Doklady Biological Sciences, Vol. 391, 2003, pp. 318–321. Translated from Doklady Akademii Nauk, Vol. 391, No.

5, 2003, pp. 707–711. Original Russian Text Copyright © 2003 by Novikov.

PHYSIOLOGY

Coordinated Expression of the Genes Gus and Mup as a Potential Basis of the Functional Activity of the Androgen-Dependent Pheromones of the House Mouse (Mus musculus L.)
S. N. Novikov
Presented by Academician A.D. Nozdrachev March 31, 2003 Received April 2, 2003

In one of the most interesting and promising models of molecular physiology, the functional activity of the androgen-dependent pheromones of house mouse Mus musculus L. (in particular, 2-sec-butyl-4,5-dihydrothiazole, 3,4-dehydro-exo-brevicomin, and E,E-α- and E-β-farnesenes) is related to the urinary proteins of the major urinary protein (MUP) complex [1]. This complex includes a group of low-molecular-weight acidic proteins (m. w., 18–19 kDa; pI, 4.2–4.7). MUPs are encoded by a cluster of Mup genes, located in chromosome 4 (linkage group VIII). To date, the Mup genes have been cloned, and the multihormonal control of the Mup expression in hepatocytes has been studied in detail. Now the Mup system is a well-developed model used in molecular genetics of mammals [2]. The existence of genotypic differences in the functional activity of pheromones is commonly attributed to the hereditarily determined deviations in the function of some enzyme systems, in particular, the kidney acidic hydrolase β-glucuronidase (GUS, EC 3.2.1.31). The latter is encoded by the Gus gene (located in chromosome 5) with participation of sex steroids and involved in the transformation of the immobilized chemosignal (propheromone) in the form of glucuronides to its physiologically active form [3]. This was the first study to present the entire picture of the functioning of these two genetic systems as a potential basis for the regulation of the physiological activity of the androgen-dependent pheromones of house mouse, based on the analysis of the characteristic features of the expression of the protein products of genes Mup and Gus as dependent on the androgen status of the organism. The study was performed in spring using mature males of two highly inbred, genealogically unrelated strains of mice with hereditarily determined differences in the physiological activity of androgen-dependent pheromones, namely, strains CBA/LacY and

C57BL/6JY (n = 48) [4]. The proteins of the MUP complex were separated by SDS-PAGE with subsequent densitometric analysis of fractions [2]. The activity of lysosomal GUS in kidney homogenate and urine was monitored by the rate of hydrolysis of phenolphthaleinβ-D-glucuronide (Sigma, United States) and expressed in Fishman’s units [3]. The testosterone content of blood plasma was determined radioimmunologically, using standard kits (Sorin, France). The results were statistically treated using standard procedures. As seen from Fig. 1, the genotype significantly affects the qualitative and quantitative composition of MUPs: fractions A, B, C, D, G, and H were found in the mice of both strains, whereas fractions E and F may serve as genetic markers of the strains CBA (fraction E) and C57BL/6 (fraction F). In general, the concentrations of the MUP fractions were 5.49 + 0.127 and 3.46 ± 0.628 mg/ml in CBA and C57BL/6 males, respectively. Correlation analysis of the level of testosterone in blood plasma, absolute content of different MUP fractions, and the activity of GUS in kidney homogenates and urine showed a significant positive correlation
Protein, mg/ml 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 A B C

Strain CBA Strain C57BL/6

D E F Protein fractions

G

H

Pavlov Institute of Physiology, Russian Academy of Sciences, nab. Makarova 6, St. Petersburg, 199034 Russia

The concentrations of the protein fractions of the MUP complex in the urine of male laboratory mice with different genotypes.

0012-4966/03/0708-0318$25.00 © 2003 MAIK “Nauka /Interperiodica”

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Table 1. Correlation between the testosterone content in blood plasma and the content of individual MUP fractions in urine of laboratory male mice of different genotypes Genotype A CBA C57BL/6
* P < 0.05.

Fraction B +0.846* +0.703 C +0.698 +0.717 D +0.122 +0.597 E –0.467 – F – +0.707 G +0.682 +0.454 H +0.116 +0.024

