Cell surface shedding—The phenomenon and its possible significance

J. theor. Biol. (1976) 62, 253-270

Cell Surface Shedding-The Phenomenon and Its Possible Significance F. DOLIANSKIAND

M. KAPELLER~

Department of Experimental Medicine and Cancer Research The Hebrew University-Hadassah Medical School Jerusalem, Israel

Surface membrane biosynthesis and turnover is reviewed focusing mainly on the fate of cell surface constituents after they terminated their sojourn as part of a functional cell structure. The different experimental approaches to study this problem are described and original data are presented on the turnover of surface membrane constituents of chicken embryo cells in culture. It is proposed that as a consequence of surface membrane turnover, certain surface macromolecules are continuously shed from cells. The size and charge of these molecules was found to be identical to molecules released from cells by mild trypsin treatment. The term shedding is proposed for this process which is assumed to occur both in vitro and in vivo. Many systems in which shedding of cell surface constituents is clearly demonstrated or can be tentatively suggested are described. The biological significance of cell surface carbohydrate containing macromolecules and the possible role of these shed cellular entities is discussed. 1. Introduction

The dynamic nature of the surface membrane can be perceived in full only by watching the living cell as it moves, divides or is engaged in other cell activities. The surface changes continuously, very often vigorously and extensively as, for example, during anaphase bubbling. Although our understanding of the molecular basis of membrane structure and dynamics has increased greatly in the last few years, we are still completely ignorant of the mechanism that enables the surface membrane to undergo such vast and instantaneous changes in surface area, topography and viscosity. Is it by rearrangement of the existing surface component, or are new components --either newly synthesized or present fully formed in intracellular poolst Fell in the Yom Kippur T.B.

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inserted into the newly-formed surface membrane, and if so, how are they removed at later stages? The present paper is concerned with a relatively neglected aspect of surface membrane dynamics, namely, membrane turnover, and our aim is to focus not on the biosynthetic pathway but on the elimination process. The problems concerns the removal and fate of cell surface constituents after they terminate their sojourn as part of a functional cell structure. The turnover of cellular proteins in general and of membrane proteins in particular, was recently the subject of excellent critical reviews by Siekevitz (1972) and Schimke (1975). These authors discuss the possible mechanisms controlling the rate of synthesis and degradation of plasma membrane proteins, and explore ideas on the nature of the linkage between these two processes, a linkage that must exist in order to allow a steady state situation. Whereas a great deal of information is available on synthetic pathways of proteins in general and on the biochemical and morphological pathways of surface membrane biosynthesis in particular (Warren & Glick, 1968; Bosmann, Hagopian & Eylar, 1969; Pasternak & Bergeron, 1970; Gahmberg, 1971; Weiser, 1973a,b, Finger, Lavanchy & Meany, 1973 ; Lodish, 1973; Lodish & Small, 1975; Leblond & Bennett, 1974; Atkinson, 1975; Kaplan & Moskowitz, 1975a,b) very little is known of the disposal path. As pointed out by Schimke (1975), a major difficulty in studying this aspect of turnover is that once a protein identifiable by some marker such as its enzyme activity or its immunological reactivity is eliminated, its fate is difficult to follow. The surface membrane, however, may be uniquely suitable for turnover studies in general, since outer surface components can be specifically and irreversibly labeled (Hynes & Humphreys, 1974; Hubbard & Cohn, 1975a; Juliano & Behar-Bannelier, 1975), thus making it possible to follow the life span of the labeled entities within the membrane and the mode and consequences of their elimination. Although concrete experimental evidence of this kind is as yet scarce and tentative, it will here be reviewed and discussed with the purpose of formulating the problems encountered in these studies and initiating interest in this aspect of cell surface activity. It will be suggested that as a result of surface membrane turnover, some surface constituents are continuously shed to the outside of the cell, both in vitro and in vivo, and that these shed molecules may affect cellular and intercellular activities. 2. Surface Membrane Characteristics

In discussing the elimination phase of surface membrane turnover, several established facts have to be taken into consideration.

