Biology of the Cell, 67 (1989) 271-279 0 Elsevier, Paris 271

Orig inal ar t ic le

Solubilization and electrophoretic studies of cyst wall proteins of a hypotrichous ciliate

Rosa Maria RIOS l, Rafael SARMIENTO 2, Antonio TORRES l and Concepcion FEDRIANI l*

~ Departamento de Microbiolog(a, Facultad de Biolog(a, Universidad de Seviila, Apdo. 1095, 41080-Seville; and 2 lnstituto de Recursos Naturales y Agrobiolog(a, CSIC, Seville, Spain

(Received 20 January 1989; accepted 10 June 1989)

A method for induetlon of synchronous encystment in a hypotrichous ciliate, Paraurostyla sp. is described. Cyst walls, isolated by shaking with glass beads, were analyzed by SDS-polyacrylamide gel electrophoresis. To test optimal con- ditions of solubilization of cyst wall proteins, different treatments using Triton X-100, EDTA, EGTA, urea, SDS and 2-mercaptoethanol were carried out. At least, 15 different proteins were identified as specific to the cyst wall. Four low molecular weight polypeptides (40, 27-26, 20 and 18 kDa represented aproximately 70% of the cyst wall proteins. The 170, 135 and 40-kDa bands exhibited a PAS-positive reaction. Hydrogen and disulphide bonds were shown to be the most important interactions involving cyst wall proteins. Amino acid composition of cyst wall proteins was also investigated by HPLC. High amounts of glyeine, cystine and proline were detected.

cyst wall --- electrophoresis --- HPLC --- ciliate protozoan

INTRODUCTION

Ciliate protozoans are the organisms that show the most elaborate and complex biological organization within the framework of unicellularity. Hypotdch ciliates represent the apex of biological differentiation at the unicellular level, but they appear to be a terminal evolutionary group. Encystment is a common phenomenological event during ciliate protozoan cell cycle and constitutes an attractive model system for studies on both cytodifferentiation (cyst wall formation) and cytodedifferentiation (loss of ciliature). Since the mid 1970's, attention has been focused on this subject and numerous publications on hypotrich ciliates encystment and excystment have appeart~l. Ultrastructural and light microscopic descriptions have in- cluded detailed studies of resting cysts [5, 8-10, 14, 26, 31, 37, 41], encystment [3, 5, 15, 20, 24, 27, 38], excystment [4, 13, 19, 30] and encystment-excystment [10, 11, 21, 39, 40].

Cyst wail formation is the most important cellular dif- ferentiation process of encystment. The study of cyst wail chemical composition is a prerequisite in order to: i) establish the structure-function relationships of dif- ferent cystic layers; ii) determine the cyst wail molecular structure, mainly in respect to the interactions between dif- ferent protein molecules or protein and polyssacharide molecules; and iii) obtain reliable markers that permit a

* Correspondence and reprints.

rigorous study of precursor genesis during the cyst wail formation process. To date, chemical composition of cyst wails has been investigated by light and electron cytochemistry [I, 2, 5, 16, 28, 32-34]. By these methods, polysaccharides and pl'oteins have been identified as their most important components. However, little biochemical information has been published on the hypotrich ciliate cyst wall and almost nothing is known about the chemical nature and structure of these macromolecules. The paucity of biochemical studies is mainly due to the difficulty in growing sufficient amounts of cells (mass cultures) and to the lack of an available technique to induce the encyst- ment process synchronically. The establishment ~f a method for inducing synchronous encystment i~i Histriculus sp. [25] made it feasible to carry out an electrophoretic analysis of Histriculus sp. cyst wail proteins [28] in which it was only possible to identify two high molecular weight proteins as specific to the cyst wail.

In this paper we describe a method for induction of syn- chronous encystment in a hypotrich ciliate mass culture Paraurostyla sp. Furthermore, we have studied the effect of several chemicals on the solubilization of cyst wall pro- teins and have made electrophoretic analyses of the solubilized molecules. The solubilization treatments car- ded out have allowed us to determine some of the chemical interactions which are established between the different protein molecules. Amino acid composition of cyst wail proteins was also investigated by HPLC. The results are discussed with the aim of furthering knowledge of the cyst wall structure in a hypotrichous ciliate.

