Major urinary proteins, α2U-globulins and aphrodisin

Biochimica et Biophysica Acta 1482 (2000) 218^228 www.elsevier.com/locate/bba Review Major urinary proteins, K2U -globulins and aphrodisin A. Cavaggioni, C. Mucignat-Caretta * Dipartimento di Anatomia e Fisiologia Umana, Universita© di Padova, Via Marzolo 3, 35131 Padova, Italy Received 14 October 1999; received in revised form 20 January 2000; accepted 10 February 2000 Abstract The major urinary proteins (MUPs) are proteins secreted by the liver and filtered by the kidneys into the urine of adult male mice and rats, the MUPs of rats being also referred to as K2U -globulins. The MUP family also comprises closely related proteins excreted by exocrine glands of rodents, independently of their sex. The MUP family is an expression of a multi-gene family. There is complex hormonal and tissue-specific regulation of MUP gene expression. The multi-gene family and its outflow are characterized by a polymorphism which extends over species, strains, sexes, and individuals. There is evidence of evolutionary conservation of the genes and their outflow within the species and evidence of change between species. MUPs share the eight-stranded L-barrel structure lining a hydrophobic pocket, common to lipocalins. There is also a high degree of structural conservation between mouse and rat MUPs. MUPs bind small natural odorant molecules in the hydrophobic pocket with medium affinity in the 104 ^105 M31 range, and are excreted in the field, with bound odorants. The odorants are then released slowly in air giving a long lasting olfactory trace to the spot. MUPs seem to play complex roles in chemosensory signalling among rodents, functioning as odorant carriers as well as proteins that prime endocrine reactions in female conspecifics. Aphrodisin is a lipocalin, found in hamster vaginal discharge, which stimulates male copulatory behaviour. Aphrodisin does not seem to bind odorants and no polymorphism has been shown. Both MUPs and aphrodisin stimulate the vomeronasal organ of conspecifics. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Pheromone; Mouse; Rat; Olfactory; Lipocalin; Vomeronasal organ 1. The rodent's physiological proteinuria Proteinuria, a secretion of plasma protein into urine, is not a normal physiological event because plasma proteins have a high molecular mass which prevents their glomerular ¢ltration in the kidney. The proteinuria of male rodents is an exception made possible because major urinary proteins (MUPs) are small monomeric proteins with Mr about 18 000 [1^15]. The major part of MUPs ¢ltered by the glo- * Corresponding author. Fax: +390 (49) 8275301; E-mail: [email protected] meruli are concentrated and excreted into the urine although some reabsorption occurs in the kidney proximal tubuli [16^18].The MUP proteinuria of an adult male mouse is about 5^10 mg protein per day, about 10% of the mouse nitrogen balance. MUPs are stable under a variety of conditions likely to be found in the ¢eld, such as freezing and thawing, drying, moderate heating and also proteolysis [14]. MUP synthesis takes place in the liver and accounts for 3.5^4% of total protein synthesis of the male mouse liver [14], and in the order of magnitude of 100 Wg/h per g of liver tissue in the isolated and perfused male rat liver [12]. MUPs expressed in the hepatocytes are cleaved of the secretion signal peptide and secreted 0167-4838 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII : S 0 1 6 7 - 4 8 3 8 ( 0 0 ) 0 0 1 4 9 - 7 A. Cavaggioni, C. Mucignat-Caretta / Biochimica et Biophysica Acta 1482 (2000) 218^228 into the bloodstream, and then ¢ltered by the kidneys [7]. The hormonal regulation of MUP synthesis in the hepatocytes operates pretranscriptionally by induction of MUP gene expression [19]. The synthesis of MUPs is sex-dependent under the in£uence of androgens [20^27]. Testosterone induces the synthesis of MUPs also in adult female mice. Other hormones, however, are needed for normal MUP synthesis, notably, the growth hormone [21^23,27], the thyroid hormone [19,20], the glucocorticoids and insulin [21]. Reciprocally, MUPs seem to in£uence the pituitary-gonadal axis and hypothalamic nervous mediator metabolism [25,26]. MUP synthesis decreases early in carcinoma transformed hepatocytes [28]. 