In search of early pregnancy factor: characterisation of active polypeptides isolated from pregnant ewes' sera

Journal of Reproductive Immunology, 6 (1984) 253-260 Elsevier

253

JRI 00310

In search of early pregnancy factor" characterisation of active polypeptides isolated from pregnant ewes' sera Shann Wilson, Robyn McCarthy and Frank Clarke * School of Science, Griffith University, Nathan, 4111 Australia

(Accepted for publication 23 November 1983)

Further biochemical characterisations of the 20 kd and 67 kd EPF active polypeptides from pregnant ewes' sera are described. Both polypeptides are shown to possess compact, disulphide linked domains which are almost totally resistant to proteolytie attack without prior reduction. In contrast to previous reports on the influence of ammonium sulphate on the expression of EPF activity, neither the biochemical nor the activity characteristics of either polypeptide is changed by ammonium sulphate fractionation. In addition, direct comparative studies have been performed which clearly distinguish the EPF active polypeptides from the known ovine hormones, prolactin, placental lactogen and growth hormone. Key words: early pregnancy factor, biochemistry, characterisation, reproductive immunology

Introduction E a r l y p r e g n a n c y factor ( E P F ) has been d e s c r i b e d as an i m m u n o s u p p r e s s i v e activity d e t e c t e d in p r e g n a n c y sera b y m e a n s of the rosette i n h i b i t i o n test ( M o r t o n et al., 1976). E P F activity can be f o u n d in p r e g n a n c y sera or urine from as early as 24 h p o s t - f e r t i l i s a t i o n a n d in m o s t species s t u d i e d it a p p e a r s to persist for at least the first t w o - t h i r d s of g e s t a t i o n ( M o r t o n et al., 1976, 1977, 1979, 1982a). P r e l i m i n a r y bioc h e m i c a l c h a r a c t e r i s a t i o n studies b a s e d solely on activity m e a s u r e m e n t s i n d i c a t e d that E P F p o s s e s s e d a c o m p l e x set of b i o c h e m i c a l characteristics. F o r example, m u l t i p l e size f o r m s were o b s e r v e d in p r e g n a n c y sera and, moreover, the c o m p l e m e n t a n d n a t u r e of these forms a p p e a r e d to vary d e p e n d i n g on the stage of g e s t a t i o n ( C l a r k e et al., 1980; C l a r k e a n d Wilson, 1982). I n furthering the b i o c h e m i c a l c h a r a c t e r i s a t i o n of E P F , we have recently d e s c r i b e d the i s o l a t i o n of the p o l y p e p t i d e s which are r e s p o n s i b l e for a large p o r t i o n of the E P F activity in p r e g n a n t ewes' sera o b t a i n e d b e t w e e n 1 a n d 2 m t h of g e s t a t i o n ( W i l s o n et al., 1983). T h e m a j o r active f o r m was c h a r a c t e r i s e d as a single p o l y p e p t i d e of m o l e c u l a r weight 20 kd. It possessed an i n t e r n a l d i s u l p h i d e l o o p structure ,as it d i s p l a y e d a m a r k e d r e d u c t i o n in m o b i l i t y on SDS p o l y a c r y l a m i d e gels following * To whom all correspondence should be addressed. 0165-0378/84/$03.00 © 1984 Elsevier Science Publishers B.V.

254 reduction with dithiothreitol. A 67 kd polypeptide was also isolated but had much less activity in the rosette inhibition test. Both appeared to be characteristic of pregnancy sera since no such active material could be isolated from non-pregnancy sera. In the present report we describe further the biochemical properties of these EPF active polypeptides. Also, because many of the properties of the major 20 kd form (its molecular weight, tendency to aggregate, presence of internal disulphide loop) are reminiscent of some of the properties of the known hormones prolactin (Wallis et al., 1980), placental lactogen (Russell et al., 1979) and growth hormone (Lewis et al., 1977), we also describe direct comparative studies which demonstrate that the EPF polypeptides are distinct from these known hormones.

