Proc. Nat. Acad. Sci. USA Vol. 72, No. 1, pp. 310-313, January 1975

Cell-Cell Interactions: Enhancement of Glycosyl Transferase Ectoenzyme Systems During Chlamydomonas Gametic Contact (cell surfaces/cell recognition and adhesion/enzyme-substrate complexes/aggregation) ROBERT J. McLEAN* AND H. BRUCE BOSMANNt * Department of Biological Sciences, State University College, Brockport, New York 14420; and t Department of Pharmacology and Toxicology, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 Communicated by Harold C. Bold November &, 1974

ABSTRACT Glycosyl transferase ectoenzyme systems flagella (9). This meant that the recognition substance was that transfer galactose, glucose, N-acetylglucqsaipine, N- small in comparison to the 108-dalton particles described by Mcetylneuraminic acid, mann6se, and fucose have been de- tected on vegetative cells and gametes of Chilamydomona4 Forster et al. (8). From this observation we tested the hy- moewusii. Gametes have higher levels of activity of the pothesis of whether glycosyl transferases and acceptors were transferase ectoenzyme systems than morphologically present on the external cell surface of the gamete, since such identical vegetative cells, as determined by transfer of ectoenzyme systems have been postulated as a potential monbsaccharide onto endogenous cell surface acceptors. cell recognition and adhesion (5-7). When (+)-and (-) gametes kire mixed, there is a significant mechanism mediating increase in the activity of transfekase ectoenzymS systems. Our data demonstrate that glycosyl transferase and ac- No enhancement in activity of transferase ectoenzyme ceptors are present on the external surfaces of the (+) and systems occurs when (+) and (7) vegetatie' cells are (-) mating types of Chlamydomonas moewusii gametes, and mixed. Flagellar membrane yesieles obtained from (+) that when flagellar membrane vesicles or gametes of the two and (-) gametes show high activity of transferase ecto- enzyme- systems per mg of protein and also demonstrate mating types are mixed, there is a significant increase in the enhanced activity upon mixing. Therefqre, glycosyl trans- activity of the glycosyl transferase ectoenzyme system$. ferases and acceptors seem to be located on ihe flagellar It would appear that external surface glycosyl transferases membrane and appear to have a function particularly re- and appropriate specific cell surface acceptors are directly im- lated to gametic cells. The mechanism of cellular adhesion plicated in a function related to cellular recognition and/or or recogkiition proposed by Roseman (1970, Chem. Phys. Lipids 5I 270-297), involving glycogyl transferases and ac- adhesion. ceptors, is strongly suggested by our data for the mating reaction in Chlamydomonas. MATERIALS AND METHODS The (+) and (-) mating types of the unicellular flagellate, Glycosyl transferase and acceptor molecules have been de- Chlamydomona2 moewusii, nos. 96 and 97, respectively (10), tected on the external cell surface of several different mam- were grown on growth medium agar (11) for 8-10 days at malian cells (1-6), but their function at this external surface 250 i 10 in north light. Cells were then suspended in liquid is uncertain. Roseman (7) has postulated that these ecto- induction medium (11) to induce gametogenesis. In order to enzyme systems may be responsible for cell recognition and/or obtain motile vegetative cells (cells not responsive in the mat- adhesiveness by the formation of an enzyme-substrate com- ing reaction), liquid growth medium was supplemented with plex. Such a specific complex formation could provide the 0.1% (w/v) NH4C1 (12). All cultures were axenic, and aseptic high degree of specificity required in such phenomena as techniques were used during induction. histotypic compatibility and egg-sperm recognition. Addi- Vegetative cells are not distinguishable from gametes ex- tionally, completion of the reaction could break the enzyme- cept that gametes of one mating type will adhere to gametes substrate complex, thus explaining