Behrooz G. Sharifi

Purification and characterization of a bovine cerebral cortex cell surface sialoglycopeptide that inhibits cell proliferation and metabolism

Abstract: A Sialoglycopeptide from bovine cerebral cortex cells was purified to apparent homogeneity by a procedure that included chloroform/methanol extraction, diethylaminoethyl ion exchange chromatography, wheat germ agglutinin affinity chromatography, size‐exclusion HPLC, and hydrophobic interaction chromatography. The cell surface inhibitor had a molecular weight of ∼18,000, no subunit composition was detectable on reduction and polyacrylamide gel electrophoresis analysis, and the glycopeptide apparently contained sialic acid, as illustrated by its ability to bind to Limulus polyhemus lectin. Deglycosylation of the molecule, however, did not reduce its protein synthesis inhibitory activity. As little as 20 ng of the Sialoglycopeptide was capable of inhibiting protein synthesis in a wide variety of fibroblast cell lines but not in transformed cells. Mice immunized with the Sialoglycopeptide produced antibodies that, when bound to protein A‐agarose gel, removed the inhibitory activity from solution. The antibodies were used to identify a single isoelectric focused band and to establish the pI of 3.0 for the molecule.

Cell surface interaction is sufficient for the biological activity of a bovine sialoglycopeptide inhibitor

A sialoglycopeptide inhibitor, isolated from bovine cerebral cortex cells, that reversibly inhibits protein and DNA synthesis, was coupled to either Sepharose or polyacrylamide beads. Whereas over 1 ng of the inhibitor was released from Sepharose beads after 30 min at 37 degrees C, less than 0.2 ng of the sialoglycopeptide was released from the polyacrylamide beads. When added to 3T3 cells, the immobilized sialoglycopeptide efficiently inhibited protein synthesis. No detectable sialoglycopeptide inhibitor was released into the assay medium in the presence or absence of 3T3 cells. Addition of [125I]sialoglycopeptide, coupled to acrylamide P100 beads, to 3T3 cells also demonstrated that the sialoglycopeptide was not internalized by the cells. Thus we conclude that an interaction of the sialoglycopeptide at the cell surface is sufficient for biological inhibitory activity.

Use of a urea and guanidine-HCl—propanol solvents system to purify a growth inhibitory glycopeptide by high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography was used to purify an inhibitory glycopeptide where resolution and recovery were enhanced by using urea or guanidine-HCl-isopropanol-water as a solvent system. Isopropanol alone or other solvent systems that have been proposed for such purification steps were not effective in eluting hydrophobic proteins from the reversed-phase column. The application of the urea or guanidine-HCl solvent systems in the separation and purification of membrane proteins, and other hydrophobic macromolecules, could greatly enhance recovery and efficiency of purification.

Abrogation of the mitogenic activity of bombesin by a cell surface sialoglycopeptide growth inhibitor

The ability of a cell surface sialoglycopeptide (SGP) growth inhibitor to abrogate the mitogenic activity of bombesin, in serum-free medium, was investigated. The SGP was shown to be a potent antagonist of bombesin-induced DNA synthesis as 2.7 nM of the inhibitor completely blocked the mitogenic activity of 5.0 nM of bombesin. Exposure of 3T3 cells to bombesin for only 10 hr was sufficient to initiate the induction of DNA synthesis 10 hr later. Kinetic studies demonstrated that the SGP was able to block bombesin-induced DNA synthesis only when added within 2 hr after exposure of the cells to the mitogen. The SGP inhibitor blocked bombesin mitogenesis at a post-receptor, intracellular, early event in the signal cascade.

A unique sialoglycopeptide growth regulator that inhibits mitogenic activity of a phorbol ester tumor promoter

The ability of a naturally occurring cell surface sialoglycopeptide growth inhibitor to antagonize the induction of DNA synthesis by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied with mouse 3T3 cells. The bovine sialoglycopeptide was shown to be a potent antagonist of TPA-induced DNA synthesis in confluent 3T3 cell cultures. Kinetic studies demonstrated that inhibition of TPA-induced DNA synthesis required the addition of the sialoglycopeptide within 15 min of TPA treatment. Addition of the sialoglycopeptide 30 min or longer after the cells were exposed to TPA did not block stimulation of DNA synthesis by TPA. The inhibition of TPA action was shown not to be restricted to DNA synthesis in 3T3 cultured cells since the sialoglycopeptide also inhibited TPA-induced ornithine decarboxylase (ODC, L-ornithine carboxylase, EC 4.1.1.17) activation in suspensions of mouse epidermal and 3T3 cells.

The effects of a calcium and a sodium ionophore on protein synthesis inhibition by a bovine cell surface sialoglycopeptide

The ability of the calcium ionophore A23187 and the sodium ionophore Monensin to antagonize the inhibition of 3T3 cell protein synthesis by a bovine cell surface sialoglycopeptide was measured. A23187, when added before and shortly after the sialoglycopeptide, significantly reduced the biological activity of the inhibitory glycopeptide. In contrast, Monensin had little, if any, influence on protein synthesis inhibition by the sialoglycopeptide. The ability of A23187 to circumvent the inhibitory action of the bovine glycopeptide was shown to be independent of the time the ionophore was incubated with the cells and the binding of the sialoglycopeptide to the 3T3 target cells. Neither the total amount of sialoglycopeptide bound to the cells, nor its affinity to the cell surface receptor, was influenced by the presence of A23187.

A monoclonal antibody to a unique cell surface growth regulatory glycopeptide

A glycopeptide, isolated from bovine cerebral cortex cells and added to cells in only nanogram/ml levels, has been shown to inhibit both cell protein synthesis and cell division. A monoclonal antibody was used to show that the inhibitory component originated from the cell surface. Incubation of the M1 IgG monoclonal antibody with partially purified bovine glycopeptide preparations and Staphyloccocus protein A removed the inhibitor from solution. Intact mouse cerebral cortex cells were found to have a similar epitope on their surfaces. In contrast, normal rat kidney cells (NRK) did not react with the monoclonal antibody. An analysis of mouse cerebral cortex membrane preparations, incubated with the monoclonal antibody, confirmed that the primary source of the antigenic determinant was the plasma membrane.

Cell agglutination by a novel cell surface sialoglycopeptide inhibitor and the relationship between its protease and biological activities.

A bovine sialoglycopeptide, purified to homogeneity and capable of inhibiting cellular protein synthesis and proliferation, was shown to agglutinate a wide variety of nontransformed and transformed cells. The cell agglutination activity was shown to be independent of the biological inhibitory action and most likely related to a protease activity that could not be physically separated during purification of the sialoglycopeptide. Samples that were completely biologically inactivated retained full protease activity and their ability to agglutinate target cells. Balb/c 3T3 cells were not agglutinated by the sialoglycopeptide and they elicited a protein that interfered with the agglutination reaction and even redispursed cells that already had been aggregated by the inhibitor.

Purification of a cell growth inhibitor from bovine cerebral cortex cells

A glycopeptide, isolated from bovine cerebral cortex cells and added in only nanogram levels to cells in culture, has been shown to inhibit both cell protein synthesis and cell division. When purified by gel filtration and Ulex europaeus lectin affinity chromatography, the radioiodinated preparation was subjected to high resolution isoelectric focusing and shown to contain three species of macromolecules. The glycopeptide focusing at pH 8.1 comprised over 75% of the radioiodinated material and possessed inhibitory activity against both cell protein synthesis and cell division. A second species that focused at pH 8.3 was also found to be inhibitory to cell metabolism and may have represented a variant of the major glycopeptide.