Stoichiometry of Wheat Germ Agglutinin As a Morphology

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STOICHIOMETRY OF WHEAT GERM AGGLUTININ AS A MORPHOLOGY CONTROLLING AGENT AND AS A MORPHOLOGY PROTECTIVE AGENT FOR THE HUMAN ERYTHROCYTE Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021

REX E . LOVRIEN and RICHARD ALLEN ANDERSON

From the Biochemistry Department, Gortner Laboratory, Biological Sciences, University of Minnesota, St . Paul, Minnesota 55108

ABSTRACT The lectin wheat germ agglutinin (WGA) is an unusually effective agent in controlling both the forward and reverse reactions of the reversible morphology conversion discocyte ~:± echinocyte for the human erythrocyte . Under conditions severe enough to drive the reactions to completion in either direction without the lectin, WGA is able to stabilize both these morphologies and to fully prevent conversion of either morphology . The lectin can quantitatively block both reactions . The ability of WGA to carry out these functions has no obvious rate limitation . Its effectiveness depends mainly on its binding stoichiometry, particularly toward the transmembrane glycoprotein, glycophorin . The critical binding stoichiometries for both the lectin and the echinocytic agent were determined in relation to the binding isotherms using "'I-labeled WGA and 35 S-labeled dodecyl sulfate . There appear to be two principal stoichiometries for WGA binding that are important in its control of erythrocyte morphology . The first stoichiometry marks the threshold of obvious protection of the discocyte against strong echinocytic agents such as detergents and, likely, is simply a 1 :1 stoichiometry of WGA : glycophorin, assuming currently recognized values of 3-5 X 101' copies of glycophorin per cell. The second important stoichiometry, whereby the cell's morphology is protected against extremely severe stress, involves binding of -4-5 WGA molecules per glycophorin . The controls that WGA exerts can be instantly abolished by added N-acetylglucosamine . However, N-acetylglucosamine ligands on the erythrocyte are of less importance than membrane neuraminic acid residues in enabling WGA to control the cell's morphology, as is shown by comparing intact cells with completely desialated cells . WGA can also be used to produce elliptocytes in vitro, but it does this at levels approaching monolayer coverage of the cell with WGA .

Lectins have long been recognized as proteins that tecting several of the blood groups (17) . Less ob- agglutinate human erythrocytes . Their agglutina- vious, however, is the extent of the control lectins tion properties have been important tools in de- exert over the discrete morphologies that may be

534 J . CELL BIOLOGY ©The Rockefeller University Press " 0021-9525/80/()6/0534/15 $1 .00 Volume 85 June 1980 534-548

obtained from the normal discocyte, if indeed they trolled morphology changes, viewed in their rela- affect erythrocyte morphology at all . tion to amounts actually bound, v, may engender The echinocyte is a common, well-recognized a different picture from that evoked simply by the morphology that can be readily produced from the total added levels, y . discocyte by echinocytic ligands such as fatty acid The most critical stoichiometries for WGA anions and alkyl sulfates . Upon removal of the binding to the erythrocyte, from a correlation of echinocytic ligands by binding them to a protein the direct binding' studies and their relation to such as serum albumin, echinocytes may be erythrocyte morphology changes, need to be com- quickly returned to discocytes . Therefore, these pared with the number of copies of glycophorin two morphology conversions may be represented per erythrocyte membrane . This is for two reasons: as the reversible reaction discocyte ;::± echinocyte . first, glycophorin has been thought to be the prin- The main emphasis of this paper is the control cipal receptor for WGA (2) ; second, glycophorin that wheat germ agglutinin (WGA) can impose on is increasingly thought to be a transmembrane Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021 both reactions in this conversion . However, WGA protein, spanning the erythrocyte membrane and can also produce a discrete morphology from nor- linked to the cytoskeleton . Inasmuch as glyco- mal erythrocytes, namely, the elliptocyte. That is, phorin is an avenue for control of at least some of WGA can drive the in vitro conversion discocyte the determinants of the cell's morphology (26), -). elliptocyte by itself without the addition of WGA might be expected to affect the cell's mor- another agent . Ordinarily, elliptocytes are pro- phology . duced only by in vivo elliptocytosis . The eleven lectins that we examined are listed There are diverse views pertaining to how the with their specificities, which are rather diverse, in human erythrocyte's morphology is controlled and Table I . None of the other lectins exhibited nearly to how the membrane lipids and proteins are the force nor the versatility that WGA can exert involved . One view is that the integral membrane as a controlling agent of erythrocyte morphology . proteins have no control of the cell's morphology, Moreover, WGA performs its functions quite rap- but that the membrane lipids are paramount (37) . idly, within 20-100 s in the concentration range of An alternative view is that the parameters that 10-'-10 -K M, unlike the other lectins . Apparently, describe the erythrocyte membrane's mechanical WGA's interaction with the erythrocyte and the properties are remote from the parameters ex- control it has over it depend simply on equilibrium pected simply from lipid bilayer behavior . There processes, not on rate processes, at 25°C . This is seems to exist a fairly tough matrix, requiring the case for both the forward and reverse reactions contributions by proteins (10) . The majority of in the discocyte ~:± echinocyte conversion . Accord- authors, considering a number of the biochemical, ingly, our work concentrated on WGA's ability to compositional, and biological aspects of the eryth- control erythrocyte morphology. rocyte membrane, believe that there is important involvement of membrane proteins ("mechano- MATERIALS AND METHODS proteins") in both the cytoskeleton and transmem- Intact Erythrocytes and General Procedures brane proteins that govern cytoskeletal behavior Erythrocytes were drawn from individuals of various blood (22) . types using procedures quite similar to those previously used We subscribe to this second view because of the (21). The cells were washed three times in isotonic Tris-saline avenues through which WGA likely exerts its (0.015 M Tris, pH 7.4) and were used for experiments within 3 h after the last washing. New stocks were generated for each control over erythrocyte morphology . In the search day's use . All components used to interact with the cells were for simple relationships, namely, the stoichiome- dissolved in the same Tris-saline buffer and rapidly mixed im- tries, between membrane morphology controlling mediately upon addition . Components were added in amounts agents and their receptors, it is necessary to gather that approximately doubled the cell suspension volume in each giving cell -1 x 10' detailed data from the regions in which the mem- case, a final standard concentration of cells/ ml, except when agglutinating cell concentrations were used . The brane's morphology changes are brought under sequence of addition of the various reagents is most important control and to determine the binding isotherms for and is described below . The standard temperature was 25 .0 ± the lectins that can exert such controls . The dis- 0.5 °C in all experiments involving discocyte z:± echinocyte equi- tinction between amounts bound, denoted v, and libria . Both 25' and 37 °C temperatures were used in the discocyte - elliptocyte conversion. The cells were fixed by the standard amounts added, denoted y, is important, particu- procedure for bright-field microscopy with a final concentrations larly when the binding constants are intermediate of 0.1% of SEM grade glutaraldehyde (Sigma Chemical Co., St . or small. The practical result is that lectin-con- Louis, Mo .), including phenol red to indicate pH 7.4 .

