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Characterization of the purified Chlamydomonas minus agglutinin

Chlamydomonas flagellar sexual agglutinins are responsible for the adhesion of opposite mating-type (plus and minus) gametes during the first stages of mating. Purification and partial characterization of the plus agglutinin was previously reported (Adair, W. S., C. J. Hwang, and U. W. Goodenough, 1983, Cell, 33:183-193). Here we characterize the purified minus molecule. We show it to be a high molecular weight, hydroxyproline-rich glycoprotein that migrates in the 3% stacking region of an SDS-polyacrylamide gel and is absent from two nonagglutinating minus mutants. Plus and minus agglutinins are remarkably similar, although nonidentical, in amino acid composition, molecular morphology, and reactivity in vivo and in vitro with monoclonal antibodies raised against the plus agglutinin. Moreover, the adhesiveness of both plus and minus agglutinins, when coupled to agarose beads, is abolished by thermolysin, trypsin, periodate, alkaline borohydride, reducing agents, or heat, but unaffected by exo- or endoglycosidases. The minus agglutinin, however, migrates just ahead of the plus molecule on SDS PAGE, is excluded from an anion-exchange (Mono Q) column, elutes earlier during hydrophobic interaction (Bio-gel TSK Phenyl 5PW) chromatography, and is sensitive to chymotrypsin digestion (unlike the plus agglutinin); therefore, it differs from the plus agglutinin in apparent molecular weight, net charge, relative hydrophobicity and proteolytic susceptibility. Nevertheless, our results generally demonstrate a high degree of homology between these complementary cell-cell recognition/adhesion molecules, which suggests that they are specified by genes that have a common evolutionary origin.

Chlamydomonas reinhardi mating-type plus (mt +) and mating-type minus (rot-) gametes recognize and adhere to one another to initiate mating via sexual agglutinins located on their flagellar surfaces (reviewed in references 1 and 2). The processes of gametic recognition and flagellar adhesive interactions are extremely specific for both the mating-type and species of Chlamydomonas (3), features attributable to the agglutinin molecules themselves (4,5). The plus and minus agglutinins can both be extracted in a biologically active form from mt ÷ and mrgametes by EDTA (4,6) and quantitated using an in vitro bioassay (4). The plus species has been purified by gel filtration chromatography and identified as a high molecular weight, hydroxyproline-containing, fibrous glycoprotein that is present as an extrinsic component of the mt+ flagellar surface (4,5,7). Recently, Saito and Matsuda (8) have followed similar protocols to extract minus agglutinin and have fractionated the activity by hydroxyapatite chromatography. They report that the minus agglutinin is also a high molecular weight glycopolypeptide.
To investigate the molecular mechanism that governs agglutinin-mediated intracellular recognition and adhesion in Chlamydomonas, it is important that both adhesins be well characterized. This paper describes the characterization of the minus agglutinin, purified by Fractogel-75 gel filtration chromatography, and its comparison with the plus agglutinin. Minus agglutinin is shown to be highly homologous to its plus counterpart in amino acid composition, structural morphology, and immunological antigenicity, with both adhesins representing high molecular weight, extrinsic, flagellar glycoproteins, rich in serine and hydroxyproline, that are absent from nonagglutinating mutants. Furthermore, many agents or treatments that perturb the adhesive nature of the plus agglutinin are shown to have a similar effect on the minus species. Despite these homologies, the two agglutinins differ with respect to net charge, hydrophobicity, electrophoretic mobility, and susceptibility to chymotrypsin digestion.

