Reuben Lotan

The fluorescence of wheat germ agglutinin and of its complexes with saccharides.

Summary Wheat germ agglutinin, upon excitation at 280 nm, presents a typical tryptophan fluorescence spectrum centered at 348 nm with a quantum yield of 0.095. Upon addition of N-acetyl-D-glucosamine or of its β(1→4) linked oligomers (GlcNAc)2,3,4, a blue shift of the fluorescence maximum and an increase in the fluorescence quantum yield of wheat germ agglutinin are observed. These are indications of the involvement of tryptophan residue(s) in the binding of saccharides. The changes with pH of the fluorescence intensity of wheat germ agglutinin and of its complexes with saccharides suggest an interaction between carboxyl and tryptophan residue(s) in the binding site which results in fluorescence quenching. Association constants for the binding to wheat germ agglutinin of (GlcNAc)2, (GlcNAc)3 and (GlcNAc)4, were determined spectrofluorimetrically, and found to be 1.3 × 104 M−1, 2.2 × 104M−1 and 3.6 × 104M−1, respectively. Addition of the oligosaccharides (GlcNAc)2,3,4 to wheat germ agglutinin produced greater enhancement of the fluorescence intensity than did addition of the corresponding monosaccharide. Our findings corroborate an earlier suggestion that the binding site of wheat germ agglutinin consists of several adjacent subsites.

Purification of wheat germ agglutinin by affinity chromatography on a sepharose-bound N-acetylglucosamine derivative.

Abstract Sepharose-2-acetamido-N-(ϵ-aminocaproyl)-2-deoxy-β-D-glucopyranosylamine was prepared by a reaction of 2-acetamido-3,4,6-tri-0-acetyl-2-deoxy-β-D-glucopyranosylamine and N-(benzyloxycarboxyl)-ϵ-aminocaproic acid, removal of the 0-acetyl and the benzyloxycarboxyl groups and coupling to Sepharose. The product was used for the purification of wheat germ agglutinin, by adsorption from a crude wheat germ extract and elution with 0.1M acetic acid. The purified agglutinin was homogeneous on SDS-polyacrylamide gel electrophoresis and had a specific hemagglutinating activity of 3000 u/mg when tested on trypsinized rabbit erythrocytes. It was rich in cysteine, cystine and glycine, and contained no sugar.

Retinoic acid-induced modifications in the growth and cell surface components of a human carcinoma (HeLa) cell line☆

Abstract The addition of retinoic acid to cultures of HeLa-S3 cells caused a reduction in cell proliferation rate which became apparent after 72 h and was linearly dependent on retinoic acid concentration in the range 10−9–10−5 M. After 72 h of exposure to retinoic acid, the cells assumed a flattened appearance and no longer formed multilayers. These changes were reversed within 48 h after removal of retinoic acid from the medium. Structural analogs of retinoic acid with a free ue5f8COOH group at C-15 were usually more potent in growth inhibition than compounds with an alcohol, aldehyde, ether or ester group. A cellular retinoic acid-binding protein was detected in cell homogenates, and the binding of [3H]retinoic acid to the binding protein was inhibited by most, but not all, analogs possessing a free terminal ue5f8COOH group. For example, the 4-oxo analog of retinoic acid, while capable of inhibiting cellular proliferation, failed to bind to the retinoic acid-binding protein. Analysis of cell surface and cellular glycoproteins by lactoperoxidase-catalysed 125I iodination and by metabolic labeling with [3H]glucosamine revealed that a 190000 D glycoprotein which was labeled by both methods and a 230000 D glycoprotein which was labeled only with [3H]glucosamine were labeled more intensely in retinoic acid-treated cells compared with untreated cells. The electrophoretic mobility of the 230000 D glycoprotein could be modified by treatment of intact cells with either neuraminidase or proteolytic enzymes, suggesting that this glycoprotein is also exposed on the cell surface. The cell surface alterations were detected much earlier than the onset of growth inhibition and appeared as early as 24 h after exposure to retinoic acid. The possible relationship between retinoic acid-induced changes in cell membrane structure, cell morphology, and cell proliferation is discussed.

A differential response elicited in macrophages on interaction with lectins.

