SIMILARITY OF VITAMIN D-BINDING PROTEIN (DBP) BETWEEN VARIOUS ANIMAL SPECIES IN DOUBLE IMMUNODIFFUSION AND WESTERN BLOTTING

Jan P. Madej¹, Janusz Boratyński², Wojciech Nowacki^1
1 Department of Veterinary Prevention and Immunology, Department and Clinic of Obsterics, Ruminant Diseases and Animal Health Care, Wroclaw University of Environmental Sciences, Poland
² Department of Experimental Oncology, Institute of Immunology and Experimental Therapy of the Polish Academy of Science, Wrocław, Poland

ABSTRACT

For the purpose of the study, self-produced polyclonal rabbit anti-DBP serum as well as sera from various animal species were used. In double immunodiffusion, a cross-reaction was demonstrated between human anti-DBP serum and sera from all the remaining animals: horse, pig, dog, cattle, cat, mouse, rat, sheep, goat. The character and intensity of the ongoing precipitation reaction were also determined. The strong cross-reaction was observed with dog and cat serum, intermediate – with pig, horse and mouse, and weak with cattle, sheep, goat and rat. The molecular mass of DBP in selected species was assessed using SDS-PAGE followed by western blotting and was estimated in horse, cat and goat sera as 51 kDa, in dog and cattle sera – 49 kDa, in human serum – 48 kDa, in pig serum – 47 kDa, in rat and mouse sera – 44 kDa).

Key words: vitamin D-binding protein, DBP, Gc globulin, double immunodiffusion, animals.

INTRODUCTION

Presence of vitamin D-binding protein (DBP), also known as Gc-globulin, has been demonstrated in numerous vertebrates [16, 17, 18]. It is also known that, in both human and animal serum, several isotypes of the protein may occur [17]. Data on the molecular mass of DBP in humans and scarce animal species can be found in publications (Table 1).

Table 1. Molecular Mass of DBP in various species

Species	Mass [kDa]	Applied Method	Publication Location
Human	60	Gel filtration (calibrated Sephadex G-200 column)	9
Human	58	SDS-PAGE	9
Human	56	SDS-PAGE	5
Human	51.2	Amino acid sequencing	8
Rat	52	SDS-PAGE and gel filtration ( calibrated Bio-Gel A-0.5m 1.5x85cm column)	1
Rat	54	SDS-PAGE	11, 12
Rat	58	Gel filtration	11
Chicken	54	Gel filtration calibrated Bio-Gel A-0.5m 1.5x85cm column)	2
Chicken	60	SDS-PAGE	2

In terms of structure, DBP is a single-chain glycoprotein, with N–acetylgalactosamin, galactose and sialic acid di- or tri-saccharides O-linked to the polypeptide chain by intermediary of threonine and serine residues [8, 22]. Gc globulin has the capacity to bind vitamin D and its derivatives to its N-end, as well as actin to its C-end [10]. It belongs to the protein-binding albumin superfamily, showing structural similarity with albumin, α-fetoprotein (AFP) [10, 23] as well as afamin (AFM) [15].

DBP, involved in many metabolic pathways, plays a crucial systemic role. Being part of the actin removal system, it binds actin G, prevents its polymerisation and transports it to the liver [6]. Involved in the transport of vitamin D and its active metabolites [16, 19], it is also a precursor of the macrophage activating factor– DBP-MAF [21, 22], also showing the ability to bind mainly mono-saturated and saturated fatty acids [7, 3]. Moreover, it is located on the surface of numerous cells and increases the chemotactic activity of complement components C5a i C5a des Arg [16].

It is highly probable that, considering its involvement in such a number of systemic processes, the structure of DBP has remained largely unmodified in the course of evolution.

Double immunodiffusion in agarose gel is a traditional method enabling the detection of antigens, precipitated by specific antibodies. Polyclonal antibodies specific for a selected polyvalent antigen, binding with its epitopes form a three-dimensional network, macroscopically visible as a precipitation arch.

The aim of the study was to demonstrate cross-reactivity between human anti-DBP serum and sera from other animal species; to determine the intensity and character of the ongoing precipitation reaction, as well as to assess the molecular mass of DBP in sera from selected animals.

