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 reac- tions . The ability of WGA to carry out these functions has no obvious rate limitation . Its effectiveness depends mainly on its binding stoichiometry, particu- larly 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 echino- cytic 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 glyco- phorin per cell. The second important stoichiometry, whereby the cell's morphol- ogy 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 .