Abstract The interaction of several lectins, both native and chemically modified, with mouse peritoneal macrophages was studied. Surface distribution and interiorization of the lectins was assessed quantitatively using their radioactively-labeled derivatives, and qualitatively by employing fluorescein-labeled lectins. On the basis of their effect on the macrophages, the lectins tested fall into two classes: lectins that induce vacuole formation in the cells (concanavalin A (ConA), wax bean agglutinin (WBA) and wheat germ agglutinin (WGA)) and lectins that in their native form do not induce vacuolation (soybean agglutinin (SBA), peanut agglutinin (PNA) and the lectin from Lotus tetragonolobus (LT)). Neuraminidase treatment of the cells did not change their response to the lectins, though in the case of SBA and PNA binding was observed only with neuraminidase-treated macrophages. Incubation of the latter cells with SBA and subsequently with ConA resulted in significantly higher vacuolation than that observed with ConA alone. Glutaraldehyde-crosslinked polymers of SBA and of PNA, which are multivalent with respect to sugar binding, induced vacuolation in neuraminidase-treated macrophages. On the other hand, succinylation of ConA, which reduces the number of sugar binding sites per mole from four to two, abolished its ability to induce vacuole formation. These data suggest that multivalency of lectins and probably also their size are important factors in inducing vacuolation, by causing extensive crosslinkage of membrane receptors which is prerequisite for triggering of vacuole formation. Quantitative binding and internalization data indicate that vacuole formation is not directly related to the number of lectin receptors on the macrophages nor to the extent of their internalization.

CHARACTERIZATION OF RETINOIC ACID‐INDUCED ALTERATIONS IN THE PROLIFERATION AND DIFFERENTIATION OF A MURINE AND A HUMAN MELANOMA CELL LINE IN CULTURE*

We employ the murine S91 and the human Hs939 melanoma cell lines for the characterization of various biochemical changes induced by retinoids. Retinoic acid (RA) causes a time-dependent, and reversible reduction in cell proliferation rate in liquid medium and inhibits growth in agar. The proportion of cells in the G1 phase of the cell cycle increases in RA-treated cells, and the uptake of TdR, UdR and Leu decreases. The growth inhibitory effect of RA is apparently not mediated via labilization of lysosomes, increase in cAMP or changes in the synthesis of prostaglandins or polyamines. Exposure to RA stimulates tyrosinase activity and increases melanin content severalfold over the levels found in untreated cells. Various retinoids exhibit the activities of RA; however, their potencies vary depending on their structure. Those possessing a free -COOH at C-15 are usually more effective than those with a different group or with a derivatized carboxyl. A positive correlation exists between the ability of retinoids with a free -COOH in C-15 to inhibit growth and to bind to an RA-binding protein found in the S91 melanoma cells. Future studies will explore recently discovered changes in the glycosylation of cell surface components and their relationship to the phenomena described here.

Aggregation and fragmentation of soybean agglutinin

Summary Upon storage in the lyophilized state, soybean agglutinin forms high molecular weight aggregates. The aggregation results in a marked increase in hemagglutinating activity, particularly when tested with untrypsinized rabbit or human erythrocytes. Highly purified preparations of soybean agglutinin contain low molecular weight polypeptide fragments, resulting probably from proteolytic cleavage of the intact subunit.

Enhancement of the biological activities of soybean agglutinin by cross-linking with glutaraldehyde.

Summary Soybean agglutinin was cross-linked with glutaraldehyde. Soluble high molecular weight complexes were isolated by affinity chromatography on a column of Sepharose-N-ɛ-aminocaproyl-β- d -galactopyranosylamine, followed by gel filtration on Sephadex G-150. The cross-linked lectin agglutinated rabbit and human erythrocytes at concentrations 100 to 200 times lower than those required for their agglutination by native soybean agglutinin. With trypsinized erythrocytes, the cross-linked agglutinin was only 3 to 15 times more active than the native protein. Mouse spleen lymphocytes were agglutinated by cross-linked soybean agglutinin at concentrations 32 times lower than those required for their agglutination by the native lectin. Maximal stimulation of neuraminidase-treated mouse spleen lymphocytes by cross-linked soybean agglutinin was obtained at concentrations 5 times lower than those of the native lectin.