MATERIAL AND METHODS

The following assays were used in the study: 1. a commercial human DBP (Gc-Globulin) – MP Biomedicals Inc. (Solon, OH, USA); 2. Freund’s Adjuvant-Complete, Freund’s Adjuvant Incomplete, goat anti-rabbit IgG HRP, TEMED, sodium dodecyl sulfate (SDS), 4-chloro-1-naphthol (C41N), Coomassie brilliant blue R-250 – Sigma-Aldrich (St. Louis, MO, USA); 3. agar-noble – Difco (Detroit, Michigan, USA); 4. nitrocellulose (Protran^® – BA85 pore size 0,45µm) – Schleicher&Schuell Inc. (Keene, USA); 5. acrylamide, bisacrylamide, tris, glycine, phosphate buffer, NaCl, HCl, acetic acid, methanol, ethanol as well as the remaining reagents – POCH (Gliwice, Polska).

Immunisation was performed by injecting rabbits with commercial human DBP, at 20µg/dose [4, 14]. DBP is a weak antigen (as confirmed by its partial homology with DBP from other mammal species [17]), therefore the 3 initial immunizing doses were suspended in 250µl of Freund’s complete adjuvant, 1:1 ratio. Subsequent immunizing doses were prepared in similar proportions using Freund’s incomplete adjuvant. The antigen was admininstered subcutaneously into the subscapular area, alternately on both sides, every 2 weeks. Previous to each injection, blood samples from the intermediate auricular vein (vena auricularis intermedia) [24] were collected into two test-tubes: 1) containing heparin (20·10³ i.u.·L^-1) as well as 2) without additives, in order to obtain serum. Immunisation was monitored by assessing: hematocrit, fibrinogen concentration, leukocyte count and leukogram. Antibody titer was periodically tested using double immunodiffusion. Immunisation was ended when a satisfying titer was reached – defined as lack of titer increase following subsequent immunisations. Rabbit blood was collected from a common carotid artery (arteria carotis communis) under general anesthesia. Animal carcasses were incinerated.

Double immunodiffusion (ID) [20] was conducted in 1.2% agarose gel in veronal buffer (pH 8.2) on microscope slides. Antigens and antibodies were deposited into selected, rosette-forming, plate-wells, at 10µl / well.

Electrophoresis was conducted in 7.5% (w/v) polyacrylamide gel in SDS-PAGE [13]. 0.5M Tris-HCl pH 6.8 with an addition of 0.4% SDS served as buffer for the concentrating gel; while 1.5M Tris-HCl pH 8.8 with 0.4% SDS was used as buffer for the separating gel. Serum samples were added to plate wells in 10µl amounts, in a 1:20 dilution ratio, having previously been boiled 5 minutes in 0.5M Tris-HCl, glicerin, SDS (37.5 : 30 : 6), bromophenol blue 0.2g/l pH 6.8. Separation was conducted in an electric field under constant 80V voltage supply (tension gradient ca. 1.33·10³ V·m^-1) in concentrating gel and 120V (tension gradient ca. 2·10³ V·m^-1) in separating gel. 0.5M Tris, 0.192M glycine, 0.1% SDS pH 8.3 was used as electrode buffer. Electrophoresis was interrupted after the lower gel edge was reached by bromophenol blue.

One gel was stained with Coomassie brilliant blue, revealing protein location, while the other gel was used for western blotting. Transfer from gel onto nitrocellulose was performed under constant tension of ca. 0.15 A·m^-2 for 1.5 h using the semidry method in a von Keutz graphite chamber. A 0.025 M Tris, 0.192 M glycin, 20% methanol pH 8.3 buffer was used for transfer. The nitrocellulose membrane was blocked in 2% casein blocking solution for 0.5h, then incubated 2h with the first antibody (rabbit anti-human DBP, 1:100 dilution ratio) in 2% casein solution. The nitrocellulose membrane was then washed thrice with TBS-T (0.054 M Tris, NaCl – 0.145 M, HCl ad pH 7.3 with an addition of 0.03% Tween 20). The next step encompassed incubation with HRP-labeled goat anti-rabbit IgG, 1:100 dilution ratio, in 2% casein solution. After washing the membrane thrice with TBS-T, the enzymatic reaction was induced with 1,4-chloro-naphthol; 0.3-0.4 g·L^-1 of 0.05M Tris-HCl buffer, pH 7.6. The reaction was halted by washing off excess of substrate with distilled water. Visual electrophoresis and immunoblotting results were scanned. Molar protein mass (kDa) was read from standard streak location, using LabImage 2006 software.