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(1) The surface membrane, although morphologically quite similar to intracellular membranes, is structurally and functionally different. Biochemical analysis clearly demonstrates the difference in protein, lipid and carbohydrate moieties (Blomberg & Perlmann, 1971; Evans & Gurd, 1971; Kawasaki & Yawashina, 1971; Lo & Ma, 1973). Furthermore the outer aspect of the surface membrane is predominantly, if not exclusively, rich in macromolecules containing carbohydrates (Cook, 1968; Kraemer, 1971; Atkinson, 1973; Hughes, 1973; Kemp, Lloyd & Cook, 1973 ; Atkinson & Summers, 1974; Cook & Stoddart, 1973; Courtois & Hughes, 1974; Leblond & Bennett, 1974; Schachter, 1974; Butters & Hughes, 1975). Some of these molecules carry out specialized (differentiated) functions of the cell, such as rhodopsin in the the rod cells (O’Brien & Muellenberg, 1973) or sucrase on the intestinal epithelium cells (Weiser, 1973~; Galand & Forstner, 1974). Although most surface glycoproteins are still “structures in search of function” (Eicholz, 1974), it is clear that at least some of them carry tissuespecific functions of the cell. The available information on the turnover of intracellular membranes may, therefore, not be relevant to surface membranes which are so markedly different from the other membranes. In addition, various areas within the surface membrane of an individual cell may also differ widely (Camilli, Puluchetti 8z Meldolesi, 1974; Fujita, Kawai, Asano & Nakao, 1973) and may possess different turnover characteristics. (2) The surface membrane is a highly asymmetrical structure (Steck, 1974). This asymmetry may affect the insertion and release of surface constituents. (3) Although the analysis of the surface membrane of eukaryotic cells is still behind that of erythrocytes (S&k, 1974), the impression gained is that some of its components are common to cells of different types and species, whereas others are characteristic of a specific cell type (Kiehn & Holland, 1970; Brown, 1971; Glossmann & Neville, 1971) such as the liver-specific surface component (Neville, 1968). As in the erythrocytes, some proteins span the whole width of the membrane while others are located exclusively on the outer or inner part of the membrane; the turnover and fate of these differently located components may be different. (4) Differences may exist in the physical state between bulk membrane lipids and the lipids in the immediate vicinity of membrane proteins (Singer & Nicolson, 1972). These lipids may also exhibit different turnovers and modes of elimination. 3. Studies on Surface Membrane Turnover in Vitro and in Viva

The first pioneer studies on surface membrane turnover were done by Warren & Glick (1968) on mouse L cells grown as suspension or as glass

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adherent cell cultures. Surface membranes, metabolically labeled with leucine, glucosamine, glucose or choline, were isolated and analyzed at different periods during and after labeling. The kinetics of loss of radioactivity from the surface membrane showed that proteins, lipids and carbohydrates turned over synchronously, and that their life span was approximately 3-4 days. The process followed first order kinetics, an indication that the molecules were eliminated not according to age but randomly. The rate of synthesis of membrane constituents in growing and non-growing cells was similar, but whereas in growing cells most of the newly synthesized material was incorporated into the newly formed surface, in non-growing cells it was synthesized and eliminated at similar rates. In contrast, recent and thorough studies by Kaplan & Moskowitz (197&b) on exponentially growing and contact-inhibited confluent cultures of a monkey epithelial cell line (MK-2) showed that whereas the rate of synthesis was higher in the growing cells versus the non-growing cells, rates of degradations of both proteins and carbohydrates were similar. Furthermore, they observed that in growing cells, proteins and glycoproteins had heterogeneous rates of turnover. This heterogeneity was much less marked in non-growing cells. A glycoprotein with an apparently high molecular weight exhibited the fastest turnover rate. Using external labeling of surface protein by enzymatic iodination, Hubbard & Cohn (19753) recently showed that the life span of these surface proteins in the L mouse cells growing in suspension is 2-3 days and all the proteins labeled exhibited similar rates of degradation. In the later two works, it was also found that rate of elimination of surface components was biphasic; part of the surface molecules was eliminated rapidly (2-3 hr), whereas the other part turned over at a much slower rate of 2-3 days. Studies on cells in vivo dealt mainly with liver cells (Evans & Gurd, 1971; Franke et al., 1971; Kawasaki & Yamashina, 1971; Gurd & Evans, 1973; Riordan, Mitranic, Slavik& Moscarello, 1974) and intestinal epithelium cells (Weiser, 1973u). It was found that both the non-growing long-lived (months) liver cells and the rapidly proliferating short-lived (days) intestinal epithelium cells actively and continuously synthesize their surface membranes. 4. Turnover of Individual Surface Components Further information on the biosynthesis and turnover of nents stems from experiments of a different design. The types of approaches were used: (1) Cell surface components were removed by exposing proteolytic enzymes or inhibitors of protein synthesis for