272 R. Rios et aL

MATERIALS AND METHODS

The organism used in the present study was a hypotrichous ciliate, Paraurostyla sp., which originated from a single organism isolated from a samp!e of water collected at Maria Luisa Park (Seville).

The ciliate was mass cultured at 20+_ 10C in a 1 1 Erlenmeyer flask containing mineral water. The green alga Chlorogonium sp. was used to feed the protozoa.

For experimental induction of encystment, the organisms were harvested by low speed centrifugation, resuspended in mineral water (6.103 cells/ml) and shaken for 72 hr at 28"C. The cysts were then harvested and centrifuged at about 100 g for 5 rain.

To separate the cysts from debris of the culture, they were washed with Percoll solutions of decreasing densities (1.091, 1.078, 1.071 and 1.065 mg/ml) by centrifugation. Finally, the cysts were washed several times with distilled water.

Isolation of cysts walls

The pellet of cysts was resuspended in an equal volume of a proteaseoinhibitor mixture (20 mM EDTA Na2, 20 mM EGTA, 10 t~g/ml leupeptine, 10 pg/ml pepstatin A, 10 mM PMSF, 5 mM O-phenantroline and 5 mM iodoacetamide in a buffer con- sisting of 20 mM hydroxymaleic acide + 3 mM EDTA pH = 7.8). The cyst suspension was transferred to a Mini-Omnimixer Sor- vail to be shaken with an equal volume of glass beads (0 = 1.0 mm) for about 20 min in an ice-bath. During shaking, aliquots were periodically examined with a light microscope in order to determine the degree of cell rupture. Immediately after the complete rupture of the cysts, the glass beads were allowed to settle for a few minutes before the cyst homogenate was col- lected. The glass beads were washed three times with a cold buffer (10 mM Tris-hydrochloride pH=7.4) and the original homogenate and rinse medium were pooled and then centrifuged at 1000 g for 5 min. The pellet consisting primarily of cyst walls was washed several times with the cold buffer by centrifugation until the supernatant was clear.

Finally, the cyst walls were treated with Triton X-100 for 20 min and washed several times with cold buffer. The cyst walls obtained were almost always 100%0 pure according to light microscope examination and retained their empty bail-like shape, with the exception of a few unbroken cysts which were eliminated by sedimentation. Purity of walls was also assessed by obser- vation of ultrathin sections of cyst walls routinarily processed by TEM.

Solubilization of cyst wall proteins

Cyst wall fraction was mixed with an equal volume of the protease-inhibitor mixture and homogenized in an ice-bath us- ing a Potter Elvehjeim-type homogenizer. To test the optimal

conditions for solubilization of cyst wall proteins different treatments were carried out using several chemicals: EDTA, EGTA, Tris-buffer, Triton X-100, urea, SDS and 2-mercapto- ethanol. The cyst wall homogenates were incubated with one or several chemicals, centrifuged (12000 g for 5 rain) and the super. natants were boiled in SDS sample buffer (Laemmli buffer with or without 2-mercaptoethanol) for five min. The different treatments employed are summarized in Table I.

Gel electrophoresis

One dimensional SDS-PAGE in 5-15 % finear polyacrylamide gradient was performed according to the method of Laemmli 1"23]. The gels were stained with Coomassie blue or with silver [29] depending on desired sensitivity. Alternatively, the gels were stained by the PAS-technique [7] to detect 1, 2-diol groups of glycoproteins. Quantification of each band in the gels was car- ried out on Coomassie blue stained gels using a DESAGA CD 60 Densitometer.

Aminoacid analysis

Isolated cyst walls were hydrolyzed in vacuo for 24 hr at 105"C in 6N HCI, the acid being removed by rotary evaporation. Then, the hydrolysate was applied to Dowex 50-H + columns: the col- umns were eluted, first with 30 mi water and afterwards with 30 ml 6N NH4OH. The NH4OH-eluted fraction (containing amino acids) was derivatized with phenylisothiocyanate accord- ing to Heinrikson and Meredith [171 and analyzed by reverse- phase HPLC. A Waters Liquid Chromatograph, equipped with a packed column housed within an oven maintained at 38"C and a variable wavelength detector at 254 nm, was used. The ehtion of amino acids was performed with acetonitrile-water mixtures.

standard mixture of amino acid was purchased from Pierce Chemical Company (Pierce Standard H). The standard mixture contained ammonium sulfate and 2.5 t~mol/ml of each of the 17 amino acids: Asp, Glu, Ser, Gly, His, Thr, Ala, Arg, Pro, Tyr, Val, Met, Leu, lie, Phe, Lys and Cys (cystine= 1.25/~mol/ml).

