Major Cat Allergen Research Articles

New therapeutic strategies: a chimeric human-cat fusion protein inhibits allergic reactivity

Summary We have undertaken to develop new therapies for allergic diseases by taking advantage of negative signalling via immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptors. Bi-functional molecules that indirectly crosslink Fcɛ receptors to FcγII receptors on targeted cells were constructed. This approach employed coaggregation of FcɛRI and FcγRII using an antigen-specific chimeric fusion protein (GFD) composed of human Fcγ and the major cat allergen Fel d 1. GFD was designed to block mediator release by directly attaching to Fcγ receptors and at the same time indirectly binding to FcɛRI via Fel d 1 antigen-specific IgE already bound to that receptor. GFD was expected to function as a safe form of antigen-specific immunotherapy by blocking acute reactivity to Fel d 1; i.e. by functioning as an immunogen but not as allergen. GFD inhibited passive cutaneous anaphylaxis (PCA) reactivity to Fel d 1 in transgenic mice but did not induce skin reactivity at sensitized sites. Balb/c mice sensitized to Fel d 1 and challenged intratracheally (IT) on days 28 and 42 showed lowered temperature and increased lung resistance, indicating systemic and pulmonary allergic reactions, respectively. On the other hand, mice given GFD 5 μg SQ on days 7, 14, and 21 showed inhibition of systemic reactivity to IT challenge on days 28 and 42. Hence GFD inhibited Fel d 1-induced allergic response in mice. This chimeric γ-allergen protein platform may provide allergen-specific therapy with an enhanced safety profile that might be critical in situations such as food allergy. Overall, we have developed new therapeutic strategies that are antigen-specific and provide novel yet distinctive approaches for the potential therapy of human allergic disease.

Epitope-specific T-cell responses and allergic phenotypes: implications for T-cell peptide therapy

In the 1990s, elucidation of the primary amino acid sequence of several major allergens using molecular cloning techniques opened the door to T-cell epitope mapping studies. Such analyses underscored the complexity of the allergen-specific T-cell repertoire and the challenges to using allergen-derived peptides to identify epitope-specific differences associated with allergic and nonallergic responses. This review highlights important factors that may influence the nature of epitope-specific T-cell responses observed in vitro. These include the properties of the allergen, genetics of the host and selection of patients with defined allergic phenotypes based on serum antibody profiles and skin test reactivity. By taking these factors into account, T-cell epitope-specific differences associated with distinct allergic phenotypes can be identified. Observations at the T-cell epitope level undermine the Th1/Th2 paradigm as a model for the development of allergic versus nonallergic responses. Instead, they support the mounting data that point to a network of interactions between T helper cells and regulatory T cells, which controls the allergic response. The ability of peptides that localize to polypeptide chain 2 of the major cat allergen, Fel d 1, to preferentially induce interleukin-10 and interferon-γ is discussed. Mechanisms whereby specific allergen-derived peptides may modify the T-cell repertoire and influence the immune outcome are also outlined. Further investigation of allergen-derived T-cell epitopes is warranted in order to optimize the design of peptide vaccines for the treatment of allergic disease.

A chimeric human-cat Fcγ-Fel d1 fusion protein inhibits systemic, pulmonary, and cutaneous allergic reactivity to intratracheal challenge in mice sensitized to Fel d1, the major cat allergen

Co-aggregation of FcεRI with FcγRIIb can block FcεRI-mediated reactivity and Fc gamma:allergen chimeric proteins, by co-crosslinking FcγRIIb to allergen-specific IgE bound to the FcεRI can block allergen-specific reactivity. We evaluated whether a human cat chimeric fusion protein (GFD) composed of part of the human Ig G1 Fc fused to the major cat allergen (Fel d1) would function as allergen immunotherapy while not inducing acute allergic reactivity in mice sensitized to Fel d1. Injection of GFD 6 h prior to Fel d1 challenge acutely blocked systemic and skin reactivity to Fel d1 challenge while mice given subcutaneous immunotherapy with GFD at days 37, 38, and 39 showed inhibition of systemic, lung, and cutaneous reactivity to Fel d1 2 weeks later. GFD immunotherapy did not induce systemic reactivity. Overall, the Fcγ-Fel d1 chimeric fusion protein blocked Fel d1-induced IgE-mediated reactivity but did not induce in vivo mediator release on its own; suggesting that this approach using allergen combined with Fc gamma1 so as to achieve inhibitory signaling may provide an enhanced form of allergen immunotherapy.