LOVRIEN AND ANDERSON Agglutinin as Controlling Agentfor Erythrocytes 535 TABLE I Lectins Surveyed

Specificity (14) Concentra- Lectins lion range Monosaccharide Erythrocyte receptor Ng/rnl Concanavalin A 0--5,000 a-D-Manp > a-D-Glcp Band 3 (12) Ricinus communis (120,000-dalton 0-1,000 13-D-Galp > a-D-Galp Band 3 and others (2) tetramer) Lens culinaris 0-200 a-D-Manp > a-D-Glcp Glycophorin, band 3 (l2) Phaseolus vulgaris 0-2,000 Oligosaccharides Of ,ß-D-Galp, /3- Glycophorin, band 3 (24) D-Manp, and ß-D-GaiNacp Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021 Arachis hypogaea 0-2,000 ß-D-Galp-(1-3)-D-Gal Nacp Limulus 0-500 NANA Solanum tuberosum 0-1,000 ß-D-GlcNAcp oligosaccharides Bandeiraea simplicifolia 0-1,000 a-D-Galp > a-D-GaINAcp Glycine max 0-3,000 a-D-GaINAcp >,8-D-GaINAcp Pisum sativum 0-500 a-D-Manp > a-D-Glcp Wheat germ agglutinin (Triticum 0-500 ,8-D-GlcNAcp, oligosaccharides Glycophorin (2) vulgaris) and NANA

WGA, 125 1-WGA, and N-acetylglucosamine Electron Microscopy Preparation (NA G) Cells were fixed for electron microscopy with a combination of 2 ml of cell suspension and 0.1 ml of 2 .1% SEM grade The lectin was prepared from raw wheat germ using the glutaraldehyde in the buffer, with the glutaraldehyde reagent method of Bouchard et al . (5), followed by affinity chromatog- adjusted to the phenol red endpoint (5 mg/liter of phenol red). raphy on Ovomucoid-Sepharose according to Marchesi (23) . The After 20 min, the concentration of glutaraldehyde was increased lectin was crystallized at pH 4.5 in an acetate-NaCI system (0.1- to 3% by the addition of 2 ml of 6% glutaraldehyde . The mixture 0.2 M concentrations of each component) at 4°C . was incubated for 2 h at 25 ° C . The cells were then washed three For 'r'1 labeling, the Fraker and Speck procedure (13) was times in water. A drop of concentrated cell suspension was placed used . This method has several distinct advantages over lactoper- on a coverslip and immersed in 25% acetone . The acetone oxidase-based methods for iodination of proteins . The' Lr'I-WGA concentrations were increased by increments from 25 to nearly prepared in this way (average specific activity, 5 x 10' dpm/mg) 100% acetone . The cells were then dried at the critical point of was >95% retained on the affinity column . The molecular weight carbon dioxide. 10-15 A ofgold wascondensed onto the mounted of WGA is taken as 36,000 . cells by evaporation with a Denton DVS02 vacuum evaporator The inhibitor N-acetylglucosamine and sialic acid were pur- (Denton Vacuum Inc., Cherry Hill, N. J .) at 10 ' torr . The cells chased from Sigma Chemical Co . on their mounts were viewed in a Hitachi SEM Model 450 .

Radiolabeled Sodium Dodecyl Sulfate Neuraminidase Desialation c "S-labeled dodecyl sulfate (sodium salt) was prepared by use Sialic acid residues (N-acetyl neuraminic acid [NANA) resi- of ["S)chlorosulfonic acid (Amersham-Searle Corp ., Arlington dues) were removed by means of Vibrio cholerae neuraminidase . Heights, 111 .) in a procedure starting with dodecyl alcohol, using The pH range was 7 .0-7 .4 in Tris-saline isoosmotic buffer, which Davidsohn and Milwidsky's general synthetic method (8) . After also contained 10 mM CaCl_ . The cell concentration was l x 10" coupling and removal of HCl, the mixture was neutralized to the cells/ml (300 Behringwerke U/ml (36)) at 37°C for 1 .2 h. The phenol red endpoint in the cold with NaOH and lyophilized . It reason for using a Tris buffer in conjunction with this enzyme is was then recrystallized three times from absolute ethanol . The that the Ca" stays in solution. Phosphate buffers, which are specific activity was 7.5 x 10" dpm/mot; mot wt, 288.7 . sometimes used, precipitate calcium (1) . The amount of released NANA was quantitated by thiobarbiturate colorimetry (42). Binding Isotherms and Scintillation Counting The general procedure used for determining the amount of RESULTS material bound has been previously described (2l) . It relies on centrifugation of the cells and calculation of the amount bound Lectins Surveyed as the difference between the amount added and the amount left Two aspects of 11 lectins were studied : their free after equilibration . Scintillation counting was carried out in change the morphology of human eryth- Patterson and Greene's fluid (28) in a Beckman LS3133 (Beck- ability to man Instruments, Inc ., Fullerton, Calif.) at an efficiency of 60- rocytes and their ability to control morphology 70%. changes imposed by echinocytic agents . The 11

