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Isolation of two odorant-binding proteins from mouse nasal tissue
Comp, Biochem. Physiol. Vol. 103B,No. 4, pp. 101!-1017, 1992 Printed in Great Britain
0305-0491/92 $5.00+ 0.00 © 1992PergamonPress Ltd
ISOLATION OF TWO ODORANT-BINDING PROTEINS FROM MOUSE NASAL TISSUE DANIELA PE$,* MA~MO DAL MONTE,~"MONICAGANNI* and PAOLOPELO$I*~ *Istituto di Industrie Agrarie della Universita' degli Studi, via S. Michele, 4-56124 Pisa, Italy (Tel. 39 50 571564); and tlstituto di Mutagenesi e Diffet~aziamento del CNR, via Svezia, 2A-56124 Pisa, Italy (Tel. 39 50 574161) (Received 16 March 1992; accepted 15 April 1992)
AIm~_et--1. Two soluble proteins, with good affinity to tritiated 2-isobutyl-3-methoxypyrazine, have been purified from mouse nasal mucosa. 2. The first protein is a heterodimer with subunits of apparent M r 18 and 19 kDa and isoeiectric point of 4.9; the second is a monomer of M r 21 kDa and isoelectric point of 4.8. 3. The characteristics of these binding proteins are compared with those of the other known OBPs and urinary proteins and their putative role is discussed.
INTRODUCTION Odorant-binding proteins (OBPs) are relatively abundant in the nasal mucus of several vertebrates. They are believed to be involved in the process of odour perception, but their specific physiological function is still unclear (Pelosi and Maida, 1990). So far, OBPs have been purified from cow (Pelosi et al., 1982; Bignetti et al., 1985; Pevsner et aL, 1985) rat (Pevsner et al., 1988b), rabbit (Dal Monte et al., 1991), pig (Dal Monte et al., 1991), and frog (Lee et al., 1987), while their presence has been demonstrated in several other species of vertebrates (Baldaccini et al., 1986). The site of production varies according to the animal species, being the tubulo-acinar glands in the cow (Avanzini et al., 1987; Pevsner et al., 1986), the lateral nasal glands in the rat (Pevsner et al., 1988b) and the Bowman's glands in the frog (Lee et al., 1987). Their amino acid sequences (Cavaggioni et al., 1987; Pevsner et al., 1988a; Tirindelli et al., 1989) show significant homology with a superfamily of carrier proteins, including some members involved in chemical communication between sexes, such as the mouse and rat urinary proteins (Finlayson et al., 1965; Dinh et al., 1965; Cavaggioni et al., 1990) and the hamster aphrodisin (Henzel et al., 1988; Singer and Macrides, 1990). Genes coding for other proteins of homologous sequence have been found in regions near the nasaloral cavity of the mouse. Because of their homology with the urinary proteins, these have also been called ~To whom all correspondence should be addreued. Abbreviations: OB~ odorant-binding protein; MUP: mouse urinary protein; HPLC: high-performance liquid chromatography; FPLC: fast-protein liquid chromatography; IEF: isoelectric focusing; BSA: bovine serum albumin.
"urinary". They are expressed in the parotid, sublingual, submaxillary and lachrymal glands, as well as in the mammary glands and in the liver; their physiological function is not yet known (Shahan et al., 1987a,b; Son eta/., 1991). In the pig, an abundant protein, called pheromaxein, has been purified from the submaxillary glands. With a Mr around 15kDa and a good binding activity to pheromonal steroids, it could be numbered in the family of odorant-binding proteins and is probably involved in chemical communication between sexes (Booth and White, 1988). Another gen¢ has been described in the lateral nasal glands of the rat, that codes for a protein similar in aminoacid sequence to the known OBP and called OBPa. The fact that more OBPs are present in the same animal species is of particular interest in that it reopens the question on the physiological function of OBPs and, in particular, whether they could be responsible to some extent for odour discrimination (Dear et al., 1991). In the mouse, we had previously indicated the presence, in the crude natal extract, of an abundant protein, migrating, in denaturing conditions, with an apparent molecular weight of about 18 kDa; the same extract also exhibited good binding activity towards tritiated 2-isobutyl-3-methoxypyrazine (Baidaccini et aL, 1986). Here we report the purification of two odorantbinding proteins from the nasal epithelium of the mouse and their preliminary characterization. MA'I'FJIAL.N AND MlgTHOI~
Materials Three-week-old, Albino CD-I mice were used for all the experiments. Tritiated 2-isobutyl-3-methoxypyrazine was prepared by tritium exchange, u reported (Pelmi et ai., 1981) and had a g~cific aztivity, at the time of this work, of 1.3 Ci/mmol. Mono-Q and Mono-P columns, polybufl'er and Ampholincs were from Pharmacia/LKB (Uppsaht, Sweden). All chemicals were of reagent grade.
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DANIELAPES et al.
Preparation of the extract
After eentrifugation, the supernatant was neutralized with Tris-base and applied to a 1 x I0 cm Whatman DE-52 column. Elution was performed, using a linear 0-0.5 M NaC1 gradient, in buffer A. Total protein was evaluated by monitoring the absorbance at 280 nm; each fraction was analyzed by SDS--PAGE on a 12% gel and assayed for binding activity to the tritiated pyrazine. The fractions with good binding activity were pooled, dialyzed against buffer A and chromatographed through a Mono-Q HR 5/5 column, using a linear 0-0.5 M NaCI gradient in buffer A. Chromatofocusing was performed on a Mono-P HR 5/20 column, equilibrated with 25 mM N-methylpiperazine/HC1 buffer, pH 5.7, and eluted with Polybuffer 74 1:10, adjusted to pH 4.0 with HCI.
Nasal tissue was obtained from mice stored at 4°C for 1-3 hr after death or for 2-4 days at -20°C. The entire mucosa of the nasal cavity was collected, including olfactory and respiratory tissue. In a typical preparation, the tissue, pooled from 20 miee, was homogenized in 2 vol of 20 mM Tris-HCl 7.4 (buffer A), using a Polytron homogenizer and then centrifuged at 20,000 g for 30 rain. The supernatant was used for the subsequent fractionations.
Purification of the binding proteins Three milliliters of extract, obtained from 20 mice, were brought to pH 4.1 with citric acid and left overnight at 4°C.
Bound "PYR (dpm x 1000) 15
12
a
6
3 0 11
r 12
I
I
I
I
I
I
13
14
15
16
17
18
19
ml of eluate MoW.