surface compofollowing three intact cells to a limited time

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period and then monitoring the re-appearance of the surface marker. In most cases full recovery of the surface molecules measured (antigen, hormone receptor, enzyme, etc.) occurred within 6-10 hr (Gasic & Gasic, 1962; Baker & Humphreys, 1972; Gordon & Cohn, 1971, 1973; Hughes, Sanford & Jeanloz, 1972; Turner, Strominger & Sanderson, 1972; Bhandari & Singal, 1973; Cullen et al., 1973; Kapeller et al., 1973; Ohnishi, 1973; Pellegrino, Ferrone, Del Villano & Reisfeld, 1973 ; Schwartz, Wickner, Rajan & Nathenson, 1973), although in a few instances shorter periods (90 min) were observed (Mauel, Rudolf, Chapuis & Brunner, 1970). The kinetics of recovery found in this type of study are dependent to a great extent on the sensitivity of the measuring system, as was elegantly demonstrated by Marcus & Schwartz (1968). It is often assumed that after stripping of different surface molecules by proteolytic enzymes, the surface “regenerates” the surface site, implying that removal stimulates the cell to replenish the missing part. Warren (1969), Hughes et al. (1972) and Plesser et al. (1976) demonstrated that the rate of biosynthesis of surface molecules after such stripping is not increased and may even be depressed. Therefore, the regeneration observed in different cell types is probably due to normal turnover processes, which seem to work at maximum level at all times and hence do not respond to need. However, a short period of increased biosynthetic activity during certain stages of the cell cycle was observed in several experimental systems (Warren, 1969; Kraemer & Tobey, 1972). (2) Cell fusion experiments-first described by Harris (1970)-in which a metabolically inert ccl1 (chicken erythrocyte) was fused with a metabolically active cell (HeLa cell). After fusion, chicken cell surface antigens disappeared within 3-4 days, an indication that the mechanism of elimination resides in the metabolically active partner. Following the re-activation of the metabolically inert nucleus, surface antigen re-appeared, reaching plateau values after 3-4 days. (3) Experiments using virally infected cells in order to follow the appearance of new surface molecules; these experiments were based on the assumption that the pattern of biosynthesis of surface constituents coded by the virus reflects the normal pattern of membrane biogenesis. This approach was designed by Marcus (1962) in experiments with myxovirus infected HeLa cells. His, to rarely cited classical experiments, were pioneering illustrations of the dynamic nature of the surface membrane, and in elegance and penetration they surpass more recent studies, Marcus demonstrated that the life span of the surface markers was about 8 hr, and he further showed that the new surface components appear at specific sites in the circumference of the cell and then move in a characteristic centripetal pattern. These

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experiments are probably the first indications that insertion and removal of new membrane components does not occur randomly but takes place at discrete sites. In Amoeba proteus, however, Szubinska (1971) believes that random insertion of new plasma membrane into old membrane takes place following membrane injury. 5. The Elimination

Phase of the Turnover Process

Taken together, all the above studies clearly established the fact that the surface membrane undergoes a rapid turnover, both in uivo and in vitro, but did not tackle the problem of the fate of membrane molecules. We began to investigate this problem in the following way (Kapeller et al., 1973): Cell surface components of chicken embryo cells (CEC) were labeled with glucosamine, known to label predominantly surface components (Hammond & Dvorak, 1972; Atkinson, 1973; Hayden, Crowley & Jamieson, 1970; Hughes, 1973; Kemp ei al., 1973; Leblond & Bennett, 1974; Schachter, 1974), following the method of Onodera & Sheinin (1970), who demonstrated that most of the labeled acid precipitable carbohydratecontaining macromolecules are located at a trypsin-removable cell site. We verified that trypsin does not penetrate into the cell (Snow & Allen, 1970) and does not make it leaky, hence the moieties stripped off by the enzyme are located in the outer side of the permeability barrier (Kapeller et al., 1975). We demonstrated (Kapeller et al., 1973) that glucosaminelabeled macromolecules appear in the medium, confirming earlier observations of Kornfeld & Ginsburg (1966), Molnar, Teegarden & Winxler (1965), Kraemer & Smith (1974) and more recent studies of others (Hughes er al., 1972; Sakaiyama & Burge, 1972; Courtois & Hughes, 1974; Kubasova, Varga & Koteles, 1974; Herrmann, Havaranis & Doetschman, 1975). We further showed that the DEAE-cellulose profiles of these molecules were identical to the profiles of the molecules stripped off from sister cultures by trypsin, subsequently referred to as “trypsinate”. The macromolecules appearing in the medium carried a characteristic surface marker-the H-2 antigenic determinant in the case of CJH mouse fibroblasts. We suggested that the macromolecules in the medium appear as a result of shedding of surface molecules as a consequence of surface membrane turnover, and proposed the term “shed” for these molecules. In further studies (Kapeller et al., 1975) we examined in detail the kinetics of turnover of these macromolecules and their shedding. It was found that the rate of incorporation of 13q glucosamine into trypsinate was rapid during the first 10 hr and proceeded at a much slower pace thereafter. No lag

