inter- and intra-specific differences in serum proteins of different species and subspecies of...

Comp. Biochem, Physiol. Vol. 103B, No. 3, pp. 589-599, 1992 0305-0491[92 $5.00 + 0.00 Printed in Great Britain © 1992 Pergamon Pre~s Ltd

INTER- AND INTRA-SPECIFIC DIFFERENCES IN SERUM PROTEINS OF DIFFERENT SPECIES

AND SUBSPECIES OF ZEBRAS

A. STRATIL,* D. (]i~ovA,* E. G~.BRXgOVA* and R. POKORN'i'~" *Czechoslovak Academy of Sciences, Institute of Animal Physiology and Genetics,

277 21 Lib6chov, Czechoslovakia; and 1"East Bohemian Zoological Garden, 544 01 Dvfr Kralov6, Czechoslovakia

(Received 22 April 1992; accepted 22 May 1992)

Abstract--1. Serum proteins of Equus grevyi, E. zebra hartmannae, E. burchelli boehmi, E. b. chapmanni and E. b. antiquorum were studied using starch-gel electrophoresis, 1-D polyacrylamide-gel electrophoresis, inhibitions of trypsin and chymotrypsin, immunoblotting, and specific staining for esterase.

2. Clear species-specific patterns were observed in albumin, transferrin, and for E. grevyi in protease inhibitor-l. Specific esterase was detected only in E. z. hartmannae.

3. Protein polymorphism was found in all studied species: E. grevyi--transferrin; E. z. hartmannae-- protease inhibitor-l; E. b. boehmi--albumin, GC, transferrin, protease inhibitor-l, protease inhibitor-T; E. b. chapmanni--albumin, GC, transferrin, protease inhibitor-l; E. b. antiquorum---CJC, transferrin, protease inhibitor- 1.

4. Phenotype patterns of the polymorphic proteins were indicative of simple codominant inheritance. Further studies of polymorphism of protease inhibitor-2 and variability of protease inhibitor-X a r e needed.

5. ~qB giycoprotein in all zebra species was monomorphic. 6. The main transferrin components and ~qB glycoprotein of zebra (E. b. boehmi) were characterized

for terminal sialic acid content.

INTRODUCTION

Living zebras are representatives of two subgenera: Dolichohippus and Hippotigris. Equus grevyi is the last representative of subgenus Dolichohippus. The other zebras, mountain zebra (two subspecies-- E.:'zebra zebra and E. z. hartmannae) and Burchell's zebra (six subspecies--E, burchelli burchelli, E. b. antiquorum, E. b. chapmanni, E. b. zambeziensis, E. b. crawshayi and E. b. boehmi), are closely interrelated and belong to subgenus Hippotigris (Groves, 1974).

The three species have different chromosome numbers: E. grevyi has 2n = 46, E. z. hartmannae has 2n = 32, and E. b. boehmi has 2n = 44 (see Ryder et al., 1978).

Electrophoretic techniques have proven to be a powerful tool in studies of genetic variation of proteins, phylogenetic relationships and population structures, and have been used extensively for domesticated animals, natural as well as captive populations (e.g. Manwell and Baker, 1970; Ferguson, 1980; Juneja, 1981; Baccus et al., 1983; Wayne et al., 1986). In this respect, data for zebras are rather scarce.

Using starch-gel electrophoresis, Osterhoff (1969) compared haemoglobin, transferrin and albumin of 38 damara zebras (E. b. antiquorum) with those of the horse and donkey. Three phenotypes of haemoglobin in the zebra appeared the same as in the horse. In transferrin eight variants were observed and three of them (H, O, R) were present in all three Equidae species studied. Albumin of the zebra was represented by two variants, the fast one being the same as

the slow variant of the horse. Weitkamp et al. (1979) studied polymorphism of vitamin D binding protein (GC) in Equidae, including two species of zebras. Two Hartmann's zebras had phenotype F, identical with the F of the horse, and two Grant's zebras had phenotype S, corresponding to the horse S. No polymorphism was observed. Kaminski et al. (1978) examined serum esterase in three species of zebras using starch-gel electrophoresis and immunoeleetro- phoresis. When using u-naphthylacetate as a substrate, Grrvy's and Grant's zebras had no esterase activity, while Hartmann's zebra showed activity that was different from esterase of the horse.

The aim of this paper was to accomplish a comparative study of serum proteins in different species and subspecies of zebras using electrophoretic techniques and specific methods for identification of proteins, and to show the differences between and within the species and subspecies. Disclosed genetic variation is, in addition to comparative biochemical aspects, important information on the genetic structure of the captive populations and can be used in breeding programmes.