RESULTS

During spontaneous Paraurostyla sp. encystment, the first cysts appeared a few hours after exhaustion of the food supply. Thereafter, the encystment rate fell to a rather low level, 100% encystment finally being achieved 3 - 4 days after the cysts first appeared. This led to the appearance, from initial encystment phases, o f cannibals which fed on

TABLE I. -- Chemical treatments employed to solubilize cyst wall proteins.

Chemical treatment Time Temperature

1.-2% Triton X-100 2.-10 mM EDTA+ 10 mM EGTA 3.-6 M urea 4.-2%0 SDS 5.-5%0 2-mercaptoethanol 6 . -2% SDS + 5% 2-mercaptoethanol 7 . -6 M urea + 5% 2-mercaptoethanol 8 . -6 M urea (30 min room temp.) and after 2% SDS + 2-mercaptoethanol 9.-10 mM Tris-HCl

10.-10 mM hydroxymaleic acid + 3 mM EDTA

30 min 30 min 30 min

5 min 30 min

5 min 30 min

5 min 30 min 30 min

room temp. room temp. room temp.

100*C room temp.

100*C room temp.

100*C room temp. room temp.

Cyst wall proteins 273

previously formed cysts, resulting in loss of a large pro- portion of cyst production. The technique for induction

Different treatments were carried out on the purified

of synchronous encystment by shaking obviates this wall homogenates in order to test optimal conditions for

phenomenon; the encystment became synchronous and solubilization of the cyst wall proteins (Table I). In con-

essentially 100% encystment was achieved in only a few trol experiments, cyst wall fragment suspensions were in-

hr. The cysts thus obtained were morphologically normal cubated with buffers used during the isolation and

and perfectly viable (Fig. la). Ultrastructurally, the resting purification processes (treatments 9 and 10). Two bands with molecular weights of 66 and 10 kDa were observed

cyst wall consists of four layers (Fig. lb) : the ectocyst, mesocyst, endocyst and granular layer as shown by

in the gels (Fig. 2). Triton X-100 and the chelating agents

paraurostyfo weissei [5] and other hypotrich ciliates [2, EDTA and EGTA, were shown not to release cyst wall

8, 10, 14, 24, 27, 37-403. proteins. Denaturing agents such as urea released a signifi-

Examination of the isolated cyst &ll fraction by light cant amount of proteins from the walls (Fig. 2). Boiling

and electron microscopy (Figs. lc, Id) showed that the in 2!70 SDS led to a similar electrophoretic pattern but

isolated cyst wall was composed of a normal ectocyst, released a major amount of proteins (Fig. 2 and Table II). In both experiments, bands with tentative molecular

mesocyst and endocyst but lacked the granular layer and that the degree of wall purification was satisfactory. Separation of mesocyst and ectocyst could be seen to take

weights of 135, 110,66,54,52,44, 37, 14, 10 and 8 kDa were observed. Sometimes, bands with molecular weights of about 220, 300 and 400 kDa could be seen.

place frequently. Urea + 2-mercaptoethanol and SDS + 2-mercaptoethanol

FIGURE 1. - Cyst and cyst wall morphology by light and electron microscopy. a. Photomicrograph of cysts washed with Perecll. X 500. b. Transmission electron micrograph of a cyst showing a four-layered cyst wall. ec, ectocyst ; me, mesocyst ; en, endowt ; IF, granular layer ; cy, cytoplasm. x 40 000. c. Photomicrograph of a isolated and purified cyst wall fraction. X 5Oh d. Transmission electron micrograph of a isolated cyst wall. ec, ectocyst ; me, mesocyst ; en, endocyst. x 40000.