Preparation of Agglutinin ED TA Extracts: Minus agglutinin was
extracted from 3 x 10 u C. reinhardi mating-type minus plate gametes (strain CC-621) using buffered EDTA (Sigma Chemical Co., St. Louis, MO) according to the protocol routinely used for plus agglutinin extraction. This procedure has been modified from that previously described (4,5) in the following ways. Washed gametes are pelleted by brief centrifugation at 8,000 g and resuspended in 15 mM EDTA, 20 mM PIPES (Research Organics, Inc., Cleveland, OH), pH 7.4, at a final cell density of 109 cells/ml. Extraction is performed at 25"C until the cells are nonagglutinative with untreated tester gametes of the opposite mating type; unlike Saito and Matsuda (6,8), we find that our minus gametes require a somewhat longer incubation period (30-45 rain) with EDTA than do plus gametes (20-30 min) for full agglutinin extraction. The suspension is then briefly centrifuged at 27,000 g to pellet cells, and the supernatant is centrifuged at 40,000 g for 20 rain. Ammonium sulfate is added to this supematant to 70% saturation for 30 min on ice. After a 15-min centrifugation at 40,000 g, the pellets are resuspended in 1.5 ml deionized water, dialyzed overnight at 4"C against the same, centrifuged at 100,000 g for 40 min to pellet large cell wall fragments, and the supernatant (crude preparation) is lyophilized and stored at -70"C. The nonagglutinating minus mutants imp-lO (CC-1147) and imp-12 (COl 149) were treated in an identical fashion to the normal minus strain. All CC numbers denote stock cultures available from the Chtamydomonas Genetics Center, Department of Botany, Duke University (Durham, NC).
In Vitro Iodination of Agglutinin: A lyophilized crude EDTA extract was resuspended in 1.0 ml of column buffer A, which consisted of 20 mM PIPES (pH 7.4), 100 mM KCI (Sigma Chemical Co.), 5 mM EDTA, and 30 mM octyl-/~-D-glucopyranoside (Calbiochem-Behring Corp., La Jolla, CA), centrifuged for l0 min at 40,000 g, and the supernatant was divided into 750 and 250-/~1 aliquots. The smaller sample was split into two portions, each of which was incubated with 200 /~Ci carrier-froe NaJ2~I (Amersham Corp., Arlington Heights, IL) and one IODO-bead (Pierce Chemical Co., Rockford, IL) for 15 min at 25"C (5). Unrcacted label was removed by a rapid desalting (9) through I ml Bio-Gel P6-DG (Bio-Rad Laboratories, Richmond, CA) into column buffer A. Both labeled samples were added to the remaining (750 ul) agglutinin aliquot and centrifuged for 10 min at 40,000 g. The supernatant (900/A), containing 1.5 x 10 9 dpm, was directly loaded onto the gel filtration column for agglutinin purification.
Denaturing PAGE: SDS PAGE, sample preparation, autoradiography, and gel stains (periodic acid-Schiff, [PAS], t Coomassie Blue, silver) were performed as described previously (4,5). Gels containing 3-4 and 4-6% gradients of acrylamide and urea, respectively, were used without stacking regions in some experiments but run at lower voltages than the above gels (40 V for -l h, then 80 V until the bromophenol blue incorporated in the gradient gel migrated from the end of the gel).
Anion-exchange chromatography was performed on a MonoQ HR5/5 column (Pharmacia Fine Chemicals, Piscataway, N J) in 20 mM HEPES (Sigma Chemical Co.), 5 mM EDTA, pH 7.2. Elution of bound proteins was accomplished with a gradient of 100-500 mM KC1 in the same buffer at a flow rate of 1.0 ml/min.
The high-pressure liquid chromatographic system employed for hydrophobic interaction and anion-exchange chromatography used a Micromeritics (Micromeritics Instrument Corp., Norcross, GA) solvent delivery system (model 750), gradient programmer (model 752), and variable wavelength ultraviolet detector (model E 54 U) on line to an IBM CS 9000 instruments computer. Aliquots (-200 #1) of column fractions were rapid-desalted through 1 ml of Bio-Gel P6-DG (9) and analyzed by SDS PAGE/autoradiography and/or PAS staining, microscopic bioassay (4), and radioactivity determinations in a Beckman Gamma 4000 Counting System (Beckman Instruments Inc., Palo Alto, CA). When required, pooled column fractions were concentrated over Aquacide 1I (Calbiochem-Behring Corp.).
Biochemical and Immunological Analyses: Amino acid analysis for minus agglutinin was conducted as described for plus agglutinin (7).
Abbreviation used in this paper: PAS, periodic acid-Schiff.

COLLIN-OSDoBY AND ADAIR
Minus agglutinin was coupled to agarose beads for inactivation studies and treated as previously described (l I).
Details of antibody production and properties of agglutinin-reactive monoclonal antibodies are presented in separate reports from our laboratory (1,12). Immunoautoradiography was performed using the spaghetti overlay procedure (13).