M A T E R I A L S A N D M E T H O D S

Animals and blood samples

All animals studied were from Zoo Dyer Kr~ilov6 n.L., Czechoslovakia. The following species and subspecies were studied: Equus grevyi (37 samples), E. zebra hartmannae (12 samples), E. burchelli boehmi (32 samples), E. b. chapmanni (11 samples) and E. b. antiquorura (31 samples). Blood samples were collected during 1986-1990; some animals

589

590 A. STRAT1L et al.

were sampled and tested twice. Serum samples were stored at -20°C until use. For comparison, blood serum samples of horse (Equus caballus) and donkey (E. asinus) were also used.

Starch-gel electrophoresis

Horizontal starch-gel electrophoresis was used to study serum albumin and transferrin. Albumin was analyzed in the discontinuous Tris-citrate (pH 6.0) and boric acid- NaOH (pH 8.6) buffer system of Tucker (I 968). Transferrin was studied in a Tris--citrate (pH 7.6) and boric acid-NaOH (pH 8.6) buffer system according to Kristjansson (1963). Autoradiography of transferrin using 59Fe citrate was performed basically according to Spooner et al. (1970).

Polyacrylamide-gel electrophoresis

One-dimensional horizontal polyacrylamide-gel electrophoresis (PAGE) was performed as described by Gahne et al. (1977) using a modification according to Juneja and Gahne (1987). Proteins were stained with Coomassie Blue G-250. Esterase was stained as described by Stratil et al. (1990).

SDS-polyacrylamide-gel electrophoresis (12.5% separation gel) was performed according to Laemmli (1970).

Detection of inhibitors of trypsin and chymotrypsin

The protease inhibitors were detected in polyacrylamide gel electrophoresis using a method described earlier (Stratil et al., 1990).

Immunoblotting

Immunoblotting was used to study GC, ~ttB glycoprotein and inter-~-trypsin inhibitor. Passive blotting (I hr for GC and A1BG; 18 hr for PI) to a nitrocellulose membrane was used. Other details were as given by Stratil et al. (1990). The anti-sera used were: rabbit anti-human GC, rabbit anti-pig ~tB glycoprotein (see Stratil et al., 1990) and sheep anti- human inter-~t-trypsin inhibitor (Serotec, U.K.). The dilu- tions of both primary and secondary antisera were I : 250 (secondary antiserum for GC typing was diluted 1:1000).

Partial purification of aqB glycoprotein and transferrin

Serum samples of E. b. boehmi, E. caballus (phenotype A1BG K) and E. asinus were fractionated by rivanol and ammonium sulphate precipitation (Stratil et al., 1987). cqB glycoprotein was precipitated between 1.5 and 2.0 M (NH4)2SO4. Further purification was accomplished on a DEAE-Sephadex column (1.76 x 15 cm) using 0.05 M Tris- HC1 buffer, pH 8.0 and a linear gradient from 0 to 0.3 M NaC1. A fraction containing ~ttB glycoprotein was eluted between 0.13 and 0.20 M NaC1.

From the same DEAE-Sephadex ehromatographies, two partially fractionated main transferrin components were obtained for the zebra and donkey.

Neuraminidase treatment Transferrin components of E. b. boehmi, E. asinus and

E. caballus (see Stratil et al., 1984) as well as ~qB glycoprotein fractions of the three species were treated with neuraminidase from Vibrio cholerae. Samples (0.5 mg) were incubated with 100 ktl neuraminidase for 24 hr at 37°C.

RESULTS AND DISCUSSION

Serum proteins of different species and subspecies of zebras were studied in starch-gel electrophoresis and one-dimensional polyacrylamide-gel electrophoresis. Proteins were identified as described in Materials and Methods. We studied albumin (ALB), vitamin D binding protein (GC), transferrin (TF), protease inhibitor-1 (PII), protease inhibitor-2 (PI2),

protease inhibitor-T (PIT), protease inhibitor-X (PIX), ~IB glycoprotein (A1BG) and esterase (ES).

Albumin (ALB)

Interspecies differences and polymorphism of albumin were studied in starch-gel electrophoresis. A comparison of albumin patterns and polymorphism in different zebras and the horse is shown in Figs 1 and 2.

