Characterization Agglutinin of the Purified Chlamydomonas minus

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 thermol,/sin, trypsin, periodate, alkaline borohydride, reducing agents, or heat, but unaffected by exoor 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. 1144 THE JOURNAL OF CELL BIOLOGY • VOLUME 101 SEPTEMBER 1985 1144-1152 © The Rockefeller University Press + 0021-9525/85/09/1144/09 $1.00 on A uust 6, 2017 jcb.rress.org D ow nladed fom MATERIALS AND METHODS 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). Chromatographic Fractionation of Agglutinin Extracts: Fractionation of minus agglutinin extracts on Fractogel TSK HW-75 (F) (EM Science, Gibbstown, N J) was performed in column buffer A (30 mM octylglucoside, 5 mM EDTA, 100 mM KCI, 20 mM PIPES, pH 7.4) as previously described for plus agglutinin (5), without prior chromatography on Sepharose 6B. For hydrophobic interaction chromatography, extracts (a crude preparation resuspended in 1.0 ml) were adsorbed to a nondenaturing hydrophobic phenyl column (Bio-Gel TSK Phenyl 5PW, Bio-Rad Laboratories), in 100 mM potassium phosphate, pH 7.0, containing 1.7 M ammonium sulfate. Bound species were eluted with a decreasing (1.7-0 M) ammonium sulfate gradient at a flow rate of 1.0 ml/min (10). 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