Anim. Blood Grps biochem. Genet. 4 (1973): 153-159

Two new sheep transferrin variants and the effect of neuraminidase

A. Stratii

Czechoslovak Academy of Sciences, Institute of Physiology and Genetics of Animals, Libgchov, Czechoslovakia

Received: 23 June 1973

Summary

Transferrin phenotypes were determined in six breeds of sheep by starch gel electrophoresis. Two new variants, Tf HCzech, and Tf KCzech,, were found and some evidence of their genetic control was obtained. Tf HCzech, was detected only in Sumava sheep; it has an intermediate mobility between Tf A and Tf B. Tf KCzech. was found only in Tsigais; it was localized between Tf B and Tf C . The frequencies of corresponding alleles were very low.

Individual transferrin variants (I, A, HCzeeh., B, KCzech., C , D, E, and P) were treated with neuraminidase. Electrophoretic mobility of the strong band was de- creased by two steps in each case. It suggests that in the strong Tf band two sialic acid residues are accessible to the enzyme.

Introduction

There have been so many variants described in the transferrin system of sheep that most letters of the alphabet, and even superscripts and subscripts have been used to describe them. However, not all the described variants really exist. For a number of them it was found that they were artefacts which were caused by some form of deterioration of the serum samples (Ashton & Ferguson, 1962; see also Cooper, 1967, and Stormont et al., 1968). Besides, several further variants were described which were detected in only a few serum samples and no evidence of their genetic control was published.

The variants where the genetic control has been proved are A, B, C , D, E (Ashton, 1958; Khattab et al., 1964), G (Ashton & Ferguson, 1962), P (Khattab et al., 1964), M (Oosterlee & BOUW, 1967; FCsiis, 1972), and I (FCsiis, 1967a). The variant I has the fastest electrophoretic mobility in alkaline buffers followed by A, G, B, C, M, D, E, in that order, and the slowest is P. All transferrin variants

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are genetically determined by a set of codominant alleles at a single locus. Variants where the genetic control has not been studied and almost no wm-

parisons have been made are: B' (Osman, 1967), B'Hongnry (Ftsiis, 1970), CHunrarv (FCsiis, 1967b), U (FCsiis & Orbinyi, 1968), N (Ashton & Ferguson, 1962), NHungsry (Fisiis & Orbbyi, 1968), Q (= C(Gil)) (Ashton, 1967; FCsiis, 1967b), R (Ashton, 1966), V (Ftsiis & Orbinyi, 1968), and E, (Stormont et aI., 1968).

Each transfenin variant is formed by one strong and one weak band, which migrates more to the anode, in starch gel electrophoresis of the whole serum. The difference between the bands of a single genetic variant is caused by different numbers of sialic acid residues bound to the protein molecule (Spooner, Oliver, Maddy, Richardson, Buttress, Feinstein & Stratil, in preparation).

In this paper are described two new sheep transferrin variants and the effect of neuraminidase on the common transfemn variants. Some further results obtained during studies of sheep polymorphic proteins (frequencies of Tf and Hb alleles, study of other polymorphic systems, and application of polymorphic proteins in parentage control of sheep) will be published elsewhere.

Materials and methods

Sheep Blood samples were collected from the following breeds in Czechoslovakia: Merino (836 animals), Askanian (176 animals), Stavropol (585 animals), Tsigai (314 animals), Improved Valachian (204 animals), and Sumava (184 animals).

Electrophoresis Serum transferrins were phenotyped in horizontal starch gel electrophoresis. A dis- continuous lithium-Tris-citrate-borate buffer system described by Ferguson & Wallace (1961) as modified by Ashton & Ferguson (1962) was used.

Partial isolation of transferrins For the study of neuraminidase effect on individual variants, partially purified transferrins and two techniques of isolation were used: 1. Fracfionation of serum on Sephadex G-100. A 7-ml sample of serum (or pooled sera) of the known Tf phenotype was fractionated on a column 5 cm? X 87 cm, and a buffer 0.01 M Tris-HC1 + 0.5 M NaCl + 0.02% NaN,, pH 8.0, was used. The flow rate was 3.8 ml cm-* h-I and 6.4-ml fractions were collected. For the further study the second peak containing transfemn was used. 2. Isolation of transferrins by rivanol-ammonium sulphate fractionation. Princi- pally, the method described by Schultze & Heremans (1966) was used. Details of the technique were presented by Stratil & Spooner (1971). Transfenin fraction after rivanol-ammonium sulphate precipitation was further purified on Sephadex G-100.

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NEW TRANSFERRIN VARIANTS IN SHEEP

Neuraminidase treatment A freeze-dried transferrin preparation (2.5 mg) obtained by the first technique was dissolved in 60 pl 0.4 M sodium acetate-acetic acid buffer + 0.08 M CaCl,, pH 5.4, and 40 p1 neuraminidase ex T'zbrio cholerae (Koch-Light; 500 units/ml) were added. In heterozygous phenotypes a 5-mg sample, 20 p1 buffer, and 80 p1 neur- aminidase were used.