the modulation between of the opposite mating type; i.e., (+) gametes will adhere to adhesion and disadhesion found in many cell interactions, e.g., (-) gametes, but (+) gametes will not adhere to (-) vegeta- release between mammalian cells observed during contact in- tive cells. Additionally, (+) and (-) vegetative cells have no hibition. affinity for each other (12). Therefore, vegetative cells were We have investigated a protist system, that of the Chlamy- used as controls in some experiments for comparison with domonas mating reaction, in which flagella of opposite gametes gametes. adhere to each other by their flagellar membrane surfaces. Membrane vesicles were isolated by differential centrifuga- After a cytoplasmic bridge has formed between the cell tion from the induction medium (9) after gametes had been bodies of two opposite gametes, the adhering flagella are re- incubated for 24-28 hr. Suspended membrane material was leased from each other and are no longer agglutinable with passed through a 1.0-jum filter (Nuclepore Corp., Pleasanton, other gametes. The mechanism of gametic adhesion and Calif.) and then layered over 40% sucrose and centrifuged for recognition in Chlamydomonas is not known but was ascribed 1 hr at 16,300 X g. Membranes were collected at the interface to large glycoprotein particles attached to the flagellar sur- and washed twice. face that were easily lost in the cell medium. Particles from the cell medium of one mating type could cause isoagglutina- t Glycosyl transferase ectoenzyme system is the term used tion (agglutination within one mating type) when added to herein to denote external surface glycosyl transferases and opposite gametes (8). It was recently found that the active surface acceptors whose activity is measured as a system. That is, factor in the cell medium was actually membrane vesicles, under the conditions of assay either glycosyl transferase or ac- and no large, active particles were observed on the vesicles or ceptor could be limiting kinetically in the reaction. 310 Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Cell Recognition and Glycosyl Ectoenzymes 311

Assay procedures for glycosyl transferase and particulars TABLE 1. Activity of the surface and total glycosyl transferase concerning substrates (preparation, specific activity, etc.) ectoenzyme system of isolated active vesicles of followed those reported (1, 2, 13). Gametes and vesicles were Chlamydomonas gametes washed in induction medium, and vegetative cells were washed in growth medium. The assay mixture for surface activity 0.5 Volume contained 10 MAl of the nucleotide phosphate [14C]mono- strain 96 plus 0.5 saccharide (about 10-9 mol), 10 of 0.1 M MgCl2, 10 of Al 1M Strain Strain 0.1 M 10 of 0.1 M Tris HCl and 50 volume MnCl2, MuI * (pH 7.6), Mul of Substrate 96(+) 97(-) strain 97 the active vesicles (about 10lg of protein) or cells suspended in 0.145 M NaCl. The final pH of the reaction was 6.9. After Surface activity 30 min of incubation at either 250 or 37°, 3 volumes of 1% UDP-galactose 3,810 4,409 16,888 phosphotungstic acid in 0.5 M HCl were added and the UDP-glucose 5,211 4,687 19,460 precipitate was centrifuged out of solution. The precipitate UDP-N-acetylglu- was washed twice with 10% trichloroacetic acid and once cosamine 5,612 5,024 21,872 with ethanol-diethyl ether (2:1, v/v), and the resultant CMP-N-acetylneur- aminic acid 6,800 6,100 49,072 precipitate was dissolved in 1 M NaOH. was Radioactivity GDP-mannose 5,321 5,916 29,112 determined in a liquid scintillation counter. Data are ex- GDP-fucose 3,507 4,111 19,100 pressed as cpm per mg of protein with background sub- tracted. Protein was determined by the method of Lowry et al. Total activity (14). For assay of the total glycosyl transferase system, a UDP-galactose 4,968 5,609 6,311 0.1% Triton X-100 homogenate of cells or vesicles (sonicated, UDP-glucose 6,409 6,802 7,119 UDP-N-acetylglu- then given 30 strokes in a Ten Broeck homogenizer) replaced cosamine 7,219 6,084 10,897 whole cells or suspended vesicles