Albumin of E. grevyi (designated Ag; g for grevyi) was monomorphic and was clearly faster than the slow, i.e. S-variant, of the horse. Albumins of E. b. boehmi and E. b. ehapmanni were polymorphic--two variants were present, which were designated A and B. The migration of the fast variant A appeared the same as that of the slow variant S of horse. E. b. antiquorum was monomorphic for phenotype B. Albumin of E. z. hartmannae (designated Bh; h for hartmannae) was just slightly faster than the B-variant of the other zebras. This difference could only be seen when albumin migrated at least 8 cm from the origin.

The phenotype patterns of albumin in E. b. boehmi and E. b. chapmanni leave no doubt as to the codominant mode of inheritance. Support for this conclusion was provided by limited family data (in E. b. boehmi), which involved matings A × A, A × B, A x AB and B x AB. Seven offspring were obtained and their phenotypes were in agreement with the codominant inheritance.

Osterhoff (1969) observed, in E. b. antiquorum, two albumin variants, one having the same mobility as horse slow albumin (S) and one being slower. In our study, E. b. antiquorum was monomorphic for the B-variant. It appears that in the population we studied the fast variant was absent.

Vitamin D binding protein (GC)

Immunoblotting with the specific antiserum to human GC, following PAGE of serum samples of zebras, revealed zones of GC in the post-albumin region that had identical mobilities with horse GC (cf. also Weitkamp et al., 1979). Two variants were observed--F and S, as in horse (Juneja et al., 1978: Weitkamp, 1978). Polymorphism was found in E. b. boehmi (9F, 19FS, 4S), E. b. chapmanni (IF, 7FS, 3S) and E. b. antiquorum (14FS, 17S). E. grevyi and E. z. hartmannae were monomorphic for variant F.

Limited family data (in E. b. boehmi), which involved matings F × F and F x FS, were compatible with the codominant mode of inheritance.

It has been known that A L B and GC loci are closely linked in all species investigated, including man (Weitkamp et al., 1966), horse (Sandberg and Juneja, 1978) and even chicken (Juneja et al., 1982). This linkage can also be studied in zebras (E. burchelli), provided suitable families are available.

Transferrin ( TF)

Although transferrin polymorphism in zebras can be studied in PAGE, we preferred starch-gel electrophoresis as the separation was better and the patterns could be interpreted more easily. With the exception of E. z. hartmannae, the transferrin of which was monomorphic, all other species studied exhibited polymorphism. Two variants were observed in E. grevyi, five in E. b. boehmi, four in E. b. chapmanni and five

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in E. b. antiquorum (Fig. 3). All transferrin variants detected are schematically shown in Fig. 4. Most of the variants appeared as two strong zones, but some had a conspicuous weaker zone localized between the two. The transferrin variants of zebras, as far as heterogeneity is concerned (i.e. the presence of two strong zones in a variant), resembled those of horse (e.g. Stratil and Glasnfik, 1981; Stratil et al., 1984). Certainly, additional weak zones can be expected after isolation.

The transferrin variants are species-specific. Some difficulties arise in the three subspecies of E. burchelli, where some of the variants appear the same in two of the subspecies (variant B of boehmi = C antiquorum; A chapmanni = B antiquorum; D chapmanni = E antiquorum). As it was not clear whether more discrimin- ating techniques would not disclose differences between these counterparts, we preferred to use an independent nomenclature for each subspecies.

The transferrin phenotype patterns clearly show a codominant mode of inheritance. In E. grevyi three expected phenotypes were detected (15 A, 19 AB, 3 B); in E. b. boehmi, of 15 possible phenotypes three were not observed (C, E, CD), in E. b. chapmanni, of 10 possible phenotypes four were not found (C, D, BD, CD), and in E. b. antiquorum, of 15 possible phenotypes four were not observed (A, C, AC, AD).

Osterhoff (1969) found eight transferrin variants in E. b. antiquorum from game reserves in South Africa, while we found only five in the zoo population.

In order to partly characterize zebra transferrin and compare it with the transferrins of horse and donkey, we accomplished a partial isolation and fractionation of zebra (E. b. boehmi) and donkey transferrin, compared reel. wt of transferrin components in SDS-PAGE with those of horse and studied the effect of neuraminidase. It was found that in SDS-PAGE the slow transferrin components of all three Equidae species had identical mol. wt (i.e. 71,000) and the fast components were identical as well (i.e. 73,000) (compare Stratil et al., 1984).

Neuraminidase treatment decreased the migration of the slow component of zebra and donkey transferrin in two charge shifts, and of the fast component in four charge shifts, indicating two and four sialic acid residues, respectively, as in horse (cf. Stratil and Glasn/tk, 1981; Chung and McKenzie, 1985).