274 R. Rios et al.

treatments released, in addition to these bands, four others with molecular weights of 170, a doublet 27-26, 20 and 18 kDa (Fig. 2). A 40-kDa band appeared in the gels when samples had been incubated with SDS+2-mercapto- ethanol but not when urea + 2-mercaptoethanol treatment had been employed (Fig. 2). 2-mercaptoethanol alone had only a minor effect on solubilizing these proteins (170, 27-26, 20 and 18 kDa) (Fig. 2). There was no variation on electrophoretic patterns when SDS- or urea-solubilized proteins were incubated with Laemmli buffer without 2-mercaptoethanol (data not shown). Finally, SDS+ urea + 2-mercaptoethanol treatment proved to be the most effective in releasing major amounts of cyst wall proteins (Fig. 2 and Table II). This treatment caused a remarkable increment of the 40-kDa band (Fig. 2).

Quantification studies of gels (Table ll) indicated that SDS treatment released double the amount of proteins as urea treatment. In the same way, SDS + urea + 2-mercapto- ethanol treatment released double the amount of proteins as SDS+2-mercaptoethanol treatment and triple the amount as urea + 2-mercaptoethanol treatment. From this study it was possible to also conclude that at least 70°/0 of cyst wall protein molecules are interlinked by disulphide bonds.

Silver staining of the gels did not reveal any new pro- minent bands with respect to Coomassie blue-stained gels. However, it was notherworthy that 135 and 110-kDa bands did not stain with this reagent (Fig. 3a). The 170 and 135 kDa bands were the only two protein bands clearly stained by PAS-reaction when cyst wall homogenates had been incubated with SDS + 2-mercaptoethanol (Fig. 4b). If samples had been treated with SDS + urea + 2-mercapto- ethanol the 40-kDa band also appeared weakly stained (Fig. 4c).

Amino acid composition of cyst wall proteins

Separation of PTC-amino acid of cyst wall proteins by reverse-phase HPLC is illustrated in Figure 5. The amount

- - 2 0 0

- - 1 1 6

_ 9 2

• i . . . . . . . . ~ : ~

- - 6 6

_ 4 5

- - 3 1

_ 2 1

_ 1 4

C UREA SOS UREA SDS 4, 4,

2HE 3HE

UREA 2Hi: 4,

SOS 4,

2Hi:

FzouRE 2. - Electrophoretic patterns of cyst wall proteins solubilized by different chemical treatments, analyzed by 5-15% linear gradient SDS-PAGE and Coomassie blue stained. Under each lane, the treatment employed is summarized. Molecular weight standards are indicated on the right. C: control; 2ME: 2-mercaptoethanol. Arrow indicates the 10 kDa band.

TABLE !I. - Quantification by densitometry of cyst wall pro- teins analyzed by SDS-PAGE.

Band U S U +

200 .- 297 448 170 - - 572 135 1 011 921 1 017 110 1 051 1 325 469 66 729 1 092 1 063 54 157 898 309 52 100 2 240 932 44 905 2 761 2 685

0 ~ -

37 834 1 016 1 546 27-26 - - 3 295

20 - - 3 915 18 - - 2 164 14 1 944 3 929 2 845

s+~

451 451 996

I 331 1 6 1 8 1 214 3 923 1 841 5 370

824 3 237 4 977 3 458 1 739

s+u+~

201 466

I 001 937 937

1 128 3 246 3 541

13 643 l 057

9 699 9 664 8 139 5 449

Cyst wall proteins were solubilized by: S: SDS treatment; U: urea treatment; U +~: urea + 2-mercaptoethanol; S +fl: SDS + 2-mercaptoethanol; S + U +~: SDS + urea + 2-mercaptoethanol. Each band is represented by its molecular weight (first lane). (-) Not detectable.

of tryptophan could not be measured in this analysis. NH4-substituents of asparagine and glutamine were lost as a consequence of acid hydrolysis producing aspartic and glutamic acid [18]. Only oxidized cysteine (as cystine) could be measured.

Table Ill shows the amino acid content of cyst walls (expressed as ~g/ml of dry weight), molar concentration of each amino acid (expressed as nmol/mg of dry weight) and the amino acid composition of cyst wall proteins based on glycine (major amino acid) = 100. Each of the 17 amino acids analyzed were found in the cyst wall proteins. Glycine, glutamic acid and/or glutamine, aspartic acid and/or asparngine and leucine were found to be the prin- cipal amino acids. Percentage-wise, acid amino acids (measured as total dicarboxilic acids detected) were 24.1%, neutral amino acids were 35.1%, polar amino acids 32.4% and basic anlino acids were only 8.4%. The relatively high amounts of glycine and proline stood out. Measured cystine indicated that cyst wall proteins contain 1.54× l0 -s mol of disulphide bonds per mg of protein (equivalent to 1 disulphide bond per 162 amino-acids).