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period was observed, indicating the absence of a pool of pre-formed surface macromolecules. Cycloheximide inhibited biosynthesis by - 50 % after a lag period of 2 hr. Incorporation kinetics, however, should be treated with caution since they are valid only if certain conditions are fulfilled: (1) the label must be present in an excess; (2) rate of entry of the isotope into the cell and pool size should not change during the labeling period; (3) the isotope should not leave the molecule once incorporated, and should not be reutilized. Only condition (1) was fulfilled with certainty, but no direct proof was available for conditions (3) and the second part of (2). Condition (2) is known not to have been fulfilled since glucosamine entry into the cell depends on glucose concentration in the medium (Molnar, Lutes & Winzler, 1965; Harris & Johnson, 1969; Plagemann & Erbe, 1973) and the latter changes continuously, causing overestimation of rates of incorporation. Therefore, determination of turnover rates from decay curves is much more reliable. A biphasic loss of radioactivity from the surface (i.e. trypsinate) with time was clearly demonstrated (Kapeller et al., 1975), indicating that at least two molecular populations are present, one exhibiting a life span of about 10 hr, the other of 4-7 days. It was also found that labeled macromolecules appeared in the medium and that their rate of accumulation was likewise biphasic. The rapid phase was identical to the rate of elimination of radioactivity from the trypsinate. This identity, taken together with the identity between the profiles of trypsinisate and shed obtained by DEAEcellulose (Kapeller et al., 1973) and SDS-polyacrilamide gel chromatographic analysis (Plesser et al., 1976), led us to the conclusion that the normal physiological consequence of surface membrane turnover is that at least some of its macromolecules are shed from the cell surface, having size and charge indistinguishable from molecules in the trypsinisate. Kraemer &Tobey(1972), working with a hamster ovary cell line, also reached a similar conclusion. In line with this conclusion are the findings of Marcus (1962) and Weiss & Coombs (1963) that cells leave “foot prints”, that is, macromolecules adhering to the substrate to which cells have been attached which carry surface determinants. Recently, Culp, 1974, 1975; Culp & Black, 1972; Gulp, Terry & Buniel, 1975; started a thorough biochemical analysis of such substrate attached material. Cell coats of microexudate described by Rosenberg (1960), Revel & Wolken (1973), Maslow & Weiss (1972), Poste, Greenham, Mallucci, Reeve & Alexander (1973) and the “carpets” studied by Yaoi & Kanaseki (1972), all seems to belong to this category of macromolecules. It is interesting that the kinetics of biosynthesis and deposition of this microexudate, its temperature dependence and its sensitivity to cycloheximide (Mallucci, Poste & Wells, 1972) are remarkably similar to our observations on shedding.

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In studies on other cell types we found that shedding can also be demonstrated in a variety of cell types including non-secretory cells such as mouse macrophages, neuroblastoma cells and a lymphoid cell line (Daudi) (Plesser et al., 1976). The possibility that cell surface components are shed from the cell surface was previously mentioned by Warren & Glick (1968). Both these authors believed that it occurs only in non-growing cells. However, they did not examine this possibility experimentally because of technical difficulties. Kaplan & Moskowitz (1975) mention in their discussion on plasma membrane turnover, unpublished observations, that a certain surface glycoprotein is continuously released into the medium, and suggest that the other membrane proteins are degraded by internal proteolysis. Hubbard & Cohn (19753) also concluded that elimination proceeds mainly by complete proteolysis of surface membrane proteins. It is interesting to note that the last-named authors found by autoradiographic analysis of enzymatically iodinated monolayer cultures that considerable radioactivity is found at the cell periphery and in the cell-free area in the dish-a type of result one would expect if shed macromolecules were present around the cells. Release of iodinated (Irz5) surface proteins into culture medium has been described by several other groups of investigators (Chiarugi & Urbana, 1972; Cone, Marchalonis & Rolley, 1971; Huang, Tsai 8z Canellakis, 1973). It can be thus provisionally concluded that some plasma membrane constituents are degraded intracellularly, whereas other membrane components are shed as macromolecules. These observations raise a number of questions: Which components of the surface membrane are shed, and which are not? What is the mechanism of their release from their parent structure? Is shedding linked to biosynthesis or it is an independent process? Are there intracellular membrane reserves? Are they released by a random process or according to age? Do these molecules have any physiological role or are they merely waste products ? Is shedding a process characteristic of most cell types, or only of certain kinds, or does it take place only during certain phases of cellular activities? 6. The Type of Molecules Shed The biochemical characterization of shed molecules is as yet at a very preliminary stage. We found (Plesser et al., 1976) that most (> 85 %) of the glucosamine labeled shed molecules can be resolved on 7.5 % -SDS-polyacrilamide gel into several (7-10) bands. When cells are labeled with either fucose, amino acid or choline in addition to glucosamine, some bands and not others are co-labeled with these precursors, indicating that the shed