536 THE JOURNAL OF CELL BIOLOGY - VOLUME 85, 1980

lectins are listed in Table I, together with their production of a discrete morphology engendered specificities . WGA turned out to be the most by WGA are displayed in Fig. 2 and are described reactive lectin in both regards . The controls which below. the other lectins seem to exert are quite diverse (a) The elliptocyte may be readily produced and merit a separate study . A salient difference from normal discocytes in vitro by proper adjust- between WGA and the other lectins is that WGA ment of WGAand cell concentration . At least 80% carries out its effects on the erythrocyte nearly as conversion to elliptocytes can be achieved at 25°C, rapidly as the cell can change shape without the and the conversion is even more complete at 37 °C. lectin, processes about which detailed knowledge Fig . 1 c shows typical results from the 25 °C con- exists (3) . The other lectins, especially concana- version . Cells from seven different individuals with valin A, are markedly time dependent in whatever various blood types were investigated. No major controls they exert, at least with respect to the differences were seen within this group in the Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021 erythrocyte at 25 °C. The properties of WGA vis- WGA-driven conversion discocyte ---), elliptocyte . à-vis the erythrocyte are apparently controlled by (b) The lectin WGA is a powerful agent, which whatever thermodynamic limitations exist, can protect or stabilize the normal human eryth- whereas the other lectins are rate limited in their rocyte in certain of its morphologies, including the overall reactions with the erythrocyte, as far as discocyte . A vivid illustration of this is provided control of morphology is concerned . Therefore, by our experiments with the strongly echinocytic our main work was confined to the WGA-eryth- agent dodecyl sulfate . The sequences of addition, rocyte system . or pathways, are shown in Fig . 2 . Echinocytes, which are readily formed by such agents from Erythrocyte Morphologies and Pathwaysfor normal discocytes, are shown in Fig. 1 d. The lectin Conversion WGA can, however, completely block formation The controls that WGA imposes on erythrocyte of echinocytes if added at the proper point in the morphology depend on cell concentration and on sequence . Fig . 1 e shows cells protected by WGA, the amount of WGA added (YWCA) . Most of the essentially retaining the discocyte morphology, in conditions that we used pertain to subagglutinat- the face of dodecyl sulfate at a concentration 50 to ing concentrations of erythrocytes on the order of 100 times that ordinarily needed to produce echin- 106-5 x 10 7 cells/ml . However, it is striking how, ocytes such as those shown in Fig . 1 d . The cells when agglutinating concentrations of cells are em- shown in Fig. 1 e are not perfect discocytes . They ployed with WGA (5 x 10" cells/ml or greater), retain the overall discocyte morphology but are extreme (and instant) distortion ensues, initiated not smooth or normal like those shown in Fig. l a . by WGA's effects on the membrane . Fig . 1 a illus- An illustration of the extreme distortion that the trates normal (control) discocytes . Fig . I b shows imperfect discocytes in Fig .I e would have pro- a typical WGA agglutinate of erythrocytes . Under duced if they had not been protected by WGA is agglutinating conditions, isolated erythrocytes still provided by Fig. If. The cells in Fig . 1f were remain normal discocytes . The extreme distortion subjected to the same concentration ofechinocytic to helmet-shaped cells, codocytes, knizocytes, etc ., agent as the cells in Fig. 1 e, but the Fig . t f cells (we use Bessis's terminology [4]) occurs only in the were not protected by WGA. Unprotected cells agglutinated clump. Therefore, it appears that are profoundly distorted and start to lyse . With such distorted morphologies, facilitated by WGA, WGA protection, there is <1% lysis according to are developed in full by cell-cell interaction . Be- hemoglobin measurements . cause in other laboratories, proteolytic enzymes As will be shown below, the lectin WGA can are often used to increase agglutinability in con- also protect the echinocyte against forces that or- junction with lectin-erythrocyte interaction (20), dinarily would completely convert it to the disco- we wish to specifically note that proteolysis is not cyte . In short, WGA can thoroughly block both necessary to obtain severe cell distortion from directions in the reversible morphology conversion WGA agglutination, as can be seen in Fig. 1 b . discocyte ;i-± echinocyte . The other morphologies in Fig . l (cf) pertain The concentration tolerances pertaining to Figs . to subagglutinating cell concentrations . The two t and 2, within which the results are readily repro- principal results that illustrate the control over ducible, are given in Table II . These tolerances or human erythrocyte morphology changes and the ranges are somewhat restricted but are not incon-

LOVRIEN AND ANDERSON Agglutinin as Controlling Agentfor Erythrocytes 53 7 Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021

COMPLETE RETENTION OF DISCOCYTE DISCOCYTE MORPHOLOGY

£CHINOCYrIC LIGANO WGA Ar FULLY ECHINOCY7/C 2105Hplml LEVEL S -100% 100 DISCOCYTES ECHINOCYTES WGA 100 to 200,aplmI

ELLIPTOCYTES

SERUM ECHINOCYrIC 10 f0 50mm 0 ALBUMIN LIGAND INTACT D15000YTE5 PONDERS 0 EXPERIMENT ALBUMIN NAG Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021 ECHINOCYrIC LIGAND

ECHINOCYTE ECHINOCYTE ECHINOCYTES 100% RESTORATION DO NOT OF DISCOCYTES REVERSE UPON DISPLACEMENT OF WGA

FIGURE 2 The principal controls that can readily be imposed on normal discocyte morphology conversions by WGA in relation to the sequences of addition and concentration ranges given in Table 11 . "Ponder's experiment" in pathway 4 refers to Ponder's original observations (30) .

veniently narrow . The sequence of addition of WGA per cell. There is no proof that such a agents to the cells must be carefully considered monolayer actually forms . Clustering of WGA at and are now described in more detail . certain receptors is quite possible . The discocyte --* elliptocyte conversion is fully Conditionsfor Production of the Various reversible by the WGA-specific ligand, N-acetyl- Morphologies and Their Control Reactions as glucosamine (NAG) . No elliptocytes are seen at a 50 mM NAG concentration . Quite discernible re- Illustrated in Fig. 2 versal is seen at -10 mM NAG . At a 50 mM NAG PATH WAY 1 : With subagglutinating amounts concentration, there are - 1,300 moles of NAG per of cells and relatively high concentrations of mole of WGA receptor sites, using Nagata and WGA, elliptocytes are rapidly formed at a level of Burger's (27) value of four receptor sites per WGA -40 x 10 -e tLg of WGA added per cell . This (mol wt 36,000) . The conditions resulting in ellip- corresponds to YWGA - 7 x 108 molecules of WGA tocyte formation do not give complete conversion . added per cell, but fewer than that are bound . The Some discocytes remain . However, the mixture is binding data indicate that PWGA is -1-2 x 10 7 dominated by elliptocytes, discocytes comprising molecules of WGA bound per cell . This is enough, only 10-20% of the population . nevertheless, to completely cover the cell surface. PATHWAY 2 : When WGA is added as a first If we use 1 .5 x 10 10 Az as the available area for step in the proper amount (Table 11), the discocyte the discocyte and a spherical radius for WGA of remains protected against the echinocytic agent 25 A, a closely packed monolayer of WGA mole- dodecyl sulfate (DOD) . It has been shown (21) cules should occur at about 1 .5 X 107 molecules of that DOD at a level of VDOD = --2 x 10 7 ligands

FIGURE 1 Morphologies of the human erythrocyte . (a) Control cells, normal discocytes . (b) Agglutination of erythrocytes by WGA ; severe distortion upon cell-cell interaction when agglutinating concentrations of cells are employed . (c) Elliptocytes produced by WGA from normal discocytes, isoosmotic buffered saline, pH 7 .4, 25 ° C . Conditions specified in Table 11 and pathway 2 of Fig. 2 . (d) Echinocytes from normal 107 levels of echinocytic agent, 2 x dodecyl sulfate anions bound per cell. (e) Erythrocytes first protected with WGA in the presence of 100 times the normal echinocytic level of dodecyl sulfate, producing moderate distortion of the discocyte . (f) Spheres and spheroechinocytes under the same conditions as in (e) but without WGA . Bar, 5 1Lm .