Peptide Immunotherapy in Fel d 1-Sensitized HLA-DR1 Transgenic Mice is Associated with Increased IL-10 but Independent of TGFbeta and Foxp3 Expression

RATIONALE: In clinical studies, cat allergic individuals treated with intradermal administration of synthetic peptides containing T cell epitopes of the major cat allergen Feld1, experienced reduced sensitivity to allergen challenge in the skin, nose and lung. To investigate immunological mechanisms we developed a model of cat allergen sensitivity in HLA DRB1∗0101-transgenic FVB/N mice, devoid of murine MHC class II (I-Eα0/β0).

Rational design of hypoallergens applied to the major cat allergen Fel d 1

Allergen-specific immunotherapy is the only treatment for allergic disease providing long-lasting symptom relief. Currently, it is mainly based on the use of crude allergen extracts. The treatment may be improved by the use of genetically engineered allergens, hypoallergens, aiming at a more effective and safer therapy. The aim of this study was to provide a rational design of hypoallergen candidates for immunotherapy by using structural information and knowledge of B and T cell epitopes of an allergen. The three-dimensional structure of the major cat allergen Fel d 1 was systematically altered by duplication of selected T cell epitopes and disruption of disulphide bonds. Seven Fel d 1 derivatives were generated and screened for allergenic reactivity in comparison with recombinant Fel d 1 in competition-ELISA. The allergenicity was further evaluated in basophil activation experiments and T cell reactivity was assessed in a lymphoproliferation assay. Three out of seven Fel d 1 derivatives, with two duplicated T cell epitopes and one or two disulphide bonds disrupted, were carefully evaluated. The three derivatives displayed a strong reduction in allergenicity with 400-900 times lower IgE-binding capacity than recombinant Fel d 1. In addition, they induced a lower degree of basophil activation and similar or stronger T cell proliferation than recombinant Fel d 1. By a rational approach, we have constructed three Fel d 1 hypoallergens with reduced IgE-binding capacities and retained T cell reactivities. This strategy may be applied to any well-characterized allergen to improve immunotherapy for allergic patients.

A chimeric human-cat fusion protein blocks cat-induced allergy

Animal allergens are an important cause of asthma and allergic rhinitis. We designed and tested a chimeric human-cat fusion protein composed of a truncated human IgG Fcgamma1 and the major cat allergen Fel d1, as a proof of concept for a new approach to allergy immunotherapy. This Fcgamma-Fel d1 protein induced dose-dependent inhibition of Fel d1-driven IgE-mediated histamine release from cat-allergic donors' basophils and sensitized human cord blood-derived mast cells. Such inhibition was associated with altered Syk and ERK signaling. The Fcgamma-Fel d1 protein also blocked in vivo reactivity in FcepsilonRIalpha transgenic mice passively sensitized with human IgE antibody to cat and in Balb/c mice actively sensitized against Fel d1. The Fcgamma-Fel d1 protein alone did not induce mediator release. Chimeric human Fcgamma-allergen fusion proteins may provide a new therapeutic platform for the immune-based therapy of allergic disease.

Molecular Characterization of Major Cat Allergen Fel d 1: EXPRESSION OF HETERODIMER BY USE OF A BACULOVIRUS EXPRESSION SYSTEM

Fel d 1 is a major cat allergen inducing allergic rhinitis and asthma in sensitized individuals. It has a more complex structure when compared with other allergens and therefore expression of recombinant Fel d 1 has been considered a challenge. The present study shows for the first time that a Baculovirus expression system is able to produce an intact rFel d 1 molecule that is glycosylated and structurally equivalent to the natural cat allergen, nFel d 1. Enzymatic digestion of rFel d 1 and further analysis by use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) resulted in a complete coverage of the amino acid sequence of rFel d 1. In addition, the three disulfide bridges at the positions alpha70-beta7, alpha44-beta48, and alpha3-beta73 were verified. The N-glycan structure of rFel d 1 was investigated by a combination of MALDI-TOF MS and monosaccharide analysis by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAC). The N-glycosylation analyses of rFel d 1 refer to a pattern of glycoforms including core alpha1.3-fucosylation that is different from nFel d 1. Further characterization by use of human serum IgE, histamine release, and lymphocyte proliferation assays demonstrated that the immunological characteristics of rFel d 1 are similar to those of nFel d 1. Detailed characterization of both natural and recombinant allergens provides tools to explore immunological mechanisms associated with allergen sensitization and desensitization.