66k
b
45k 29k 20k
14k
28
29
30
31
32
33
34
35
36
fractions Fig. I. Chromatography on Mono-Q column of a partially purified extract of mouse nasal tissue. The gradient was 0-0.5 M NaC1 in 20raM Tris-HCl buffer, pH 7.4. Fractions of 0.Sml were assayed for binding to tritiated 2-isobutyl-3-methoxypyrazine (a) and analyzed by SDS-PAGE Co), Molecular weight markers (not shown) were (from top): BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and = -lactalbumin.
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DANIELA PES e t al.
Preparation of the extract Nasal tissue was obtained from mice stored at 4°C for 1-3 hr after death or for 2-4 days at -20°C. The entire mucosa of the nasal cavity was collected, including olfactory and respiratory tissue. In a typical preparation, the tissue, pooled from 20 mice, was homogenized in 2 vol of 20 mM Tris-HCl 7.4 (buffer A), using a Polytron homogenizer and then centrifuged at 20,000 g for 30 rain. The supernatant was used for the subsequent fractionations.
Purification of the binding proteins Three milliliters of extract, obtained from 20 mice, were brought to pH 4.1 with citric acid and left overnight at 4°C.
After centrifugation, the supernatant was neutralized with Tris-base and applied to a 1 × 10cm Whatman DE-52 column. Elution was performed, using a linear 0-0.5 M NaCI gradient, in buffer A. Total protein was evaluated by monitoring the absorbance at 280 nm; each fraction was analyzed by SDS-PAGE on a 12% gel and assayed for binding activity to the tritiated pyrazine. The fractions with good binding activity were pooled, dialyzed against buffer A and chromatographed through a Mono-Q HR 5/5 column, using a linear 0-0.5 M NaCI gradient in buffer A. Chromatofocusing was performed on a Mono-P HR 5/20 column, equilibrated with 25 mM N-methylpiperazine/HCl buffer, pH 5.7, and eluted with Polybuffer 74 1: I0, adjusted to pH 4.0 with HC1.
Bound "PYR (dpm x 1000) 15
12
a
J 0 11
I 12
w 13
= 14
I
I
I
I
15
16
17
18
19
ml of eluate M°W,
66k
b
45k 29k
20k
14k
28
29
30
31
32
33
34
35
36
fractions Fig. 1. Chromatography on Mono-Q column of a partially purified extract of mouse nasal tissue. The gradient was 0-0.5 M NaCI in 20mM Tris-HCl buffer, pH 7.4. Fractions of 0.5 ml were assayed for binding to tritiated 2-isobutyl-3-methoxypyrazine (a) and analyzed by SDS-PAGE (b). Molecular weight markers (not shown) were (from top): BSA, ovalburnin, carbonic anhydrase, trypsin inhibitor and c(-lactalbumin.
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DANIELA PES et al.
Fig. 3. Isoelectric focusing of purified samples of the two mouse odorant-binding proteins (lane h mouse OBP]I , lane 3: mouse OBP]) and of bovine OBP (lane 2) as a reference. Isoelectric point markers (last lane on the right) were soy bean trypsin inhibitor (4.6) and carbonic anhydrase (5.4).
RESULTS
The fractionation protocol used in this work was similar to that successfully employed for the purification of other OBPs and involved the use of anionexchange chromatography, monitored by binding assay with tritiated 2-isobutyl-3-methoxypyrazine and SDS-PAGE. Binding assay performed with tritiated 2-isobutyl3-methoxypyrazine on the fraction obtained from the first step on DE-52 showed two major peaks, eluted at ionic strength around 0.3 M. The relative fractions were pooled and further chromatographed on Mono-Q (Fig. 1). The binding profile shows again two peaks, although not well resolved, the first at 16 ml, the second at 17 ml of eluate. A better separation was obtained with the same eluate from the DE-52, when chromatofocussed on a Mono-P column (Fig. 2). The fractions eluted from this column still exhibited two peaks of binding and indicate the presence of two different proteins, with affinity towards 2-isobutyl-3-methoxypyrazine, that we call OBPj and OBPu, respectively. The S D S - P A G E analysis reveals bands in the fractions corresponding to both peaks, with molecular weights around 20 kDa as the putative OBPs. The two proteins, however, were still accompanied by minor impurities. This experiment also allowed a good estimation of the isoelectric points, that afforded values of 4.9 and 4.8 for OBPt and OBPn, respectively. Final purification of the two proteins was accomplished by combining the two chromatographic steps on Mono-Q and Mono-P. The purified sample of OBP~ still showed two bands of the same intensity, when analyzed by SDS-PAGE, with Mr = 18 and 19 kDa, respectively; based on the observation that the Mono-P column can easily separate proteins differing by only 0.05 units in their isoelectric point, we can interpret this fact as an indication that OBP~ is a beterodimer. The hypothesis is further supported by other exper-
imental data: the protein migrates as a single band in native electrophoresis, gives a single sharp line, when electrofocused in a gradient of Ampholines (Fig. 3) and shows a molecular weight of about 45 kDa, when measured by gel filtration on Sephadex G-100 (Fig. 4). OBPn, on the contrary, is eluted on a Sephadex G-100 column with an apparent molecular weight of 21 kDa and appears, therefore, to be a monomeric species (Fig. 4).
M.W. (xlO00) 100
OBPII
10 6
~ 10
i 14
18
ml of eluate
Fig. 4. Gel filtration on Scphadex G-100 o f the two purified
mouse OBPs. OBPx, that shows two bands in SDS-PAGE (M~ = 18 and 19 kDa), migrates with an apparent molecular weight of about 45 kDa. O B P n, that gives a single band in SDS-PAGE (Mr = 21 kDa) is elutcd from the G-100 at a molecular weight just above 20 kDa. Molecular weight markers were: BSA (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.3 kDa).
