Animal Genetics 1992,23,457-462

SHORT COMMUNICATION

Genetic polymorphism of plasma vitamin D-binding protein (GC) in Australian goats

D. M. VANKAN & K. BELL

Department of Physiology and Pharmacology, The University of Queensland, St Lucia, Queensland, 4072, Australia

Summary. Polymorphism at the GC locus in goats was detected using isoelectric focusing (pH 4.5-5.4) and immunoblotting with antiserum to human GC. Three variants, designated A, B and C in order of decreasing mobility to the anode, were detected and were shown to be controlled by three codominant alleles, GCA, GCB and GCc. GCA and GCB occurred in all four breeds (Australian and Texan Angora, Cashmere and Dairy) with GCA being the most common and having gene frequencies ranging from 0.851 to 0.993. GCC was found only in Australian Angora and Cashmere animals. The products of the three GC alleles had isoelectric points in the range pH 4.63-4.95 and M , of approximately 54375. The major isoforms of the three alleles were shown to contain sialic acid. Linkage between the GC and albumin loci was unable to be demonstrated due to the low frequency of ALBA (0.02) in the Cashmere breed. Keywords: GC, vitamin D-binding protein, polymorphism, goats, isoelectric focusing, immunoblotting

Genetic polymorphism of plasma GC (vitamin D binding protein) has been reported in humans, several other mammals and in chickens (for references see Kalab et al. 1990). GC polymorphism was reported recently in sheep (Kalab et al. 1990; Erhardt 1991). There was no evidence for GC polymorphism in a limited number of goats studied in some previous reports (Van de Weghe et al. 1982; Ogata et al. 1988; Kalab et al. 1990). By using isoelectric focusing and immunoblotting we describe here GC polymorphism in goats.

Plasmas from heparinized bloods of 3665 goats of the Australian and Texan Angora (989 and 66 respectively), Cashmere (2372) and Dairy breeds (238) were stored at -20°C until examined by electrophoresis. The Dairy classification comprised Saanen, Toggenberg, British Alpine and Anglo-Nubian animals and their crosses as insufficient numbers were available from the individual breeds.

Isoelectric focusing gels (240mm x llOmm x 0*5mm), pH 4-5-54, were cast between two glass plates in a manner similar to that described by Pollitt & Bell

Correspondence: Dr K. Bell, Department of Physiology and Pharmacology, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia. Accepted 4 March 1992

457

458 D. M. Vankan & K . Bell

(1983). Isoelectric focusing was performed on a flatbed apparatus (LKB Ultrophor, model 2217, Pharmacia, Yeronga, Queensland, Australia) cooled to 10°C. The anolyte and catholyte were 0 . 0 4 ~ glutamic acid and 0.1 M NaOH respectively. Gels were prefocused for 30min at 12 W constant power before applying the filter paper inserts (Whatman 17Chr, Selby Scientific & Medical, Milton, Queensland, Australia; 4mm x 3mm X 1 mm) soaked with plasmas. The inserts were applied 2cm from the cathode for 20min at 12 W constant power and focusing was continued for a further 145 min. The GC bands were identified by print blottingof proteins onto nitrocellulose (Trans-Blot@ transfer medium,Bio-Rad, North Ryde, New South Wales, Australia, 0.45 p,; 20min) and immunoblotting with rabbit antiserum to human GC (1:1000, Dako, Bioscientific, Gymea, New South Wales, Australia) as outlined in the BioRad Bulletin (goat anti-rabbit IgG alkaline phosphatase conjugate kit).

Albumin phenotyping was performed using 12% T horizontal polyacrylamide gel electrophoresis (pH 7.9) as described by Bell et al. (1988). Filter paper inserts (Whatman 17Chr; 4mm x 3mm X lmm) soaked with the plasmas were elec- trophoresed into the gel at a constant current of 45mA for 15s. The inserts were removed and electrophoresis was continued at 60W constant power until the tracking dye had reached the anode wick (approximately 3.5 h).

