Defective glycoproteins in the plasma membrane of an aggregation minus mutant of Dictyostelium discoideum uith abnormal cellular interactions.

Experiments involving the co-incubation of wild type (A3) cells of Dictyostelium discoideum and a spontaneous aggregation-minus mutant (HW 2) suggested that the mutant was defective in cellular interactions. The inhibition of A3 development by HW 2 cells and the differentiation of a small fraction of HW 2 cells which is allowed by A3 cells, both depend on cell contact. Therefore, we compared cell surface molecules in vegetative A3 and HW 2 cells by a variety of techniques to determine whether defects in HW 2 could be found prior to the inhibition of development in vegetative amoebae. Antigenic defects, or differences in binding of concanavalin A, or both, were localized to three plasma membrane macromolecules using glutaraldehyde-fixed sodium dodecyl sulfate gels of plasma membranes. Two periodic acid-Schiff-positive glycoproteins, and one glycolipid also differed in HW 2. Three glycoproteins had an increased sensitivity to pronase in isolated plasma membranes suggesting an alteration in their topography. Glycoprotein E, the major glycoprotein of vegetative plasma membranes is abnormal in topography, altered as a concanavalin A receptor, and is antigenically abnormal.

When development is blocked many normal changes do not occur including the accumulation of stage-specific enzymes (11, the appearance of CAMP receptor and phosphodiesterase (2,3), and of contact sites A (4). Thirteen normal developmental alterations in the spectrum of macromolecules and five developmental changes in polypeptide topographical location in the plasma membrane of D. discoideum are blocked in a developmental mutant.' Although several biochemical failures are associated with developmental mutants, many of these effects could be secondary results of the inhibition of the developmental program.
* This paper is Number 6 in a series entitled "The Role of the Plasma Membrane in the Development of Dictyostelium discoideum." Paper 5 is Footnote 1. This work was supported by United States Public Health Service Grant GM 06965. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisemerit" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' S. Hoffman  to diffusible molecules, the interactions are frequently blocked (5)(6)(7)(8). One interpretation of these data is that these phenomena are mediated by cell surface molecules that must contact each other for an interaction to take place (9 ated with  particulate  cellular  components  found only on  aggregating  cells (18, 19). An antigen exists that is found specifically on spore cells (20). Gregg  test for the ability of cells to form contact sites A (4), 5 ml of A3 or HW 2 cells in suspension at 1 x lo7 cells/ml in aggregation buffer (22) are rotated at 20 to 24 rpm in a screw-top culture tube (25 x 200 mm) about the tube's long axis in a device built according to Gerisch's description (23). After 12 h at 22", aliquots of cell suspension were photographed in the presence or absence of 10 rnM EDTA. If cells remained aggregated in the presence of EDTA, they were assumed to have formed contact sites A (4). Development of Cells on Filters ~ A3, or HW 2 cells, or both, were washed and resuspended in lower pad solution (24). The indicated numbers of A3 cells, HW 2 cells, or a combination of the two were uniformally spread on a 47-mm diameter Millipore filter on a pad saturated with lower pad solution and incubated for development at 22".
In experiments where cells were incubated on opposite sides of Millipore filters from each other, the appropriate number of A3 or HW 2 cells were spread onto each filter and the filters were clamped together back to back with a stainless steel ring and suspended horizontally 5 mm above a pad soaked in lower pad solution.
Triton X-100 Treatment Spores were treated with 0.2% Triton X-100, and suitable dilutions were mixed with bacteria, and plated on SM agar (25). Fewer than 1 in 10' amoebae survive this treatment although it has no effect on the viability of spores.

Plasma Membrane Preparation and SDS-Gel Electrophoresis
Cells were grown, plasma membranes prepared (261, and analytical SDS-gels were run and stained as previously described (14). The Coomassie blue-stained gel in Fig. 10 contains 50 pg of protein per lane and the PAS-stained gel in Fig. 8 contains 200 pg of protein per lane as estimated by the Lowry method (27). Pronase treatment of intact cells and isolated plasma membrane was also as previously described (14).

Scanning
Electron Microscopy Cells were fixed with glutaraldehyde, postfixed with 0~0, dehydrated with an ethanol series, and critical-pointed dried. Before viewing the samples were shadowed with gold.