O d o r a n t - b i n d i n g p r o t e i n s f r o m m o u s e n a s a l tissue
No cross reactivity was detected between the two mouse OBPg and antibodies against both the bovine and the pig OBP, when checked by immun0affinity chromatography. This result is in agreement with the negative or slightly positive data obtained so far in experiments of immuno-cross reactivity between OBPs from different species of vertebrates and indicates a poor homology in their amino acid sequences. DISCUSSION
The characteristics of the two proteins purified from mouse nasal tissue indicate both as members of the OBP family, on the basis of the following data: (a) they bind 2-isobutyl-3-methoxypyrazine with good affinity; (b) their molecular weights and isoelectric points are similar to the other known OBPs; (c) they are very abundant in the extract of nasal tissue. In Table 1, the properties of the two mouse OBPs are compared with those of other OBPs, as well as with urinary proteins of rat and mouse and aphrodisin from the vaginal discharge of the hamster. Two findings show particular interest for their novelty and deserve to be discussed in more detail: (a) two different OBPs are purified from the nasal mucosa of the same species; (b) the mouse OBP~ is a heterodimer, unlike the other OBPs so far purified, that are either homodimers or monomers. The presence of two different odorant-binding proteins in the same animal raises the question of whether they bind different classes of compounds. Although both have affinity for 2-isobutyl-3methoxypyrazine, they could still present different, but overlapping spectra of binding. The fact that more than one type of OBPs are found in the nasal mucosa of the same animal species is directly related to the physiological role of these binding proteins. A recent paper (Dear et al., 1991) reports the identification of a gene, in the nasal tissue of the rat, coding for a protein of Mr 23 kDa, homologous to the previously discovered OBP and called OBPH. In addition to the OBPs, six more proteins are synthesized around the nasal-oral areas in the mouse, as shown in experiments of molecular biology (Shahan et al., 1987a,b). The site of production and
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secretion of these proteins are the parotid, sublingual, submaxillary and lachrymal glands. Because of their homology to the proteins, identified several years ago in the urine of the mouse (MUPs), they have been called "urinary proteins". Their physiological role is unknown, however their tissue localization suggest a closer similarity, as long as their function is concerned, to OBPs rather than to MUPs. On the other hand, MUPs have been shown recently to share a functional property with OBPs, the ability of binding 2-isobutyl-3-methoxypyrazine and other odorants (Cavaggioni et al., 1990). Therefore, they are "odorant-binding proteins", probably keeping lipophilic odorants and pheromones in the aqueous urine, much in the same way as OBPs are suggested to help the solubilization of hydrophobic odorants in the nasal mucus. Moreover, more types of MUPs have been identified, similar in their amino acid sequence, but probably different in their binding specificity. A parallel picture is being discovered in the olfactory system of insects. At least in certain species of moths, the presence of two classes of binding proteins for pheromones (PBPs) and two for general odorants (GOBPs) has been clearly demonstrated (Vogt et al., 1991a, 1991b). When approaching the problem of understanding the physiological role of OBPs, a comparison with parallel systems, such as that of the urinary proteins or those of insects' binding proteins, can suggest hypothesis, on the basis of similar features. In the above systems, all using hydrophobic ligands, the presence of carrier proteins certainly increases the concentration of odorants and pheromones; however, it would be desirable to understand in better detail the interaction between these proteins and their substrates; in particular, the question is whether there is any specificity in the binding and therefore whether odour discrimination could occur, at least partially, at the level of OBPs. Competitive binding experiments, performed on the purified OBP from bovine nasal tissue, indicate that several types of odorants bind the protein with similar strength (Pelosi and Tirindelli, 1989; Pevsner et al., 1990). This is in agreement with the broad specificity generally assumed for the olfactory system and still supports the hypothesis that OBPs could be the odour-discriminating elements. This question can be approached only now that a second type of OBPs seems to be present in the rat (Dear et al., 1991), in the mouse (this report)
Table 1. Purified odorant-binding proteins of vertebrates, probably involved in chemical communication Protein bov-OBP rab-OBP pig-OBP rat-OBP~ rat-OBPll mus-OBP~ mus-OBP u MUP-I a-2u Aphrodisin
Animal species
M.W.
pl
/~ (#M)
Localization
Ref.
Cow Rabbit Pig Rat Rat Mouse Mouse Mouse Rat Hamster
2 × 19 k 2 × 19 k 22 k 2 x 18 k 23 k 18 + 19 k 21 k 19 k 19 k 17 k
4.7 4.7 4.2 --4.9 4.8 4.2 5.4 --
3.0 0.8 0.5 20 ---0.3 1--4 --
Tubulo-acinar glands Nasal mucosa Nasal mucosa Lateral nasal glands Lateral nasal glands Nasal mucosa Nasal mucosa Urine, liver Urine, liver Vaginal discharge
1 2 2 3 4 5 5 6 7 8
I. Pelosi et al., 1982; Bignetti et aL, 1985; Pevsner et al., 1985. 2. Dal Monte et aL, 1991.3. Pevsner et aL, 1988b. 4. Dear et al., 1991.5. This report. 6. Finlayson et aL, 1965; Cavaggioni et al., 1990. 7. Dinh et al., 1965; Cavaggioni et al., 1990. 8. Henzel et aL, 1988; Singer and Macrides, 1990.
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DANmLAPES et al.
and perhaps in other vertebrates (Pelosi et al., unpublished results). The data, that have recently been accumulating on odorant-binding proteins provide indication for the characteristics of the two classes of OBP. The proteins of the first class (OBPI) are dimers with subunits of Mr 18-19 kDa, those of the second class (OBPn) are monomers of Mr 21-23 kDa; their isoelectric points are in the range 4-5, being the members of the second class slightly more acidic than those of the first class. According to these criteria, the bovine and rabbit OBPs, as well as the first one isolated from the rat and the heterodimer of the mouse, are to be classified as OBPx, while the odorant-binding protein purified from the pig represents the first example of OBPu, being more similar to the mouse monomeric OBP and to the expression product of the second gene found in the rat. The definition of the action spectra of different OBPs, obtained by competitive binding studies with several odorants, will provide useful information towards the solution of the physiological problem. In the mouse, an additional difficulty derives from the presence of several proteins of similar structure in the proximity of the nasal cavity. Distinguishing among the various elements of this protein family is not an easy task; in fact we expect some degree of crossreactivity, when using polyclonal antibodies in immunocytochemical experiments. Monoclonal antibodies, in this case, would be very valuable tools, together with techniques of molecular biology. A second point that deserves attention, is the finding of an OBP composed of two different subunits. The study of this protein offers the possibility, for the first time, to investigate the two polypeptide chains separately, particularly with respect to the odorant-binding site. We are currently characterizing these two new proteins both in terms of ligand-binding specificity and amino acid sequence. are grateful to the Istituto Zooprofilattico, Pisa and Istituto Gcntili S.p.A., Pisa for supplying the mouse-heads, and to Isabella Andreini, Istituto di Mutagenesi e Differenziamento, CNR, Pisa, for a sample of rabbit antiserum against the pig OBP. We also thank Ambretta Navarrini for technical assistance. Research supported by National Research Council of Italy, Special Project RAISA, Sub-project N.4, Paper N454. Acknowledgements--We
REFERENCES
Andreini I., Dal Monte M., Pelosi P. and Revoltella R. (1990) Solid phase enzyme immunoassay for the odorant binding protein. J. Immanol. Res. 2, 117-125. Avanzini F., Bignetti E., Bordi C., Carfagna G., Cavaggioni A., Ferrari G., Sorbi R. T. and Tirindeili R. (1987) Immunocytochemical localization of pyrazinebinding protein in the cow nasal mucosa. Cell Tissue Res. 247, 461-464. Baldaccini N. E., Gagliardo A., Peiosi P. and Topazzini A. (1986) Occurrence of a pyrazine binding protein in the nasal mucosa of some vertebrates. Comp. Biochem. Physiol. 84B, 249-253. Bignetti E., Cavaggioni A., Peiosi P., Persaud K. C., Sorbi R. T. and Tirindelli R. (1985) Purification and characterization of an odorant binding protein from cow nasal tissue. Eur. J. Biochem. 149, 227-231.