The results of mol. wt comparisons and neuramin- idas¢ treatment of transferrin components of the three Equidae species indicate that the oligosaccharide structures of zebra and donkey transferrin may be the same as in horse, i.e. the fast component may contain two biantennary glycans, and the slow component one biantennary glycan (cf. Coddeville et al., 1989).

Protease inhibitor-1 (PII )

After PAGE of zebra serum samples, protease inhibitor-l, inhibiting both trypsin and chymotrypsin, was localized in the prealbumin region (Figs 5 and 6). The patterns were simple--each variant appeared as a single zone and apparent beterozygotes displayed two zones of equal intensity. Variation in PI1 could be accounted for by codominant autosomal alleles (supported by limited inheritance data from six families of E. b. boehmi). In some animals, in the prealbumin region, some further zones were present that did not inhibit proteases.

Polymorphism of PII was detected in E. z. hartmannae (three variants), E. b. boehmi (seven variants), E. b. chapmanni (six variants) and E. b. antiquorum (six variants). In E. grevyi a weak zone of PI1 was localized near the albumin and was monomorphic. It was better seen on inhibition gels after 2-D agarose gel-PAGE.

A schematic diagram of PI1 variants in different species and subspecies of zebras is shown in Fig. 7. Some of the variants in the three subspecies of E. burchelli appeared to have similar mobilities, but in this paper we use an independent nomenclature for each subspecies.

Protease inhibitor-2 ( PI2 )

In the albumin region after PAGE of zebra serum samples strong inhibition activity of both trypsin and chymotrypsin was detected. Variation of the inhibitors in this region was observed, but could not be studied under these conditions.

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Fig. 6. Polyacrylamide-gel electrophoresis to show inhibitors of trypsin in E. b. boehmi. Under these conditions polymorphism of protease inhibitor-2 (PI2) cannot be studied. Note polymorphism of PI 1 and PIT; polymorphism of the latter was observed only in E. b. boehmi. A zone marked with a dot appears to represent a heterogeneity, as it is also present in numerous other samples in varying intensity. PIX is

strongest and most variable in E. b. boehmi. Lane 1 is horse serum.

each allele being responsible for the presence of two strong zones. Here we designate this system as protease inhibitor-2 (PI2).

In addition to the strong zones some weak zones with protease-inhibiting activities were also observed. At present it is not clear whether they represent heterogeneity of PI2, or belong to another system of protease inhibitors.

A rabbit anti-serum to horse ~ protease inhibitor AT (prepared in our Institute) reacted in immunoblotting with both PI 1 and PI2 (data not given). This fact indicates that the zebra protease inhibitors are homologues of horse ~1 AT.

Numerous studies of ~ protease inhibitors were performed in horses (see e.g. Juneja et al., 1979; Bell et al., 1984; Patterson et aL, 1991b). Four loci of P I (,qAT) were revealed in horse, and it appears that in donkey at least three P I (~AT) genes are present

(Patterson, 1991). In zebras we can be sure of two 1)1 (~AT) genes, but this study cannot be considered conclusive.

Protease inhibitor-T (PIT) and protease inhibitor-X (PIX)

In the region between albumin and transfvrrin a zone is present inhibiting trypsin, but not chymotrypsin. Its position appeared to correspond to a zone of horse serum that had been designated as inter-~- trypsin inhibitor (Pollitt and Bell, 1983). However, on immunoblotting with the use of sheep anti-serum to inter-~-trypsin inhibitor this zone did not react (neither did the horse protein supposed to be inter-~-trypsin inhibitor). Therefore, we prefer to use the designation PIT.

PIT appeared to have the same mobility and w a s

monomorphic in all zebras except E. b. boehmi, where

596 A. STRATIL et al.

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Fig. 8. Polyacrylamide gel to show the effect of neuraminidase on serum ~)B glycoprotein (A 1BG) of horse, donkey and zebra. Lanes 1, 2, 3 and 10 are serum samples; lanes 4, 5, 6, 7, 8 and 9 are partially isolated AIBG. - , untreated; + , treated with neuraminidase, ca--E, caballus; as---E, asinus; b--E. b. boehmi.

three variants were observed (Fig. 6). Phenotype patterns were easy to interpret and were indicative of simple codominant inheritance.

After PAGE further regions of trypsin- and chymotrypsin-inhibiting zones were present between PIT and the origin. In one of them (PIX) a variability occurred, but could not be interpreted.

gIB glycoprotein ( A I BG )

In Fig. 8 it can be seen that in PAGE a zone of zebra AIBG had a more anodic mobility than AIBG of horse and donkey. AIBG of all three Equidae species reacted in immunoblotting with rabbit anti- pig A1BG. There were neither inter-species differences nor polymorphism in zebras.