Lectin and Antibody
Labeling of Gels SDS slab gels were run (14) and then cut into 0.7-cm-wide gel strips containing 120 pg of plasma membrane protein. The strips were then fixed as described in Ref. 28. For lectin binding, gel strips were incubated for 3 days in 8 ml of infusion solution (28) per gel strip plus 0.25 mg of FITC-lectin per gel strip. They were then washed for 2 days in phosphate-buffered saline (0.9% NaCl solution) (28) with two changes of solution and photographed over a short wavelength (predominantly 254 nm) UV light box through a Wratten type 65 filter (14). For antibody binding, gel strips were incubated for 3 days with 5 ml of rabbit serum plus 3 ml of 2.67 times infusion solution per gel strip. The gels strips were then washed as above to remove unbound antibody and incubated for 3 days with 8 ml of infusion solution plus 5 mg of FITC-goat anti-rabbit immunoglobulin per gel strip. Finally the gels were washed and photographed as above.

Gel Scanning
Photographic negatives of gels were scanned on a Syntex AD-1 Autodensitometer.

Preparation,
Titering, and Adsorption of Sera Rabbits were inoculated with A3 vegetative plasma membrane (10 mg of protein content) suspended 1:l (v/v) with complete Freund's adjuvant in a final volume of about 2 ml. Equal aliquots were injected intramuscularly, intradermally, and subcutaneously. Similar inoculations were given 4 weeks later and weekly for 3 weeks thereafter.
Starting at Week 5, the rabbits were bled about 25 ml weekly from the ear. After 3 h at room temperature the serum was separated from the blood clot by centrifugation for 10 min at 4200 x g.
To titer sera, vegetative A3 cells were washed and resuspended in 0.15 N NaCl plus 2 rnM EDTA (pH 6.0) at 4 x 10' cells/ml. Onetenth milliliter of cells were mixed with 0.1 ml of a range of serum dilutions and the highest dilution to give complete agglutination was determined. Typically titers were 64 for immune serum and 2 for preimmune serum.
Adsorption of a titer 64 serum with 3.2 x 10H A3 or HW 2 cells/ml for 30 min completely removes all agglutinating activity. However, when pooled immune sera from two rabbits (average titer 64) was adsorbed with a sufficient number of A3 cells to completely adsorb 128 units of agglutinating titer, detectable antibody binding to fixed gels of A3 vegetative plasma membranes remained (data not presented). Repeating this adsorption a total of three times removed almost all antibody binding to fixed gels. Therefore, A3 and HW 2 adsorption of anti-A3 vegetative plasma membrane antiserum with cells of A3 or HW 2 was routinely done by three rounds of adsorption, each with 6.4 x 10" cells/ml of serum.

Preparation of Lipids and Thin Layer Chromatography
Total cell or plasma membrane lipids were prepared as described in Ref. 29  Experiments in which A3 and HW 2 cells are mixed suggest that a biochemical defect in HW 2 is expressed at the cell surface. When HW 2 cells are co-incubated with A3 cells, the development of the A3 cells is inhibited. When A3 cells are plated at the standard concentration, 4.7 x 10' spores developed in Experiment 1A (Table I). Similarly incubated HW 2 cells formed no spores (Experiment 1G). As might be expected, replacement of 50% of the A3 cells with HW 2 cells resulted in about a 50% decrease in number of spores formed (Experiment 1D). However, as the percentage of HW 2 cells were further increased, the number of spores formed fell drastically.
Inclusion of 75% (Experiment 1E) or 87.5% HW 2 (Experiment 1F) cells decreased the number of spores found, respectively, to 0.78% and 0.04% of the number formed by a pure A3 culture. Control experiments with 25% (Experiment 1B) or 12.5% (Experiment 1C) of the standard number of A3 cells incubated alone indicated that, for the most part, the decrease in spores formed by co-incubation A3 and HW 2 cells is due to the presence of HW 2 cells and not the lower density of A3 cells present. Co-incubation of A3 and HW 2 reduce both the number and size of sorocarps which are produced and slows their development (data not shown).  Procedures." The fact that the number of spores recovered under optimal conditions is greater than the original number of cells plated is consistent with previous results (38) indicating increase in cell number during development.
In Experiment 2, the spores from 12 sorocarps from Experiment 1E were harvested and suitable dilutions incubated with bacteria on SM agar and the development of the plaques formed were scored. Some spores were treated with 0.2% Triton X-100 before olatina to destrov anv contaminating amoebae. which allow development of A3. Therefore, the rescue of HW 2 also appears to depend on cell contact.