Booth W. D. and White C. A. (1988) The isolation, purification and some properties of pheromaxein, the pheromonal steroid-binding protein, in porcine submaxillary glands and saliva. J. Endoer. 118, 47-57. Bruns R. F., Lawson-Wendfing K. and Pugsley T. A. (1983) A rapid filtration assay for soluble receptors using polyethylenimine-treated flters. Analyt. Biochem. 132, 74-81. Cavaggioni A., Sorbi R. T., Keen J. B., Pappin D. J. and Findlay J. B. C. (1987) Homology between the pyrazine binding protein from nasal mucosa and major urinary proteins. FEBS Lett. 212, 225-228. Cavaggioni A., Findlay J. B. C. and Tirindeili R. (1990) Ligand binding characteristics of homologous rat and mouse urinary proteins and pyrazine binding protein of the calf. Comp. Biochem. Physiol. 96B, 513-520. Dal Monte M., Andreini I., Revoltella R. and Pelosi P. (1991) Purification and characterization of two odorant binding proteins from nasal tissue of rabbit and pig. Comp. Biochem. Physiol. 99B, 445-451. Dear T. N., Campbell K. and Rabbitts T. H. (1991) Molecular cloning of putative odorant-binding and odorant-metabolizing proteins. Biochemistry 30, 10376-10382. Dinh B. L., Tremblay A. and Dufour D. (1965) Immunochemical study of rat urinary proteins: their relation to serum and kidney proteins. J. lmmunol. 95, 574-582. Finlayson J. S., Asofsky R., Potter M. and Runner C. C. (1965) Major urinary protein complex of normal mice: origin. Science 149, 981-982. Henzel W. J., Rodriguez H., Singer A. G., Stults J. R., Macrides F., Agosta W. C. and Niall H. (1988) The primary structure of aphrodisin. J. biol. Chem. 263, 16682-16687. Krieger J., Raining K. and Brecr H. (1991) Cloning of genomic and complementary DNA encoding insect pheromone binding proteins: evidence for microdiversity. Biochim. Biophys. Acta 1068, 277-284. Laemmli U. K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lee H. K., Wells R. G. and Reed R. R. (1987) Isolation of an olfactory cDNA: similarity to retinol binding protein suggests a role in olfaction. Science 253, 1053-1056. Pelosi P., Pisanelli A. M., Baldaccini N. E. and Gagliardo A. (1981) Binding of [3H]-2-isobutyl-3-methoxypyrazine to cow olfactory mucosa. Chem. Senses 6, 77-85. Pelosi P., Baldaccini N. E. and Pisanelli A. M. (1982) Identification of a specific olfactory receptor for 2-isobutyl-3-methoxypyrazine. Biochem. J. 201, 245-248. Pelosi P. and Maida R. (1990) Odorant binding proteins in vertebrates and insects: similarities and possible common function. Chem. Senses 15, 205-215. Pelosi P. and Tirindeili R. (1989) Structure/activity studies and characterization of an odorant-binding protein. In Chemical Senses: Vol. 1. Receptor Events and Transduction in Taste and Olfaction. (Edited by Brand J. G., Teeter J. H., Cagan R. H. and Kate M. R.), pp. 207-226. Marcel Dekker, New York. Pevsner J., Trifiletti R. R., Strittmatter S. M. and Snyder S. H. (1985) Isolation and characterization of an olfactory receptor protein for odorant pyrazines. Proc. hath. Acad. Sci. USA 82, 3050-3054. Pevsner J., Hou V., Snowman A. M. and Snyder S. H. (1990) Odorant-binding protein, characterization of ligand binding. J. biol. Chem. 265, 6118-6125. Pevsner J., Sklar P. B. and Snyder S. H. (1986) Odorantbinding protein: localization to nasal glands and secretions. Proc. nam. Acad. Sei. USA 83, 4942-4946. Pevsner J., Reed R. R., Feinstein P. G. and Snyder S. H. (1988a) Molecular cloning of odorant-binding protein: member of a ligand carrier family. Science 241, 336-339.
Odorant-binding proteins from mouse nasal tissue Pevsner J., Hwang P. M., Sklar P. B., Venable J. C. and Snyder S. H. (19881)) Odorant-binding protein and its mRNA are localized to lateral nasal glands implying a carrier function. Proc. hath. Acad. Sci. USA 85, 2383-2387. Shahan K. M., Denaro M., Gilmartin M., Shi Y. and Derman E. (1987a) Expression of six mouse major urinary protein genes in the mammary, parotid, sublingnal, submaxillary and lachrymal glands and in the liver. Molec. cell. Biol. 7, 19471954. Shahan K. M., Gilmartin M. and Derman E. (1987b) Nucleotide sequences of liver, lachrymal and submaxillary gland mouse major urinary protein mRNAs: mosaic structure and construction of panels of genespecific synthetic olignnucleotide probes. Molec. cell. Biol. 7, 1938-1946. Singer A. G. and Macrides F. (1990) Aphrodisin: pheromone or transducer7 Chem. Senses 15, 199-203.
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Son H. J., Shahan K., Rodriguez M., Derman E. and Costantini F. (1991) Identification of an enhancer required for expression of a mouse major urinary protein gene in the submaxillary gland. Molec. cell. Biol. 11, 4244-4252. Tirindelli R., Keen J. N., Cavaggioni A., Eliopoulos E. E. and Findlay J. B. C. (1989) Complete amino acid sequence of pyrazine-binding protein from cow nasal mucosa. Fur. J. 8iochem. 185, 569-572. Vogt R. G., Pr~twich G. D. and Lerner M. R. (1991a) Odorant-binding-protein subfamilies associate with distinct classes of olfactory receptor neurons in insects. J. Neurobiol. 22, 74-84. Vogt R. G., Rybezynski R. and Lerner M. R. (1991b) Molecular cloning and sequencing of general odorantbinding proteins GOBPI and GOBP2 from the tobacco hawk moth Manduca sexta: comparison with other insect OBPs and their signal peptides. J. Neurosci. 11, 2972-2984.