RESULTS

Rabbit anti-human DBP serum was obtained as a result of rabbit hiperimmunisation. Its titer, determined with immunodiffusion was 1:64 (photos show dilutions up to 1:32) (Fig. 1).

Fig. 1. Rabbit anti-human DBP Titer in Double Immunodiffusion. The central well contains the antigen (Ag) – human serum

Cross-reactivity was demonstrated between rabbit anti-human DBP and sera from the following animals: horse, pig, dog, cattle, cat, mouse, rat, sheep, goat. No precipitation arch was observed in the instance of chicken serum due to a large, intense “halo” around the plate well.

In order to compare obtained precipitates, sera obtained from different animal species were placed in adjacent plate wells (Fig. 2, Table 2). This pattern enabled the simultaneous observation of precipitation in all varied-species sera pairs in reaction to immunoserum. There are three basic types of reactions in double immunodiffusion. The identity reaction (“I”) (continued precipitation lines), indicating that the antibody precipitates identical epitopes in each of the compared antigens. This does not imply that both antigens are indeed identical, meaning only that they are indistinguishable in the ID assay. The non-identity (“N”) reaction (cross-cut precipitation lines) is observed, when independent precipitation arches cross-cut each other, which indicates presence of different epitopes, typical for each of the antigens. In the partial identity reaction both antigens hold a common epitope, however one of them also carries another, different epitope, revealed by the presence of a “spur” (“S”). In this case, the used antibody (or another clone specifically reacting with the antigen) interacts with both antigen determinants. Results of observations concerning the character and intensity of precipitation between polyclonal anti-human DBP and sera from different species of animals are located in Table 2.

Fig. 2. Cross-reaction of polyclonal anti-human DBP serum (Ab) with sera from: Human (Ho), cattle (Bo), horse (Eq), cat (Fe), goat (Cap), sheep (Ov), mouse (Mu), dog (Ca), rat (Ra) and pig (Su)

Table 2. Type of cross-reactions between anti-human DBP serum and sera from various animal species. S – “spur” (top of column, species side) , I – identity reaction, N – non-identity reaction

Species	Human	Pig	Horse	Cattle	Sheep	Goat	Dog	Cat	Rat	Mouse	Reaction Intensity:
Human	–										++++
Pig	S	–	I	I		I	S	S	N	N	++
Horse	S	I	–				S	S	N	N	++
Cattle	S	I	I	–	I	I	S	S	S	N	+
Sheep	S	S	S	I	–	I	S	S	S	N	+
Goat	S	I	S	I	I	–	S	S	S	N	+
Dog	S						–	I			+++
Cat	S						I	–			+++
Rat	S	N	N				S	S	–	I	+
Mouse	S	N	N	N	N	N	S	S	I	–	++

DBP mass was also assessed in various animal species (Fig. 3). Sera from selected mammalian species were separated in SDS-PAGE and transferred onto nitrocellulose membranes, then subjected to western blotting (Fig. 3a). Protein masses in different species were determined using LabImage 2006 software (Table 3). Differences in immunoblotting colour intensity arise from varied cross-reactivity between anti-human DBP serum and Gc-globulin from other animal species.