Plasma Membrane
Antigens -Two plasma membrane macromolecules bear antigens that are qualitatively, or quantitatively different in A3 and HW 2, or both. One of these macromolecules appears to be glycoprotein E (14). Antigens associated with glycoprotein E will be referred to as E(Ag). E(Ag) from HW 2 plasma membranes (Fig. 3B) binds at least twice as much unadsorbed antibody as does E(Ag) from A3 plasma membranes (Fig. 3A). However, HW 2 adsorption of the antiserum decreases E(Ag) antibody binding to an undetectable level in HW 2 plasma membranes (Fig. 4B), but has no effect on E(Ag) antibody binding to E(gp) from A3 plasma membranes (Fig. 4A). This differential effect of HW 2 adsorption clearly indicates that the E(Ag) of HW 2 plasma membranes is exposed at the surface of HW 2 cells but that the E(Ag) of A3 plasma membranes differs from the HW 2 E(Ag) so that its antibody binding is not affected by HW 2 adsorption. A3 adsorption of antiserum removes the antibodies that bind both to E(Ag) from A3 (Fig. 4C), and HW 2 cells (Fig.  40). Therefore, both the A3 and HW 2 forms of E(Ag) are present on the A3 cell surface.
Another antigenic difference between A3 and HW 2 is associated with a macromolecule of molecular weight 150,000, referred to as 150(Ag). The 150(Ag) in A3 plasma membranes (Fig. 3A) binds more antibody from unadsorbed serum against A3 plasma membrane than does the 150(Ag) in HW 2 plasma membranes (Fig. 3B). The data using adsorbed sera are also consistent with the possibility that the only difference in 150(Ag) between A3 and HW 2 is quantitative (more in A3) although a qualitative difference cannot be ruled out. A3 adsorption reduces 150(Ag) antibody binding at least 80% in both A3 (Fig. 4C) and HW 2 (Fig. 40) 4A) or HW 2 (Fig. 4B) plasma membranes.
The molecule bearing 150(Ag) and glycoprotein D (14) have similar molecular weights.
However, there is no other evidence for their identity.
However, antibodies were uniformly adsorbable by either A3 or HW 2 cells (Fig. 4). Unadsorbed serum indicated one further difference between A3 and HW 2 plasma membranes.
A broad, continuous band of antibody binding was observed at a lesser migration in HW 2 (Fig. 3B) than in A3 (Fig. 3A)  were separated by SDS-gel electrophoresis, fixed in the gels, and reacted with either preimmune rabbit serum or immune serum prepared against vegetative A3 plasma membranes.
The antigens were then detected with fluorescent goat anti-rabbit antibodies.
The details of this technique are presented under "Experimental Procedures." A, A3 plasma membrane macromolecules reacted with immune serum; B, HW 2 plasma membrane macromolecules reacted with immune serum. The two major discrete antigenic differences between A3 and HW 2 plasma membranes are indicated as E(Ag) because this antigen co-migrates with E(gp) (13)  n-mannose or n-glucose, N-acetyl-n-glucosamine, L-fucose, and n-galactose or N-acetyl-n-galactosamine. This method can detect about lo4 receptors per cell (17) and is therefore more sensitive than the analysis of sugars by gasliquid chromatography.
With the exception of FBP, the lectinbinding patterns shown here are inhibited by the appropriate monosaccharide haptens (17). There are two differences in lectin receptors between A3 and HW 2 detectable with Con A (Fig. 5A)  the difference in E(Con A) was accentuated and the difference was still obvious at one-fifth the routine concentration of Con A (data not shown).
No difference between A3 and HW 2 plasma membranes were detected with the other lectins used (Fig. 5 6 and 7). J&p) and K(gp) are present in lesser amounts in the HW 2 plasma membrane.
Although E&p) was defective antigenically and in Con A binding in HW 2, it was not altered in PAS staining. Therefore, the defect in E(gp) in HW 2 does not significantly affect the number of periodate-oxidizable carbohydrate residues.
We have previously (14) used treatment with pronase to assay the topography of plasma membrane molecules during development.
Topographical differences between A3 and HW 2 PAS-positive glycoproteins are also detectable. As in A3 (14) no glycoproteins are sensitive to pronase treatment of intact cells (Fig. 6, Lane 2). However, glycoproteins C, E, and G are sensitive to pronase treatment of isolated HW 2 plasma membranes (Fig. 6, Lane 31, although they are relatively insensitive to similar treatment of A3 plasma membranes (Fig, 4, Lane 8, Ref. 14). Therefore, E&I), besides being defective in Con A binding and antigenicity in HW 2, is also in an aberrant topographical location or configuration within the HW 2 plasma membrane.
At the present we do not know whether the altered sensitivity of glycoproteins C and G represents an alternation in the nature of their insertion into the plasma membrane, their structure, or a change in shielding of these components by an altered molecule such as E&p).
Plasma' Membrane Polypeptide Composition and Topography -We were concerned with the possibility that the defects observed above in the HW 2 plasma membrane were secondary effects of a general disruption in plasma membrane composition or organization. Therefore, plasma membrane polypeptide composition and topography were examined. No significant differences were found between A3 (Fig. 8, Lane 5) and HW 2 vegetative plasma membranes (Fig. 8 2, plasma membranes from pronase-treated vegetative HW 2 cells; 3, pronase-treated vegetative HW 2 plasma membranes; 4, as in Lane 1; 5, vegetative A3 plasma membranes. White letters indicate glycoproteins deficient in HW 2. Black letters indicate glycoproteins sensitive to pronase treatment of isolated HW 2 plasma membranes that were insensitive to similar treatment in A3 (13). The lettering system is the same as used in Ref. 13.

