Purification and characterization of two odorant-binding proteins from nasal tissue of rabbit and pig

Comp. Biochem. Physiol. Vol. 99B, No. 2, pp. 445-451, 1991 Printed in Great Britain

0305-0491/91 $3.00 + 0.00 © 1991 Pergamon Press pie

PURIFICATION A N D CHARACTERIZATION OF TWO ODORANT-BINDING PROTEINS FROM NASAL TISSUE OF RABBIT A N D PIG MASSIMO DAL MONTE, ISABELLAANDREINI,* ROBERTO REVOLTELLA* and PAOLO PELOSIt Istituto di Industrie Agrarie della Universita' degli Studi, via S. Michele, 4, 1-56100 Pisa, Italy; and *Istituto di Mutagenesi e Differenziamento, CNR, Pisa, Italy (Tel: 39 50 571564) (Received 12 December 1990) Abstract--1. Soluble proteins showing binding activity to 2-isobutyl-3-methoxypyrazine have been purified to homogeneity from rabbit and pig nasal tissue; their characteristics are similar to the bovine odorant-binding protein and are to be considered members of the same family. 2. The rabbit protein is a homodimer with subunits of Mr 19k and an isoelectric point of 4.7, whereas the pig protein appears to consist of a single polypeptide chain of Mr 22k and an isoelectric point of 4.2. 3. Both proteins bind 2-isobutyl-3-methoxypyrazine with dissociation constants in the micromolar range. 4. Antibodies against the bovine OBP react well with the rabbit protein, and slightly with the porcine one.

INTRODUCTION The search for olfactory receptor proteins led several years ago to the identification of the first ordorantbinding protein (OBP) (Pelosi et al., 1981; Pelosi et al., 1982) from bovine nasal tissue. This protein was subsequently purified (Bignetti et al., 1985; Pevsner et al., 1985) and characterized both in terms of binding specificity (Pevsner et al., 1985; Topazzini et al., 1985; Pelosi and Tirindelli, 1989) and molecular structure (Cavaggioni et al., 1987; Pevsner et al., 1988a; Tirindelli et al., 1989). Its broad specificity towards several classes of odorants, together with its soluble nature and high concentration in the nasal mucosa do not support a role of olfactory receptor, as first proposed. Sequence homology with several binding proteins, such as mouse and rat urinary proteins, fl-lactoglobulin and retinol-binding protein suggest a carrier function, although its specific role in odour perception remains still unknown. A similar protein was purified from rat (Pevsner et al., 1986; Pevsner et al., 1988b) and a protein showing sequence homology with the bovine OBP was identified in frog olfactory tissue (Lee et al., 1987). Recently, an odorant binding protein has been purified from human nasal mucosa, but its characterization is still at an early stage (Pelosi et al., 1990). Immunohistochemical techniques showed that the bovine protein is localized in the tubulo-acinar glands underlining the nasal respiratory epithelium (Pevsner et al., 1986; Avanzini et al., 1987), whereas in the rat it is present in the lateral-nasal glands (Pevsner et al., 1988b). Early experiments had shown binding activity for 2-isobutyl-3-methoxypyrazine together with protein tAuthor to whom all correspondence should be addressed. Abbreviations used--HPLC, high pressure liquid chromatography; SDS--PAGE, sodium dodecyl sulphatepolyacrylamide gel electrophoresis; BSA, bovine serum albumin; OBP, odorant binding protein. 445

bands in the S D S - P A G E of crude extracts of nasal tissue from several mammal species (Pelosi et al., 1982; Baldaccini et aL, 1986). This paper reports the purification and characterization of two additional OBPs from rabbit and pig. MATERIALS AND METHODS Tritium labelled 2-isobutyl-3-methoxypyrazine was prepared as previously described (Pelosi et al., 1981) and had a spec. act., at the time of the experiments, of 1.38 Ci/mmol. All chemicals used were of reagent grade. Tissue extraction Rabbit nasal mucosa was collected from heads stored 2~5 hr after death at 4°C. Pig nasal mucosa was obtained within 1 hr from death. In both cases, olfactory and respiratory epithelium were pooled and quickly frozen to -30°C. Extracts were prepared within one or two days by homogenruing in two vols of 20 mM Tris-HCl pH 7.4 (buffer A), using a Polytron homogenizer. After centrifugation at 20,000g for 30 min, the clear supernatant was brought to pH 4.1 with citric acid and left overnight at 4°C, according to the described protocol (Tirindelli et al., 1989). After a second centrifugation, in the same conditions, the extract was neutralized to pH 7.4 with Tris base and used for the purification. Purification of the binding proteins A similar procedure was used for the purification of the odorant-binding proteins from the two animal species. In both cases, about 50 ml of extract were chromatographed 2-3 times through a Whatman DE-52 anion exchange column (16 x 1.5 cm). A linear gradient of 04).5 M NaC1 (or a narrower gradient, where more appropriate) in buffer A was used, collecting fractions of 2 ml. Each fraction was assayed for binding activity and analyzed by SDS-PAGE, while total protein content was evaluated by monitoring the absorbance at 280 nm. Final purification was accomplished in both cases by chromatography on a Pharmacia Mono-Q column (0.5 x 5 cm), using a linear gradient from 0.2 to 0.4 M NaC1 in buffer A and a Waters HPLC system.