0305-0491/92 $5.00+ 0.00 © 1992PergamonPress Ltd
ISOLATION OF TWO ODORANT-BINDING PROTEINS FROM MOUSE NASAL TISSUE DANIELA PE$,* MA~MO DAL MONTE,~"MONICAGANNI* and PAOLOPELO$I*~ *Istituto di Industrie Agrarie della Universita' degli Studi, via S. Michele, 4-56124 Pisa, Italy (Tel. 39 50 571564); and tlstituto di Mutagenesi e Diffet~aziamento del CNR, via Svezia, 2A-56124 Pisa, Italy (Tel. 39 50 574161) (Received 16 March 1992; accepted 15 April 1992)
AIm~_et--1. Two soluble proteins, with good affinity to tritiated 2-isobutyl-3-methoxypyrazine, have been purified from mouse nasal mucosa. 2. The first protein is a heterodimer with subunits of apparent M r 18 and 19 kDa and isoeiectric point of 4.9; the second is a monomer of M r 21 kDa and isoelectric point of 4.8. 3. The characteristics of these binding proteins are compared with those of the other known OBPs and urinary proteins and their putative role is discussed.
INTRODUCTION Odorant-binding proteins (OBPs) are relatively abundant in the nasal mucus of several vertebrates. They are believed to be involved in the process of odour perception, but their specific physiological function is still unclear (Pelosi and Maida, 1990). So far, OBPs have been purified from cow (Pelosi et al., 1982; Bignetti et al., 1985; Pevsner et aL, 1985) rat (Pevsner et al., 1988b), rabbit (Dal Monte et al., 1991), pig (Dal Monte et al., 1991), and frog (Lee et al., 1987), while their presence has been demonstrated in several other species of vertebrates (Baldaccini et al., 1986). The site of production varies according to the animal species, being the tubulo-acinar glands in the cow (Avanzini et al., 1987; Pevsner et al., 1986), the lateral nasal glands in the rat (Pevsner et al., 1988b) and the Bowman's glands in the frog (Lee et al., 1987). Their amino acid sequences (Cavaggioni et al., 1987; Pevsner et al., 1988a; Tirindelli et al., 1989) show significant homology with a superfamily of carrier proteins, including some members involved in chemical communication between sexes, such as the mouse and rat urinary proteins (Finlayson et al., 1965; Dinh et al., 1965; Cavaggioni et al., 1990) and the hamster aphrodisin (Henzel et al., 1988; Singer and Macrides, 1990). Genes coding for other proteins of homologous sequence have been found in regions near the nasaloral cavity of the mouse. Because of their homology with the urinary proteins, these have also been called ~To whom all correspondence should be addreued. Abbreviations: OB~ odorant-binding protein; MUP: mouse urinary protein; HPLC: high-performance liquid chromatography; FPLC: fast-protein liquid chromatography; IEF: isoelectric focusing; BSA: bovine serum albumin.
"urinary". They are expressed in the parotid, sublingual, submaxillary and lachrymal glands, as well as in the mammary glands and in the liver; their physiological function is not yet known (Shahan et al., 1987a,b; Son eta/., 1991). In the pig, an abundant protein, called pheromaxein, has been purified from the submaxillary glands. With a Mr around 15kDa and a good binding activity to pheromonal steroids, it could be numbered in the family of odorant-binding proteins and is probably involved in chemical communication between sexes (Booth and White, 1988). Another gen¢ has been described in the lateral nasal glands of the rat, that codes for a protein similar in aminoacid sequence to the known OBP and called OBPa. The fact that more OBPs are present in the same animal species is of particular interest in that it reopens the question on the physiological function of OBPs and, in particular, whether they could be responsible to some extent for odour discrimination (Dear et al., 1991). In the mouse, we had previously indicated the presence, in the crude natal extract, of an abundant protein, migrating, in denaturing conditions, with an apparent molecular weight of about 18 kDa; the same extract also exhibited good binding activity towards tritiated 2-isobutyl-3-methoxypyrazine (Baidaccini et aL, 1986). Here we report the purification of two odorantbinding proteins from the nasal epithelium of the mouse and their preliminary characterization. MA'I'FJIAL.N AND MlgTHOI~
Materials Three-week-old, Albino CD-I mice were used for all the experiments. Tritiated 2-isobutyl-3-methoxypyrazine was prepared by tritium exchange, u reported (Pelmi et ai., 1981) and had a g~cific aztivity, at the time of this work, of 1.3 Ci/mmol. Mono-Q and Mono-P columns, polybufl'er and Ampholincs were from Pharmacia/LKB (Uppsaht, Sweden). All chemicals were of reagent grade.
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Preparation of the extract
After eentrifugation, the supernatant was neutralized with Tris-base and applied to a 1 x I0 cm Whatman DE-52 column. Elution was performed, using a linear 0-0.5 M NaC1 gradient, in buffer A. Total protein was evaluated by monitoring the absorbance at 280 nm; each fraction was analyzed by SDS--PAGE on a 12% gel and assayed for binding activity to the tritiated pyrazine. The fractions with good binding activity were pooled, dialyzed against buffer A and chromatographed through a Mono-Q HR 5/5 column, using a linear 0-0.5 M NaCI gradient in buffer A. Chromatofocusing was performed on a Mono-P HR 5/20 column, equilibrated with 25 mM N-methylpiperazine/HC1 buffer, pH 5.7, and eluted with Polybuffer 74 1:10, adjusted to pH 4.0 with HCI.
Nasal tissue was obtained from mice stored at 4°C for 1-3 hr after death or for 2-4 days at -20°C. The entire mucosa of the nasal cavity was collected, including olfactory and respiratory tissue. In a typical preparation, the tissue, pooled from 20 miee, was homogenized in 2 vol of 20 mM Tris-HCl 7.4 (buffer A), using a Polytron homogenizer and then centrifuged at 20,000 g for 30 rain. The supernatant was used for the subsequent fractionations.
Purification of the binding proteins Three milliliters of extract, obtained from 20 mice, were brought to pH 4.1 with citric acid and left overnight at 4°C.
Bound "PYR (dpm x 1000) 15
12
a
6
3 0 11
r 12
I
I
I
I
I
I
13
14
15
16
17
18
19
ml of eluate MoW.