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molecules are a heterogeneous population comprising glycoproteins, glycosaminoglycans, and phospholipids. Release of molecules of this type was recently shown in several types of cultured cells (Kraemer & Smith, 1974; Suzuki, 1970; Hughes et al., 1972; Roblin, Albert, Gelb & Black, 1975; Peterson & Rubin, 1969). 7. Mechanism of Release of Surface Components Nothing is known about the mechanism of the release. The identity in size and charge between the molecules in the trypsinisate and the shed raises the possibility that membrane-bound proteolytic activity may be involved. Surface protease activity has been demonstrated in a number of cell types (Ossowski, Quigley, Kellerman & Reich, 1973a,I?) and may be quite a genera1 surface membrane characteristic. No evidence exists at the moment of its involvement in shedding. The fact that shedding is a temperature-dependent process (Kapeller et al., 1975) does not necessarily imply that it involves metabolic or enzymatic activity. We have recently observed in preliminary experiments that soyabean trypsin inhibitor (100 pg/ml) inhibits shedding by 30-50x. Another possibility that may be considered is based on the observation that cells send out long microvilli by which they attach themselves to the substrate. As the cell moves, the tips of the microfilaments are pinched off. This may be a process akin to clasmatosis occurring in certain cells in vim. It could be one of the processes by which surface membrane is shed from the cell. The low surface membrane curvature of the tip may facilitate the dissociation of the departing molecules from their neighboring molecules within the membrane structure. 8. Is Shedding a Random Process Most studies on the decay curves of labeled plasma membranes make it clear that the process of elimination is random (Warren & Glick, 1968; Kaplan & Moskowitz, 1975a; Hubbard & Cohn, 1975b). In our experiments, however, where the carbohydrate-containing surface macromolecules were studied, the decay curves did not provide clear-cut answers as to whether elimination is a random process. 9. Shedding of Surface Components io Different Systems? If the shedding of certain surface components is indeed the normal physiological consequence of surface membrane turnover, not only in vitro but also in tissue cells in vim, it follows that we may expect to find surface

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components in interstitial fluid and serum. The recent interesting observation of Ruoslahti, Vaheri, Kuusela & Linder (1973) that an antigenic determinant found on the surface of cultured chicken fibroblasts is also present in chicken serum, supports these expectations. Similar results were obtained with human fibroblasts and serum (Ruoslahti & Vaheri, 1974). This fibroblast surface antigen was also released into the culture medium. The finding that Beta,-microglobulin present in human serum and urine can be detected immunologically on the surface of human leucocytes (Peterson, Cunningham, Berggard & Edelman, 1972) as well as in culture medium (Cejka, Peterson & Belamaric, 1975) may be interpreted in a similar manner. It should be emphasized that the presence of surface molecules in medium or serum does not necessarily mean that cells shed surface components. Their presence could be due to cell death and disintegration, in which case -obviously-surface components may be released. It could also be due to secretion of intracellular products which may have common properties, or antigen determinants with surface components. The first possibility can be excluded in experiments with cell culture. It was shown (Kapeller et al., 1975) that cell number and viability remain essentially constant. The latter possibility can only be excluded definitely in the type of experiments where surface molecules are irreversibly marked and then their appearance in medium with characteristic kinetics can be demonstrated. This approach was adopted by several groups (Huang et al., 1973; Hubbard & Cohn, 197%) using surface labeling by enzymatic iodination. Most studies were done on B-lymphocytes (Vitteta & Uhr, 1972; Melchers & Cone, 1975; Melchers, Cone, Von Boehmer & Sprent, 1975). The shedding of a specific surface component of B-lymphocytes-the IgM-was clearly demonstrated. The IgM monomer is primarily a protein of the surface membrane with its Fc portion immersed in the lipid layer and its Fab portion facing outwards. It contains the core sugars (glucosamine and mannose) but not the penultimate (galactose), and the terminal sugars (fucose and N-glycolyl-neuraminic acid) (Melchers & Andersson, 1973). IgM synthesis is part of the overall process of membrane biogenesis. Stimulation of the B-lymphocytes results in preferential increase in the rate of IgM synthesis over that of other proteins. These IgM molecules show biphasic rates of shedding: IgM molecules that are rapidly shed (half-life 4 h) and IgM molecules that are released more slowly (Z&SO hr). Melcher & Cone (1975) demonstrated that different lymphoid cell populations exhibit different rates of IgM shedding. The H-2 surface determinants do not show this rapid shedding. This illustrates that differential rate of shedding may exist for different surface constituents. In the case of surface iodinated HeLa cells, Huang et al. (1973) have shown that iodinated surface proteins are released into the medium in both acid-