Cyst wall proteins 275

T,\~;LE III. -- Amino acid composition of cyst wall proteins.

Aa I II II1

Gly Glx Asx Leu Ser Ala Pro Thr Val Tyr Lys Phe lie Arg His Net Cis T

27.5 50.6 34.3 32.0 18.6 14.9 17.8 15.8 14.9 21.1 13.6 13.0 10.3 12.5 6.7 2.8 3.7

314.5

366.3 344.6 258.4 244.5 177.6 168.2 155.2 132.8 128.0 116.7 93.0 79.2 79.1 71.8 43.2 19.0 15.4

2493.4

100 94 70 67 48 46 42 36 35 32 25 22 22 20 12 5 4

solubilization, several chemical treatments were carried out on homogenates of isolated and purified cyst walls. These treatments allow us to determine some of the interactions involving cyst wall proteins.

In control experiments, buffers used to isolate and purify cyst walls showed a release of two protein bands with molecular weights of 66 and 10 kDa. This fact seems to indicate that these proteins might be anchored to the cyst wall by weak ionic interactions. A shift in pH or ionic strength conditions, caused by buffers, might modify these interactions and solubilize both proteins. Preliminary studies using an antiserum against 66 kDa-band indicate that this protein is located on the outermost layer of the cyst wall. Both observations might explain the varia- tions observed in 66-kDa protein concentration on samples resulting in differences of staining intensity on Coomassie-stained gels. Sometimes this band does not even appear in gels stained with this dye, but it is always possible to detect it in silver-stained gels or by immuno- blotting assays.

Lane !: amino acid content expressed as/.tg/mg of dry weight; lane if: molar concentration of each amino acid expressed as iimol/mg of dry weight; lane Ill: amino acid composition of cyst wall proteins based on gly= 100.

a b

DISCUSSION

The most important problem in studying biochemically the cyst walls of hypotrich ciliates is being able to obtain large quantities of cysts. Thus, it is necessary to cultivate beforehand large amounts of vegetative cells (mass cultures) and to have available a method to synchronical- ly induce the encystment process. Matsusaka (1977) described a technique to synchronyze the encystment of Histriculus sp., based on the importance of osmolarity or ionic strength on synchrony and rate of encystment. However, this method requires several washes of cell cultures and was designed for small volumes of cultures (30 ml). Besides, it has not been demonstrated if this technique obviates cannibalism as this phenomenon, usual- ly observed in other hypotrich cultures, only occurred in exceptional cases in Histriculus sp. cultures.

The technique of encystment by shaking described by us has several advantages:i) it has been employed with large volumes of cultures since only one centrifugation step is required (this step can be ommitted if the species used is too fragile; high density cultures can be shaken directly); ii) this technique obviates cannibalism; and iii) the cysts do not adhere to flasks walls and are easy to collect and clean.

Several workers, who have studied cell walls of different biological systems, have reported the difficulty of solubHiT-

iing the proteins making up these resistant structures. We ! have analyzed, by electrophoresis, proteins of a hypotrich ciliate cyst wall. To test the optimal conditions of

_ 2 0 0

, i ~

- - 1 1 6

- - 9 2

_ 6 6

_ 4 5

_ 3 1

_ 2 1

UREA UREA 4, 4,

SOS SOS 4, 4,

2HE 2HE

FIOURE 3. -- Paraurostyla sp. cyst wall proteins, separated by SDS-PAGE and stained with silver (lane a) 8~nd Coomassie blue (lane b).

276 R. Rios et al.

Non-ionic detergent Triton X-100 and chelating agents had no effect on the solubilization of cyst wall proteins. Although this result does not permit the ruling out of the presence of lipids or divalent cations in cyst walls, it is possible to suppose that they do not play an important role in interlinking individual species of proteins. Both SDS and urea released 8 prominent bands. Although SDS proved to be a more useful agent to solubilize cyst wall proteins, urea released a significant amount of these pro- teins, indicating that numerous hydrogen bonds are estabfished between different protein molecules in the cyst wall.

