The concanavalin a agglutinating system of cell membranes

BioSystems 6 ( 197 5) 209-216 0 North-Holland Publishing Company, Amsterdam - Printed in The Netherlands

A

OF CELL

AMES

NEJAT DtiZGmE;S Department of Biophysical Sciences and Center for Theoretical Biology, State Uniuersity of New York at Buffalo, Amherst, N.Y. 24226, U.S.A. The hopes raised by the finding of differential ConcanavaIin A (Con A) agglutinability formed cells in efforts to quantify differences between these cell lines seem not to have paper, the development of information on the ism of action of Con A on the cell theories put forward at each stage of this dev ent are surveyed. In this respect, constitute a case hiat ‘n biological research. involvement of glycoproteins and c -cell interactions an eir relations to Con A are also reviewed.

of normal and trans- ’ been justified. In this surface, as well as the stigations 0x1 Con A actoayitransferases in

210 II.

Glyeoproteins

The presence of carbohydrate-containing materials at the cell surface has been demonstrated by electrophoresis of intact ceh, by immunolo&al studies and by microscopical techniques (Kemp et al., 1973). Glycoproteins may be broadly defined as those proteins containing carbohydrate covalently linked to the polypeptide portion. The major sugars found in glycoproteins are D-ghcase, D-galactose, D-mannose, L-fucose, Nglucosamine, N-acetylgalactosamineV neuraminic acid (sialic acid), L-arabinose and D-xylose (Sharon, age between amino acids and. s Q- and N-glycosidic bonds; limited to certain amino acids and however. The carbohydrate in structure and coproteins 0 molecule altho from mole peptide portion is invariant. This is due to the mechanism of synthesis of glycoproteins, the ing added at sites on polypeptide has been nthesized (Bretscher, 1973). This ed out by enzymes called glyc rases, specific for each saccharide. ation is possible in the oligosacchaosition of a ~~ic~~~~g~ycoprotein. sted that these macromolecules tion surface and could be the basis for ecificity in cellul on due to the

s, enzymes capable of ar residues from glYcoproon in the adhesion of cells

b

a wide variety of interactive phenomena (phagocytosis, bacterial attachment) (Kemp et al., 1973). (ii) Siiple sugars in growth medium affect the interactive behavior of cells; ghzcosamine, for example, inhibits the ion of trypsin-dissociated embryonic cells in vitro, either due to its effect on glucose metabolism or on the biosynthesis of cell surface materials. Other complications are also p ever (Kemp et al,, 1973). ation indi(iii) Studies on sponge cell cate that the “ is a multicomponent, multivalent glycoprotein and that carbohydrates can provide specificity to its Burger, Weinbaum arid

A ~onc~v~n A bind~g protein is0

sYs

211

The information presently available is not sufficient to discriminate between these two hypothesis; nor is there any detailed study of surface alterations under the influence of Con A. Our understanding of ea?n the large scale action of Con A (at the cellular level) has been partially achieved through several controversies over the past five years. We shall now describe some of these studies with reference to the working hypoth,eses developed as e work in this field progressed. Inbar and Sachs (1969) and B parent cells are not.

A, whereas the normal treatment of northem ~glut~able

(BHK) cells and polyoma virus transformed BNK (PyBHK) cells are also similar (8.&--6.8 X 10’ sites, respectively) (H&l-Wallach, 19’72). Inbar and Sachs (1969) indicate that .about 85 per cent of the ar-methyl-D-glucopyranoside binding sites of Con A are in a cryptic form on normal cells. At cell division, however, the lectin sites of normal cells are exposed; fluorescein tagged wheat germ agglutinin (another plant lectin) specifically labels 3T3 cells in mitosis as well as virally 971). There is a

212

inhibition of growth in cultures of polyoma transformed 3T3 cells which normally do not exhibit contact inhibition of growth. The higher the concentration of monovalent Con A in the medium, the more the growth behavior of the transformed cells approached that of cells. Thus, there seems to be a quantitative re;ationship between the covering of glutinin receptor layer and the degree to which contact inhibition of growth is expressed. For the monovalent Con A to be efhowever, a particular cell density has reached in the culture. Burger and t that the loss of contact inNonnan be ascribed to either the abhibition sence of a “cover layer” or the exposure of inin receptor site, which when covthe split lectin forms an artificial cover layer. This covering may affect the physicochemical surface properties like adhesiveness or membrane flexibility which is important for cell mobility and cell division; it may influence m attachment or pe

changes in the membrane brought about by the transformation process or of the incorpsome virus-inoration into the membrane duced gene product, which would then act as a nucleus for aggregation. In the “semi-cryptic sites” model (see Nicolson, 1971) the presence of surface structures prevents agglutination although Co (or any other lectin) can bind these s These surface structures can be proteolytic treatment or malign mation. This is, then, an alternative explanation i&r the proteolysis experiments mentioned above. Alternatively, the results of electron microscope studies using ferritin labeled Con A on 3T3 and SV3T3 cells indicate that there is a 3-3.5 fold ’ density of Fer-Con A sites on the SV3T3 membrane surface and Con A is more clustered in its dis (Nicolson, 1971). Since SV3T3