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soluble and acid insoluble forms. Antibodies prepared against isolated whole surface membranes, as well as antibodies against released surface components, inhibited growth of cells in culture. There are several other systems where shedding was demonstrated. In some systems, the evidence that the released component is derived from the surface is clear, in others it can be only tentatively suggested. Cooper, Codington & Brown (1974), for example, have shown a unique surface membrane glycoprotein on cells of a non-strain specific ascites tumor Iine (TA,-Ha), originally derived from a mouse mammary adenocarcinoma, appearing in the ascites fluid and serum of mice bearing this tumor. The authors believe that it is not released from dead cells but is a result of surface membrane turnover. The released molecule is larger than the trypsin cleaved glycoprotein. Since shedding of this molecule was observed only in the tumor cell line able to grow in an allogeneic recipient, whereas another cell line (TA,-St) which can grow only in a syngeneic host, shedding of this component into the serum does not occur, they assume that circulating membrane-derived molecules may play a role in blocking the immune response to the tumor. Several investigators (Sjogren, Hellstrom & Hellstrom, 1971; Baldwin, Bowen & Price, 1973; Anderson & Coggin, 1974; Graber, 1974; Rim, Baumler, Carruthers & Bielat, 1975) have recently considered the possible immunological consequences (such as blocking, enhancement, tolerance) of shedding of cell surface antigens. The interesting finding of Terasaki’s group that HL-A antigens can be isolated from serum (Miyajma, Hirata & Terasaki, 1972) also strongly suggests that shedding of the surface constituents takes place in uiuo. Ramseier’s (1974, 1975) experiments showing that normal mouse T lymphocytes rapidly shed receptor (recognition molecule) for H-2 antigens in an orderly manner (as a result of a temperature-dependent process) into the surrounding medium, are also in line with the above observations. The shedding proceeds in several 8-h waves. Ramsier also found that antigen-induced receptor release was faster than receptor shedding in the absence of antigen. In studies on another lymphoid cell surface antigen-the TL antigen-it was found (Yu & Cohen, 1974) that synthesis and shedding of this antigen can be demonstrated in TL+ but not in TL- lymphoid cells from TL+ or TL- mice. Shedding of 3H-fucose or rz51 labeled TL antigen into the medium was similar in TL+ lymphocyte in the presence of normal syngeneic serum as well as in medium containing anti-TL serum. The rate of synthesis of the TL+ antigen was found to be identical in modulating and non-modulating lymphocytes. The fact that the rate of biosynthesis and the release of a surface component may not alter following its specific interaction with an antiserum, suggests that the rate of membrane turnover must not necessarily be affected as a

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result of such interaction. Macrophages were also shown to shed surface components into the medium as demonstrated by Schroit, Geiger & Gallily (1973). 10. PossibleRole of Shed Macromoecules

In order to elucidate the possible roles of shed molecules, we examined their effect on the shedding processes (Kapeller et al., 1975). Medium of 48 hr confluent CEC cultures was filtered through Diaflow membranes to increase the concentration of the shed molecules IO-40 fold. It was found that the higher the concentration of shed, the slower the shedding process. Cell population density had a similar effect. These results lend support to Rubin’s concent of the dynamic nature of the plasma membrane (1966). He suggests that plasma membrane components (sub-units) are released from the cells and can be re-inserted into the membrane. At low cell densities, moving out of membrane components predominates, imposing a state of instability in membrane structure and preventing the proliferation of cells. Increasing the concentration of membrane components in the cell periphery by the use of conditioning factor or feeder layer restores physiological equilibrium and allows cells to proliferate. In line with this are the recent findings of Yamada & Weston (1975) demonstrating that a large surface glycoprotein of chick embryo cells, isolated from celIs can be progressively readsorbed on cells depleted of this component by cycloheximide. Apart from contributing to the maintenance of surface membrane integrity, at least under certain conditions, these shed molecules may influence growth and development of cells. This suggestion is based on the following consideration: All these molecules, as stated earlier, contain carbohydrates, most of them probably being glycoproteins, glycosaminoglycans, glycolipid or lipoglycoprotein complexes. The special property the carbohydrate moiety confers on a macromolecule was stressed by Winzler (1973). These are characterized by their high informational content-thus, whereas a disaccharide unit is capable of forming 16 alternative conformations, two amino acids have only one possibility. Obviously, such macromolecules may be suspected of carrying highly specific functions. It is a widely accepted notion that cell surface sugar components play a role in sped& cell-cell and cellsubstrate interactions (Cook, 1968; Kemp et al., 1973; Roseman, 1970). The fact that these molecules can be stripped off the cell while the cell remains viable and capable of growth leads to the assumption that these molecules are luxury molecules in Ephrussi’s (1972) and Holtzer’s (1968) sense, i.e. molecules serving specialized, differentiated functions and not basic and maintenance functions.