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Fig. 1. Elution profile of a partially purified extract of rabbit nasal mucosa on DE-52 column. Crude extract was chromatographed twice through DE-52 anion exchanger; each time the fractions with binding activity to tritiated 2-isobutyl-3-methoxypyrazine were pooled and dialyzed against 20 mM Tris-HCl buffer, pH 7.4 before the next passage. The graph shows the third purification step. Elution gradient was 0.2 to 0.5 NaCI in the above buffer and 2 ml fractions were collected. Absorbance at 280 nm was monitored continuously (unbroken line) and single fractions were assayed for binding activity (solid circles).

Proteins were considered pure when they migrated as single bands on a 12% SDS-PAGE gel.

RESULTS

Purification o f rabbit OBP Gel electrophoresis Electrophoresis was run in 1% SDS, using 12% polyacrylamide gels cast with a Bio-Rad Mini-Protean apparatus, according to Laemmli (1970). Gels were stained with Coomassie Brilliant Blue R250. Reagents for electrophoresis were from Bio-Rad Laboratories.

lsoelectric focusing Isoelectric points of the purified proteins were evaluated by isoelectric focusing on a gradient of Ampholines (pH 3.5-9.5), run on a 5% polyacrylamide gel and using an LKB Multiphor apparatus.

Gel filtration Molecular weight determination in non-denaturing conditions was accomplished by gel filtration of the purified proteins through a Sephadex G-100 column (30 x 1.0 cm), using BSA, carbonic anhydrase and cytochrome-c as standards.

Binding assay A filtration method was used to measure the binding activity to tritiated 2-isobutyl-3-methoxypyrazine. The same method was applied to screen the chromatographic fractions of each experiment, as well as to determine the dissociation constants of the purified proteins. According to the described procedure (Bruns et al., 1983), glass fiber filters Whatman GF/B 2.5cm were soaked for l h r in a 1% aqueous solution of polyethylenimine (Sigma), then drained and placed on a I0 place filtration manyfold (Hoefer FH-225V). The protein solution, incubated at 4°C with the appropraite amount of radioactive ligand, was placed onto the filters and a vacuum applied. The filters were then washed with 5 ml of cold buffer A and counted, using a scintillation cocktail Hydro-Luma (Lumac) and a Packard counter mod. Tri-carb 2000 CA. Non-specific binding was measured in the presence of a 1000-fold excess of cold 2-isobutyl-3-methoxypyrazine, and accounted for not more than 2-3*/0 of total binding.

Rabbit OBP was purified to homogeneity by three chromatographic separations on a DE-52 anion exchange column, followed by a passage on a M o n o - Q column (Pharmacia). After each chromatographic step, fractions yielding a positive binding assay were pooled and dialyzed against low ionic strength buffer. Figure 1 shows the elution profile relative to the third chromatography on DE-52. SDS electrophoretical analysis of the single fractions (Fig. 2) indicates an abundant protein of apparent tool. wt of about 19k, and it eluted at the ionic strength of 0.3 M NaC1 as the most likely protein responsible for the observed binding activity. The protein at this stage appears nearly pure, contaminated by traces of the serum albumin or other protein species of the same electrophoretic mobility.

Purification o f pig OBP Figure 3 shows the elution profile of a crude extract, after acid fractionation, as described, of pig nasal mucosa when chromatographed on DE-52. The maximum of binding activity coincides with the presence of a protein of Mr 22k, as revealed by S D S - P A G E (Fig. 4, fractions 31 and 33), which is eluted at higher ionic strength (0.35 M NaC1) with respect to the rabbit OBP. The fractions containing the protein were further enriched by a second passage through the same column. Final purification was also accomplished in this case on a M o n o - Q column.

Characterization of the two OBPs Molecular weight in denaturing conditions, measured in 12% S D S - P A G E , indicated a value of M, 19k for the rabbit OBP and 22k for the pig protein. Figure 5 shows the electrophoretic pattern of the two purified proteins, together with a sample of

Fig. 2. SDS-PAGE of selected fractions relative to the chromatography of Fig. 1. Total concentration of polyacrylamide was 12%. Lane numbers refer to fractions of Fig. 1. Molecular weight standards (ST) were (from the top): BSA (66k), ovalbumin (45k), carbonic anhydrase (29k), trypsin inhibitor (20k) and ct-lactalbumin (14k). Rabbit OBP is the major protein band in lanes 11 and 13, with an apparent M, of 19k.