2. MUP polymorphism Several forms of MUPs are expressed in the liver and excreted in urine of rodents. The forms di¡er in the deduced amino acid sequence. Some of the MUP forms can be separated by charge or mass di¡erence [29^33]. Isoelectric focussing of male mouse urine resolves up to 15 MUP forms with pI ranging from 4.6 to 5.3 [30]. Adult female rats excrete a smaller amount of MUPs, about one sixth that of male rats, and these are distinct from male MUPs with pI values of about 4.6 [31]. There are interline di¡erences in the MUP excreted by di¡erent mouse inbred strains [7], e.g., Balb/c and C57BL/6 give forms differing in pI and mass [30,34^36]; forms with Mr 18 645 and 18 710 dominate in C57BL/6 male mice and Mr 18 694 in Balb/c male mice. There is also considerable di¡erence in the pattern of MUP secretion among individuals of wild type populations, and wild individuals excrete MUPs not previously observed in inbred strains [37]. Lines of wild-derived mice express in the liver amounts of MUP mRNA that di¡er considerably from the inbred C57BL/6 line, and male to female MUP mRNA ratios range from one to several hundred, a ¢gure to be compared to the 5^10 ratio normally observed in inbred mouse strains [32]. It has been proposed that MUP polymorphism is a protein compatibility system involved in chemosensory allorecognition among rodents [38]. Allorecognition aimed at maintaining the genetic identity or to 219 avoid inbreeding may be su¤cient for the evolution of polymorphisms, the ultimate degree of which is equal to the phenotype diversity in the population [39]. Interestingly a well known compatibility group of proteins, the highly polymorphic MHC, has been shown to be involved in intraspeci¢c recognition of mice and rats based on urinary olfactory cues [40^ 42]. Testing whether also MUPs represent a compatibility system used for allorecognition would require the use of chemically de¢ned MUP forms as adequate stimuli. The expression and high secretion yield of recombinant MUP by the yeast Pichia pastoris host system pave the way for testing this hypothesis [43]. Comparing mouse and rat MUPs, the amino acid sequence reveals protein homology. Identity of the aligned residues is about 65% and characteristic motifs conserved in the MUPs of the mouse are conserved in the rat. A signature motif of MUPs is the amino-terminal hexapeptide Glu-Glu-Ala-Ser-SerThr [13]. A major di¡erence between species is that MUPs are glycosylated in rats [12,44] but not in mice. 3. MUP genetics MUP genes form a polymorphic multi-gene family [45^59]. MUP genes are clustered in the MUP locus on chromosome 4 of the mouse [46,54,56] upstream from the brown locus from the centromere, and on the homologous rat chromosome 5 [59]. The multigene family is composed of at least 35 distinct MUP genes per haploid mouse genome [48]. MUP genes fall into two groups, group 1 made of active genes and group 2 of pseudogenes containing inappropriate stop codons and frameshift mutations [44,47,52]. The MUP genes contain six coding exons. The predominant arrangement of the genes is peculiar. Group 1 and group 2 genes are arranged in pairs each containing a group 1 and a group 2 gene in divergent transcriptional orientation with 15 kb of DNA between the cap sites, forming a unit of about 45 kb [53,55]. There is base conservation between units, coupled with divergence within the unit. The evolutionary rate of divergence of MUPs of mice with a homologous MUP of rats is high, whilst there has been a selective pressure against change within 220 A. Cavaggioni, C. Mucignat-Caretta / Biochimica et Biophysica Acta 1482 (2000) 218^228 the species [49]. It has been proposed that the evolutionary ampli¢cation of these tandemly arranged genes could be brought out either by unequal crossing over or by gene conversion [48,53]. These observations suggest that MUPs must have important species-related functions. The possibility of epigenetic inheritance has been investigated. MUP DNA sequences are poorly methylated in diplotene oocytes and highly methylated in pachytene spermatocytes [57]. Repression of MUP gene methylation in adult mice can be experimentally induced by epigenetic manipulations in the early embryo such as nuclear transplantation [60]. Strikingly, the low methylation phenotype acquired through epigenetic manipulations is transmitted to most of the o¡spring of the manipulated parent mouse [61]. The MUP locus on chromosome 4 of mice has been linked genetically to some factors responsible for sperm morphology [62]. 4. Constitutively expressed MUPs and control of expression Forms of the MUP family are expressed in exocrine glands of mice and rats independently of sex, age, and state, i.e., constitutively [63^79]. Beside the liver, MUPs are constitutively expressed in the mammary, parotid, sublingual, submaxillary, lachrymal, nasal, and modi¢ed sebaceous glands. In the mouse, MUP mRNAs conventionally referred to forms from I to VI show a loose speci¢city with exocrine glands. MUPs I, II and III are expressed in the liver, MUP II also in the mammary gland, MUP IV in the parotid, lachrymal and nasal glands, MUP V in the submaxillary and MUP VI in the parotid gland [65,67,69,70]. In the rat, MUP mRNAs have been detected, beside the above glands, also in modi¢ed sebaceous glands, namely, preputial and perianal