In addition* to the three hypotheses considered by Burger (1969), Singer and Nicolson cells) is responsible for

tinab~~ty .

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agglutinable at the lowered temperature. It is suggested that *he component which determines agglutination may be associated with a specific metabolic activity, presumably an enzyme which is active only in the transformed cell membrane and which may change the surface charge (Inbar et aL, 19 Recent results, however, have considerably ged our view of the Concanavalin A agating system. The experiments by Micoln (1973) on the temperature dependence of the mob~ity of Con A sites on tumour cells have shown that it is the tetravalent Con A molecule which causes the clustering of the escein thioisocyanate

are labelled with incubated at 22°C or cooled at 0°C for label-

vent the reorganization of k&in-binding sites, for example in phagocytosis (Oliver et 1974). Similar results have been obtained by Rosenbhth et al. (1973). Chemical fixation of cells before labeling with Con A and hemocyanin (which binds &n A and is readily visible by electron microscopy in conventional shadowcast replicas), or labeling exclusively at 4” C allows one to d&tin tween the original top0 hical distribution of Con A binding sites rangement of these sites b ~~nte~~ce of the eel tion of the fore, Con Al the i~~he~e~t to It is observed

cona, 1971), much like transformed cells. The agglutina;iion of EDTAdissociated cells is much faster and more extensive than that of trypsin dissociated cells. Embryonic cells from these two different tissues can therefore react with Con A without proteolytic treatment, unlike culture cells from adult cell lines. Moscona (1971) offers the interpretation that the embryonic cells have unmasked binding sites for Con A on their surfaces and that these sites may function in morphogenetic cell contacts, cell mobilitjl and tissue anization durin en cells reach an orphogenesis is completed, come masked. The neoplastic transformation would then implicate a “retrogression” to the e Con A sites to

components to account tinability. The use of E in inducing an incre

shown that the

Another aspect of the cell surface with which Concanavalin A has been shown to have a relationship is the gaJa&osyltransferase activity. Roth and White (1972) have shown that 3T3 cells have a transferase enzyme which is capable of hansferring galactose from uridine diphosphate galactose to galactosyl acceptors on adjacent cells after intercellular contact is made. However 3T12 cells, -which are not contact inhib not require contact with cation between cells tions between cell s

and communi-

215

Con A to a specific glycoprotein, in this case galactosyltransferase, which leads to agglutination, then the difference between agglutinable and non-agglutinable cells may be the amount of a specific membrane galactosyl transferase present (Podolsky et al., 1974). In the experiments mentioned above, changes in surface structure has been determined from an agglutination reaction and not from any specific chemical change accompanying lectin binding. It is not clear that the lectin receptor site is identical in normal and transformed cells (Kemp et al., 1973). The interpretations given by Nicolson (1973) and Rosenblith et al. (1973), only imply a change in the nature of the membrane, not specifically in the Con A binding sites. The hopes raised by the finding of differential Con A agglutinability of normal and transformed cells in trying to quantify differences between these cell lines seem not to have been justified. The mechanism of action of Con A is still controversial; however if the fluid dynamic interpretation is correct, five years of Con A studies will have taught us little more than the more mobile nature of the malignant cell membrane components. The problem of the associations between this increased fluidity and the control of cell proliferation, perhaps through microfilaments and microtubules, has more intriguing prospects at this time. References Aldrich, H.C. and J.H. Gregg, 1973, ‘Unit Membrane Structural Changes following Cell Association in Dictyostelium”, Exptl. Cell Res. 81,407. Auerbach, R,, 1971, “Contacts and Communications between Cells in Their Relationship to Morphogenesis and Differentiation”, in The Dynamic Structure of Cell Membranes (D.F. H&l-Wallach, H. Fischer, eds.) p. 37, Springer-Verlag, Berlin. Bretscher, MS., 1973, “Membrane Structure: Some General Principles”, Science, 181,622.

The author was supported by NIGMS Grant Ii-TGlGM00718 to the Departraent of Biophysical Sciences.

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