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Their possible functions in growth, development and differentiation can be inferred from recent studies on widely different experimental systems. The aggregation-promoting factor (APF) can serve as one such example. It enhances tissue-specific association of isolated embryonic cells to form organized and developing tissues (Lilien, 1969; Moscona, 1974). It was shown to be a glycoprotein, derived from the cell surface and accumulating in the culture medium. Recently APF was isolated and partially characterized (Balsam0 & Lilien, 1974; Hausman & Moscona, 1975), and it was suggested that its highly specific tissue afEnity is determined, at least in part, in the sequence of sugars in an oligosaccharide residue of the glycoprotein (Balsam0 & Lilien, 1975). Hausman & Moscona (1975), on the other hand, found that the cell aggregating effect of the factor did not require the integrity of the carbohydrate part of the molecule. Another interesting example which may be relevant to the problem of the role of shed molecules is the colony-stimulating factor (CSF). Formation of colonies by bone marrow cells in cultures in viscid media is enhanced by a factor found in the growth media of a variety of cell types. Recent studies on the biochemical characterization of this factor have shown that the cell surface membrane is the reservoir for the colony-stimulating activity (Price, McCulloch & Till, 1975). The macrophage growth factor also produced and released into the medium by certain cell types in culture was found to be a trypsin-removable cell surface moiety (Cifone & Defendi, 1974). Several groups of investigators (Culp, 1974, 1975; Weiss, Poste, MacKearin & Willet, 1975; Butters & Hughes, 1975) have recently described the effect on the growth pattern of cultured cells of the substrate attached material produced by cells in culture. The morphogenetic influences of glycosaminoglycans (Hay & Meier, 1974; Meier & Hay, 1975; Moran & Rice, 1975) as well as the characteristic changes occurring in the cell-surface carbohydrate containing macromolecules during cell differentiation (Pinsker & Mintz, 1973; Zalik & Scott, 1973; Galand & Forstner, 1974) further suggest that these types of molecules are involved in the expression of differentiative functions of cells and may serve after being released from the cells, as mediators of inductive stimuli during development. 11. Concluding Remarks Multicellular organisms evolved from unicellular organisms. All cells possess carbohydrate-containing macromolecules as part of their surface structure. These macromolecules form a border area which is the first to react with the environment, and they have to protect the cell from its hazards.

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During the emergence of multicellularity a new biological property evolved -cell-cell adhesion-making a multicellular organism possible. This cell-cell adhesion is necessarily mediated through the carbohydrate-rich area and it is reasonable to suspect that the latter plays a part in cell associations. In this new biological situation, some form of information transfer from cell to cell allowing the unit to function as a whole must exist. Following Carter’s (1968) ideas on control of cell movement and cell division, we propose that the specific cell adhesion and control of proliferation are mediated by the same molecular surface entities. Furthermore, many of the new functions developing as a result of multicellularity may be expected to be associated with these entities. As we have seen, these surface carbohydrate-containing macromolecules undergo active turnover, and at least some of them are shed. The shedding of these molecules, carrying receptors, antigens (what is their function?), enzymes and other constituents keeps the cell surface in a virgin state, freed from all those complexes formed between surface sites and environmental factors, whether hormones or antibodies, and free to accept new stimuli or messages. This process seems to work continuously and at a maximum pace. As to the shed molecules, their role after having left the cell is still obscure. Several possible roles can be envisaged: First, they may play a part in a dynamic interchange between shed constituents and their parent surface structure. Second, the various unidentified factors in culture media and sera such as the aggregation-promoting factor, inducer of morphogenetic development, colony stimulating molecules or chalons may turn out to be surface shed molecules. Third, shed molecules carrying surface antigens may play an immunological role, such as allowing a state of tolerance in a system where cellular recognition is present. Finally, the dynamic nature of surface membrane (expressed in many of its properties, including turnover and shedding) is part of the universal property of living matter-the continued replacement of parts-molecules or cells. To maintain such a high turnover and shedding of macromolecules and cells, the organism has to invest an enormous amount of energy. This raises the question: “What is the physiological raison d’t?tre of this continuous replacement ?” Siekewitz (1972) recently proposed several models that may explain the usefulness of this turnover for the organism. We propose a further conjecture based on Szent-Gyorgi’s (1972) ideas on living matter. In his book The Living State he writes: “In inanimate systems the most stable state is at the minimum of free energy and maximum of entropy. This is ‘physical stability’. In living systems the opposite is true. The greatest stability is at the maximum of free energy and minimum of entropy, which corresponds to the best working order. This is ‘biological stability’. In