glands [63,64,66,71]. The endocrine status of the rodent organism controls the MUP gene transcription in the liver, whereas the gland cell speci¢cally controls the constitutive MUP gene expression. Epigenetic factors may be into play [61]. The high degree of homology between the £anking regions of MUP genes suggests that some of the DNA response elements are located in these regions [72]. Inserting 7 kb of DNA comprising a rat MUP gene with its £anking regions into the mouse germ line has been reported to result in transgenic mice with a high heterologous MUP expression in the liver suggesting that the transgene comprised the regulatory regions; there was expression also in preputial glands, a fact not observed in natural mice, indicating that the transgene had tissue-speci¢c response elements [73]. Cis-acting regulatory sequences involved in the di¡erential tissue-speci¢c expression have been described [74]. Beside cis-activity, there is evidence that a trans-acting gene regulates expression in a MUP variant observed in a substrain of Balb/c mice [75]. A MUP-tk reporter gene exhibits many of the characteristics of endogenous MUP genes but shows expression also in the preputial glands and testis of transgenic mice and, curiously, confers male sterility [76]. Three cis-response elements and one element located within intron 4 bind the glucocorticoid receptor in vitro and are required for inducing MUP expression with dexamethasone [77]. Three response elements for GH-dependent induction of MUP expression, two of which are involved in sexually dimorphic expression have been localized [27,78]. Finally, the mute gene MUP 1.5 b is silenced probably by steric e¡ects hampering its transcription rather than by cis- or trans-inhibiting elements [79,80]. 5. Binding of odorant molecules Besides MUPs, a variety of volatile molecules found in adult male mouse urine, that are not present or present in smaller quantity in female or castrated mouse urine, have been characterized by gas chromatography and mass spectrometry [81]. A reason of this di¡erence between sexes is that MUPs bind and help concentrate odorants in male urine. MUPs are thus odorant binding proteins [82^89]. Among the variety of odorant molecules bound to MUPs in urine, 2-sec-butyl-4,5-dihydrothiazole and dehydro-exo-brevicomin are most abundant. The binding a¤nity of MUPs for these molecules is in the order of 104 ^105 M31 , as determined by competitive displacement of the ligand 2-[3 H]isobutyl-3-methoxypyrazine. Mouse and rat MUPs bind with different a¤nity and di¡erent forms of MUPs of the A. Cavaggioni, C. Mucignat-Caretta / Biochimica et Biophysica Acta 1482 (2000) 218^228 same species bind with di¡erent a¤nity. Precise kinetic data on the binding are not available but the dissociation of the bound odorant is a slow process. Not surprisingly, therefore, MUPs retain the odorants bound when chromatographed. Changes in the short range forces between the odorant and the protein moiety, associated with small conformational changes are likely to help retain the odorant bound for long periods although the binding a¤nity is not particularly high. It is conceivable that MUPs bind the odorants in the bloodstream and convey them into the urine. The volatile molecules would then slowly di¡use in air once urine has been released in the ¢eld to mark a spot. There is direct evidence that MUPs evince a slow release of olfactory signals from scent marks that extends the longevity of olfactory signals [90,91]. The release of odorants is a complex phenomenon. Both the dissociation constant as well as the lifetime of the odorants are into play. The racemization, e.g., of the 2-sec-butyl-4,5-dihydrothiazole stereoisomers is likely to provide a cue for the mouse to date the time of deposition of the scent mark, a task that mice are very good at doing [92]. 6. MUP structure and relation to other lipocalins The structure of MUP of mice and rats has been studied by X-ray di¡raction of MUP crystals and the peptide backbone has been resolved [86]. The map of the binding site occupied by 2-sec-butyl-dihydrothiazole [89] and the solution structure of recombinant MUP [93] have been obtained with NMR spectroscopy (Fig. 1). MUPs share the overall folding pattern of lipocalins characterized by eight L-sheets de¢ning a L-barrel structure. The interior of the L-barrel forms a pocket with a highly apolar lining, ideal for the binding and transportation of small hydrophobic molecules through hydrophilic media like plasma and urine. There are, however, di¡erences in both the identity and positions of amino acid residues forming the binding pocket. Most noticeable are the substitution of Val 54 and Ala 104 of the mouse MUP by phenylalanine in rat MUP. In the mouse MUP structure there is a hydrogen bond to one of two water