Plasma
Membrane and Whole Cell Lipid Composition-Since defects in several glycoproteins occur in HW 2, we thought .it possible that glycolipids might also be defective.
Therefore, we examined plasma membrane lipid composition. HW 2 plasma membranes contain a glycolipid not found in A3 plasma membranes (Fig. 9, Lane 6). Since it has a similar R, to cerebroside (data not shown) and since it partitions into the lower phase (21) it may be cerebroside but it has not been characterized. The remaining glycolipids found in both A3 (Fig. 9, Lane 3) and HW 2 (Fig. 9, Lane 5) probably have more extensive carbohydrate domains since they partition primarily into the upper phase (29) (in some preparations a minor portion partitioned into the lower phase) and have smaller RF values indicating they are more polar. Therefore, it is possible that the HW 2-specific glycolipid is an incomplete form of the glycolipids common to both A3 and HW 2. These low RF glycolipids are found only in the plasma membrane as would be expected if the lipids bear complex oligosaccharide chains (29). Whole cells and plasma membranes from a similar number of cells contain a similar amount of glycolipids ( Fig.  9, Lanes 1 and 3). Glycolipids were below detection (Fig. 9, Lane 2) in the whole cell protein equivalent to the protein content of the plasma membranes shown in Fig. 9, Lane 3.
Although the low RF glycolipids are identical in lipid prepa-  Fig. 8 were scanned on a Syntex AD-l Autodensitometer.
The two major differences, J&p) and K(gp) are indicated. The apparent difference about molecular weight 65,000 was not reproducible. present at the cell surface, must be altered in structure in HW 2 since HW 2 adsorbed anti-A3 plasma membrane antiserum binds to E(Ag) in A3 but not in HW 2. HW 2 E(Ag) does bind unadsorbed antiserum, in fact, it binds more antibody from unadsorbed anti-A3 plasma membrane antiserum than does A3 E(Ag). Two independent models have been constructed for the general nature of the antigenic relationships between E(Ag) and 150(Ag) from A3 and HW 2 cells. Other, more complicated, but supportable models are possible. In the first model, glycoprotein E from A3 cells is suggested to bear a unique antigen not found elsewhere on A3 cells or at all on HW 2 cells. Glycoprotein E in HW 2 cells bears an antigen that is also found on another molecule (X) in HW 2 cells and on X in A3 cells. The 150(Ag) is structurally similar in A3 and HW 2 cells but a greater amount of the antigen is expressed on A3 cells. Therefore, A3 adsorption removes antibody against the A3 E(Ag), the X antigen, and the 150(Ag). HW 2 adsorption removes antibody against the X antigen (or HW 2 E(Ag)) and a small part of the antibody against the 150(Ag). The model thus predicts that A3 adsorption will remove all antibodies against glycoprotein E and the 150,000-dalton macromolecule, while HW 2 adsorption will leave antibodies against the glycoprotein E in A3 cells and the 150,000-dalton macromolecule in A3 and HW 2 cells.
The second model suggests that both macromolecules detectable on A3 plasma membranes by HW 2 adsorbed serum (glycoprotein E and the 150,000-dalton macromolecule) bear a common antigen (the 150-E antigen) and that glycoprotein E from A3 cells has an additional unique antigenic site (the E antigen) in close proximity to the location of the 150-E site on E&I).
In this model, glycoprotein E from A3 and HW 2 cells bear the E antigen in common. In addition, glycoprotein E plasma membranes from 2 x lOa A3 cells; 4, lower phase lipids from plasma membranes from 2 x 10" A3 cells; 5, upper phase lipids from plasma membranes from 2 x lOa HW 2 cells; and 6, lower phase lipids from plasma membranes from 2 x lOa HW 2 cells. The positions of the sample origins and solvent front are indicated.