LOVRIEN AND ANDERSON Agglutinin as Controlling Agentfor Erythrocytes 539

TABLE Il Range of Conditions Used in the Pathways for Figure 2 Cell concentration WGA NAG (GIuNAc) reversal Pathway 1 1-5 X 106 cells/ml 100-300 ttg/ml 10-50 mM Cell concentration WGA Ligand GIuNAc reversal Pathway 2 106-5 X 10' cells/ml 2-50 wg/ml 2-10 X 10' DOD bound/cell 0.5-5 mM Cell concentration Ligand WGA Plasma albumin GIuNAc reversal Pathway 3 106-5 X l0' cells/ml -4 X 10' DOD bound/cell 2-5 irg/ml -200yg BSA/ml 0.5-5 mM Cell concentration Ligand Plasma albumin Pathway 4 106-5 X 10' cells/ml 10-20 X 10' DOD bound/cell -100Pg/ml Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021

bound per cell is ordinarily sufficient to convert from the cells? A distinction should be made be- all discocytes to echinocytes. Under conditions tween echinocytes stabilized by WGA and free of under which the WGA is bound first, not only are DOD and echinocytes with both WGA and DOD no echinocytes formed but the cells can readily bound in which WGA somehow prevents serum withstand DOD at considerably greater levels than albumin removal of DOD . The distribution of PDOD = 2 X 10' without switching morphology . DOD was measured using 'SS-labeled DOD. This reaction is referred to as a protection reaction . Echinocytes were formed by subjecting discocytes The level ofWGA needed to protect the cells as to cold and [' S S]DOD, then stabilized by WGA. discocytes is far lower than that needed to convert Serum albumin was then added in various concen- them to elliptocytes . About 0.2 X 10 -'ttg ofWGA trations as part of pathway 3 . After equilibration, per cell is needed, 100-200 times less than in the system was centrifuged, and the supernate was pathway 1 . counted . Serum albumin remaining in the super- PATHWAY 3 : Addition of DOD to discocytes nate would be expected to gain the "S-labeled as a first step instantly resulted in the formation of DOD if it removed DOD from the echinocytes . In echinocytes (discocyte ---> echinocyte) as expected . control experiments, the same dilutions were car- Ordinarily, addition of serum albumin removes ried out without protein . Hence, the net amount the ligand DOD from such elliptocytes, and the of DOD removed by serum albumin from the cells readily revert to discocytes without delay echinocytes was measured as a function of serum (echinocyte -* discocyte) . This commonly inves- albumin concentration . The slope, in units of(mol- tigated reaction is illustrated in pathway 4 . But if ecules DOD bound) (ml) (cell) - ' (molecules serum the echinocytes are treated with WGA before se- albumin)- ' was 3 .3 X 10- ''', negative in sign . This rum albumin is added, serum albumin, when it is corresponds, taking into account the stoichiometry added as a third step, is completely unable to of serum albumin for DOD, which is between 5 reverse the echinocyte to the discocyte . Hence, and 10 (33), to quantitative transfer of the DOD WGA performs a function very similar to its func- that originally produced the echinocytes to the tion in pathway 2 : it quantitatively locks the cells serum albumin . Yet the cells remain echinocytes into whatever morphology they have, be they so long as the WGA concentration is maintained . echinocytes or be they discocytes, at the point of This clearly demonstrates that WGA does not addition of WGA. The morphologies are com- prevent the echinocyte ---> discocyte conversion by pletely stable over long periods (hours) . simply preventing removal ofthe DOD. The DOD However, if NAG is then added as a fourth step, is exhaustively removed by stoichiometric the cells instantly and quantitatively revert to nor- amounts of serum albumin . mal discocytes . Thus, NAG displaces WGA. Be- PATHWAY 4 : This pathway was established cause serum albumin is already present in suffi- some time ago by Ponder (30) . It is illustrated here cient quantity at this stage to bind all the echino- for completeness and to emphasize again the ex- cytic ligand, the final result is that all the cells cellent reversibility of the discocyte ~ echinocyte immediately return to discocytes . conversion . However, there remains a question : does WGA The lectin WGA is indeed effective in control- merely prevent serum albumin's removal of DOD ling erythrocyte morphology and erythrocyte re-

540 THE JOURNAL OF CELL BIOLOGY " VOLUME 85, 1980

sponse to ligands . At this point, there are two main discocyte --+ echinocyte titration plot relative to questions : what is the apparent number of critical the amount ofligand needed when ywGA = 0 . The sites for WGA binding at which control is exerted magnitude of Lw is directly dependent on WGA's and what relationship do they have to the number powers as a protective agent for the discocyte . The of membrane components of which the membrane Lw values from families of such plots, dependent is composed? on WGA, are shown in Fig . 4 . Fig. 4 indicates two levels of WGA on the Binding Stoichoimetries and Binding abscissa with brackets . The first bracket designates Isotherms the region Of ywGA at which WGA's protection is first made evident (Lw~ 0) . It marks the threshold The number of critical binding sites for WGA range of ywGA at which WGA obviously starts control of erythrocyte morphology was sought by to protect the discocyte against echinocytic agents .

direct determination of binding isotherms ofWGA Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021 The threshold level is 3-5 x 105 molecules of in relation to the morphology changes it controls WGA per cell. With increasing WGA in native and by quantitating the discocyte -* echinocyte cells, there occurs a second, higher level of WGA titration plots that are governed by WGA. Fig . 3 (shown by the second bracket of Fig . 4 ; - 18-20 shows how the fraction of cells that are echino- x 105 molecules of WGA per cell) where Lw cytes, r, varies with yDEC, the added amount of sharply increases . Further addition of WGA be- decyl sulfate . Fig. 3 also shows how WGA shifts yond this level does not further change the behav- such a plot at a concentration of 1 .9 x 10 -8 M ior of the system until elliptocytes are formed. In WGA. A major shift in the plot is caused by the range 18-20 x 105 molecules WGA per cell, WGA, as was expected from its behavior in path- of way 2 . The displacement resulting from WGA's the discocytes are maximally protected (in the way indicated by Fig . 1 e), even with huge amounts of protective effect is measured by the area between echinocytic ligand . With lesser, but still fully the two plots: r vs . yDEC with and without WGA echinocytic amounts ligand, _ 8 for each prescribed level of WGA. Such an area, of 10'-10 molecules of ligand per cell, the protected discocytes hatchmarked in Fig . 3, is denoted Lw . It is equiv- are entirely like the control cells (Fig .I a) . These alent to a circular integration of r dy between the two levels, x 10' molecules WGA two plots . Area Lw has the dimensions of mole- 3-5 of per cell (threshold level for displacement of the r vs . yDEC cules of ligand added per cell . Area Lw is a meas- plot) and 18-20 x 10 5 molecules of WGA per cell ure of the extra amount of echinocytic ligand (maximum useful WGA level), are consistent with needed when WGA is present to generate the

16 14 as 12 u °10 0.6 O G X 8 Wu 3 0.4 Native RBC J 6 2 with WGA, 0.7 í ,q WGA/107 cells 4, 1 4 02 '9 \ OC-6 0 0 2 ^ 4 6 8 10 12 14 16 18 20 22 24 0 5 10 15 20 25 30 35 40 y, Molecules Decyl Sulfats added/Call x 10-* v x 10 -5, Molecules WGA bound /Cell FIGURE 3 Plot of data. r, fraction of cells converted to FIGURE 4 Plot of Lk values as a function of amount of echinocytes as a function of y, i .e ., the amount of echin- WGA bound to the cell, from use of ' ZSI-WGA . Bracket ocytic agent added (decyl sulfate), dependent on WGA . at the lower level : range of bound WGA, which is the Native cells (1 x 10' cells/ml) are markedly protected threshold level at which WGA obviously protects the by WGA (1 .9 x 10 -8 M; mol wt, 36,000), producing area cell against echinocyte formation . Upper level bracket: Lw, which is a measure of WGA's ability to forestall range of WGA past which extreme (lytic) levels of echinocyte formation . Desialated cells, initially in normal echinocytic agent must be employed to force the cells out discocyte morphology, fail to be protected by WGA . of the discocyte morphology .