molecules in the binding pocket from the side chain hydroxyl group of Tyr 120. In rat MUP this group points directly to the bound 221 ligand. These di¡erences help explain why these highly homologous proteins bind a variety of ligands with di¡erent a¤nities. NMR data on a recombinant MUP-racemic 2-sec-butyl-dihydrothiazole complex show that in the hydrophobic pocket the dihydrothiazole ring is adjacent to Leu 40 and the sec-butyl chain approaches the side chains of residues 42, 54, 90, 103 and 120 in one or both diastereoisomeric complexes [89]. Classi¢cation of the lipocalins using a pro¢le search method shows that more than half of the lipocalin amino acid sequences form a large group, with signi¢cant scores for excretory MUPs, including odorant binding proteins, proteins involved in the reproductive system and the L-lactoglobulin family [94^99]. In these proteins an `open' and a `closed' end leading to the hydrophobic cavity seems well adapted to binding small ligands and interaction with cell surface elements respectively [97,98]. 7. MUPs and chemical signalling The odour of male mouse urine has a profound e¡ect on the biology [100], the endocrine system and behaviour of conspeci¢cs [101]. In an early report Vandenbergh and his colleagues [102] attributed a major role to urinary volatile molecules in the test of acceleration of female puberty induced by male urine. In subsequent studies [103,104] they noticed that the factor responsible for this activity could not be extracted in ether, was destroyed by boiling and digestion but not by organic extraction of the aqueous phase and could be chromatographically resolved in two peaks, a protein peak corresponding to Mr about 18 000 and a small molecular weight component with Mr about 800. The Mr 18 000 corresponds to the mass of MUP and Mr 800 has been suggested to correspond to the hexapeptide Glu-GluAla-Arg-Ser-Met which is a potential out£ow of a MUP pseudogene [52]. There is no report, however, that a peptide corresponding to this amino acid sequence has been isolated from male mouse urine. Later studies con¢rmed that MUP partially puri¢ed from male mouse urine and dissolved in juvenile mouse urine is active on the puberty acceleration test, even after stripping o¡ of natural ligands by organic extraction or competition displacement, 222 A. Cavaggioni, C. Mucignat-Caretta / Biochimica et Biophysica Acta 1482 (2000) 218^228 Fig. 1. Graphical representation of the hydrophobic binding pocket inside recombinant MUP as resolved by NMR spectroscopy. The side chains of all residues in the interior of the L-barrel are displayed as rods. The non-polar side chains are coloured in green (Trp 19) and yellow, while polar residues are shown in red. From [93]. A. Cavaggioni, C. Mucignat-Caretta / Biochimica et Biophysica Acta 1482 (2000) 218^228 whereas the volatile fraction is inactive [105]. Very similar results were obtained with the oestrus synchronization test (Whitten e¡ect) showing that MUP with the ligands is active in water but MUP without ligands is not active in water but is active in urine naturally excreted without MUP, namely, in urine of a male mouse castrated early in life or of juvenile male mouse or in urine of castrated female mouse (A. Marchlewska-Koj, A. Cavaggioni, P. Olejniczak, unpublished data). These results suggest that MUPs require the bound ligands or a urinary context and that the volatiles require MUPs to express their endocrine priming activity. At variance of previous observations [106] recent results repropose a role for the volatile fraction and tend to exclude a role for the protein fraction in the puberty acceleration test [107]; the majority of the positive results, however, were obtained with the volatiles in a carrier medium containing MUP rather than in air or in an organic phase, while the negative results were obtained with water as a carrier. A detailed analysis of this protocol versus the former experimental protocols may help clarify the complex issue of MUPs, volatiles and MUPs with bound volatiles in the endocrine priming e¡ects of male mouse urine. The role of volatiles on behaviour is clearer. MUP ligands play a role in chemical signalling, as the volatile odorants from MUPs have a di¡erent impact on environment exploration of male and female mice [108], females being attracted whilst male mice are repelled [109] to such an extent that they ultimately display aggressive behaviour even against receptive females anointed with MUP-borne molecules [110]. These results highlight the complexity of chemical signalling by means of urine in mice which is still largely unravelled. MUPs play an important role in chemical communication as they bind, concentrate and slowly release odorants [90] in the air like £ags [111] to mark a spot. In a urinary context they seem to play