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Fig. 3. Elution profile of a crude extract of pig nasal mucosa on DE-52 column. A gradient of 0.2 to 0.5 NaC1 in 20 mM Tris-HC1 buffer, pH 7.4, was used and 2 ml fractions were collected. Absorbance at 280 nm was monitored continuously (unbroken line) and single fractions were assayed for binding activity (solid circles).

bovine OBP as a reference. In gel filtration on Sephadex G-100 the rabbit OBP was eluted at an apparent mol. wt of 45k, while for the pig OBP we measured a value of 25k; as a reference, the bovine OBP, which is known to be a homodimer, gave, in the same conditions, a value of 47k. The rabbit OBP, therefore appears to be a homodimer, like the bovine one, whereas the pig protein is more likely to consist of a single polypeptide chain. Isoelectric points, as determined by isoelectric focusing on Ampholine gradient pH 3.5-9.5 were 4.7 for the rabbit OBP and 4.2 for the pig OBP. Binding curves were determined for both proteins, using tritiated 2-isobutyl-3-methoxypyrazine as a ligand, along with the assay described in Materials and Methods. Figure 6 shows the binding curves together with the relative Scatchard plots. Dissociation constants, calculated from these data, were 0.8 micromolar for rabbit and 0.5 micromolar for pig OBP. The low capacity of binding observed with the rabbit preparation is probably an effect of the rather long storage of rabbit heads at 4°C, as reported in Materials and Methods.

DISCUSSION A comparison of the two purified proteins with bovine OBP is summarized in Table 1 and indicates a strong similarity between the rabbit and the bovine OBPs. In particular both proteins are constituted by homodimers of Mr 19k and present the same

Table I. Characteristicsof rabbit and pig odorant binding proteins compared with those of bovineOBP Rabbit Pig Bovine M, (in SDS-PAGE) No. of subunits Isoelectric point KD (,u M)*

19k 2 4.7 0.8

22k 1 4.2 0.5

19k 2 4.7 3.0

*Measured with tritiated 2-isobutyl-3-methoxypyrazine.

isoelectric point of 4.7. The pig protein shows clear, although not dramatic, differences: its Mr (22k) is sensibly higher than the other species and, unlike the other members of the family, it appears to be a monomer rather than a dimer; the isoelectric point is also noticeably lower (4.2), a characteristic that made its purification much easier. Concerning the presence of these proteins in other tissues, early experiments (Pelosi et al., 1982) had shown that in the rabbit binding activity to the tritiated pyrazine was confined to the nasal tissue. Similar experiments have not yet been done in the pig. Cross reactivity between the anti-bovine OBP antibodies and OBPs from other species, including rabbit and pig, was reported not to occur (Bignetti et al., 1987). However, when an antiserum, obtained from rabbits hyperimmunized with bovine OBP, was used in immunoaffinity experiments, the rabbit OBP was found to be strongly retained by the affinity column, being eluted only with acidic buffer; the pig OBP was only retarded on the column, showing a lower, but positive degree of affinity (Andreini et al., 1991). These data confirm that the rabbit OBP is much more similar to the bovine protein than the pig OBP and suggest a simple way for screening other animal species for the presence of OBPs, providing at the same time a rapid method of purification. Polyclonal antibodies to the purified OBPs have been raised and should shortly provide information on the localization of the two new proteins in the nasal tissue. We hope such information will help to clarify the function of OBP in odour perception. Amino acid sequence determination is being carried out; preliminary results show strong homology with the bovine OBP and the other members of the superfamily of binding proteins.

thank Dr. Luciano Tarquini of F. B. M., Calci (Pisa) for having supplied pig nasal tissue and Cuniavicola Gianro', Montecarlo (Lucca) for the rabbit heads. Research supported by National Research Council of Italy, Special Project RAISA, Sub-project N.4, Paper N.6. Acknowledgements--We

Odorant binding proteins from nasal tissue

Fig. 4. SDS-PAGE of odd number fractions relative to the chromatography of Fig. 2. Total concentration of polyacrylamide was 12%. Lane numbers refer to fractions of Fig. 1. Molecular weight standards (ST) were the same as in Fig. 2. Pig OBP is the protein band with an apparent M r of 22k in lanes 31 and 33.

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Fig. 5. Comparison of purified rabbit (R), bovine (B) and pig (P) OBPs on 12% SDS-PAGE. Molecular weight standards (ST) were the same as in Fig. 2.

REFERENCES

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Fig. 6. Binding curves and Scatchard plots of the purified rabbit (R) and pig (P) OBPs measured with tritiated 2isobutyl-3-methoxypyrazine. Each point is the average of two determinations. Non-specific binding, as measured in the presence of a 1000-fold excess of non-radioactive ligand accounted for 2-3% of total binding and was subtracted. Dissociation constants evaluated from these data were 0.8 micromolar for rabbit OBP and 0.5 micromolar for pig OBP.

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