2-mercaptoethanol alone had only a minor effect. However, addition of 2-mercaptoethanol to samples treated with SDS or urea released the four most abundant polypeptides (40, 27-26, 20 and 18 kDa) representing 23, 16.5, 16.4 and 13.8%o respectively of the total cyst wall proteins. Both results indicate: i) that numerous hydrogen bonds are also established between these polypeptides, since a previous or simultaneous action of a denaturating agent to break these interactions and provide the reduc- ing agent with free access to disulphide bonds is required to solubilize them; and ii) that disulphide bridges are the most important interactions involving cyst wall proteins, since more than 70% of them are interlinked by this type of bond.

SDS+2-mercaptoethanol treatment also released a 40-kDa protein band that did not solubilize by the action of urea + 2-mercaptoethanol. SDS requirement in order to release this protein might suggest that it establishes hydrophobic interactions with neighbouring molecules. Treatment with SDS + urea + 2-mercaptoethanol proved to be the best way to release cyst wall proteins. This treat- ment caused a remarkable increment of the 40 kDa band. Data obtained from solubilization studies do not permit us to know if this treatment releases a major amount of 40-kDa band solubilized by the action of SDS + 2-mercaptoethanol treatment or if it releases a new pro- tein of 40 kDa. Preliminary studies using polyclonal an- tibodies, obtained by us against cyst wall proteins, have demonstrated that on the cyst wall of Paraurostyla sp. there exist two different 40 kDa proteins (unpublished data).

The existence of intrachain disulphide bonds could not be detected in these polypeptides (170, 40, 27-26, 20 and 18 kDa) since assayed concentrations of the thiol agent cleave all disulphide bonds indiscriminately. However, it was possible to determine that proteins solubilized by SDS did not contain intrachain disulphide bonds, since supres- sign of 2-mercaptoethanol of Laemmli s~tmple buffer did not alter the electrophoretic pattern, tt is well known that reduced and non-reduced proteins have different elec- trophoretic migration rates, with the ~tonreduced proteins exhibiting lower apparent M r values.

In conclusion, from solubilization studies it is possible to infer that only 4 low molecular weight polypeptides which are interlinked by disulphide bonds represent more than 70% of cyst wall proteins. In coa~trast, 10 species of proteins (including high molecular weight proteins), not covalently linked to other wail components, represent less than 30%. On the other hand, the results obtained indicate that the conventional method for soiubilizing proteins (Laemmli buffer) might not to be sufficient to release pro- teins making up resistant structures.

When gels were stained with silver, it was possible to observe that the 135 and 110 kDa bands did not stain at all (negative staining). According to several workers [6,

a b c d

_ _ 2 0 0

. + .. . . . . ,+. _ _ 1 1 6

_ _ 9 2

+++ ,+~+ ++~+ ++ + _ 6 6

_ 4 5

;'+~++++ _ 3 1

. _ 2 1

SOS SOS 4, 4,

2 M E . 2ME

UREA UREA 4, 4,

SOS SOS 4, 4,

2ME 2ME

FIGURE 4. -- Paraurostyla sp. cyst wall proteins, separated by SDS-PAGE and stained with PAS (lanes b and c) and Coomassie blue (lane a and d).

12, 42] high amounts of aspartic acid, glutamic acid or serine in a protein may determine a negative response of the protein to silver staining. Therefore, it is possible to suppose that 135 and 110 kDa bands may contain a high mol percent of these amino acids.

Protein bands of 170 and 135 kDa, which were stained diffusely and rather weakly by Coomassie blue, showed an intense PAS reaction, clearly indicating that they are glycoproteins with a high content of neutral or acidic sugars. This high sugar content can be considered respon- sible for the anomalous behaviour of these glycoproteins towards Coomassie blue. Therefore, these protein concen- trations in the cyst wall could be higher than those detected by us in quantification studies. The 40-kDa band solubiliz- POt by the action of SDS + urea + 2-mercantoethanol show- ed a weak PAS positive reaction. The 40-kDa band

Cyst wall proteins 277

soi,abilized by the action of SDS + 2-mercaptoethanol did not stain with Schiff's reagent. This result in conjunction with the behaviour of both proteins against the polyclonal antibodies above mentioned confirms that in the cyst wall of Paraurostyla sp. there exist two different 40-kDa pro- teins: one of them is glycosylated and the other is not.