Light, Fluorescence, and Electron Microscopy: Agglutinin
bioassay by light microscopy was performed according to the method of Adair et at. (4). Quick-frozen, deep-etched samples of purified agglutinins were prepared for electron microscopy according to Heuser (14) and visualized as described previously (5,7,15).
Immunofluorescence microscopy of agglutinin-reactive monoclonal antibodies bound to cells was performed as follows. Cells were washed once with cold buffer of 10 mM HEPES, pH 7.5, 5 mM MgSO4, 0.5 mM EGTA, 25 mM KCI, fixed for at least 2 h in the same buffer containing 2% paraformaldehyde (Eastman Laboratory and Specialty Chemicals, Rochester, NY), and washed three times with phosphate-buffered saline (PBS) before incubation for 30 rain with monoclonal antibodies (25 t~g/ml of a 50% ammonium sulfate cut of ascites fluids). Control samples were incubated with nonimmune mouse serum (Sigma Chemical Co.) or an irrelevant monoclonal antibody (D3, raised against Chlamydomonas dyneins, Mehard, B. B., and W. S. Adair, unpublished observation). After primary antibody incubations, cells were washed three times with PBS and incubated for 15 min with 5 ~g/ml biotinylated horse anti-mouse Ig (Vector Laboratories, Burlingame, CA). After they were washed twice with PBS and once with 50 mM Na2CO3, 0.15 M Nacl, the cells were incubated for 5 min in the latter buffer containing 20 #g/ml fluorescein isothiocyanate-avidin D (Vector Laboratories). The labeled cells were then washed three times with 50 mM Na2CO3, 0.15 M NaCI and suspended in mounting buffer (25% glycerol in PBS). A Zeiss Photomicroscope I equipped with a III RS epifluorescence condenser was employed to visualize and photograph the labeled cells through a K550 barrier filter using Kodak Tri-X film, shot at ASA 400 and developed at ASA 800.

Purification of Mating-type Minus Agglutinin
Quantitative release of the minus agglutinin from gametic flagella is readily accomplished by incubation of living gametes with buffered 15 mM EDTA, analogous to the protocol employed for extraction of plus agglutinin (4, 5). Fig. 1 illustrates the purification of an in vitro 125I-labeled minus agglutinin EDTA extract by Fractogel-75 sizing chromatography. Elution of labeled extract components was monitored by radioactive determination and a quantitative bioassay (4) was used to detect agglutinin biological activity. In the first cycle through the column matrix, a major portion of the labeled extract eluted in one broad peak (Fig. l A). Maximum minus agglutinin activity was predominantly associated with the shoulder of this peak. Autoradiography of column fractions after SDS PAGE revealed a number of labeled bands in the biologically active fractions.
To more fully resolve components eluting in this region, the most active fractions (fractions 20-26) were pooled, concentrated, and rechromatographed. The major agglutinin activity was now found within the peak of remaining 125I-labeled EDTA-extracted material (Fig. 1 B). Peak minus agglutinin activity co-fractionated with two high molecular weight polypeptides, and these fractions otherwise contained only relatively minor amounts of other labeled components. When these two high molecular weight molecules were separated by high-performance liquid chromatography hydrophobic interaction chromatography, minus agglutinin activity was invariably associated with the slower-migrating, higher molecular weight gel band (as described below); this molecule is therefore considered to be the active minus agglutinin. The gel-band species has been named polypeptide A-, by analogy with the similarly migrating gel band of active plus agglutinin (5), which we henceforth designate polypeptide A ÷.