66k
b
45k 29k 20k
14k
28
29
30
31
32
33
34
35
36
fractions Fig. I. Chromatography on Mono-Q column of a partially purified extract of mouse nasal tissue. The gradient was 0-0.5 M NaC1 in 20raM Tris-HCl buffer, pH 7.4. Fractions of 0.Sml were assayed for binding to tritiated 2-isobutyl-3-methoxypyrazine (a) and analyzed by SDS-PAGE Co), Molecular weight markers (not shown) were (from top): BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and = -lactalbumin.
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Preparation of the extract Nasal tissue was obtained from mice stored at 4°C for 1-3 hr after death or for 2-4 days at -20°C. The entire mucosa of the nasal cavity was collected, including olfactory and respiratory tissue. In a typical preparation, the tissue, pooled from 20 mice, was homogenized in 2 vol of 20 mM Tris-HCl 7.4 (buffer A), using a Polytron homogenizer and then centrifuged at 20,000 g for 30 rain. The supernatant was used for the subsequent fractionations.
Purification of the binding proteins Three milliliters of extract, obtained from 20 mice, were brought to pH 4.1 with citric acid and left overnight at 4°C.
After centrifugation, the supernatant was neutralized with Tris-base and applied to a 1 × 10cm Whatman DE-52 column. Elution was performed, using a linear 0-0.5 M NaCI gradient, in buffer A. Total protein was evaluated by monitoring the absorbance at 280 nm; each fraction was analyzed by SDS-PAGE on a 12% gel and assayed for binding activity to the tritiated pyrazine. The fractions with good binding activity were pooled, dialyzed against buffer A and chromatographed through a Mono-Q HR 5/5 column, using a linear 0-0.5 M NaCI gradient in buffer A. Chromatofocusing was performed on a Mono-P HR 5/20 column, equilibrated with 25 mM N-methylpiperazine/HCl buffer, pH 5.7, and eluted with Polybuffer 74 1: I0, adjusted to pH 4.0 with HC1.
Bound "PYR (dpm x 1000) 15
12
a
J 0 11
I 12
w 13
= 14
I
I
I
I
15
16
17
18
19
ml of eluate M°W,
66k
b
45k 29k
20k
14k
28
29
30
31
32
33
34
35
36
fractions Fig. 1. Chromatography on Mono-Q column of a partially purified extract of mouse nasal tissue. The gradient was 0-0.5 M NaCI in 20mM Tris-HCl buffer, pH 7.4. Fractions of 0.5 ml were assayed for binding to tritiated 2-isobutyl-3-methoxypyrazine (a) and analyzed by SDS-PAGE (b). Molecular weight markers (not shown) were (from top): BSA, ovalburnin, carbonic anhydrase, trypsin inhibitor and c(-lactalbumin.
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Fig. 3. Isoelectric focusing of purified samples of the two mouse odorant-binding proteins (lane h mouse OBP]I , lane 3: mouse OBP]) and of bovine OBP (lane 2) as a reference. Isoelectric point markers (last lane on the right) were soy bean trypsin inhibitor (4.6) and carbonic anhydrase (5.4).
RESULTS
The fractionation protocol used in this work was similar to that successfully employed for the purification of other OBPs and involved the use of anionexchange chromatography, monitored by binding assay with tritiated 2-isobutyl-3-methoxypyrazine and SDS-PAGE. Binding assay performed with tritiated 2-isobutyl3-methoxypyrazine on the fraction obtained from the first step on DE-52 showed two major peaks, eluted at ionic strength around 0.3 M. The relative fractions were pooled and further chromatographed on Mono-Q (Fig. 1). The binding profile shows again two peaks, although not well resolved, the first at 16 ml, the second at 17 ml of eluate. A better separation was obtained with the same eluate from the DE-52, when chromatofocussed on a Mono-P column (Fig. 2). The fractions eluted from this column still exhibited two peaks of binding and indicate the presence of two different proteins, with affinity towards 2-isobutyl-3-methoxypyrazine, that we call OBPj and OBPu, respectively. The S D S - P A G E analysis reveals bands in the fractions corresponding to both peaks, with molecular weights around 20 kDa as the putative OBPs. The two proteins, however, were still accompanied by minor impurities. This experiment also allowed a good estimation of the isoelectric points, that afforded values of 4.9 and 4.8 for OBPt and OBPn, respectively. Final purification of the two proteins was accomplished by combining the two chromatographic steps on Mono-Q and Mono-P. The purified sample of OBP~ still showed two bands of the same intensity, when analyzed by SDS-PAGE, with Mr = 18 and 19 kDa, respectively; based on the observation that the Mono-P column can easily separate proteins differing by only 0.05 units in their isoelectric point, we can interpret this fact as an indication that OBP~ is a beterodimer. The hypothesis is further supported by other exper-
imental data: the protein migrates as a single band in native electrophoresis, gives a single sharp line, when electrofocused in a gradient of Ampholines (Fig. 3) and shows a molecular weight of about 45 kDa, when measured by gel filtration on Sephadex G-100 (Fig. 4). OBPn, on the contrary, is eluted on a Sephadex G-100 column with an apparent molecular weight of 21 kDa and appears, therefore, to be a monomeric species (Fig. 4).
M.W. (xlO00) 100
OBPII
10 6
~ 10
i 14
18
ml of eluate
Fig. 4. Gel filtration on Scphadex G-100 o f the two purified
mouse OBPs. OBPx, that shows two bands in SDS-PAGE (M~ = 18 and 19 kDa), migrates with an apparent molecular weight of about 45 kDa. O B P n, that gives a single band in SDS-PAGE (Mr = 21 kDa) is elutcd from the G-100 at a molecular weight just above 20 kDa. Molecular weight markers were: BSA (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.3 kDa).