Fig. 3a. Western Blotting results of sera from diverse animal species with the use of Rabbit anti–human DBP antibodies

Fig. 3b. The same gel stained with Coomassie brilliant blue

Table 3. Molecular Masses of DBP in Animal Sera

Path No:	Assessed Mass: [kDa]
1 – Dog Serum 2 – Human Serum 3 – Pool of Horse Sera 4 – Cat Serum 5 – Pig Serum 6 – Cattle Serum 7 – Goat Serum 8 – Rat Serum 9 – Mouse Serum	49 48 51 51 47 49 51 44 44

DISCUSSION

In our study, we noted cross-reactivity between rabbit anti-human Gc and sera from numerous animal species. Presence of such a reaction is due to the immunised rabbit’s immune response to an antigen determinant common for many species. Considering that the obtained immunoserum contains polyclonal antibodies reacting with human DBP, we may assume that cross-reactivity with DBP from other species will not depend solely on antigen similarity (number of common antigen determinants), but also on the diversification of antigen-specific antibodies in the obtained immunoserum. Cross-reactivity with serum DBP of such a number of mammalian species speaks for considerable conservatism of the studied antigen. Simultaneously, the possibility to obtain serum reacting with xenogenic DBP through immunisation, basing on a single available commercial human Gc-protein, greatly widens research prospectives.

In the above study, inter-species DBP similarity has also been demonstrated basing on ID results in the form of defined precipitation line patterns in all serum pairs. Mouse serum DBP showed a particularly strong dissimilarity with remaining mammalian species, interreacting only with rat serum Gc-globulin. Human Gc-globulin precipitate was also found of particular interest as it showed presence of a “spur”, indicating existence of an isotype lacking in the remaining mammalian species.

Ogata et al. also used the ID method to investigate inter-species DBP similarity. Using anti-human Gc serum, they demonstrated antigen similarity between Gc-globulin from humans and various animal species [17]. This similarity, reflected in the level of cross-reactivity between anti-human Gc serum and Gc from a given species, expressed as percentage, amounted to (in comparison with human serum): dog 93%, cat 82%, rat 74%, pig 48%, cattle 42%, goat 40%. A precipitation line was also observed in the case of horse serum [18], however it did not occur in sera from: chicken [9, 17], pigeon, duck, goose, newt nor fish [17]. Identity was also demonstrated in ID in sera from: dog, cat, pig, cow and goat. Rat and giunea pig showed partial identity with Gc from: dog, cat, pig, cow and goat, while also having homologous antigens for anti-human Gc serum [17].

In other studies, it has been proven that, anti-rat Gc immunoserum cross-reacts with mouse serum, showing partial indentity [1], while not reacting with sera from: human, monkey (Cebus appella), dog, cow nor rabbit [1, 11].

In our own studies, we assessed DBP mass in selected mammalian species using western blotting. The conducted analysis has shown that rat and mouse Gc-globulin has the lowest mass (ca. 44 kDa). DBP from pig, human, dog and bovine sera hold a slightly higher mass (47-49 kDa), while highest masses were noted in horse, cat and goat sera.

CONCLUSIONS

Produced rabbit polyclonal serum anti-human DBP cross-reacts with serum Gc-globulin from all the studied mammals. Some differences of molecular weight and intensity of reaction were determined using immunodiffusion and western blotting.

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This project is financially supported by research grants for the years 2006-2007 (Research Project Nr 2P06K 03030) as well as by the European Social Fund from the European Union.

 

 

Accepted for print: 21.06.2007

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Jan P. Madej
Department of Veterinary Prevention and Immunology,
Department and Clinic of Obsterics, Ruminant Diseases and Animal Health Care,
Wroclaw University of Environmental Sciences, Poland
Norwida 31, 50-375 Wrocław, Poland
email: jan_madej@interia.pl

Janusz Boratyński
Department of Experimental Oncology,
Institute of Immunology and Experimental Therapy
of the Polish Academy of Science, Wrocław, Poland
Weigla 12, 53-114 Wrocław, Poland
Jan Dlugosz, Czestochowa University,
Institute of Chemistry and Environmental Protection,
Al. Armii Krajowej 13/15, 42-201 Czestochowa, Poland

Wojciech Nowacki
Department of Veterinary Prevention and Immunology,
Department and Clinic of Obsterics, Ruminant Diseases and Animal Health Care,
Wroclaw University of Environmental Sciences, Poland
Norwida 31, 50-375 Wrocław, Poland

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