Minus Agglutination Mutants
Chlamydomonas mutants unable to agglutinate sexually with gametes of the opposite mating-type have been isolated in both plus and minus strains and genetically characterized (22,23). Adair et al. (5) have shown that EDTA extracts from nonagglutinating plus mutants lack the agglutinin polypeptide A ÷. To learn whether minus nonagglutinating mutants lack polypeptide A-, EDTA extracts (inactive) were prepared from imp-lO and imp-12 strains, and subjected to Fractogel-75 purification in a manner analogous to that employed for active minus extracts, and the fractions were analyzed by SDS PAGE. Fig. 2 (lanes 2 and 3) demonstrates that both mutants lack a polypeptide A-component, which supports the identification of this high molecular weight glycoprotein as the active minus agglutinin molecule. In addition, molecules with the morphology of A-are present on the surface of gametic, but not vegetative, minus cells when observed in situ (15).
The mutants do, however, produce glycopolypeptides that migrate farther into the stacker region of the gel (Fig. 2). These species, which are also produced by wild-type gametes, derive from the flagellum but are not involved in adhesion; they are described in detail elsewhere (15).  terminal hook. The minus agglutinin however, displays a "shepherd's crook" conformation near the head region, and its head is somewhat larger. A detailed structural comparison of these proteins is provided in a separate report (l 5).  (7) and for 2BI, one of the major C. reinhardi cell wall glycoproteins (24). The three Chlamydomonas high molecular weight glycoproteins are remarkably similar in overall composition. Furthermore, values for the C. reinhardi sexual agglutinins closely resemble those reported for the minus agglutinin of C. eugamatos (25). As expected, the plus and minus agglutinins are more similar to one another than to the cell wall glycoprotein, presumably reflecting their more similar evolutionary histories. Note that the minus agglutinin, like its plus homologue and 2BI, contains high levels of hydroxyproline and serine residues. As discussed previously, these two amino acids serve as points FIGURE 1 Purification of mt-agglutinin. A crude EDTA extract, obtained from 3 x 1011 mt-gametes, was iodinated in vitro and fractionated by two cycles of Fractogel-75 chromatography (see Materials and Methods). Agglutinin activity was determined by quantitative bioassay (4). Radioactivity of 100 ~1 of each fraction (2.6 ml total fraction volume for cycle 1, 2.2 ml for cycle 2) was determined by gamma-counting. For SDS PAGE, 50 #1 of each fraction was rapid-desalted into dH20 (9), lyophilized to dryness, and resuspended in 10/~1 SDS sample buffer (4).  for oligosaccharide attachment in a variety of plant glycoproteins, including Chlamydomonas cell wall glycoproteins, and the hydroxyproline residues are thought to confer on the protein its fibrous properties (7).

Differential Electrophoretic M o b i l i t y of Plus and Minus Agglutinins
Both plus and minus agglutinins migrate slowly in SDS PAGE gels (Fig. 4, lanes 1 and 4). As described in detail elsewhere (15), one or more faster-migrating glycopolypeptides, collectively designated B (Fig. 4, lanes 2-4), often contaminate peak agglutinin fractions; since the B material alone has no adhesive activity, however, it is not relevant to the present study. Agglutinin fractions enriched for A ÷ (Fig. 4, lane 1) consistently display maximal activity, whereas fractions containing abundant band B (lane 2) are much less active. Of interest here is that the polypeptide A-band reproducibly migrates farther into the 3% stacking region of SDS PAGE gels than does polypeptide A ÷, regardless of the purification method. This difference is most easily observed with longer electrophoretic runs on urea/polyacrylamide gels containing gradients of 3-4% acrylamide and 4-6% urea, conditions that provide improved separation as well as increased migration (without loss of resolution) of both bands A and B (Fig. 4, lanes 3 and 4). The increased migration of active minus agglutinin relative to active plus agglutinin presumably reflects a structural disparity under denaturing conditions between the plus and minus sexual adhesins.

Chromatographic Behavior of Plus and Minus Agglutinins
Comparable elution profiles are obtained for plus or minus agglutinin preparations fractionated on a variety of gel-filtration sizing columns, including Fractogel-75. In contrast, the adhesins behave quite differently on anion-exchange (MonoQ) and hydrophobic interaction (Bio-Gel TSK Phenyl 5PW) columns. Whereas plus activity is retained on a MonoQ column (20 mM HEPES, 5 mM EDTA, pH 7.2) and subsequently eluted with a 100-500 mM KC1 gradient (Fig. 5), minus activity is excluded from the anion-exchange resin.
Therefore, it appears that the agglutinins differ in net charge, a feature that might facilitate their interaction in vivo. Note that this column is also useful for separating bands A and B of plus agglutinin. Although both plus and minus agglutinins are efficiently adsorbed to the hydrophobic phenyl matrix (1.7 M ammonium sulfate, 100 mM potassium phosphate, pH 7.0), each elutes (with decreasing ammonium sulfate concentration) at an independent region of the gradient (Fig. 6), a characteristic retained when plus and minus extracts are combined and cochromatographed (not shown). Because minus activity elutes before the major extract contaminants (Fig. 6A), and plus activity is retained until after most of the wall material has been released (Fig. 6B), both agglutinins can be effectively purified to near homogeneity on the nondenaturing hydrophobic phenyl column. In addition, the high molecular weight gel band that co-elutes during F-75 purification with the minus agglutinin can be separated from the active adhesin by such hydrophobic interaction chromatography, as it elutes at a region intermediate to the elution positions of the minus Note that polypeptides A + and B are resolved and that agglutinin activity is associated with A + alone. The arrowhead denotes the stacking gel interface. and plus agglutinins (not shown). Since selective desorption depends on the strength of the hydrophobic interaction between the nonpolar amino acid groups of the protein and the phenyl substituents of the matrix (10), the lengthy retention time of plus agglutinin on this column is suggestive of its being relatively more hydrophobic than the minus agglutinin.