in the assay. This procedure CMP-N-acetylneur- allowed for a total assay of both internal and surface glycosyl aminic acid 12,462 10,492 16,471 transferase systems. GDP-mannose 7,996 8,142 12,489 Measurements of electrophoretic mobilities were done ac- GDP-fucose 4,079 4,822 5,967 cording to procedures outlined (2), with a whole-cell particle electrophoresis chamber obtained from Rank Brothers, Vesicles were prepared as described in the text. Data are cpm/ Bottisham, U.K. Mobilities of the particles were calculated mg of protein. The surface activity system contained 10 Ml of the in (,Mm/sec) V- cm-'. At least 160 individual cells were nucleotide phosphate [14C]monosaccharide (about 10-9 mol), 10 analyzed for each cell type for the electrophoretic mobility MAl of 0.1 M MgCl2, lOAl of 0.1 M MnCl2, 1O Il of 0.1 M Tris.HCl measurements. (pH 7.6), and 50 Ml of the active vesicles (about 10 g of protein) suspended in 0.145 M NaCl. The final pH of the reaction was 6.9. After 30 min of incubation at (reaction at RESULTS 370 250 gave quanti- tatively similar results), macromolecular-bound radioactivity was Isolated flagellar membrane vesicles from gametes were as- determined. For the total system, 50 MAl (about 10 ug of protein) sayed for activity of the glycosyl transferase ectoenzyme sys- of a 0.1% Triton X-100 homogenate (sonicated, then given 30 tem. Table 1 shows activity of surface glycosyl transferase strokes in a Ten Broeck homogenizer) replaced the suspended ectoenzyme of vesicles and total transferase activity of vesicles vesicles in the assay. Since only small amounts of protein were available, each assay resulted in solubilized with 0.1% Triton X-100. Total activity of in- cpm that ranged from 3 to 50 dividual transferases is usually less than double that of the times background (19 cpm). All data have background sub- tracted. Data are means from five determinations. For the mixed enzyme monitored for vesicle surface activity, indicating that experiments, 25 ul of strain 96(+) and 25 Mul of strain 97(-) at least 50% of the activity is located on the external mem- vesicles were the enzyme/acceptor fraction in the assay. brane surface. When vesicles of strain 96 (+) and 97 (-) are mixed, there is a 4- to 8-fold increase in the activity of the six glycosyl transferase ectoenzyme systems that were de- enzyme systems for each of the monosaccharides may have tected, indicating that when they are mixed there is a pre- different significance based on whether they catalyze transfer ferred reaction between the surface transferases and acceptors. of internal or terminal monosaccharide residues. Furthermore, this probably means that a threshold number of Gametes and vegetative (nonsexual) cells were also assayed reactions must be completed for adhesion or recognition to for activity of surface and total glycosyl transferase system occur, i.e., the low levels of system activity in the iso-strain are (Table 2). Total activities of gametes and vegetative cells not sufficient for mediation of recognition or adhesion, but treated with 0.1% Triton X-100 are about the same as the the high levels of system activity in the complementary assay surface transferase activity of these respective cells. This must be sufficient to mediate cell-cell recognition or adhesion. would suggest that most of the activity of the glycosyl trans- Total activity from mixing vesicles of strains 96 and 97 ferase system of the cells is on the surface. When (+) and (-) treated with the nonionic detergent Triton X-100 is not more whole gametes are mixed, a 2- to 3-fold increase in activity of than double the activity of the individual strains treated with the glycosyl transferase ectoenzyme system (except for the the detergent, which solubilizes most enzyme activity. Thus, GDP-fucose system) is detected. There is no enhanced activity the additive reaction of the surface system is not seen in the when (+) and (-) whole vegetative cells or (+) and (-) total transferase system. It is of interest to note that all six gametes and (+) and (-) vegetative cells treated with Triton glycosyl transferase ectoenzyme systems were found in X-100 are mixed. It would appear that only whole cells that Chlamydomonas, since in most cellular glycoproteins some of have differentiated into gametes have sufficient comple- the monosaccharides are internal while others are predomi- mentary enzymes and acceptors to effect higher activity, pos- nantly terminal residues. Thus the glycosyl transferase ecto- sibly resulting in adhesion between opposite gametes. Downloaded by guest on September 29, 2021 312 Cell Biology: McLean and Bosmann Proc. Nat. Acad. Sci. USA 72 (1976)

TABLE 2. Activity of surface and total glycosyl transferase ectoenzyme system of gametes and vegetative cells of Chlamydomonas

Gametes Vegetative cells 0.5 Volume 0.5 Volume 96 plus 0.5 96 plus 0.5 Strain 96 Strain 97 volune 97 Strain 96 Strain 97 volume 97 Surface activity UDP-galactose 1390 1348 3378 680 720 760 UDP-glucose 1260 1294 2753 740 860 890 UDP-N-acetylglucosamine 670 1381 2806 880 260 810 CMP-N-acetylneuraminic acid 610 890 2280 600 720 700 GDP-mannose 870 1305 2319 710 720 810 GDP-fucose 1000 1140 1170 460 480 400 Tctal activity UDP-galactose 1590 1460 1346 800 840 840 UDP-glucose 1660 1392 1329 760 210 560 UDP-N-acetylglucosamine 890 1408 1290 820 860 840 CMP-N-acetylneuraminic acid 890 1104 1210 700 810 760 GDP-mannose 1140 1385 1316 810 840 840 GDP-fucose 1260 1204 1208 520 530 590

Systems of assay were exactly as given in Table 1 except that active gametes or vegetative cells (about 1 mg of protein) replaced the active vesicles in the assay. Data are cpm/mg of protein. Since ample amounts of protein were available, actual cpm were 30 to 100 times background (19 cpm). All data have background subtracted. Data are means from five independent determinations.

The enzymatic nature of one of the active vesicle glycosyl DISCUSSION transferase ectoenzyme systems is demonstrated in Table 3 by The detection of glycosyl transferase ectoenzyme systems on the dependence of the reaction on temperature, incubation cells of Chlamydomonas moewusii and the enhancement of time, divalent cations, and non-heat-denatured enzyme. enzyme activity when opposite gametes are mixed strongly Furthermore, fetuin minus N-acetylneuraminic acid added suggest that glycosyl transferases and acceptors may be a exogenously to the reaction produced enhanced activity, in- contributing mechanism in the phenomenon of gametic recog- dicating that this glycoprotein acted as a substrate for the nition and adhesion in the present cell system. Furthermore, surface glycosyl transferases. since glycoproteins have been found at the Chlamydomonas Electrophoretic mobility data (Table 4) reveal a net dif- cell surface (8), some of the total enzyme activity may be ference of -0.35 (am/sec) * V-1 * cm-' between the two mating responsible for synthesis of these membrane components. types of C. moewusii gametes. These data clearly demonstrate Only the flagellar membrane of the gamete or vegetative cell a rather striking difference in the electrokinetic and presum- is exposed, while the remainder of the cell body and plasma ably biochemical nature of these surfaces. Since apparently membrane is covered by the cell wall. When placed in the con- the only genetic difference between the two mating types is text of the Roseman hypothesis (7), the flagella of opposite sexual, it would appear that the electrokinetic difference is gametes would adhere to each other when the enzyme-sub- related to their function as gametes. strate complex is formed. After plasmogamy is initiated be- tween the cell bodies of the two opposite gametes, nucleotide TABLE 3. Characterization of reaction of glycosyl transferase phosphates would be released to complete the reaction and ectoenzyme system for active vesicles break the enzyme-substrate complexes, thereby breaking flagellar adhesion. The separated flagella are not susceptible to epm/mg adhesion by other gametes (12) possibly because the glyco- System of protein protein