Materials and Methods

EPF-active polypeptides and other hormones The purification of the EPF-active polypeptides from pregnant ewes' sera obtained at 1-2 mth gestation has been fully described in the preceding paper (Wilson et al., 1983). HPLC pool B (containing the 67 kd polypeptide) and HPLC pool C (containing the 20 kd polypeptide) described in that report were the preparations used in the present study. The hormones ovine prolactin (oPRL S-12, NIH), growth hormone (oGH 0986C, NIH) and placental lactogen (oPL) were a generous gift from Dr. Michael Waters, University of Queensland. All samples were in PBS and stored at - 3 0 ° C until used. All experiments described here have been performed using iodinated polypeptides. Iodinations were carried out either using the lactoperoxidase-glucose oxidase method of Hubbard and Cohn (1972) with the solid phase Bio Rad Enzymobead reagent or by using the Bolton-Hunter reagent (Bolton and Hunter, 1973). Following iodination the samples were diluted in PBS containing 200 /~g/ml BSA and stored in aliquots at -30°C. Measurement and E P F activity EPF activity was assessed using the rosette inhibition test as described previously (Wilson et al., 1983). Gel electrophores& Electrophoresis under dissociating conditions was performed in 7% polyacrylamide slab gels prepared and run in 0.06 M Tris-glycine, pH 9.0 containing 6 M urea, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), calibration of the gels and autoradiography were carried out using the methods reported previously (Wilson et al., 1983). Direct silver staining of SDS-polyacrylamide gels was performed as described by Morrissey (1981). Protease digestion Iodinated pools B and C, and the hormones prolactin, placental lactogen and growth hormone, were subjected to proteolytic digestion by trypsin and chymotryp-

255 sin, in a medium containing 200 # g / m l BSA in PBS. Trypsin and chymotrypsin were used at a final concentration of 1 mg/ml. The reactions were stopped by the addition of trypsin inhibitor at a final concentration of 2 m g / m l for the trypsin incubations, and by 25 mM phenylmethylsulphonyl fluoride (PMSF) for the chymotrypsin incubations. Control incubations were performed in the absence of proteases. Samples were digested (i) in the absence of DTT, (ii) in the presence of 50 m M DTT, (iii) in the presence of 50 mM DTT, 0.1% SDS. After stopping the reactions the samples were mixed with an equal volume of an SDS sample buffer containing 50 mM DTT, placed in a boiling water bath for 2 rain and loaded on an 18% SDS-polyacrylamide gel on the basis of equal counts.

Ammonium sulphate fractionation Ammonium sulphate ranging from 10 to 90% in 10% increments was added to 20,000 cpm of labelled pool B and C material in normal sheep serum which was added to facilitate protein precipitation. After standing on ice for 15 rain the samples were centrifuged at 20,000 rpm for 15 min in a Sorvall centrifuge. The supernatants were removed and both supernatant and pellet fraction were counted. After subtraction of a tube binding control (the cpm bound to the centrifuge tube in a sample minus ammonium sulphate) the % cpm in each pellet was calculated.

Results

In the preceding paper (Wilson et al., 1983) we described the isolation of two RIT active polypeptide fractions from pregnant ewes' sera. These were designated pools B and C and the S D S - P A G E patterns of these fractions revealed by a direct protein silver stain are shown in Fig. 1. Pool C is seen as a homogeneous 20 kd polypeptide which displays a pronounced reduction in mobility following treatment with DTI'. Pool B contains a major 67 kd polypeptide; a trace of disulphide linked dimer is also seen at 140 kd, but this disappears following D T T treatment and the major band shows a characteristic reduction in mobility. These results (Fig. 1) obtained with a direct protein silver stain confirm the characterisation of these polypeptide fractions previously obtained using only iodinated material (Wilson et al., 1983).

