Male-specific submaxillary gland protein, a lipocalin allergen of the golden hamster, differs from the lipocalin allergens of Siberian and Roborovski dwarf hamsters.

BACKGROUND: An increasing number of asthma cases upon exposure to hamsters and anaphylactic reactions following hamster bites are being reported, but the allergens responsible are still poorly characterized. In the Golden hamster, male-specific submaxillary gland protein (MSP), a lipocalin expressed in a sex- and tissue-specific manner in the submaxillary and lacrimal glands, is secreted in the saliva, tears and urine. The purpose of this study was to determine if MSP is an allergen, to identify IgE-reactive proteins of different hamster species and to analyse potential cross-reactivities. METHODS: Fur extracts were prepared from four hamster species. Hamster-allergic patients were selected based on a history of positive IgE-test to hamster epithelium. The IgE-reactivity of patients' sera was investigated by means of immunoblot and ELISA. IgE-reactive proteins in fur extracts and the submaxillary gland were identified using anti-MSP antibodies, Edman sequencing or mass spectrometry. MSP was purified from Golden hamster and recombinant MSP was expressed in E. coli. RESULTS: Four patients had IgE-antibodies against 20.5-kDa and 24-kDa proteins of Golden hamster fur extract, which were identified as MSP. IgE-reactive MSP-like proteins were detected in European hamster fur extract. Three patient sera showed IgE-reactive bands at 17-21 kDa in Siberian and Roborovski hamster fur extracts. These proteins were identified as two closely related lipocalins. Immunoblot inhibition experiments showed that they are cross-reactive and are different from MSP. CONCLUSION: MSP lipocalin of the Golden hamster was identified as an allergen, and it is different from the cross-reactive lipocalin allergens of Siberian and Roborovski hamsters. Our findings highlight the need for specific tools for the in vitro and in vivo diagnosis of allergy to different hamster species.

Purification, cloning and regulation of a novel acid-lipase-like protein of hamster expressed in lacrimal glands and tears during lactation.

We report a novel 48-kDa tear acid-lipase-like protein (TALLP), which is markedly induced in lacrimal glands (LG) and secreted in tears of hamster dams during lactation. TALLP is undetectable in LG and tears of normal hamsters, but is also induced after gonadectomy in both sexes and this is prevented by androgen, estrogen or thyroid hormone treatment. These observations and the obliteration of TALLP upon cessation of lactation suggest that endogenous estrogens (in females) and androgens (in males) completely repress TALLP expression. Purified TALLP is monomeric, contains approximately 18% N-glycosylation and several pI isoforms. TALLP expression was tissue-specific and immunolocalized in LG acinar cells. The cDNA deduced amino-acid sequence of TALLP precursor (398 residue, containing a 19 residues signal-peptide) showed only 43-48% identity with all known mammalian acid-lipases, including even those of other rodents, suggesting that TALLP is a prototype of a new category, within the acid-lipase family. Surprisingly, although the catalytic triad residues and other sequence features important for lipolytic activity are conserved in TALLP, it has no detectable lipase activity. However, TALLP binds the polarity sensitive hydrophobic probe, 1-aminoanthracene (K(d)=12 microM). TALLP might have a unique substrate-specificity or a lipid-binding/carrier function in tears of hamster dams. This is the first report of an acid-lipase-like protein secreted in tears of any species. Since TALLP lacks the usual lipase activity, it can be an excellent model to understand better what other structural features in acid-lipases influence their catalytic activity.

Sex differences in expression and differential regulation by androgen and estrogen of two odorant-binding tear lipocalins in lacrimal glands of immature hamsters.

In adults of several mammalian species, lacrimal glands (LG) have sex differences but there is no report of any sexual dimorphism in LG of immatures. In LG and tears of adult hamsters, we found female-specific expression of two closely related odorant-/pheromone-binding lipocalins, FLP (female lacrimal protein) and MSP (male-specific protein; initially identified in salivary glands of males). Although, both androgens and estrogens markedly repress FLP and MSP in LG of adults, the expression of these lipocalins in females is due to their incomplete repression by endogenous estrogens. Here we report a marked sexual dimorphism in the expression of FLP and MSP in LG and tears of 20-day-old immature hamsters. The age-dependant expression of these lipocalins and effect of neonatal-gonadectomy and sex hormone treatments on their expression in immatures was investigated. FLP and MSP are detectable in LG at 10-day age in both sexes of hamster but by 20-day age levels of both lipocalins show sex differences wherein FLP is several fold higher in males and MSP is obliterated in males. Thereafter, FLP declines in male LG and is obliterated by 36-day age, resulting in female-specific expression of both LG lipocalins as seen in adults. In LG of 20-day-old immatures, FLP and MSP are insensitive to repression by androgen and estrogen, respectively, which was unlike the androgen/estrogen-repressed regulation of both lipocalins in adult LG. The estrogenic repression of FLP and androgenic repression of MSP in LG of immature hamsters could be prevented by treatment with tamoxifen and flutamide, respectively. Our studies indicate that (i) presence of gonads in immatures can have significant effects on LG lipocalins resulting in their sexually dimorphic expression, (ii) in immatures, unlike adults, the repressive effects of estrogen and androgen on LG lipocalins are selective for FLP and MSP, respectively, and (iii) these repressions are likely to be mediated by sex hormone receptors.