O d o r a n t - b i n d i n g p r o t e i n s f r o m m o u s e n a s a l tissue
No cross reactivity was detected between the two mouse OBPg and antibodies against both the bovine and the pig OBP, when checked by immun0affinity chromatography. This result is in agreement with the negative or slightly positive data obtained so far in experiments of immuno-cross reactivity between OBPs from different species of vertebrates and indicates a poor homology in their amino acid sequences. DISCUSSION
The characteristics of the two proteins purified from mouse nasal tissue indicate both as members of the OBP family, on the basis of the following data: (a) they bind 2-isobutyl-3-methoxypyrazine with good affinity; (b) their molecular weights and isoelectric points are similar to the other known OBPs; (c) they are very abundant in the extract of nasal tissue. In Table 1, the properties of the two mouse OBPs are compared with those of other OBPs, as well as with urinary proteins of rat and mouse and aphrodisin from the vaginal discharge of the hamster. Two findings show particular interest for their novelty and deserve to be discussed in more detail: (a) two different OBPs are purified from the nasal mucosa of the same species; (b) the mouse OBP~ is a heterodimer, unlike the other OBPs so far purified, that are either homodimers or monomers. The presence of two different odorant-binding proteins in the same animal raises the question of whether they bind different classes of compounds. Although both have affinity for 2-isobutyl-3methoxypyrazine, they could still present different, but overlapping spectra of binding. The fact that more than one type of OBPs are found in the nasal mucosa of the same animal species is directly related to the physiological role of these binding proteins. A recent paper (Dear et al., 1991) reports the identification of a gene, in the nasal tissue of the rat, coding for a protein of Mr 23 kDa, homologous to the previously discovered OBP and called OBPH. In addition to the OBPs, six more proteins are synthesized around the nasal-oral areas in the mouse, as shown in experiments of molecular biology (Shahan et al., 1987a,b). The site of production and
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secretion of these proteins are the parotid, sublingual, submaxillary and lachrymal glands. Because of their homology to the proteins, identified several years ago in the urine of the mouse (MUPs), they have been called "urinary proteins". Their physiological role is unknown, however their tissue localization suggest a closer similarity, as long as their function is concerned, to OBPs rather than to MUPs. On the other hand, MUPs have been shown recently to share a functional property with OBPs, the ability of binding 2-isobutyl-3-methoxypyrazine and other odorants (Cavaggioni et al., 1990). Therefore, they are "odorant-binding proteins", probably keeping lipophilic odorants and pheromones in the aqueous urine, much in the same way as OBPs are suggested to help the solubilization of hydrophobic odorants in the nasal mucus. Moreover, more types of MUPs have been identified, similar in their amino acid sequence, but probably different in their binding specificity. A parallel picture is being discovered in the olfactory system of insects. At least in certain species of moths, the presence of two classes of binding proteins for pheromones (PBPs) and two for general odorants (GOBPs) has been clearly demonstrated (Vogt et al., 1991a, 1991b). When approaching the problem of understanding the physiological role of OBPs, a comparison with parallel systems, such as that of the urinary proteins or those of insects' binding proteins, can suggest hypothesis, on the basis of similar features. In the above systems, all using hydrophobic ligands, the presence of carrier proteins certainly increases the concentration of odorants and pheromones; however, it would be desirable to understand in better detail the interaction between these proteins and their substrates; in particular, the question is whether there is any specificity in the binding and therefore whether odour discrimination could occur, at least partially, at the level of OBPs. Competitive binding experiments, performed on the purified OBP from bovine nasal tissue, indicate that several types of odorants bind the protein with similar strength (Pelosi and Tirindelli, 1989; Pevsner et al., 1990). This is in agreement with the broad specificity generally assumed for the olfactory system and still supports the hypothesis that OBPs could be the odour-discriminating elements. This question can be approached only now that a second type of OBPs seems to be present in the rat (Dear et al., 1991), in the mouse (this report)
Table 1. Purified odorant-binding proteins of vertebrates, probably involved in chemical communication Protein bov-OBP rab-OBP pig-OBP rat-OBP~ rat-OBPll mus-OBP~ mus-OBP u MUP-I a-2u Aphrodisin
Animal species
M.W.
pl
/~ (#M)
Localization
Ref.
Cow Rabbit Pig Rat Rat Mouse Mouse Mouse Rat Hamster
2 × 19 k 2 × 19 k 22 k 2 x 18 k 23 k 18 + 19 k 21 k 19 k 19 k 17 k
4.7 4.7 4.2 --4.9 4.8 4.2 5.4 --
3.0 0.8 0.5 20 ---0.3 1--4 --
Tubulo-acinar glands Nasal mucosa Nasal mucosa Lateral nasal glands Lateral nasal glands Nasal mucosa Nasal mucosa Urine, liver Urine, liver Vaginal discharge
1 2 2 3 4 5 5 6 7 8
I. Pelosi et al., 1982; Bignetti et aL, 1985; Pevsner et al., 1985. 2. Dal Monte et aL, 1991.3. Pevsner et aL, 1988b. 4. Dear et al., 1991.5. This report. 6. Finlayson et aL, 1965; Cavaggioni et al., 1990. 7. Dinh et al., 1965; Cavaggioni et al., 1990. 8. Henzel et aL, 1988; Singer and Macrides, 1990.
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DANmLAPES et al.
and perhaps in other vertebrates (Pelosi et al., unpublished results). The data, that have recently been accumulating on odorant-binding proteins provide indication for the characteristics of the two classes of OBP. The proteins of the first class (OBPI) are dimers with subunits of Mr 18-19 kDa, those of the second class (OBPn) are monomers of Mr 21-23 kDa; their isoelectric points are in the range 4-5, being the members of the second class slightly more acidic than those of the first class. According to these criteria, the bovine and rabbit OBPs, as well as the first one isolated from the rat and the heterodimer of the mouse, are to be classified as OBPx, while the odorant-binding protein purified from the pig represents the first example of OBPu, being more similar to the mouse monomeric OBP and to the expression product of the second gene found in the rat. The definition of the action spectra of different OBPs, obtained by competitive binding studies with several odorants, will provide useful information towards the solution of the physiological problem. In the mouse, an additional difficulty derives from the presence of several proteins of similar structure in the proximity of the nasal cavity. Distinguishing among the various elements of this protein family is not an easy task; in fact we expect some degree of crossreactivity, when using polyclonal antibodies in immunocytochemical experiments. Monoclonal antibodies, in this case, would be very valuable tools, together with techniques of molecular biology. A second point that deserves attention, is the finding of an OBP composed of two different subunits. The study of this protein offers the possibility, for the first time, to investigate the two polypeptide chains separately, particularly with respect to the odorant-binding site. We are currently characterizing these two new proteins both in terms of ligand-binding specificity and amino acid sequence. are grateful to the Istituto Zooprofilattico, Pisa and Istituto Gcntili S.p.A., Pisa for supplying the mouse-heads, and to Isabella Andreini, Istituto di Mutagenesi e Differenziamento, CNR, Pisa, for a sample of rabbit antiserum against the pig OBP. We also thank Ambretta Navarrini for technical assistance. Research supported by National Research Council of Italy, Special Project RAISA, Sub-project N.4, Paper N454. Acknowledgements--We
REFERENCES
Andreini I., Dal Monte M., Pelosi P. and Revoltella R. (1990) Solid phase enzyme immunoassay for the odorant binding protein. J. Immanol. Res. 2, 117-125. Avanzini F., Bignetti E., Bordi C., Carfagna G., Cavaggioni A., Ferrari G., Sorbi R. T. and Tirindeili R. (1987) Immunocytochemical localization of pyrazinebinding protein in the cow nasal mucosa. Cell Tissue Res. 247, 461-464. Baldaccini N. E., Gagliardo A., Peiosi P. and Topazzini A. (1986) Occurrence of a pyrazine binding protein in the nasal mucosa of some vertebrates. Comp. Biochem. Physiol. 84B, 249-253. Bignetti E., Cavaggioni A., Peiosi P., Persaud K. C., Sorbi R. T. and Tirindelli R. (1985) Purification and characterization of an odorant binding protein from cow nasal tissue. Eur. J. Biochem. 149, 227-231.