–0.604 +0.295

between the testosterone level and the content of the fraction B in CBA males (Table 1) and a positive correlation between the testosterone content and the GUS activity, also characteristic solely of CBA males (Table 2). It was shown earlier that a change in the endogenous testosterone level in mature male mice, induced by castration, resulted in a drop of the absolute content of all seven fractions in C57BL/6 mice and six fractions in CBA mice [2]. Therapy with testosterone led to the restoration of the relative (partial) content of the fractions to the level characteristic of sham-operated animals. In this aspect, the expression of the proteins of three fractions (B and C in CBA mice and D in C57BL/6 mice) is of particular interest. These three fractions are completely absent in MUPs of females, disappear after castration of mature males, and are restored to the preoperational level after testosterone therapy [2]. Therefore, the biosynthesis of proteins comprising these fractions completely depends on the level of endogenous testosterone in the organism. It can be assumed the fractions B, C, and D are the most interesting in terms of studying the characteristics of affinity binding of MUPs with the androgen-dependent pheromones [1]. The data presented in this study indicate that the expression of two different proteins (the androgendependent fraction B of the MUP complex and β-glucuronidase), coordinated with the testosterone level, is characteristic of adult male CBA mice (with highly active sex pheromones [4]), but not C57BL/6 mice. Thus, we found a significant correlation between the level of the hormone regulating the biosynthesis of sex pheromones in house mouse [4] and the activity of GUS involved in the transformation of immobilized (latent) propheromone in the form of glucuronides in the physiologically active form [3]. These data may considerably broaden our notion on the molecular genetic mechanisms regulating the physiological activity of natural compounds of this group, under the action of steroids on genetic material (genes Gus and Mup). A low activity of the androgen-dependent pheromones affecting the reproduction of C57BL/6 mice is apparently related to the low testosterone level and the genetically determined characteristics of steroidogenesis and hormonal reception [4]. Earlier, using the models of the pheromonal regulation of aggressive behavior and spermatogenesis, we showed that excreted urine of
DOKLADY BIOLOGICAL SCIENCES Vol. 391 2003

mature males of this strain contained a large amount of glucuronides: the incubation of the specimens with commercial preparations of β-glucuronidase in vitro resulted in a significant increase in the physiological activity of the pheromone [4]. On the other hand, as was shown in [3], the activity of native enzyme in C57BL/6 males was decreased by five and ten times in the kidneys and urine, respectively, compared to CBA males. However, a possible universal role of GUS in the regulation of the physiological activity of androgendependent pheromones is questionable. A marked correlation between the enzyme activity in the kidney and urine of CBA males and a low correlation in C57BL/6 males (Table 2) may be indicative of not only heterogenic composition of the isozyme pool of GUS in excreted urine, but also a high substrate specificity of the enzyme in vivo, as well as the effect of the genotype on its physical and chemical characteristics (in particular, the resistance to the specific inhibitor, glucaro-1,4lactone, a product of glucuronic acid metabolism [3]). In this aspect, a comparative biochemical study of the isozyme forms of this hydrolase in the kidney and preputial gland (the main producers of a series of chemically identified androgen-dependent pheromones of house mouse [1]) would be especially interesting. Based on preliminary data on the separation of the proteins of the MUP complex using ion-exchange chromatography on DEAE cellulose, we assumed that each of the electrophoretic fractions A–H is a mixture of isomorphic proteins, differing in the charge of the molecule. This assumption is corroborated by the results of a long-term work performed by scientists from the United Kingdom, who studied the genetic polymorTable 2. Correlation between the testosterone content in blood plasma and the activity of β-glucuronidase in kidneys and urine of male laboratory mice of different genotypes Genotype TA CBA C57BL/6 +0.670* +0.158 SA +0.702* +0.026 +0.851** +0.382 Kidneys Urine

* P < 0.05, ** P < 0.01. TA, total enzyme activity, expressed in Fishman’s units; SA, specific enzyme activity, expressed in Fishman’s units per mg tissue

320

NOVIKOV

phisms of MUPs in the populations of the wild form of house mouse [5]. Using HPLC and mass-spectrometry, it was shown that the proteins of the MUP complex, which do not differ in molecular weight, may be present in different chromatographic fractions. This may be accounted for by their amino acid composition (in particular, the ratio between glycine, glutamine, and lysine). In this case, insignificant amino acid substitutions may result in significant conformational changes, thereby affecting the functional properties of the protein molecule, the basis of the ligand-binding pocket of which is formed by Val58, Ala107, Leu44, and Ala122 [6]. The authors of [6] assumed that the substitution of Val58 and Ala107 with phenylalanine and Leu44 and Ala122 with valine may reflect the characteristics of the protein binding with one or another ligand. A good experimental confirmation of this assumption was the determination, in the MUP molecule, of the high-affinity site for the known androgen-dependent pheromones (2,3-dehydro-exo-brevicomin and 2-sec-butyl-4,5dihydrothiazole) and the creation, on the basis of these data, of an artificial physiologically active hexapeptide N-Glu–Glu–Ala–Arg–Ser–Met, which accelerates sex maturation of female laboratory mice [7]. Therefore, it can be assumed that the proteins contained in the electrophoretic fractions A–H (Fig. 1) will differ, on the one hand, in charge; on the other hand, in the composition of the aforementioned amino acids. Taken together, the above data and our results suggest that the mechanisms of regulation of the functional activity of the androgen-dependent pheromones in laboratory mice may be the following. The expression of the genes coding for the MUP cluster is controlled by sex steroids (primarily, testosterone). The level, balance, and effectiveness of steroid hormones depend on the genetically determined characteristics of steroidogenesis (first of all, the activity of 3β-hydroxysteroid dehydrogenase/isomerse (EC 1.1.1.145) and the hormonal reception), as well as environmental factors (in particular, the nature of the intrapopulation relations). The aforementioned factors affect the qualitative and quantitative composition of MUPs, which are synthesized in the liver and excreted from the organism with urine. The differences in the characteristics of the protein binding with physiologically active ligands (determined, on one hand, by its structural and conformational characteristics and, on the other hand, by the activity of β-glucuronidase) will affect the absolute and partial concentrations of these compounds in the solution and the pheromone composition and determine the functional and informational properties of the pheromonal “bouquet” on the whole (as a vector with targeted effect on the recipient organism). The central place in this scheme belongs to the idea on key roles of (a) the ratio between different MUP fractions; (b) differential expression of these proteins in hepatocytes [2]; and (c) significant differences in the nature of affinity binding of the protein molecule with one or another ligand [1]. Thus, according to our scheme, a limited