Inactivation of Minus Agglutinin Conjugated to Agarose Beads
Purified agglutinin, when covalently attached to an inert support such as agarose beads, retains potent biological activity in assays using living gametes of the complementary mating type (l 1). Such immobilized preparations provide a sensitive and rapid in vitro assay system for assessing the effects of various agents (chemical, enzymatic, etc.) on agglutinin activity (11). Table II compares data obtained for immobilized minus agglutinin subjected to various treatments with results previously published (11) for the effects of these treatments on beads coupled with plus agglutinin. Most of the treatments have a similar effect on both aggiutinins. Thus, both species are sensitive to digestion by thermolysin or trypsin, alkaline borohydride reductive modification of carbohydrate, heating above 65"C (although minus may be slightly more heat labile than plus), periodate oxidation (see also reference 6), and thiol reduction. Unlike C. eugamatos (16)(17)(18), minus agglutinin is not inactivated by a-galactosidase, nor is plus activity destroyed by a-mannosidase. An interesting difference is seen with a-chymotrypsin: minus agglutinin activity demonstrates a partial loss of activity at 0.1 mg/ml and a complete loss of activity at 1 mg/ml, whereas plus activity is unaffected by COLLIN-OSDOBY AND ADAIR even 10 mg/ml of this protease. Since the overall amino acid composition of minus agglutinin, as compared with plus agglutinin, is not enriched for aromatic amino acids (see Table  I), it is unlikely that the chymotrypsin-induced loss of minus biological activity results from a generalized degradation of minus agglutinin by this protease. If the selective inactivation of minus agglutinin results from chymotryptic cleavage at or near the minus adhesive site, this enzyme should be useful for probing the differences in the plus and minus mating-typespecific adhesion sites.

Immunologic Relatedness of Plus and Minus Agglutinins
Monoclonal antibodies directed against determinants found on the plus agglutinin have been generated in this laboratory and are described in detail elsewhere (1,12). Three classes of agglutinin-reactive monoclonal antibodies have been identified. Two are partially (class III) or extensively (class I) crossreactive with other Chlamydomonas flagellar or wall components; the third (class II) reacts specifically with polypeptide A and the polypeptide B family (l, 12, 15).
All three monoclonal antibody classes, although initially selected for their ability to isoagglutinate plus gametes by their flagella (1), prove to be equally effective at isoagglutinating minus gametes. Fig. 7 illustrates this cross-reactivity for two of these antibodies using immunofluorescence microscopy. Like the patterns observed for plus (1,12), the minus gametes display highly labeled flagella and cell walls with class I antibody (Fig. 7A), and punctate flagellar staining and unlabeled cell walls with class II antibody (Fig. 7B). Similar mating-type cross-reactivity is also observed with Class III

monoclonal antibodies (not shown).
To learn whether the antibodies were recognizing the same surface molecules on both plus and minus gametes, class I and II monoclonal antibodies were used to probe a partially purified minus agglutinin preparation, after SDS PAGE, by spaghetti overlay immunoautoradiography (25). Antigen-antibody complexes were detected using 12Sl-sheep anti-mouse Ig, F(ab)2, followed by autoradiography. Fig. 8 shows that both class I (lane 1) and class II (lane 2) monoclonal antibodies recognize the minus agglutinin. Since recent immunotopographical mapping studies have demonstrated that the class II monoclonal antibodies bind to a repeating determinant along the length of the plus agglutinin rod (1, 12), the plus and minus agglutinins may have common features in their polypeptide backbones, consistent with their highly similar amino acid compositions and morphologies.
In conclusion the minus sexual agglutinin has been extracted from C. reinhardi minus gametes with 15 mM EDTA, and purified by Fractogel-75 sizing chromatography, and the purified adhesin was characterized. These studies have demonstrated that, overall, minus agglutinin is remarkably similar to the plus sexual adhesin in amino acid composition, immunological antigenicity, structural morphology, and sensitivity or resistance to various agents or treatments.
Such homology between the C. reinhardi sexual agglutinins 1