Booth W. D. and White C. A. (1988) The isolation, purification and some properties of pheromaxein, the pheromonal steroid-binding protein, in porcine submaxillary glands and saliva. J. Endoer. 118, 47-57. Bruns R. F., Lawson-Wendfing K. and Pugsley T. A. (1983) A rapid filtration assay for soluble receptors using polyethylenimine-treated flters. Analyt. Biochem. 132, 74-81. Cavaggioni A., Sorbi R. T., Keen J. B., Pappin D. J. and Findlay J. B. C. (1987) Homology between the pyrazine binding protein from nasal mucosa and major urinary proteins. FEBS Lett. 212, 225-228. Cavaggioni A., Findlay J. B. C. and Tirindeili R. (1990) Ligand binding characteristics of homologous rat and mouse urinary proteins and pyrazine binding protein of the calf. Comp. Biochem. Physiol. 96B, 513-520. Dal Monte M., Andreini I., Revoltella R. and Pelosi P. (1991) Purification and characterization of two odorant binding proteins from nasal tissue of rabbit and pig. Comp. Biochem. Physiol. 99B, 445-451. Dear T. N., Campbell K. and Rabbitts T. H. (1991) Molecular cloning of putative odorant-binding and odorant-metabolizing proteins. Biochemistry 30, 10376-10382. Dinh B. L., Tremblay A. and Dufour D. (1965) Immunochemical study of rat urinary proteins: their relation to serum and kidney proteins. J. lmmunol. 95, 574-582. Finlayson J. S., Asofsky R., Potter M. and Runner C. C. (1965) Major urinary protein complex of normal mice: origin. Science 149, 981-982. Henzel W. J., Rodriguez H., Singer A. G., Stults J. R., Macrides F., Agosta W. C. and Niall H. (1988) The primary structure of aphrodisin. J. biol. Chem. 263, 16682-16687. Krieger J., Raining K. and Brecr H. (1991) Cloning of genomic and complementary DNA encoding insect pheromone binding proteins: evidence for microdiversity. Biochim. Biophys. Acta 1068, 277-284. Laemmli U. K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lee H. K., Wells R. G. and Reed R. R. (1987) Isolation of an olfactory cDNA: similarity to retinol binding protein suggests a role in olfaction. Science 253, 1053-1056. Pelosi P., Pisanelli A. M., Baldaccini N. E. and Gagliardo A. (1981) Binding of [3H]-2-isobutyl-3-methoxypyrazine to cow olfactory mucosa. Chem. Senses 6, 77-85. Pelosi P., Baldaccini N. E. and Pisanelli A. M. (1982) Identification of a specific olfactory receptor for 2-isobutyl-3-methoxypyrazine. Biochem. J. 201, 245-248. Pelosi P. and Maida R. (1990) Odorant binding proteins in vertebrates and insects: similarities and possible common function. Chem. Senses 15, 205-215. Pelosi P. and Tirindeili R. (1989) Structure/activity studies and characterization of an odorant-binding protein. In Chemical Senses: Vol. 1. Receptor Events and Transduction in Taste and Olfaction. (Edited by Brand J. G., Teeter J. H., Cagan R. H. and Kate M. R.), pp. 207-226. Marcel Dekker, New York. Pevsner J., Trifiletti R. R., Strittmatter S. M. and Snyder S. H. (1985) Isolation and characterization of an olfactory receptor protein for odorant pyrazines. Proc. hath. Acad. Sci. USA 82, 3050-3054. Pevsner J., Hou V., Snowman A. M. and Snyder S. H. (1990) Odorant-binding protein, characterization of ligand binding. J. biol. Chem. 265, 6118-6125. Pevsner J., Sklar P. B. and Snyder S. H. (1986) Odorantbinding protein: localization to nasal glands and secretions. Proc. nam. Acad. Sei. USA 83, 4942-4946. Pevsner J., Reed R. R., Feinstein P. G. and Snyder S. H. (1988a) Molecular cloning of odorant-binding protein: member of a ligand carrier family. Science 241, 336-339.
Odorant-binding proteins from mouse nasal tissue Pevsner J., Hwang P. M., Sklar P. B., Venable J. C. and Snyder S. H. (19881)) Odorant-binding protein and its mRNA are localized to lateral nasal glands implying a carrier function. Proc. hath. Acad. Sci. USA 85, 2383-2387. Shahan K. M., Denaro M., Gilmartin M., Shi Y. and Derman E. (1987a) Expression of six mouse major urinary protein genes in the mammary, parotid, sublingnal, submaxillary and lachrymal glands and in the liver. Molec. cell. Biol. 7, 19471954. Shahan K. M., Gilmartin M. and Derman E. (1987b) Nucleotide sequences of liver, lachrymal and submaxillary gland mouse major urinary protein mRNAs: mosaic structure and construction of panels of genespecific synthetic olignnucleotide probes. Molec. cell. Biol. 7, 1938-1946. Singer A. G. and Macrides F. (1990) Aphrodisin: pheromone or transducer7 Chem. Senses 15, 199-203.
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Son H. J., Shahan K., Rodriguez M., Derman E. and Costantini F. (1991) Identification of an enhancer required for expression of a mouse major urinary protein gene in the submaxillary gland. Molec. cell. Biol. 11, 4244-4252. Tirindelli R., Keen J. N., Cavaggioni A., Eliopoulos E. E. and Findlay J. B. C. (1989) Complete amino acid sequence of pyrazine-binding protein from cow nasal mucosa. Fur. J. 8iochem. 185, 569-572. Vogt R. G., Pr~twich G. D. and Lerner M. R. (1991a) Odorant-binding-protein subfamilies associate with distinct classes of olfactory receptor neurons in insects. J. Neurobiol. 22, 74-84. Vogt R. G., Rybezynski R. and Lerner M. R. (1991b) Molecular cloning and sequencing of general odorantbinding proteins GOBPI and GOBP2 from the tobacco hawk moth Manduca sexta: comparison with other insect OBPs and their signal peptides. J. Neurosci. 11, 2972-2984.