The only electrophoretic analysis of cyst wall proteins of a hypotrich ciliate has been carried out in Histriculus muscorum [28]. In this study, two bands with molecular weights of 190 and 140 kDa specific to the cyst wall were identified. Another six bands with tentative molecular weights of 60, 48, 40, 20, 16 and 15 kDa were also appar- ently on the cyst wall. Sometimes they observed a band with a molecular weight of about 250 kDa. The 190-kDa band clearly stained by a PAS-reaction. These results do not differ markedly from those obtained by us: a notice- able heterogeneity of cyst wall proteins and the existence of glycoproteins of high molecular weight seem to be com- mon characteristics of the cyst walls of both species.

HPLC analysis of isolated cyst wails revealed that all 17 amino acids analyzed are present in cyst wall proteins. Detected values of polar and neutral amino acid are nor- mal. Low values detected of histidine and methionine are also usual. However, low proportions of basic amino acid and high contents of glycine and proline are exceptional. Although it is well known that there exist proteins with a high glycine content ~-keratin or collagen helix), a dif- ferent possibility can be considered in order to explain the high content of glycine detected in Paraurostyla sp. cyst wall proteins. For example, Kottel et al. [22] have report- ed that the myxospore coat of Myxococcus xanthus con- tains glycine that is not a part of the protein. They suggest that a portion of glycine might serve to cross-link individ- ual polysaccharide polymers. In Paraurostyla sp. cyst wall, it is possible that a portion of the glycine plays a similar

role. Support for this idea arises from the results obtain- ed when cyst walls were subjected to mild acid hydrolysis. Under these conditions, when only 60% of the proteins had been solubilized, we could detect 80% glycine, indi- cating a selective solub'flization of this amino acid. This fact is particularly notable if we take into account the fact that glycine is an amino acid that can be found in sequences which are extremely resistant to acid hydrolysis [18].

The cystine content detected indicates that a high pro- portion of protein molecules are interlinked by disulphide bonds, thus agreeing with results obtained by electropho- resis. It must be considered that a part of cystine could be lost during the hydrolysis process (cystine is oxidized to cysteic acid) and, therefore the real cystine content of cyst wall must be still higher. The significance of disul- phide bonds linking proteins that form resistant structures has been reported extensively. In general, the existence of disulphide bonds is related both to resistance of these structures and to their insolubility. For example, Colpoda steinii cyst wall was resistant to the most drastic chemical treatments, but when the walls were oxidized with performic acid (all disulphide bonds were broken) about 60% of cyst wall proteins were made soluble [35]. It has been reported that in Saccharomyces cerevisiae, at least 300 of wall mannoproteins are interlinked by disulphide bonds [36].

In conclusion, the present study demonstrates that more than 15 different proteins, some of them glycosylated, constitute a part of the cyst wall. Four low molecular weight polypeptides represent 70% of cyst wall proteins. Hydrogen and disulphide bonds have been found as the most important interactions involving cyst wail proteins. More such comprehensive studies are still necessary to elucidate and understand which mechanisms ciliated proto- zoa have developed in order to construct a cyst wail that

I

4 .88 m:s

_ f _

I,,75 ASP 1 . 9 5 GLU

3. ]'4 SP-R

8 . g 4 . _ - . .

• ~ ILEU

.... 1e.87 P a B

5 5 -r

I ~_ I! I LYS i m

. ~ = . ~:)

4.11 GL¥

ALA PRO

TYR VAL

LED

FIGURE 5. -- Separation of amino acids of cyst walls by reverse-phase HPLC. Digits indicate retention time of each amino acid. Abbreviations used are standard, except ¢is= cystine.

278 R. Rios et al.

is extremely resistant to adverse environmental conditions. At the moment, we have three polyclonal antibodies avail- able against cyst wall proteins. These antibodies will per- mit us to carry out a rigorous study of precursors genesis during encystment, in an attempt to understand cyst wall protein structure and finally to use molecular character- istics as criteria to establish taxonomic and phylogenetic relationships in a group as controversial as the hypotrich ciliates.

ACKNOWLEDGEMENTS

This investigation was supported by grams from the DGICYT (PB 87/0939) and from the Junta de Andalucia (Spain).

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