Ammonium sulphate fractionation Previous studies on the EPF activity present in ewes' sera obtained within 1-2 days of fertilisation indicated that it is susceptible to ammonium sulphate fractionation, specifically treatment of such sera with 40% ammonium sulphate produced pellet and supernatant fractions neither of which displayed any activity in the rosette inhibition test (Clarke et al., 1980). Activity was recovered, however, on recombination of the fractions, suggesting that 40% ammonium sulphate had separated EPF into two components (these were designated EPF-A, present in 40% supernatant, and EPF-B, present in 40% pellet) both of which were necessary for expression of activity. Consequently, it was of interest to examine the ammonium sulphate solubility properties of the presently isolated polypeptides, which it should be noted have been isolated from sera obtained at 1-2 mth gestation.

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Fig. 1. Silver stained S D S - P A G E patterns of RIT active polypeptides in pools B and C of Wilson et al. (1983). - , Samples run without DTT treatment; + , samples run after DTT treatment; St, molecular weight standards.

Samples of the isolated 20 kd and 67 kd iodinated polypeptides were individually mixed with normal sheep serum and subjected to ammonium sulphate fractionation as described in Materials and Methods. The resulting ammonium sulphate solubility profiles are shown in Fig. 2. The 20 kd and 67 kd polypeptides displayed very 10(~ 90 86

°/oPrecipit at ion

76 6C 5( 4( 3C 2C 10

/ 10

2030405060708090

~saturation

ammonium sulphate

Fig. 2. Ammonium sulphate precipitation profiles of pool B ( . . . . . .

) and pool C (

) polypeptides.

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Fig. 3. Comparative SDS-PAGE patterns of pool B, pool C, ovine placental lactogen (oPL), ovine growth hormone (oGH) and ovine prolactin (oPRL) run under reducing (+) and non-reducing ( - ) conditions. When applied, reduction was accomplished by treating samples with 50 mM DTT for 30 min at 37°C prior to electrophoresis. Molecular markers are as indicated.

similar profiles. Very little of either was precipitated before 30% saturation and relatively high saturations ( < 60%) were required to bring about maximum precipitation. At 40% a m m o n i u m sulphate saturation approximately one-third of the 20 kd polypeptide was found in the pellet fraction and both the 40% pellet and 40% supernatant displayed activity in the rosette inhibition test. Moreover, the S D S - P A G E patterns with and without prior D T T reductions revealed no differences in the properties of the 40% pellet and supernatant fraction. Similar results were observed for the 67 kd polypeptide following 40% a m m o n i u m sulphate fractionation.

Comparison with known hormones and susceptibility to proteolytic digestion Figure 3 compares the S D S - P A G E patterns of iodinated pool B and C polypeptides with those of the known ovine hormones, placental lactogen, growth hormone and prolactin. These hormones all have molecular weights in the 20 kd region and display characteristic changes in mobility on S D S - P A G E following D T T treatment due to reduction of internal disulphide bonds. It is evident from the comparisons provided in Fig. 3, however, that the 20 kd RIT-active polypeptide isolated from pregnant ewes' sera is quite distinct from these known ovine hormones. It should be noted that these experiments and those reported in Fig. 4 were performed with 20 kd material which had been stored at least 3 mth since isolation and consequently little or no fragmentation was noted on D T T reduction (Wilson et al., 1983). Of course the D T T induced fragmentation observed in fresh preparations is a further property which distinguishes the 20 kd material from the previously described hormones. Another notable characteristic shared by the 20 kd and 67 kd polypeptides of pregnancy sera which further distinguishes them from the known hormones was the DTT-dependency of their susceptibility to proteolytic digestion. As shown in Fig. 4,

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Fig. 4. Comparative SDS-PAGE patterns of pool B, pool C, ovine placental lactogen (oPL), ovine growth hormone (oGH), and ovine prolactin (oPRL) following incubation with trypsin in the presence or absence of DTT. A, control samples; B, samples incubated with trypsin in PBS; C, samples incubated with trypsin in PBS, 50 mM DTT; D, samples incubated with trypsin in PBS, 50 mM DTT; 0.1% SDS. both the 20 kd and 67 kd polypeptides were totally resistant to digestion by trypsin unless they were previously reduced with DT1L In contrast, the hormones placental lactogen, growth hormone and prolactin (Fig. 4) were readily cleaved by trypsin regardless of whether they had been reduced or not. Similar results have been obtained with a number of other proteolytic enzymes such as chymotrypsin and papain which have different peptide bond specificity. In all cases the proteolytic cleavage of the 20 kd and 67 kd polypeptides was largely dependent on prior reduction with DTT, but this was not so for the hormones.