The isoeIectric points (PI) of the major isoforms of the G C variants were determined by calibrating the pH gradient of the gel using the Pharmacia Low PI Calibration Kit (pH 2.5-6.0) and the relative molecular mass (M,) determinations were performed by 2D-SDS-PAGE (IEF pH 3.5-6.0 and SDS-PAGE pH 8.8) (Laemmli 1970; Poliitt & Bell 1983). 2D-SDS-PAGE was necessary to separate GC from albumin.

Appropriate plasmas were pre-incubated for 30min at 37°C in an agitating water bath before the addition of 0.075 ml neuraminidase (from Vibrio cholerae : 1 U/ml activity, Calbiochem-Behring, Alexandria, New South Wales, Australia) to 0.75 ml plasma. After 6 and 24h at 37"C, 0.lml aliquots were removed and the reactions stopped with 0.02ml of 0.012M tetrasodium EDTA. These were stored at -20°C until subjected to isoelectric focusing.

Frequencies of the GC and albumin alleles were determined by simple gene counting and the exclusion probabilities (PE) were calculated according to Jamieson (1965).

Isoelectric focusing of caprine plasmas followed by immunoblotting with antiserum to human G C revealed six different GC patterns which could be explained by the existence of three variants, designated A , B and C in order of decreasing mobility to the anode (Fig. 1). The patterns for each variant were characterized by three major bands and one minor band migrating cathodally to the major bands. Another minor band migrating between the cathodal minor band and the major bands was observed in the B type. The central major band stained most intensely and the anodal band was the weakest of the three major bands. Combinations of two of the variants produced patterns identical to simple mixtures.

The distribution of G C phenotypes in offspring from 1810 matings are detailed in Table 1. The family data were consistent with a mode of inheritance of three codominant GC alleles, GCA, CCB and G c .

Allele frequencies in Australian and Texan Angoras, Cashmere and Dairy

GC polymorphism in goats 459

Figure 1. IEF (pH 4.5-5.4) and immunoblot (1:lOOO rabbit antiserum to human GC) patterns of normal caprine plasmas showing GC types (from left to right) A, C, BC, B, AC, AB and A.

breeds are listed in Table 2 along with the exclusion probabilities (PE). GCA was the most frequent allele in all four breeds with frequencies ranging from 0.851 to 0.993. GCB was present in fairly low frequencies in all four breeds and GCC was only observed in Australian Angora and Cashmere goats.

There was close agreement between the observed and expected GC phenotypic frequencies in each breed assuming Hardy-Weinberg equilibrium ( P > 0.05) but in the overall population a significant deviation ( P < 0.01) existed due to an excess of homozygous and heterozygous B types (data not shown). This reflected that the population was not panmictic.

The isoelectric points for all the isoforms of the caprine GC variants were estimated from a minimum of six IEF runs and ranged from pH 4.63-4-95. The PI’S of the major isoforms were between pH 4-63-4075, which concurs with the findings of Ogata et al. (1988), who demonstrated that all of the strong GC bands in goats migrated anodally to the human fast band which has a PI of 4.9. Neuraminidase treatment of plasmas followed by electrophoresis produced cathodal shifts of all major GC bands and very slight shifts in the minor cathodal bands. However, the relative migration rates of the major bands of the different alleles remained unchanged at all time-points, indicating that the observed differences in the major isoforms of the three alleles may be due to differences in the polypeptide chains. The relative molecular mass of G C estimated from three two-dimensional electrophore- tic runs was 54375 k 1312, which is comparable to the estimates recorded for both humans and rats (Petrini et al. 1984; Imawari et al. 1980).

Two albumin phenotypes were observed in Cashmere goats (n = 1339) and the frequencies of ALBA and ALBB were 0.02 and 0.98 respectively. Due to the low frequency of ALBA in families, there were insufficient data to determine whether the GC and ALB loci are linked in the goat.

