ingenol esters which stimulate cell growth in culture and have tumor-promoting activity in vivo inhibit the binding of labeled PDBu, while the biologically inactive derivatives fail to do so. Other nonditerpene tumor promoters, retinoids, steroids, and prostaglandins do not interfere with PDBu-protein interaction. Epidermal growth factor, insulin, bovine serum albumin, hemoglobin, ovalbumin, ferritin, myoglobin, fetuin, and lipase do not interact directly with PDBu. The purified binding protein competitively inhibits the binding of PDBu to its specific receptors. It is nonglycosylated and slightly hydrophobic. The protein is heat- and acid-labile and is present in sera of various mammalian species. Its concentration in murine sera is age-, sex-, and strain-independent.

Distribution of bovine fetuin and albumin in plasma, allantoic and amniotic fluids during development.

A radioimmunoassay for bovine fetuin was developed and its specificity and validity established. Albumin was measured by radial-immunodiffusion assay. Fetuin levels in fetal plasma increased from 10 to 15 mg/ml between 4 and 8 months of gestation; albumin levels remained higher than fetuin. Neonatal plasma fetuin levels rapidly declined during the first 14 days post partum, coincident with a marked reciprocal increase in albumin levels. In allantoic fluid fetuin and albumin concentrations reached a peak at 7 months but fetuin values were always higher. In amniotic fluid both proteins peaked at 8 months; albumin levels were similar to those in allantoic fluid but fetuin values remained consistently lower than those in allantoic fluid throughout gestation. Fetuin levels in maternal plasma declined from 0.7 to 0.4 mg/ml between 1 month and term. We conclude that (1) at term there is an abrupt change from fetuin synthesis to increased albumin synthesis by the neonatal liver; (2) fetuin appears to be preferentially accumulated in the allantois whereas albumin is equally concentrated in the allantois and amnion.

Immunological studies on ovine fetuin: Radioimmunoassay and comparison with related antigens

A radioimmunoassay for fetuin is described. It uses [ 125I]fetuin and rabbit antisera. The bound antigens are precipitated by anti-rabbit globulins coupled to Sepharose. This assay has been used to compare the immunological properties of ovine and bovine fetuin. It allows to detect a serum antigen cross-reacting with fetuin in pig species. A very faint reaction is observed with human fetal serum, that does not support the existence of a fetuin like antigen in human.

Improved basal medium for Y-1 mouse adrenal cortex tumor cells in culture

An improved basal medium is presented that required only minimal supplementation with dialyzed fetal bovine serum or bovine serum albumin and fetuin to be comparable to Ham's F-10, which requires 15% horse serum (HS) and 2.5% fetal bovine serum (FBS) for the growth and function of Y-1, mouse adrenal cortex tumor, cells. Cell monolayers maintained for up to 2 weeks without any protein supplementation have retained their steroid response to ACTH. The medium differs from Ham's F-10 in its buffer composition and higher calcium-ion concentration. This medium should be a useful adjunct to studies pertaining to steroid and lipid intermediary metabolism, the retention of a specialized physiological function in a chemically defined medium, and the mechanism of hormonal response.

Development and differentiation of mouse blastocysts in serum-free medium

Numerous studies have shown that the in vitro development and differentiation of mouse blastocysts require serum, but the number and nature of serum factors involved remains unclear. In this article, we describe a culture medium, EM-2, containing as a source of protein only commercially purified bovine serum albumin (BSA) and fetuin. This medium supports hatching, attachment and outgrowth of mouse blastocysts. Although attachment and outgrowth are delayed in EM-2 medium 12–15 and 5–8 h, respectively, these events occur at frequencies comparable to those observed in serum-containing media. Trophoblast cells are capable of differentiating in this medium: they synthesize Δ 5,3β-hydroxysteroid dehydrogenase and their nuclei become polyploid. The inner cell mass also appears to differentiate to some extent in EM-2 medium as evidenced by the appearance of cells with characteristics of parietal endoderm. The fetuin factor is necessary at least for trophoblast outgrowth and the albumin factor is required for the survival and/or growth of the inner cell mass. It is, however, not evident from these studies whether the serum fractions used are actually involved in the induction of differentiation, or whether the early differentiative steps in the mouse blastocyst are preprogrammed and require for expression only a normal cellular metabolic rate.

3H]Raffinose, a novel radioactive label for determining organ sites of catabolism of proteins in the circulation.