Discussion The further characterisation studies reported here have revealed a number of interesting features about the molecules responsible for the expression of EPF activity in 1 - 2 mth pregnancy sera. At least to the resolution of the techniques applied, the material in the main active fraction ( H P L C pool C, Wilson et al., 1983) appears to be a single polypeptide with a molecular weight of approximately 20 kd. The evidence indicates that it possesses an internal disulphide loop structure which maintains the molecule in a tight conformational domain as it is almost totally resistant to proteolytic attack without prior reduction of the internal disulphide(s). The presence of disulphide linked domain(s) is reminiscent of the structure of m a n y biologically active peptides, including, for example, the hormones prolactin, placental lactogen and growth hormone, some or all of which would be expected to be present in the pregnancy sera used for the isolation of the 20 kd polypeptide, However, it is clear on the basis of the biochemical comparisons provided in this

259 paper that the 20 kd RIT-active polypeptide is quite distinct from these known hormones. Moreover, it may be noted that these purified hormones have no activity in the rosette inhibition test. As described in the preceding paper (Wilson et al., 1983), a second less active fraction (HPLC fraction B) has also been isolated from pregnancy sera. It contains a single 67 kd polypeptide which, apart from its apparent activity in the rosette inhibition test, also shares a number of biochemical properties with the major 20 kd polypeptide. Namely, it also possesses an internal disulphide loop structure and is also almost totally resistant to proteolytic cleavage without prior reduction. Although it shares these overt biochemical characteristics with the 20 kd polypeptide any structural relationship between these molecules is not yet established and must await further study. The final aspect which should be clarified is the relationship of the present studies to the previous work (Clarke et al., 1980) on EPF characterisation. These original studies, which provided a preliminary characterisation of EPF activity, were performed mainly with sera obtained within the first 7 days post-fertilisation, i.e. well before implantation. The most interesting observation from these original studies, which has since become established in the literature (Morton et al., 1982a, b; Cavanagh et al., 1982), was that two components appeared to be necessary for the expression of EPF activity. For simplicity these were designated EPF-A (soluble in 40% ammonium sulphate) and EPF-B (insoluble in 40% ammonium sulphate). Neither was capable of inducing an increase in the rosette inhibition titre, only when they acted together was such activity observed. Other studies (Morton et al., 1980; Cavanagh et al., 1982) have indicated that these ammonium sulphate derived components from very early pregnancy sera have their in vivo counterparts. EPF-A has been equated with the material produced by the oestrous oviduct while EPF-B may be equated with the material produced by the corpus luteum of early pregnancy. Two points about these previous studies should be emphasised. Firstly, these characterisations were based solely on activity measurements using the rosette inhibition test, and we have already discussed (Clarke and Wilson, 1982) the inherent limitations on interpretation which this imposes. Secondly, as already noted above, the studies which indicated the existence of two components were performed using only very early (1-7 day) pregnancy sera. In contrast, the present biochemically characterised RIT active polypeptides have been isolated from 1-2 ruth pregnancy sera. Ammonium sulphate fractionation does not result in the separation of two inactive molecules, rather the sum total of evidence indicates that only one component is necessary for the expression of activity. In keeping with these biochemical studies on purified material we have observed that the EPF activity in unfractionated 1-2 mth pregnancy sera or placental extracts is not influenced by ammonium sulphate fractionation. Thus we conclude that there may be "early" and "late" forms of EPF activity. Whether the presently described molecules which are responsible for the expression of 'late' EPF activity are related to the molecule(s) responsible for the expression of 'early' EPF activity remains to be seen. Only the isolation and biochemical characterisation of the RIT-active molecules present in early pregnancy sera will answer this question, and studies directed towards that end are now in progress.

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Acknowledgements This work was supported by grants from the National Health and Medical Research Council of Australia and by the Griffith University Research Committee.

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