acceptors, each with an additional monosaccharide Complete 49,000 unit, would no longer be suitable substrates. The method by Complete, 00 2,700 which nucleotide phosphate monosaccharide becomes avail- Complete, 200 44,000 able to complete the reaction is uncertain. These substrates Complete, 80° 10,100 Complete + 10-8 mol of CMP-N-[14C] acetyl- TABLE 4. Electrophoretic mobility of active gametes of neuraminic acid 49,100 Chlamydomonas Complete + fetuin minus N-acetylneuraminic acid (0.5 mg per assay) 63,400 Electrophoretic mobility, 0 time 4,710 Mating type (Am/sec) *V-1 cm1 Complete minus Mn + + and Mg + + 9,710 Strain 96(+ ) -0.89 di 0.02 ± CMP-N-[14C] acetylneuraminic acid was used as substrate with Strain 97(-) -1.23 0.03 equal aliquots (25 1A) of vesicle preparations from strains 96 and 97 as enzyme/acceptor source. The complete system was exactly All data were performed in 0.0145 M NaCl, 4.5% sorbitol (pH as given in Table 1 (i.e., third column, line 4) and contained about 7.4), 0.6 mM NaHCO3 at 250 as described in text. Data are means 10 j.zg of protein. Radioactivity was recovered as 97% N-acetyl- i SD. At least 160 individual particles were analyzed for each neuraminic acid. mating type. Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Cell Recognition and Glycosyl Ectoenzymes 313

could be released from the cell; alternatively, they might be- the purposeful occurrence of these enzymes and their sub- come bound to that part of the enzyme that transcends the strates on the plasma membrane of only gametic cells. membrane and is positioned on the cytoplasmic surface (15). It is difficult to determine what relationship this work has The significant enhancement of transferase activity that with the work of Moscona (23) and Lilien (24), in which cell occurs when opposite gametes, or flagellar membrane vesicles aggregating factors were isolated from the cell medium. The from opposite gametes are mixed, would certainly point to a factors were not assayed for glycosyltransferase activity nor role for transferases and acceptors in the sexual function of were they examined electron-microscopically. It would be of Chlamydomonas gametes. This is further supported by the interest to determine if the aggregating factors are similar to lack of enhanced transferase activity when the morphologi- the membrane vesicles that were isolated from the medium of cally identical, sexually incompetent vegetative cells of the Chlamydomonas. Warren and Glick (25) suggested that mem- respective mating types are mixed. Higher activity on the brane components may be released into the medium as a vesicles indicates that the enzymes and substrates have been normal turnover mechanism. purified. From the data presented herein, the mechanism for cell The precise role of the individual glycosyl transferases and recognition-adhesion would seem to involve the glycopro- acceptors is not known. Collectively they could function in tein: glycosyltransferase ectoenzyme systems as proposed by gametic recognition and/or adhesion. Individually, one or Roseman (7). Such a mechanism may relate not only to the more might participate as messengers in initiating growth of Chlamydomonas mating reaction, but also to egg-sperm recog- the papilla which results in fusion of the gametes. This event nition, tumor cell systems (2, 3), and embryonic development is known to begin almost immediately after membrane con- (6). tact between opposite gametes is made (16). Such a mecha- nism for cell-cell communications through membrane surface This work was supported by NSF Grant BO 39881 to R.J.M. receptor-cytoplasmic interactions has been proposed by Edel- and an American Cancer Society Grant, Grant CA-13220 and GM-15190 (NIH), to H.B.B. During the initial aspects of this man et al. (17). The presence of glycosyl transferases at re- work, H.B.B. was a Research Career Development Awardee of duced levels on vegetative cells indicates either that the trans- NIGMS; he currently is a Scholar of the Leukemia Society of ferases are incidental on the surface