LOVRIEN AND ANDERSON Agglutinin as Controlling Agentfor Erythrocytes 541

other stoichiometries involving morphology con- 5 quantitates some of the behavior qualitatively version reactions that are discussed below . outlined by pathway 2 in Fig . 2 . Isotherms of WGA binding to the erythrocyte The ranges of the steeper parts of the isoclines membrane were determined in two ways : (a) In in Fig . 5 extend to PWGA values of -8-18 x 10'' relation to the binding isotherms of the ligand molecules of WGA bound per cell. In the lower DOD and in relation to the boundaries between ranges at which protection against echinocyte for- the discocyte morphology and the echinocyte mor- mation by bound DOD is elicited by bound WGA, phologies . This leads to a "phase diagram," or a the isoclines are sloped and lie roughly (region a) morphology behavior diagram, as illustrated in in the range PWGA = 0-5 x 105 . This range corre- Fig. 5 . (b) In a conventional binding isotherm sponds to the threshold range for WGA protection, involving only WGA in Scatchard plot, as shown as seen from the Lw plot method in Fig. 4 . Another in Fig . 6 . This figure clearly shows that the early point, which is expressed by the steeply rising stages of WGA binding are sharply cooperative, nature of the isoclines can clearly be seen in Fig. Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021 of positive sign, for the native erythrocyte . 5 : the binding levels of WGA, PWGA, are apparently Fig . 5 shows how DOD-induced conversion independent of PLOD . This is seen more directly in from the normal discocyte proceeds through echin- Fig . 7, which shows how, in a plot of PW(;A vs. ocyte morphology substages I and II (4) . Fig . 5 is YDOD, spanning the critical (echinocytic) range of plotted with respect to amounts of WGA bound, y = 0-10 x 10' molecules of DOD per cell, PWGA PWGA, and with respect to amounts of DOD bound, increases with increasing overall level ofWGA, as 125 [35S]_ PLOD, as determined with 1-WGA and one might expect . However, the plots are flat, DOD, respectively . With no WGA, PWGA, of within experimental error, confirming PWGA's in- course, equals zero . The critical level for DOD dependence of DOD level . Thus, although the binding in complete conversion to echinocytes is binding levels of WGA and of the echinocytic PROD = 2-8 x 10' molecules of DOD ligand per ligand are surprisingly independent of one an- cell, as shown by the intercepts on the abscissa of other, the consequences of binding on erythrocyte Fig . 5 . This reinforces our previous results (21) . morphology are strongly dependent on: (a) bind- The range over which the discocyte is stabilized is increased as PWGA increases . Thus, the data of Fig . 42

36 d s w E 24 U v 16

o_ 12 X U 6 á Neuraminidosel~_ ä treated Red Cells o - 0 4 B 12 16 20 0 10 20 300 400 50 60 'D0o,molecules bound/cell x 10 -7 v x 10-s , Molecules bound /Cell

FIGURE 5 Production of principal echinocyte mor- FIGURE 6 Binding isotherms, Scatchardplots, of WGA phologies dependent on P, i .e ., amounts bound, for both to native and to desialated cells, showing marked positive echinocytic agent (dodecyl sulfate) and WGA (1 X 10' cooperativity by the native cells up to the Pw point and cells/ml, 25°C. "Region a" marks the areas of the more abolishment of positive cooperativity in WGA binding critical stoichiometries for WGA binding in the protec- by removal of sialic acid residues . Dashed line and later tion reaction in relation to binding levels of the echino- sectors of native erythrocyte isotherm are in agreement cytic agent at which substages of morphologies are seen with Adair and Kornfeld's earlier data (2) . P, order of and in relation to intercepts of the plots on the abscissa. magnitude of the ordinate .

542 THE JOURNAL OF, CELL BIOLOGY " VOLUME 85, 1980

Sialic Acid Residues (NANA) as Ligandsfor

IO »g/ml of WGA WGA Binding. Consequences ofDesialation in WGA's Control of Morphology

whether sialic 2 wg/ml of WGA To determine acid residues might primary ligands for WGA interaction 0---0-- 0 0 be the with its erythrocyte receptor, the sialic acid groups were 114/ml of WGA x-x---X --y -c--- c-x-x-z- removed by neuraminidase treatment and quantitated . In four determinations, using neuraminidase in the pH range 7 .0-7 .4 (25°C), 4 .1, 4.4, 5 .4, L 2 4 6 s 10 and 3 .7 x 10' sialic residues per cell, for an average MOLECULES DODECYL SULFATE value of 4 .4 x 10' residues per cell, were measured . Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021 ADDED PER RED CELL, x10 -7 Seaman et al. (36) obtained an average of 3 .5 x FIGURE 7 Increase of WGA added to native erythro- 10' residues per cell under somewhat different cytes increases the levels of WGA binding (ordinate) . conditions involving added Ca" . However, the ability of WGA to bind at various levels is The morphologies and the morphology inter- independent of the amount of echinocytic agent added (dodecyl sulfate, abscissa) . This behavior is consistent conversion reactions of these desialated human over the range over which the cells become echinocytes, erythrocytes were examined, as illustrated by Fig. i .e., between 2 and 8 x 107 DOD added per cell . 8 . First, excellent discocyte morphologies are retained by completely desialated cells. Second, the ing levels ; (b) the sequence of addition of the reversible conversion discocyte ;~-_ echinocyte is compounds which produce the behavior illustrated retained to a remarkable degree. Echinocytic lig- in pathways 2, 3, and 4 of Fig. 2 . Nevertheless, ands drive the conversion completely to the right, there is no discernible time dependence in any of and serum albumin completely reverses it . The these reactions involving WGA . third result is dramatic : WGA's control over both Fig . 6 shows the results from direct determina- reactions of the desialated cells is completely abol- tion of WGA's binding isotherm to the erythro- ished . WGA exerts no control at all, even at WGA cyte, plotted according to Scatchard's method . The levels of -r60 Itg/ml. This third result is illustrated sharp maximum in the Scatchard plot of Fig . 6, by Fig . 3, where echinocyte formation behavior v, is similar to plots from some of our earlier work for desialated cells in the presence of WGA is in which erythrocyte binding isotherms exhibited plotted . The plot is congruent with that for intact sharp, positive cooperativity (21) . A transition is cells without WGA . This third result is strong involved near the maximum, which is made visible evidence that, indeed, WGA's control over the because of a gross morphology change, in the case intact cell is mediated via sialic acid ligands . The of certain ligands . Here, the ability to bind WGA lectin is best displaced from the cells by NAG, not increases rather sharply up to v , and a "normal" by added sialic acid, however . The reason for this appearing (negatively sloping) isotherm follows . likely lies in the anomeric equilibria that prevail In the region of PWGA - 40 X 10 5 , Adair and in free sialic acid, as discussed below. In any case, Kornfeld (2) also determined a WGA isotherm for complete desialation does not affect either of the binding to erythrocytes, but they gathered no data two morphologies nor the interconversion that below the level Of IhvGA - 15 x 105 molecules usually occurs in the absence of the lectin . bound per cell . In the range in which there is The isotherms for 1251-WGA binding were re- overlap between their results and ours, there is determined for completely desialated cells, and the reasonable agreement . plot is shown in Fig. 6. The entire positively The region of binding of WGA in excess of cooperative branch of the isotherm was destroyed about VWGA = 30 X 105 , is marked by a much as shown . Even so, the "normal," or negative flatter, only gently sloping portion of the Scat- sloping branch of the plot still intercepts on the chard plot . This is consistent with an ongoing abscissa at about 30 x 10 5 molecules bound per binding of WGA molecules, of much less affinity, cell, as is the case for native erythrocytes and as also seen by Adair and Komfeld (2) . This was Adair and Kornfeld also found (2) . There is a 32- considered consistent with binding of WGA via fold decrease in the apparent association constant sialic acid ligands, because of their large numbers. of the negative sloping branch, K (-Kn = n - (the