This is the first report of genetic variation at the GC locus in goats. IEF in the narrow pH range, 4.5-5-4, was necessary to demonstrate the polymorphism as print blotting of horizontal 12% T PAGE, pH 7.9 patterns did not reveal any polymorphism. In contrast to the Australian and Texan Angoras and Dairy breeds,

Tab

le 1

. O

bser

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and

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cted

num

bers

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from

181

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capr

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ass

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les,

G

CA

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CC

Num

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C P

heno

type

A

AB

A

C

B

BC

c

Mat

ing

n O

bser

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Exp

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d xL

df

AX

A

A X

AB

A

X A

C

AX

B

A x

BC

A

xC

A

B X

A

B

AB

X A

C

AB

x B

A

B x

BC

A

C x

AC

A

Cx

B

1234

12

34

359

179

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32

32 9 7 48

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9

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1

1234

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180 32 4

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0

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GC polymorphism in goats 461

Table 2. Frequencies of the GC alleles, GC", GCB and GCc, in the Australian and Texan Angora, Cashmere and Dairy breeds and in the total goat population and exclusion probabilities (PE)

Breed

Australian Texan Angora Angora Cashmere Dairy Total

Allele (n = 660) (n = 66) (n = 926) (n = 194) (n = 1846) ~ ~ ~

A 0.993 0.977 0.851 0.979 0.918 B 0.006 0.023 0.110 0.021 0.062 C 0~001 O.Oo0 0.039 O.Oo0 0.020 P E 0.007 0.022 0.131 0.020 0.076

Cashmere goats demonstrated greater variability at the GC locus. It is possible that GCC is specifically associated with Cashmere goats and the appearance of GCC in Australian Angoras reflects the common practice of 'upgrading' of feral Cashmere goats to purebred types through successive backcrosses with pedigree Angora bucks.

The electrophoretic pattern of caprine GC reported by Ogata et al. (1988) after IEF and immunofixation is very similar to those described here. As the IEF pH range which they used (either 4-6 or 4-5-54) is not clear, their monomorphic type may in fact correspond to either the AB or AC phenotype described here.

The exclusion probability of 0.131 in Cashmere goats indicates the potential utility of GC in parentage testing and as a genetic marker in this breed. It remains to be seen whether substantial polymorphism also exists in goat populations in other parts of the world.

Acknowledgements

This work was supported by a grant from the Australian Stud Book, Alison Road, Randwick, New South Wales 2031, Australia. We wish to thank Dr C. Bunn for supplying the Texan goat samples.

References

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Erhardt G. (1991) Genetic polymorphism of vitamin D binding protein and serum albumin in sheep. Animal Genetics 22, (Suppl. l), 28-9.

Imawari M., Akanuma Y., Muto Y., Itakura H. & Kosaka K. (1980) Isolation and partial characterization of two immunologically similar vitamin D-binding proteins in rat serum. Journal of Biochemistry 88, 349-60.

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Jamieson A. (1965) The genetics of transferrins in cattle. Heredity 20, 419-41. Kalab P., Strati1 A. & Glasnak V. (1990) Genetic polymorphism of serum vitamin D-binding protein

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462 D. M . Vankan & K . Bell

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Ogata M., Nakasono I . , Iwasaki M., Kubo S. & Suyama H. (1988) Comparative immunological and electrophoretic analysis of Gc protein in sera from various animals. Comparative Biochemistry and Physiology WB, 193-9.

Petrini M., Galbraith R.M.. Werner P.A.M., Emerson D.L. & Arnaud P. (1984) Gc (vitamin D binding protein) bind to cytoplasm of all human lymphocytes and is expressed on B-cell membranes. Clinical lmmunology and Immunopathology 31, 282-95.

Pollitt C.C. & Bell K. (1983) Characterization of the ul-protease inhibitory system in throughbred horse plasma by horizontal two dimensional (ISO-DALT) electrophoresis. 1. Protein staining. Animal Blood Groups and Biochemical Genetics 14, 83-105.

Van de Weghe A, , Van Zeveren A. & Bouquet Y. (1982) Vitamin D binding protein in domestic animals. Comparative Biochemistry and Physiology 73B, 977-82.