A 1BG of human serum is a glycoprotein containing four N-linked oligosaccharide chains (Ishioka et aL, 1986). In the pig eight sialic acid residues accessible to neuraminidase treatment were present (Stratil et al., 1987), also indicating the presence of four biantennary glycans. In horse and donkey A1BG, after neuraminidase treatment of plasma samples, Patterson et al. (1991a) observed charge shifts indicating four sialic

acid residues in horse A1BG variants B, K, S, throe residues in variant M, and five residues in A1BG of donkey.

We performed neuraminidase treatment of partially purified A1BG of horse, donkey and zebra (E. b. boehmi). In the horse and donkey protein, there were six charge shifts, while in the zebra there were only four (Fig. 8). The difference between the results of Patterson et al. (1991a) and those we obtained is evidently due to their use of plasmas while we used partially purified proteins.

Although the samples were incubated with a high concentration of neuraminidase for a long time, the splitting of sialic acid did not result in the appearance of a single zone after PAGE. This result is in agreement with the suggestion of Patterson et al. (1991a) that horse and donkey A1BG contain, in addition to N-acetylneuraminic acid, also N-acetyl- 4-O-acetylneuraminic acid, which is not accessible to neuraminidase.

Supposing that biantennary glycans are present in A1BG of Equidae, three glycans could then be present in horse and donkey proteins, and two

598 A. STRATIL et al.

glycans in zebra AIBG. Further studies are needed to provide conclusive evidence on the number and primary structures of glycans in different species of Equidae.

Esterase (ES)

After separation of serum samples in PAGE and staining for esterase using ~-naphthylacetate as a substrate, esterase zones were revealed only in E. z. hartmannae, and not in the other zebra species. The esterases were different from that of the horse. These results are in agreement with the findings of Kaminski et al. (1978).

CONCLUSIONS

Using various methods we identified and studied some serum proteins in different species of zebras: albumin (ALB), vitamin D binding protein (GC), transferrin (TF), protease inhibitors (PI1, PI2, PIT, PIX), ~B glycoprotein (A1BG) and esterase (ES). Species-specific patterns were observed in albumin, transferrin and, for E. grevyi, protease inhibitor-l. Specific esterase was detected only in E. z. hartmannae.

Polymorphism was observed in albumin, vitamin D binding protein, transferrin, protease inhibitor-1 and protease inhibitor-T. The results of genetic variation for the zebra species and subspecies studied are summarized in Table 1. Polymorphism was also observed in protease inhibitor-2 (this will require a more detailed study) and a variability in protease inhibitor-X. Subspecies of E. burchelli have both identical (or similar) and different variants of TF and PI1. The great number of variants in these two systems makes it difficult to clearly differentiate between the three subspecies of E. burchelli.

Although a limited number of animals was studied, it can be seen that in some zoo populations the extent of genetic variation (polymorphism) is very high, especially in E. b. boehrni, E. b. chapmanni and E. b. antiquorum. These polymorphisms can be directly applied to breeding programmes of zoo zebra populations as well as to studies of genetic diversity of both captive and wild populations. Once we know that there is genetic variation in a species, changes in gene frequencies can be followed to study the changes in genetic structure of the populations.

At present we do not know whether wild populations of E. grevyi and E. z. hartmannae have such low levels of genetic variation in the studied systems, or whether the results we obtained just reflect the present status of the captive populations we analyzed.

New information was obtained about some biochemical properties of zebra and donkey transferrin and alB glycoprotein. On neuraminidase treatment

Table 1. A summary of variants of serum polymorphic proteins in zebras

Number of variants

Taxon ALB GC TF PI1 PIT

E. grevyi 1 1 2 I 1 E. z. har tmannae 1 1 1 3 1 E. b. boehmi 2 2 5 7 3 E. b. chapmanni 2 2 4 6 1 E. b. ant iquorum I 2 5 6 1

main transferrin components of a single variant behaved similarly to the horse transferrin components, i.e. the two components in each species differed in moi. wt. A1BG of zebra appeared to have four sialic acid residues while horse and donkey proteins appeared to have six. The results indicate the presence of two glycans in zebra A1BG and three glycans in horse and donkey protein.

Acknowledgements--We thank Drs J. Vfihala and P. Moucha for collecting blood samples of zebras.

REFERENCES

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