of these cells as a result of America. We thank Carmen Laurendi, Glen F. Bieber, Kenneth Golgi vesicle fusion with the plasma membrane during plasma R. Case, and Roger Gutheil for technical assistance. membrane biogenesis or exocytosis (4), or that their surface 1. Bosmann, H. B. (1972) Biochim. Biophys. Acta 279, 456- function may be unrelated to the sexual process. Their pres- 474. ence in the vegetative cells is unrelated to mating, since there 2. Bosmann, H. B., Case, K. R. & Morgan, H. R. (1974) Exp. is not even a slight enhancement when (+) and (-) are mixed Cell Res. 83, 15-24. (Table 2). However, their role as ectoenzymes is uncertain and 3. Lloyd, C. W. & Cook, G. M. W. (1974) J. Cell Sci. 15, 575- may be related to the threshold number of 590. enzyme/acceptors 4. Patt, L. M. & Grimes, W. J. (1974) J. Biol. Chem. 249, necessary for true genetic recognition adhesion. 4157-4165. The species and sex-specific nature of the Chlamydomonas 5. Roth, S. & White, D. (1972) Proc. Nat. Acad. Sci. USA 69, mating reaction (12, 18) is certainly explicable by the highly 485-489. specific nature of an enzyme-substrate complex. 6. Roth, S., McGuire, E. J. & Roseman, S. (1971) J. Cell Biol. Sexually 51, 536-547. incompatible species such as C. moewusii and C. reinhardtii 7. Roseman, S. (1970) Chem. Phys. Lipids 5, 270-297. (18) may not have sufficient genetic similarities to synthesize 8. Forster, H., Wiese, L. & Braunitzer, G. (1956) Z. Natur- appropriate enzymes and substrates. Further sexual isolation forsch. B 11, 315-317. of these two species is demonstrated by the different mecha- 9. McLean, R. J., Laurendi, C. J. & Brown, R. M. (1974) nisms for gametic fusion Proc. Nat. Acad. Sci. USA 71, 2610-2613. (16, 19). On the other hand, inter- 10. Starr, R. C. (1964) Amer. J. Bot. 51, 1013-1044. fertile species such as C. eugametos and C. moewusii (18) 11. McLean, R. J. & Brown, R. M. (1974) Develop. Biol. 36, could have similar genetic information to allow synthesis of 279-285. compatible transferases and acceptors. 12. Wiese, L. (1965) J. Phycol. 1, 46-54. The presence of enhanced transferase activity during the 13. Bosmann, H. B. (1971) Biochem. Biophys. Res. Commun. 43, 1118-1124. mating reaction in Chlamydomonas could be coincidental. The 14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, function of these enzymes may be only indirectly related to R. T. (1951) J. Biol. Chem. 193, 265-275. gametogenesis, since their synthetic function could be in the 15. Singer, S. J. (1972) Ann. N.Y. Acad. Sci. 195, 16-23. cell interior for catalysis or complex carbohydrate biosyn- 16. Brown, R. M., Johnson, C. & Bold, H. C. (1968) J. Phycol. thesis in the Golgi apparatus (4). This would 4, 100-120. require that a 17. Edelman, G. M., Yahara, I. & Wang, J. L. (1973) Proc. Nat. different adhesive mechanism is involved in the Chlamydo- Acad. Sci. USA 70, 1442-1446. monas system, possibly similar to the mating factors present 18. Trainor, F. R. (1959) Amer. J. Bot.'46, 65-70. on yeast (20). 19. Friedmann, I., Colwin, A. L. & Colwin, L. H. (1968) J. Cell Glycosyl transferases are known to occur in Golgi bodies Sci. 3, 115-128. 20. Crandall, M., Lawrence, L. M. & Saunders, R. M. (1974) (21). Flagellar membrane appears to derive also from the Proc. Nat. Acad. Sci. USA 71, 26-29. Golgi, since Bouck (22) demonstrated that mastigonemes 21. Schachter, H., Jabbal, I., Hudgin, R. L., Pinteric, L., (flagellar appendages) are placed on the membrane surface McGuire, E. J. & Roseman, S. (1970) J. Biol. Chem. 245, by Golgi vesicles. Patt and Grimes (4) suggested that glycosyl 1090-1100. transferase occurrence on the plasma membrane may be 22. Bouck, G. B. (1971) J. Cell Biol. 50, 362-384. due 23. Moscona, A. A. (1968) Develop. Biol. 18, 250-277. to the fact that they are found in the Golgi and, therefore, are 24. Lilien, J. E. (1968) Develop. Biol. 17, 657-678. incidentally secreted via Golgi vesicles. Our data emphasize 25. Warren, L. & Glick, M. C. (1968) J. Cell Biol. 37, 720-746. Downloaded by guest on September 29, 2021