The primary tissue sites of catabolism of plasma proteins with long circulating half-lives are unknown. It has been difficult to identify these sites because plasma proteins are delivered to tissues at relatively slow rates but are rapidly degraded intracellularly within lysosomes. Therefore, a tracer attached to protein is lost from the site of uptake before an amount sufficient for quantitation can accumulate. We hypothesized that sucrose (Glupal-2 /3Fruf) would be a useful label to circumvent this difficulty because of the stability of sucrose in lysosomes; and thus, sucrose should remain in tissue long after the protein to which it was attached had been degraded to products released from the lysosome. [G-3HJRaffinose (RAF, GalpalGlupcwl2flFruf) was selected as the vehicle for attaching sucrose to protein. [31XjRAF was converted to the C-6 aldehydogalactose form with galactose oxidase and then covalently coupled to protein by reductive amination using NaBH3CN. [‘H]RAF was coupled first to two relatively long lived plasma proteins, bovine serum albumin and fetuin. The half-lives of these proteins in the rat circulation (&,* = -24 h) was unchanged, suggesting that RAF did not alter the normal mechanisms of protein clearance. When attached to short lived proteins with known sites of catabolism, such as asialofetuin, RNase B, and heatdenatured albumin, neither the tissue nor cellular sites of uptake of the proteins were altered. Thus, 13H]RAFasialofetuin was recovered almost exclusively (>90%) in the liver parenchymal cell fraction, while both [JHJRAF-labeled RNase B and heat-denatured albumin were recovered primarily (>85%) in nonparenchymal cells. In addition, the RAF label was observed to reside stably (tl,2 > 100 h) in the liver following degradation of the carrier protein; in contrast, radioactivity from “‘Ilabeled asialofetuin or RNase B was rapidly (tl,z < 30 min) lost from liver. Radioactivity from 13H]RAF-proteins was recovered in a lysosomally enriched subcellular fraction in liver and consisted of a low molecular weight species (-llOO), containing both glucose and fructose in a ratio similar to that in the original protein. The results of these studies establish that 13H]RAF

Human serum and epithelial spread in tissue culture.

The dependence of epithelial spread on human serum in culture has been studied. Using measured pieces of mouse ear skin epithelial outgrowth about floating explants (epiboly) and from adherent explants was studied. Where compared directly the two systems show similar results. Because of its ease of quantitation, the adherent explant culture was studied in greater detail. In this system in the presence of serum appreciable spread was found only after 48 h but spread continued for at least the next 4 days. In the absence of serum only minimal epithelial spread occurred. Adding serum to deficient media enhanced spreading and removing serum from media depressed spreading. The extent of spread appeared independent of mouse age for the first 4-10 weeks though 2-week-old mouse skin showed quantitatively greater spreading. The activity in human serum responsible for epithelial spread acts under conditions of minimal DNA synthesis and is not reproduced by bovine serum albumin, fetuin, or serum that had been exposed to 100 degrees C for 5 min. The activity is not dialyzable and it is resistant to the protease inhibitors DFP and PMSF. These studies suggest that a specific serum component(s) serves to support epithelial spread in vitro.

Bovine transferrin glycopeptide: the relevance of its structure to interaction with the mammalian hepatic lectin that binds asialoglycoproteins.

After proteolytic digestion of bovine transferrin (phenotype AA), a glycopeptide fraction was isolated by gel filtration and high-voltage electrophoresis. Two glycopeptide bands were recovered, each of which contained one residue of aspartic acid, two of serine, two of galactose, three of mannose, and four of N-acetyl glucosamine together with some fucose. However, the bands differed with respect to sialic acid content (two and three residues respectively). Using the same isolation procedures, a commercial sample of human transferrin yielded four glycopeptide bands (two major, two minor) on high-voltage electrophoresis. On analysis, the two major bands differed in amino-acid residues but their carbohydrate compositions were very similar. The analyses largely resembled previously published analytical data for two human transferrin glycopeptides (Jamieson, G. A., Jett, M. &amp;DeBernardo, S. L. (1971) J. Biol. Chem. 246, 3686–3693; Spik, G., Bayard, B., Fournet, B., Strecker. G., Bouquelet, S. &amp;Montreuil, J. (1975) FEBS Lett. 50, 296–299) and the carbohydrate analyses showed many similarities to the bovine transferrin glycopeptide. From these data and earlier observations made on bovine and human transferrins (Hatton, M. W. C, Regoeczi, E. &amp;Wong, K.-L. (1974) Can. J. Biochem. 52, 845–853), we have concluded that, in contrast with human transferrin, bovine transferrin contains only one heterosaccharide chain per molecule.Following tritiation, bovine transferrin asialoglycopeptide was compared with asialoglycopeptides from other serum proteins for their affinity to the rat liver in vivo. Considerable differences were observed and the following order of binding was established for the asialoglycopeptide preparations: bovine transferrin &lt; human transferrin [Formula: see text] bovine fetuin [Formula: see text] human α1-acid glycoprotein.