a role on the endocrine system of female conspeci¢cs conveying information on the sex and hormonal status of the releaser and on its phenotype as well. Volatile odorants of urine are likely to stimulate the main olfactory epithelium and the vomeronasal organ (VNO), and the latter is thought to be accessible to proteins [112]. Interestingly, acceleration of female puberty onset by male pheromones is medi- 223 ated by the VNO [113] and so is the oestrus synchronization e¡ect driven by MUPs (Marchlewska-Koj et al., unpublished data). Two types of cells have been described in the VNO, a cell type which expresses putative olfactory receptor molecules of class V1R homologous to the main olfactory epithelium, and a second cell type peculiar of the VNO which expresses receptors of class V2R homologous to a metabotropic glutamate receptor molecule [114]. It is not known whether the two cell types project to different parts of the accessory olfactory bulb (AOB), the ¢rst central relay nucleus of the VNO. A di¡erent spatial pattern of activation, however, has been described [115] in AOB structure following VNO exposure to MUPs stripped of volatile odorants or to the odorants alone. Taken together this evidence suggests that the VNO is a complex receptor with an organized sensory system [116]. It will be interesting to see whether MUPs and the volatile odorants stimulate di¡erent VNO receptor cells and activate di¡erent sensory pathways to the brain [38]. 8. Aphrodisin In the golden hamster a proteinaceous factor present in the vaginal discharge has been found to elicit male mounting behaviour in conspeci¢cs [117,118]. A protein has been isolated from the vaginal discharge which retains this biological activity and has been appropriately termed aphrodisin. The protein, made of 151 amino acids and glycosylated at two asparagine sites, is homologous to the lipocalin family as deduced from amino acid sequence analysis [119^121]. The similarity with excretory lipocalins like MUPs, odorant binding proteins and L-lactoglobulin is high. The highest similarity found so far is to both subunits of the heterodimer odorant protein I of the mouse nasal glands [70,122]. Aphrodisin gene expression is highest in the glands of the cervix uteri of the hamster and in the lower part of the uterus [123]. Immunohistochemical methods con¢rm this localization and detect aphrodisin extracellularly on the surface of the anterior vaginal pluristrati¢ed epithelium resulting from excretion in the vaginal discharge. Aphrodisin, however, is also excreted in female hamster parotid gland [124]. Aphrodisin-elicited sexual behaviour is lost by heating and proteolysis 224 A. Cavaggioni, C. Mucignat-Caretta / Biochimica et Biophysica Acta 1482 (2000) 218^228 but is retained after procedures for removing all possible ligands such as volatile odorants, steroids and peptides from the protein. This evidence strongly suggests that aphrodisin is a reproductive pheromone [117]. At variance with MUPs, there is no published evidence that aphrodisin carries volatile pheromones, and no aphrodisin polymorphism has been so far described based on amino acid sequence, mRNA and gene base sequences. The receptor organ for aphrodisin is the vomeronasal organ of the hamster and vomeronasalectomy abolishes the behavioural response. The intracellular transduction mechanism activated by aphrodisin in the vomeronasal receptor neurones seems to be the IP3 cascades. Puri¢ed aphrodisin modulates IP3 production of vomeronasal membranes of male hamster, while the production of cAMP is not altered [125]. The vomeronasal speci¢city of aphrodisin transduction is demonstrated by the fact that aphrodisin does not change the concentration of either second messenger in a preparation of membranes from the main olfactory mucosa. 9. Conclusions Mice, rats and hamsters have been shown to communicate sexual information among conspeci¢cs by means of chemical systems based on the excretion of lipocalins. Mice and rats communicate by means of a complex system based on a polymorphic system of lipocalins (MUPs) and their volatile ligands excreted in urine by male conspeci¢cs, ultimately releasing both behavioural as well as endocrine responses. Hamsters make use of a single lipocalin (aphrodisin) excreted in the vaginal discharge of receptive hamsters which triggers male mounting behaviour. In general, lipocalins are characterized by the diversity of the functional roles despite conservation of the structure. The MUP and aphrodisin systems illustrate that lipocalins subserve di¡erent roles in the sexual communication among conspeci¢cs. References [1] J.A. Parfentjer, Calcium and nitrogen content in urine of normal and cancer mice, Proc. Soc. Exp. Biol. Med. 29 (1932) 1285^1287. [2] P.J. 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