number of individual pheromone-like compounds exists in the nature, and the mechanisms forming the olfactory image are based on the combination of the ratios between these physiologically active compounds in the solution. The genetic model based on a coordinated expression of the genes Gus and Mup under the action of testosterone probably represents the first and yet the only example of a specific gene net whose nucleus consists of these genes interacting at the level of their products and thus affecting the physiological activity of the androgen-dependent pheromone in rodents. Within the framework of the genetic physiological model put forward, particularly interesting are the data on the presence, in excreted urine of house mouse, of meprine, an androgen- and genotype-dependent metallopeptidase (EC 3.4.24.18), the main function of which is degradation of the proteins of the MUP complex [8]. The process of MUP degradation to individual oligopeptides is the necessary and, possibly, the key stage in the mechanism of effective action of the ligand–protein complex on the recipient organism. A similar conclusion on a pivotal role of the oligopeptides from urine of male rats, weighing less than 5 kDa, in the regulation of the activity of the c-Fos genes in the cells of additional olfactory bulb in females was made in [9]. In the context of the hypothesis on the possible role of the protein product of the Gus gene, β-glucuronidase, in the regulation of the functional activity of house mouse pheromones, the study on uridine 5'-diphosphoglucuronyltransferase (UDP-glucuronosyltransferase, EC 2.4.1.17) from olfactory lining (UGTolf) is especially interesting [10]. The authors of [10] reported on the key role of this enzyme in the mechanisms of detoxification of xenobiotics and a broad range of physiologically active volatile compounds. Thus, UDP-glucuronosyltransferase fulfils a protective function, on one hand, and is involved in biotransformation of chemical compounds, on the other hand. One of the characteristic features of the uroproteins of the MUP complex is related to their striking structural similarity with odorant-binding protein (OBP), which carries the odorant molecules to the receptor cell of the olfactory lining and plays a key role in the mechanisms of olfactory reception in terrestrial vertebrates [11]. Interestingly, in addition to its protective and transport functions, OBP is also involved in the primary “recognition” (decoding) of the odorant. The data that mRNA of the Mup genes is expressed in the cells of mouse olfactory lining in the prepubertal period are also interesting [12]. When comparing the data reported in [1, 5, 6, 8, 11, 12] with our results, taking into account the logic of this line of reasoning, a question arises as to whether the MUP and OBP proteins may be units of the same phylogenetically ancient cascade, transducing informative chemosignals (pheromones) from one organism to another, and whether the
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process of information exchange between OBPs and MUPs may be a sort of a dialogue in the language of chemosignals. It should be added that the discovery, in the mid-1990s, of high-affinity membrane receptors for OBP in the cells of mammalian olfactory lining and pulmonary tissue is suggestive of the existence of specific mechanisms of targeted axonal transport of physiologically active ligands in the form of complex with OBP or its polypeptide fragments as a result of endocytosis. The effect of sex pheromones on the reproduction and various behavioral patterns in the majority of mammalian species is directly related to the function of the vomeronasal (Jacobson’s) organ, a phylogenetically ancient pair structure, located in the base of nasal septum [1, 4, 7]. This neuroanatomic structure is directly projected via an additional olfactory bulb on three most important parts of hypothalamus (the preoptical region, as well as the ventromedial and premammilary nuclei) [4]. Taking into account the existence of tight neuronal connections between the vomeronasal organ and the hypothalamic–pituitary complex, on the one hand, and its key role in the regulation of the effect of sex pheromones on the organism, on the other hand, we can suggest that further development of the experimental model suggested in this study will lead to the development of a highly effective system of vector control of the reproductive function and sex behavior of mammals, based on the principles of transnasal targeted transport of physiologically and pharmacologically active compounds to the brain in the form of artificial protein complexes. The development of the methods of intranasal introduction of oligopeptides and drugs, associated with a protein matrix [13–15], is a good empirical argument in favor of this conclusion. ACKNOWLEDGMENTS I am grateful to G.A. Churakov, A.A. Filimonenko, and V.E. Sukonina, who kindly provided the experimental data necessary for the analysis, and I.A. Burkot, who prepared the manuscript for publishing.

The study was supported by the Russian State Science and Technology Program “Frontiers in Genetics” (project no. 2.152) and the Russian Foundation for Basic Research (project no. 02-04-49273). REFERENCES
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