LOVRIEN AND ANDERSON Agglutinin as Controlling Agentfor Erythrocytes 543 Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021

FIGURE 8 Morphology conversion of completely desialated cells: the normal, reversible conversion, discocyte ~-_ echinocyte is still observed with erythrocytes stripped of surface sialic acids . However, WGA imposes no control over this reaction, in contrast to native cells . 1 x 10' cells/ml, 6011g WGA/ml .

slope); n - 30 x l0 5), for desialated cells relative The lectin does not produce echinocytes or dis- to intact cells . However, neither of these large cocytes at any binding level . Rather, it locks or changes in the binding isotherms, namely, loss of protects either morphology such that further mor- positive cooperativity and decrease in K after phology changes are blocked in the face of con- desialation, occur at the cost of overt alterations in ditions which ordinarily would readily force such cell morphology, as Fig. 8 shows . This behavior is changes . The control that WGA exerts does not consistent with good retention ofnormal discocyte occur by competition, such as prevention of echin- morphology in native cells, up to and beyond the ocytic agent binding or unbinding, as is shown by v4 , point as WGA progressively binds. the experiments with radiolabeled detergents that were designed to answer the question of competi- DISCUSSION tion . It might have been thought that such controls, The critical binding level, or the threshold number even if they applied to the discocyte, might not for WGA binding at which WGA imposes ob- apply to the echinocyte if the distribution of the vious, strong influence over the discocyte - echin- receptors for WGA in the echinocyte were differ- ocyte equilibrium, has the value PWGA = 3-4 x 10 5 ent from that in the discocyte . However, the mem- molecules of WGA bound per cell . The best earlier brane cytoplasmic protein spectrin, which is fun- estimate of the number ofcopies ofglycophorin is damental in regulating cell shape, has been shown that of Fairbanks et al. (11), who found 2.6 x 10 5 by Ziparo et al . (45) to maintain the distribution copies per cell membrane . The latest measure- in the echinocyte that it apparently had in the ments are by Sadler et al . (35) . They lead to close discocyte . to 6-7 x 10 5 chains per membrane . It is likely that The discrete morphology that WGA can inde- glycophorin tends to exist as the dimer in the pendently produce is the elliptocyte . It can be membrane (25) . Therefore, WGA's interaction estimated that elliptocytes are produced at levels with the human erythrocyte in the region of the corresponding to a monolayer ofWGA molecules critical stoichiometry at which WGA imposes bound to the cell surface, using the approximation strong control over the cell's morphology changes that the cell surface areas of the elliptocyte and of might be mediated through a glycophorin dimer the discocyte are equal. We know little of the rather than simply through a monomer . mechanism for production of the elliptocyte, ex-

544 THE JOURNAL OF CELL BIOLOGY " VOLUME 85, 1980

cept that it is likely very different from production tainly have to be contiguous in groups of three or of the echinocyte because the conditions for for- four, judging by the behavior of WGA with chi- mation in vitro are profoundly unlike . Because the tooligosaccharides (14) . Moreover, in the carbo- discocyte --+ elliptocyte conversion can take place hydrate sequences, the position ofG1cNAc groups in vitro, there is a possibility that elliptocytes could is almost always "endo," or interior within the form from normal discocytes in vivo, but this is type II glycophorin oligosaccharides (35) . Yet far from established . WGA's interaction with oligomers of GIcNAc is The sharply increasing branch of Fig. 4, the best developed when at least one of the GIcNAc "hyperprotecting" region that marks WGA's more groups is a terminal, or "exo" group . Summarizing forceful abilities in controlling the cell's morphol- what has been discussed so far, the density and ogy, occurs at PWGA = 18-20 x 10 5 . At this level, arrangement of GIcNAc in glycophorin is not the discocyte is stabilized, almost regardless of advantageous for binding WGA, even though free

how much echinocytic agent is present (Fig. 1 e GIcNAc or NAG in millimolar concentrations can Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021 andf pathway 2 of Fig. 2). When thNGA values are displace WGA . produced that have an apparent stoichiometry of In contrast, the density and arrangement of 4:1 or 5 :1 with respect to glycophorin, there arises sialic acid (NANA) in glycophorin fills the re- the question of whether some WGA molecules quirements seen to be emerging (43) for WGA- might be distributed elsewhere, even though the l : NANA binding . Quite recently, WGA was found I stoichiometry (threshold protection) indeed to use NANA as ligands, and WGA often has helps confirm (2) glycophorin as a principal recep- more affinity for NANA than for GlcNAc (16) . tor . There is a connected question . Is WGA likely The results summarized by Fig. 8 strongly support acting as a cross-link between remote receptors, the view that NANA is particularly important in e .g ., different glycophorins or other glycoproteins? WGA's control of erythrocyte morphology . This These questions need to be gauged with respect to is not to say that NANA is the exclusive ligand the numbers, affinities, spacing, total valency, and for WGA in the erythrocyte surface ; GIcNAc pos- valence directional properties of such groups . For- sibly becomes involved under some conditions . tunately, Wright (43) has developed a 2.8 A reso- Nevertheless, viewing the composition of the lution of WGA's structure complexed with whole membrane with respect to NANA, both NANA, illustrating how such ligands are oriented glycophorin and the remainder of the membrane in binding . have enough NANA to account for WGA binding, The molecular weight of glycophorin is 29,000- even at the "hyperprotecting" level, MGA = 18-20 31,000 (15), and it is 60-64% carbohydrate by x 10 5 molecules per cell. This requires --0 .8 x 10' weight. It has 8 N-acetylglucosamine residues NANA residues if WGA is tetravalent for NANA . (G1eNAcp) and 28-33 N-acetylneuraminic acid Glycophorin alone carries about 1 .8 x 10' NANA residues (NANA), with some variation between on the whole cell . The cell's total NANA is 3 .0- laboratories (19, 35, 40) . Until recently, most au- 4 .0 x 10', and glycophorin contributes 40-60% . thors thought that WGA uses GIcNAc groups (in Therefore, glycophorin's composition and ar- the,ß-anomeric form) almost exclusively for bind- rangement with respect to NANA's sequence are ing to cell surfaces . However, the monosaccharide favorable for both the 1 :1 and the 1 :5 ratios for of GIcNAc is a ligand of much lower affinity than WGA interaction, in contrast to G1cNAc . the oligosaccharides of N-acetylglucosamine (32) . In our experiments, free or monomeric NANA Although there are enough GIcNAc groups on did not displace WGA, in contrast to NAG . How- glycophorin to accommodate a single WGA mol- ever, WGA binding is specific for the a-anomer of ecule, there are not nearly enough GIcNAc resi- the saccharide (43), whereas the free, unattached dues to accommodate four or five WGA mole- sugar in water mutarotates to a mixture that is cules, even if the GIcNAc residues were arranged 90% /3-anomer (43) . In this respect, and in certain in the most advantageous sequence for generation others involving O-glycosidic linkages available in of large binding affinities, for WGA is tetravalent glycophorin but lacking in free NANA, free (27), and it has to exert at least a trivalency, NANA does not adequately duplicate the sialyl preferably a tetravalency, for best binding (14) . group stereochemistry that prevails in glycophorin . Approximately 15-20 GIcNAc residues per gly- It is most unlikely that "cross-linking" (à la cophorin would have to be present for tight bind- glutaraldehyde), wherein WGA simultaneously ing of five WGA molecules, and they would cer- binds two glycophorin receptors, is responsible for