cDNA cloning and regulation of two sex-hormone-repressed hamster tear lipocalins having homology with odorant/pheromone-binding proteins.

A major 20-kDa protein is female-specifically expressed in exorbital lacrimal gland (LG) of hamsters and secreted in tears. Here, we identify this female-specific LG protein (FLP) as a lipocalin, having 85% protein sequence identity with male-specific submandibular salivary gland proteins (MSP) secreted in saliva and urine of male hamsters. MSP is also female-specifically expressed in LG and secreted in tears but FLP was undetectable in submandibular gland (SMG). FLP and MSP have similar sex-hormonal regulation in LG, which is different from regulation of MSP in SMG. Female-specific expression of FLP and MSP in LG is due to their incomplete repression by endogenous estrogens and gonadectomy in both sexes and lactation in females resulted in their marked induction, which was prevented by estrogen or androgen treatment. FLP and MSP show best sequence identity with odorant/pheromone-binding lipocalins (58-29%). Maximum identity (58%) is with rat odorant-binding protein (OBP) expressed in lateral nasal glands, followed by aphrodisin of hamster vaginal discharge (39%). Cognate transcript and a cross-reacting 20-kDa protein were detected in nasal glands of rat in both sexes but not in hamsters. Results suggest that two closely related lipocalin genes encode FLP and MSP, which are evolutionarily closer to rat OBP than to hamster aphrodisin and these have evolved different tissue-specificity and sex-hormonal regulation. Possible functions for FLP and MSP are suggested, considering their homology to odorant/pheromone-binding lipocalins, their presence in tears, saliva and urine as well as their sex-specific and lactation-induced expression.

Estrogen and androgen repression of two female specific lacrimal lipocalins in hamster: Pituitary independent and sex hormone receptor mediated action.

Sexual dimorphism in lacrimal gland (LG) gene expression is believed to be due to direct inductive effects of androgens mediated by androgen receptors (AR) but hypophysectomy dramatically curtails these inductive effects. Since, functional estrogen receptors (ER) could not be detected in LG, estrogen effects on LG are believed to be indirectly mediated by changes in levels of pituitary hormones. We found that two lipocalins expressed in female hamster LG display an unusual and marked repression by both androgens and estrogens, which could be detected both at the level of transcripts and proteins. Here, we investigate whether these repressions, (i) require presence of pituitary and (ii) are mediated by androgen and estrogen receptors. Pituitary-ablation but not gonadectomy reduced LG weights in hamster. However, both pituitary-ablation and gonadectomy induced abundant expression of the LG lipocalins, which were markedly repressed by androgen or estrogen treatment. AR- and ER-antagonists prevented these repressions and only ER-alpha- but not ER-beta-specific agonist could mimic the estrogenic repression. AR transcript and protein and ER-alpha transcript were also detected in hamster LG. Thus, pituitary factors are neither essential for the expression of these LG lipocalins nor for their estrogenic or androgenic repressions and these repressions are very likely mediated by functional ER and AR present in LG.

Marked sexual dimorphism of lacrimal gland peroxidase in hamster: repression by androgens and estrogens.

Peroxidase secreted in tears by the lacrimal glands is a marker of secretory activity of these glands and is believed to have an antimicrobial function. We report for the first time a marked sex difference in lacrimal gland (LG) peroxidase in hamsters ( approximately 3.4-fold higher activity in females), which is due to an unusual repression by physiological levels of androgens in males. LG peroxidase activity was markedly induced in a time-dependent manner after gonadectomy in males and also females ( approximately 8- and 2-fold, respectively) and was strongly repressed by androgen treatment in a dose- and time-dependent manner. Estrogen treatment of gonadectomized hamsters could also repress LG peroxidase but not below female levels. These repressions by androgens and estrogens were significantly prevented upon co-treatment with their respective receptor antagonists. Western blotting showed that differences in LG peroxidase specific activity, in different sex hormonal states and treatments were due to changes in the levels of peroxidase protein in LG. A tear peroxidase with a clear sex difference suggests that it might also have other novel function(s) in hamster tears.