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terms of biology, physical stability means death. Life tends toward biological stability which can be maintained only by function, and if there is no function the system slips towards physical stability.” Inactivity brings atrophy. Structure is maintained by function, which maintains itself by work. The physiological significance of turnover may be to maintain “good working order” that will help to preserve the stability of the biological system. REFERENCES ANDERSON, N. G. & COGGIN, J. H. (1974). In The Cell Surface in Development (Moscona, A. A.. ed.). D. 297. New York: John Wiley & Sons. AT&N, P.* h. (1973). In Methodr in Cell-Biology, vol. 8, p. 158 (Prescott, D., ed.), New York: Academic Press. ATKINSON, P. H. (1975). J. biol. Chem. 250, 2123. ATKINSON, P. H. & SUMMERS, D. F. (1974). J. biol. Chem. 246, 5162. BAKER, J. B. & HUMPHREYS, T. (1972). Science, N. Y. 175, 90.5. BALDWM, R. W., BO~EN, J. G. & PRICE, M. R. (1973). Br. J. Cancer 28, 16. BALSAMO, J. & LIL~N, J. (1974). Proc. natn. Acad. Sci. U.S.A. 71, 727. BALSAMO, J. & LILIEN, J. (1975). Biochemistry 14, 167. BHANDARI, S. C. & SINGAL, D. P. (1973). Tissue Antigens 3, 140. BLOMBERG, F. & PERL.MANN, P. (1971). Expl. Cell Res. 66, 104. Bosm~~. H. B., HAGOPIAN, A. & EYLAR, E. H. (1969). Archs. biochem. Biophys. 130, 573. BROWN, J. C. (1971). fipl. Cell Res. 69, 440. Bumms, T. D. & HUGHES, R. C. (1975). Biochem. J. 150, 59. CAMILLX, P. D., Pu~.ucmm, D. & MELDOLFSI, J. (1974). Nature. Land. 248, 245. CARTER, S. B. (1968). Nature, Land. 220,970. C%XCA, J., PETERSON, W. D., BELAMARIC, J. 8c KITHIER, K. (1975). Devel. Biol. 43, 200. CHURUGI. V. P. & URBANA, P. (1972). J. gen. Virol. 14, 133. CIFONE, M. & DEFENDI, V. (1974). Nature, Land. 252, 151. CONE, R. E., MARCHALONIS, J. J. & ROLLEY, R. T. (1971). J. expl. Med. 134, 1373. COOK, G. M. W. (1968). Bill. rev. Cambridge 43, 363. COOK, G. M. W. & STODDART, R. W. (1973). In Surface Carbohydrates of Eukaryotic Cells. London-New York: Academic Press. COOPER, A. G., CODINGTON, J. F. & BROWN, M. C. (1974). Proc. natn. Acad. Sci. U.S.A. 71, 1224. GXJRTOIS, Y. & HUGHES, R. C. (1974). Eur. J. Biochem. 44, 131. CULLEN, S. E., BERNARD, D., CARBONARA, A. 0.. JAIZ~T-GUILLARMOD, H., TRINCHIERI, G. & CEPPELLINI. R. (1973). Transplant. Proc. V, 1835. CULP, L. A. (1974). J. Cell Biol. 63, 71. CULP, L. A. (1975). Expl. Cell Res. 92,467. CULP, L. A. & BLACK, P. H. (1972). Biochemistry 11, 2161. CULP, L. A., TERRY, A. H. & BIJNIEL, Y. F. (1975). Biochemistry 14, 406. EICHOLZ, Z. (1974). Science, Wash. 186, 1109. EPHRUSSI, B. (1972). In Hybridization of Somatic Cells. p. 53. New Jersey: Princeton University Press. EVANS, W. H. & GURD, J. W. (1971). Biochem. J. 12.5, 615. FINGER, I., LAVANCHY, P. & MEANY, A. (1973). J. Cell Biol. 56. 434. FRANKE. W. W., MORRE, D. Y., DEIJMLING, B.,CHEE~~M, R. D., ~TENBECK, J., JARAXH, E. D. & ZJLNTQRAF.H. W. (1971). Zeitschr Natarforschww 26b. 1031. FUJITA, M., KAWAI, K., AWNS, S. & NAKAO, M. (1573). Bioihim..Biophys. Acta 307,141. GAHMBERG, C. G. (1971). Biochtm. Biophys. Acta 249, 81.

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