Defective
Glycoproteins in Mutant Strain of D. discoideum from A3 cells has an antigen which is found only on the 150,000-dalton macromolecule of HW 2 cells (the 150-E antigen). Since there appears to be less 150,000-dalton macromolecule exposed in HW 2 than in A3 cells, A3 adsorption is assumed to remove antibody against both of these antigens, while HW 2 adsorption leaves most of the antibody against the 150-E antigen.
We also detected several defects in HW 2 using techniques sensitive only to carbohydrate structure. Two Con A receptors were different in A3 and HW 2 plasma membrane. E(Con A), although detectable in A3, bound more lectin in HW 2. 113(Con A) was detectable only in A3. No differences were detected between A3 and HW 2 plasma membrane in receptors for the lectin WGA RCA-60, or FBP. Two PAS-positive glycoproteins were diminished in amount in HW 2. Three other PAS-positive glycoproteins, including glycoprotein E, which was also detected as an antigenic and Con A-binding defect, were more sensitive to pronase treatment of isolated plasma membrane in HW 2 than in A3. A glycolipid was present in HW 2 that was not detectable in A3. This glycolipid may be an incomplete form of more complex glycolipids common to both A3 and HW 2 but must be examined in more detail.
can also explain the difference in Con A binding between A3 and HW 2 E&p). Con A binds to glycoproteins containing LYn-glucose or a-o-mannose residues with unsubstituted hydroxyl groups at C-3, C-4, and C-6 (33). These residues need not be terminal.
However, glycopeptides that do bind Con A can be fractionated on the basis of their avidity for the lectin (34). The less avid class of receptors contains less mannose and a greater variety of other monosaccharides (35). It seems unlikely that the difference in E(gp) Con A binding between A3 and HW 2 is due to more receptors in HW 2 since in that case PAS staining of glycoprotein E in HW 2 and the adsorption of antibody against E might have increased. Therefore, HW 2 E&p) may bear an oligosaccharide chain(s) terminating in a-n-mannose or a-n-glucose, while A3 E&J) may have 1 additional carbohydrate molecule not recognized by Con A at the end of an otherwise identical carbohydrate chain. This configuration could cause A3 and HW 2 E&p) to be antigenitally distinct and cause E(gp) from HW 2 cells to bind more Con A than E(gp) from A3 cells.
These studies indicate that an examination of plasma membrane composition only by one or two methods may mask many important independent structural defects.
To survey fully plasma membrane composition, a variety of independent analytical techniques may generally be necessary. With one exception, the defects found in the HW 2 plasma membrane were detectable only with one of the techniques used. This may be due to different structural requirements for antigenicity, lectin binding, and the PAS reaction. Therefore, 113(Con A) J&p), and K&p) may not be antigenic in rabbits or may share their antigenic sites with other membrane molecules and so would not be observed with adsorbed sera. Antibody binding to J&p) and K(gp) by unadsorbed serum also might have been obscured by antibody binding to the broad, continuous band of antigen observed. The defects in 150(Ag), E(Con A), llJ(Con A) need not involve changing the number of PAS-positive carbohydrate residues on a glycoprotein.
The fact that no differences in lectin binding were seen at the position of J(gp) and K(gp) suggests that although fewer PAS-positive carbohydrate residues are present in HW 2 than in A3, an equal number of lectin binding sites with equal affinities are available. Alternatively, they may only be co-migrating with other lectin receptors. Acknowledgements-We wish to thank Doctors J. Shively and M. Wrann for performing the gas-liquid chromatographic analysis of plasma membrane carbohydrate composition. We also wish to thank Dr. J.-P. Revel for the use of his electron microscopy facilities and Dr. R. Stroud for the use of his autodensitometer.
Thanks also go to Chris West for help in antiserum preparation and electrophoresis.

REFERENCES
The defect in E&p) in HW 2 was detected both antigenically and by Con A binding.
Subsequently we have purified E(gp) and found that both defects seem to be associated with E&p) and not a co-migrating contaminant. E(gp)s from A3 and HW 2 cells have a very similar amino acid composition. 5 Therefore, both the antigenic and lectin-binding defects in HW 2 E&p) may result from the same defect in carbohydrate structure.