Almost pure transferrin obtained by the second procedure (0.5 mg) was dis- solved in 40 p1 acetate buffer and 60 ,LJ neuraminidase were added.

Samples were incubated with neuraminidase at 37°C for 24 h. Treated samples were analysed by starch gel electrophoresis.

Nomenclature of traitsferrin variants Some transferrin phenotypes (A, B, C , D, and E) were compared with standard serum samples kindly supplied by Dr E. M. Tucker. A variant migrating faster than A was designated I, and a variant slower than E was designated P. It is sup- posed that these two variants correspond with phenotypes described by FCsiis (1967a) and Khattab et al. (1964), respectively, but the direct comparison has not been carried out yet. Also the symbol G for a variant slightly slower than A was chosen only on basis of published data. Tf M and Tf D were not differentiated.

Results

New variants In breeds studied in OUT Institute variants I, A, G, B, C, D, E and P were found; they were already described and their genetic control was determined. In addition to these, two new variants have been detected for which symbols HCzech, and KCzeeh, have been used (Fig. 1). These are different from Tf H and Tf K described by Ashton & Ferguson (1962) which appear to be the deteriorated Tf C and Tf E, respectively, in the original nomenclature of Ashton (1958). (See Oosterlee & BOUW, 1967, and Stormont et al., 1968.)

Tf HCzech, has an intermediate position between A and B, and has been found only in the Sumava breed. Tf KCzech. is localized between B and'C; it has been detected only in the Tsigai breed.

Four animals with the new variants were repeatedly blood-sampled and tested. The phenotypes were always identical.

Distribution of Tf phenotypes in offspring from matings involving a heterozygous ram (Tf HCzeeh. C ) and different ewes is summarized in Table 1.

Two lambs (6 and 9) were obtained from one ewe which had phenotype Tf AKcZech.. Both of them had phenotype Tf KCzeeh.D. The Tf phenotype of the ram used for mating was not known, but the resulting phenotypes of lambs suggest that both animals inherited the allele T f K z e c h . from their mother.

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Fig. 1. Starch gel showing the new variants of sheep trans- femn (Tf HCzech. and Tf KCzech.). H*C = HCzech.C; K*D = KCzeeh.D.

Although the results from the matings are limited, there is no reason to doubt that these two new variants are inherited as codominant characters. Both new alleles had very low frequencies in studied breeds. The frequency of

TfKzech . was 0.0160 in parents (94 animals) and 0.0278 in their progeny (90 animals). The frequency of TfVzech . was 0.0029 in parents (174 animals) and 0.0036 in their progeny (140 animals).

Neuraminidase treatment In order to contribute to the study of nature of differences between individual

Table 1. Distribution of Tf variants derived from the ram (Tf HCzech.C) in offspring from different matings.

Type of mating Number of Number of Distribution of Tf ewes offspring variants in offspring

* Tf + + represents various Tf phenotypes of ewes.

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NEW TRANSFERRLN VARIANTS IN SHEEP

Fig. Za, b. Starch gel showing the effect of neuraminidase on electrophoretic mobility of different transferrin phenotypes. For electrophoresis and neuraminidase treatment samples after fractionation on Sephadex G-100 were used. D, ID, etc. are untreated samples; D+, U3+, etc. are samples treated with neuraminidase.

transferrin variants, partially purified transferrins were treated with neuraminidase. The results are shown in Fig. Za, b. The same study was also performed with the newly discovered variants (Fig. 3).

In all cases, the effect of neuraminidase on the individual transferrin variants was identical. Electrophoretic mobility of the strong band was decreased by two steps, with a simultaneous slowing down of the weak band. This result suggests that neuraminidase splits two residues of sialic acid from the transferrin molecule.

Fig. 3. The effect of neuraminidase on the newly discovered transfenin vanants. For electrophoresis and neuraminidase treatment samples of isolated transfernns were used (rivanol-ammonium sulphate precipitation; gel filtration on Sephadex G-100). Samples 1-5 and 11 are untreated; samples 6-10 are treated. H*C = HCzech.C; K*D = KCzech.D.

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Discussion

The only sheep transferrin variants that could be considered as ‘accepted’ were I, A, G, B, C, M, D, E and P. The new variants, Tf HCzech. and Tf KCaeeh., described in this paper appear to be genetically controlled and so the number of variants with known inheritance has increased. Remaining variants can only be considered as ‘reported’ until their genetic nature is proved, and/or comparison with variants described by other authors is carried out.

Earlier studies performed with sheep transferrin B and D (Spooner et al., in preparation) showed that the strong transferrin band contains three sialic acid residues and the band localized more to the anode contains four sialic acid residues per protein molecule. After treatment of the strong band with neuraminidase its mobility was slowed by two steps. The results presented in this paper indicate that the situation is comparable to other sheep transferrins studied. There has been no evidence in sheep transferrins of a situation like that in cattle transferrins, where the difference between ‘normal’ and ‘abnormal’ type is caused by a presumed polymorphism in a sialotransferase, a difference which disappears after neurami- nidase treatment (Spooner & Baxter, 1969; Strati1 & Spooner, 1971).

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