LOVRIEN AND ANDERSON Agglutinin as Controlling Agentfor Erythrocytes 54 5 WGA's ability to protect . It has already been negatively sloped .) The discocyte is not only pres- noted that WGA needs immediately adjacent, con- ent but is well stabilized at the v point shown in tiguous ligands for best binding . Glycophorin has Fig . 6. This favors assignment of positive cooper- an average radius of ^25 A (25) . Hence, the ativity to molecular association-dissociation equi- surface density of NANA in glycophorin is -160 libria, probably in glycophorin, rather than mem- x 10' molecules of NANA per square angstrom . brane rearrangement . Positive cooperativity of this The glycophorin receptors are, on the average, kind is increasingly found in lectin binding to ^220 A distant . If WGA could successfully func- membranes other than in the erythrocyte (31, 38) tion merely using two intermolecular NANA, the but is also found for drug binding in the human ligand density would be 0.13 x 10' molecules of erythrocyte (21) . NANA per square angstrom, 1,200 times lower In the human erythrocyte, the best evidence for than for the intramolecular case. The lectin likely positive binding cooperativity involves inner has to orient the requisite ligands for proper val- membrane receptors for glyceraldehyde-3-p-de- Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021 ence direction in intermolecular binding (43) . hydrogenase (44) . The G3PD receptor is the band Moreover, the observed stoichiometry is incorrect 3 protein, which also binds aldolase on the cyto- for interglycophorin contribution : if WGA does plasmic side, perhaps with positive cooperativity span two glycophorin receptors at the point of the in its initial stages (39) . Thus, band 3's binding observed morphology control, a 0.5 :1 .0 stoichi- isotherms for these two proteins is kindred to ometry would occur . If WGA is tetravalent and glycophorin's behavior in binding WGA in overall operates intermolecularly, the ratio 0.25 :1 for outline . Glycophorin and band 3 are both trans- WGA:Glycophorin is expected . Glycophorin membrane proteins, but they are asymmetrically likely is a dimer or a multimer in the membrane placed. Edwards et al . (9) clearly showed that (25) . This reinforces the foregoing arguments . glycophorin is exposed to the outside, whereas There are four salient aspects of the isotherms band 3 is mainly on the cytoplasmic or inner side . for binding WGA to the membrane that we would The relationship of both proteins to the "Intra- like to discuss: (a) the positive cooperativity of membrane Particle Assembly" (IMP) merits care- lectin binding to the discocyte in the early stages ful focus . There are 3-5 x 10 5 IMP per human (PW( .A < vw ) of the association and its erasure upon erythrocyte . The IMP may harbor both glyco- desialylation; (b) the parallels between what is phorin and band 3 . Rothstein (34) thinks that seen here and binding isotherms of other intracel- band 3 may be "covered" by the sialoglycoprotein, lular proteins using erythrocyte inner membrane glycophorin. It is likely that there are two copies receptors; (c) the independence of the binding of band 3 monomer/IMP (one glycophorin per isotherms of the lectin and of echinocytic ligands IMP), so that band 3 monomers are possibly di- with respect to one another; (d) the expression and mers in the IMP . As noted above, concanavalin A confirmation of previous work (21) in the results can control some of the discocyte -), echinocyte reported in this paper . conversions, but much less forcefully than can Positive cooperativity of the kind seen in Fig. 6 WGA. Concanavalin A's receptor is band 3 (29) . may arise in three general ways (7) : a change in The binding isotherms for the membrane in- the state of association of the binding molecule, a volving the lectin and the echinocytic agent show change in state of the receptor molecule, and that the two binding processes are independent rearrangement or exposure of "cryptic" sites as (see Fig. 7) . This holds through ranges of binding binding develops. Both WGA and glycophorin in which a morphology change occurs . Neverthe- may exist as dimers, so that the first two possibil- less, in control of the cell's morphology, there is ities, changes in state of association at the molec- an intimate mutual dependence of lectin and ular level, are good possibilities. Cryptic sites not echinocytic agent in the consequences of binding, initially available or an increase in affinity of the as is vividly seen in Fig. 2. The binding units first sites which are forced into availability as themselves appear to be independent, but after the binding progress fit equally well. (Initially, v is complexes form between the lectin and its receptor small but Cf, >> v, so that v/Cf is relatively small . and the echinocytic agent and its binding unit, the Later, v increases relatively rapidly as ligand is filled units interact strongly . The principal controls added, so that v/Cf climbs, and the slope of v/Cf appear to be thermodynamic and not rate limited . vs . v is positive in sign . Eventually, remaining sites It was found earlier (21) that for all echinocytic become exposed, so that v/Cf is forced to become ligands studied, regardless of their structure, mo-