Distribution of the T-antigen on erythroid cell surfaces. Studies with peanut agglutinin, an anti-T specific lectin

Abstract The interaction of peanut agglutinin, a lectin with a specificity similar to that of serum T-agglutinin, with human, guinea pig and rabbit young and old red blood cells and bone marrow erythroid cells, both before and after neuraminidase treatment, was investigated. Distribution of the peanut agglutinin receptors was determined by electron microscopical analysis with the ferritin conjugated lectin. In addition, kinetics of agglutination by the lectin were examined with the aid of the Fragiligraph. Rabbit red blood cells of all ages bound peanut agglutinin without neuraminidase treatment. Untreated human and guinea pig red blood cells, as well as immature erythroid cells of guinea pig bone marrow were not agglutinated by the lectin, nor was binding of the lectin to these cells observed by electron microscopy. Treatment with neuraminidase resulted in exposure of peanut agglutinin binding sites on erythroid cells of all stages of maturation and aging. Our findings show that peanut agglutinin receptors are absent from the surface of old circulating red blood cells and extruded erythroid nuclei, in spite of a remarkable in vivo loss of surface sialyl residues. The use of neuraminidase-treated red blood cells, as a model for the study of the clearance of aged red blood cells from circulation seems, therefore, not to be entirely justified.

Stimulation of sialyltransferase activity of melanoma cells by retinoic acid

Retinoic acid (RA) treatment of murine S91-C2 melanoma cells has been found to augment the activity of glycoprotein: sialyltransferase in a dose-dependent and time-dependent process. The enzymatic activity in cells treated with 10 microM RA reached a maximal level, 3-fold higher than in untreated cells, 72 h after initiation of treatment. In contrast, the addition of RA directly into the reaction mixture had no stimulatory effect on sialyltransferase. The endogenous glycoproteins to which sialic acid is transferred from cytidine monophosphate (CMP)-[14C] sialic acid by the action of sialyltransferase have been identified by fluorography after polyacrylamide gel electrophoresis. One of these acceptors, a glycoprotein of Mr 160 000, comigrated in gel electrophoresis with a cell surface sialoglycoprotein that can be labeled by the periodate-tritiated borohydrate procedure more intensely on intact RA-treated than on untreated cells. Removal of sialic acid residues exposed on the surface of either control or RA-treated cells enhanced 2- to 3-fold the transfer of sialic acid to endogenous acceptors. These results suggest that the increased sialyltransferase activity in RA-treated melanoma cells may be responsible for the enhanced sialylation of certain cell surface glycoproteins. RA treatment of several other tumor cell lines also resulted in stimulation of sialyltransferase activity indicating that this effect of RA is not limited to the S91-C2 melanoma cells.

Comparison of retinoic acid effects on anchorage-dependent growth, anchorage-independent growth and fibrinolytic activity of neoplastic cells.

Abstract We have compared the abilities of retinoic acid to modify the fibrinolytic activity, the anchorage-dependent growth and the anchorage-independent growth of various cultured tumor cells. Treatment with 10 μM retinoic acid for 3 or 6 days increased 2- to 4-fold the level of cell-associated plasminogen activator in S91-C2 and B16 (cell lines F1, F10, F10-B2, BL6 and LR6) mouse melanoma cells and in a human osteosarcoma (Hs781). In a few of the B16 sublines there was a similar increase in fibrinolytic activity secreted into harvest fluids. In contrast, there was no significant effect of retinoic acid on the fibrinolytic activity of five human melanomas (A375, Hs294, Hs852, Hs939 and SH4), two human carcinomas (HeLa-S3 and A431), two human osteosarcomas (Te85 and Hs791) and a murine fibrosarcoma (UV-2237P). Both the anchorage-dependent growth and the anchorage-independent growth were inhibited by retinoic acid to varying degrees in most of the cells employed (A375, Hs939, the B16 sublines, S91-C2, HeLa-S3, Te85 and Hs781), whereas only the anchorage-independent growth of the Hs852, the SH4 and the UV2237P cells was inhibited. The anchorage-dependent growth of the Hs294 cells was stimulated, whereas their anchorage-independent growth was not altered by retinoic acid. These results show that there is no correlation between the effects of retinoic acid on plasminogen activator and on cell growth. The data also indicate that retinoic acid can inhibit the anchorage-independent growth of cells which are resistant to retinoic acid under anchorage-dependent conditions.