THE JOURNAL OF CELL BIOLOGY " VOLUME 85, 1980

lecular weight, or total ligand concentration REFERENCES (yLigand), that discocyte -. echinocyte conversion 1 . RDA, G. L., E. L. FRENCH, and P . E. LIND. 1967. Purification and always becomes developed when PLigand - 2 ± 1 properties of neuraminidase from V. cholerae. J. Gen. Microbial. 24 : x 10' ligands bound per erythrocyte . This includes 409421 . 2 . ADAIR, W. L., and S. KORNFELD . 1974, Isolation of receptors for wheat decyl and dodecyl sulfate, ligands used for this germ agglutinin and the Ricinus communis lectins from human eryth- work . Fig . 5 confirms the earlier work : the isocline rocytes . J. Biol. Chem. 249:46964704. 3 . ANDERSON, P. C., and R. E. LOVRIEN . 1977 . Human red cell hemolysis for conversion to the first discernible echinocyte, rates in the subsecond to seconds range . Biophys. J. 20:181-191 . echinocyte I, intercepts the abscissa at PDDD - 2 4 . BEssls, M. 1973 . Living Blood Cells and Their Ultrastructure. Springer- Verlag, New York . 197-216 . x 10' . This points out an avenue for echinocyte 5 . BOUCHARD, P.. Y. MOROUx, R. TIMER, J . P. PRIVAT, and M. MONSIGNY . formation . 1976 . An improved method for purification of wheat germ agglutinin (lectin) by affinity chromatography . Biochimie(Paris). 58:1247--1256. It has commonly been thought that the discocyte 6 . COHEN, C. 1979 . Cell architecture and morphogenesis . The cytoskeletal -. echinocyte conversion simply represents proteins. Trends in Biochem. Set. 4:73-77. an iso- 7 . DAHLQUIST, F. W. 1978. The meaning of Scatchard and Hill plots . volumetric cell change with membrane area ex- Methods Enzymol. 48:270-299 . Downloaded from http://rupress.org/jcb/article-pdf/85/3/534/1388797/534.pdf by guest on 29 September 2021 8 . DAVIDSOHN, A.. and B. WILWIDSKY . 1967 . Synthetic Detergents . Leon- pansion . The hydrocarbon chains insert into the ard Hill Publishers, London. 114-118 . membrane lipids, and the increased volume re- 9 . EDWARDS, H. H., T. J . MUELLER, and M. MORRISON . 1979 . Distribution of transmembrane polypeptides in freeze fracture . Science (Wash, D. quirement, plus electrostatic repulsion from the C.) .203:1343-1345 . anions, presumably are the main driving forces 10 . EVANS, C. A., and R. M. HOCHMUTH . 1977 . A solid-liquid composite model of the red cell membrane . J. Membr . BioL 30:351-362. (37) . However, in such morphology changes the 11 . FAIRBANKS, G. . T. L . STICK, and D. F . WALLACH. 1971 . Electrophoretic cell cytoskeleton must at least conform to, if not analysis of the major polypeptides ofthe human erythrocyte membrane . Biochemistry . 10-2606-2617. help produce, the morphology change . Cytoskel- 12 . FINDLAY, J . B . 1974 . The receptor protein for concanavalin A and Lens culinaris phytohemagglutinin in the membrane of the human erythro- eton-controlling assemblies must be reckoned with cyte. J. Biol. Chem. 249:43984403 . in many cells (6) . For the erythrocyte, several 13 . FRAKER, P . 1 ., and J . C. SPECK . 1978 . Protei n and cell membrane inclinations with a sparingly soluble chloroamide . Biochem. Biophys. papers (we cite only two, Van Zoelen et al . [41] Res. Commun . 80:849-853 . and Kahane and Gitler [18]) show that glyco- 14 . GOLDSTEIN, I . J., and C. E . HAYES . 1979 . Th e lectins : carbohydrate binding proteins of plants and animals . Adv. Carbohydr. Chem. Bio- phorin is in intimate, extensive contact with mem- chem . 35:128-340 . brane lipids . Van Zoelen et al. give a range of 15 . GREFRATH, S., andJ . A. REYNOLDS. 1974. The molecular weight of the major glycoprotein from thehuman erythrocyte membrane . Proc. NaIL from 9 to as many as 80 membrane lipid molecules Acad Sci. U. S . A. 71 :3913-3916 . interacting with each glycophorin. Taking a rough 16 . GREENAWAY, P. J ., and D. LEVINE . 1973 . Binding of N-acetylneuraminic acid by wheat germ agglutinin . Nature New Biology. 241:191-- average of 50 lipids per glycophorin and ^4 x 10 5 192 . 17. IsSITT, P. D., and C. H. IssirT. 1975 . Applie d Blood Group Serology . glycophorins per membrane, the product is 2 x Becton, Dickinson & Co., Oxnard, Calif 183-199 . 10 7 lipids per membrane . This may be merely 18 . KAHANE, I., and C. GITLER . 1978. Red cell membrane glycophorin labeling from within the lipid bilayer. Science ( Wash . D. C. ) 201:351- fortuitously close to our critical range, ItLigand = 2 352 . ± 1 x 10' . However, it appears that when the 19. KORNFELD, S., and R. KORNFELD . 1976. Comparative aspects of glycoprotein structure . Annu. Rev. Biochem. 45:217-237 . "lipid bilayer" is invaded, with a number of in- 20. KUETTNER, C. A., L. A. STAEHELIN, and J . A. GORDON . 1976. Receptor truding ligands comparable distribution and the mechanism of enhanced agglutination by soybean to the number with agglutinin. Biochim . Biophys. Acta. 448:114-120 . which glycophorin already interacts, not one but 21 . LovRIEN, R., W. TISEI., AND P . PESCHECK . 1975 . Stoichiometry of two events follow . compounds bound to human erythrocytes in relation to morphology . J. First, membrane expansion and BioL Chem. 250:3136-3141 . electrostatic repulsion may indeed occur. But, in 22. Lux, S . E. 1979 . Spectrin-actin membrane skeleton of normal and addition, abnormal red blood cells . Semm. ffematoL 16 :21-51 . glycophorin becomes perturbed, in turn 23 . MARCHESI,V .1972.Wheat germ (Traicumvulgaris)agglutinin. Methods allowing the cytoskeletal change necessary to form EnzymoL 28B, 355-357 . 24 . MARCHES], V., T. TILLACK, R. JACKSON, J . SEGREST, and R. SCOTT . a new morphology. Not only are the forces in the 1972. Chemical characterization and surface orientation of the major morphology change at stake but the governance glycoprotein of the human erythrocyte membrane. Proc . NaIL Acad. Sci. U. S. A. 641442-1449 . of the consequences of dissipating the forces may 25 . MARCHESI, V. T. 1979. Functional proteins of the humanred blood cell be of equal importance . Glycophorin provides membrane. Sem. flematoL 16:3-20 . 26 . MARCHESI, V. T., and H. FURTHMAYER . 1976. The red cell membrane . such governance . Annu. Rev. Biochem . 45:667-698 . 27 . NAGATA, Y., and M. BURGER. 1974 . Wheat germ agglutinin. molecular characteristics and specificity for sugar binding. J. BioL Chem. 249 : 3116-3122. 28 . PATTERSON, M, S ., and R. C. GREEN . 1965 . Measurementof low energy This work was supported by the University of Minnesota beta emitters in aqueous solution by liquid scintillation counting of Graduate School and National Institutes of Health grant emulsions. Anal. Chem. 37:854-857. GM 18807 . 29 . PINTO DE SILVA, P., and G. L. NICOLSON. 1974. Freezeetch localization of concanavalin A receptors to the membrane intercalated particles of humanerythrocyteghost membranes. Biochim. Biophyss. Acia. 363:311- Receivedfor publication 13 319 . September 1979, and in revised 30 . PONDER, E . 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LOVRIEN AND ANDERSON Agglutinin as Controlling Agentfor Erythrocytes 547

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54$ THE JOURNAL OF CELL BIOLOGY - VOLUME 85, 1980