The effect of pet ownership on the risk of allergic sensitisation and bronchial asthma

An increasing volume of evidence suggests that early contact of children with the allergens of furred pets (especially those produced by cats) may determine a lower risk of developing allergic sensitisation to these materials. A possible explanation of this data is that an early inhalation of high levels of the major cat allergen Fel d 1 induces the production of IgG and IgG4 antibodies with a “protective” effect.Other authors have shown that the prevalence of allergic sensitisation to cats, in adults, is reduced in those patients exposed to the lowest and highest levels of the allergens. On the contrary, the risk of developing sensitisation to cats is significantly higher when the patients were exposed to intermediate levels of Fel d 1. Moreover, epidemiological studies have demonstrated a relatively low prevalence of cat allergy (about 10%) in some countries where rates of cat ownership are high.This data confirms the role of indirect exposure to pet allergens in inducing allergic sensitisation. Clothes of pet owners have been indicated as the carriers for the dispersal of these allergens in pet-free environments. However, it is important to point out that exposure of highly sensitised patients to relevant amounts of pet allergens (such as in a pet shows/shops) may determine a dramatic exacerbation of nasal and/or bronchial symptoms.

The effects of T cell peptides in patients sensitive to cats

SummarySynthetic peptides representing T cell epitopes of the major cat allergen Fel d 1 were administered by intradermal injection or inhalation to cat allergic asthmatic volunteers. Both routes of administration were associated with the induction of IgE‐independent, MHC‐restricted isolated late asthmatic reactions (LAR; prolonged bronchoconstriction initiating 2–4 hours after peptide challenge) in a proportion of individuals. Administration via the intradermal, but not the inhaled route, was associated with the induction of antigen‐specific hyporesponsiveness or “tolerance”, both in vivo and in vitro. Following intradermal peptide administration, the magnitude of both the early‐ and late‐phase skin reaction to intradermal challenge with whole allergen extract were significantly reduced. In vitro, proliferative responses of peripheral blood mononuclear cells (PBMC) were reduced together with both Th1 and Th2 cytokines. Production of IL‐10 was increased. LAR were not a pre‐requisite for the induction of tolerance. Hyporesponsiveness was transient but several months were required to return to basal reactivity.

A novel adjuvant-allergen complex, CBP-rFel d 1, induces up-regulation of CD86 expression and enhances cytokine release by human dendritic cells in vitro.

Allergen-specific immunotherapy is commonly performed with allergen extracts adsorbed to aluminium hydroxide (alum). The undesirable effects associated with the use of alum, including granuloma formation at the site of injection and stimulation of T helper 2 (Th2) cytokine production, has generated interest in alternative allergen carriers, one being carbohydrate-based particles (CBPs). Here, we have investigated the in vitro effects of the recombinant major cat allergen Fel d 1 (rFel d 1) coupled to CBPs (CBP-rFel d 1) on human monocyte-derived dendritic cells (MDDCs) obtained from healthy blood donors. A majority of the CD1a(+) MDDCs internalized fluorescein isothiocyanate-labelled CBP-rFel d 1, as demonstrated by flow cytometry and confocal laser-scanning microscopy. Furthermore, an up-regulation of the expression of the costimulatory molecule, CD86, on the MDDCs was induced by CBP-rFel d 1, but not by rFel d 1 or CBPs alone. Finally, three- and fourfold increases in the release of interleukin-8 and tumour necrosis factor-alpha, respectively, were observed when MDDCs were cultured in the presence of CBP-rFel d 1. Altogether, our results indicate that the use of CBPs as an allergen carrier and adjuvant is a promising candidate for the improvement of allergen-specific immunotherapy.

A Novel Adjuvant Allergen Complex, CBP‐Fel d 1, Induces Upregulation of CD86 and Cytokine Release in Human Dendritic Cells

Allergen‐specific immunotherapy (SIT) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). Drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. An alternative carrier is 2 μm carbohydrate‐based particles (CBPs). In mouse, allergen‐coupled CBPs have been demonstrated to skew the allergen‐specific immune response towards a Th1‐like activity (Grönlund et al. Immunology, 2002). We here coupled the recombinant major cat allergen Fel d 1 to CBPs (CBP‐Fel d 1) by cyanogen‐bromide activation, resulting in covalent binding. The effect of CBP‐Fel d 1 on monocyte‐derived dendritic cells (MDDCs) from healthy human blood donors was studied. We found that the majority of the CD1a+ MDDCs were capable of taking up FITC‐labelled CBP‐Fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. Furthermore, incubation with CBP‐Fel d 1 resulted in an upregulation of the costimulatory molecule CD86 on the MDDCs, which was not observed with Fel d 1 or CBPs alone. Finally, CBP‐Fel d 1 induced a fivefold increase in the release of the pro‐inflammatory cytokine tumour necrosis factor (TNF)‐α and a fourfold increase in the release of the chemokine interleukin‐8 from MDDCs. Taken together, the effects CBPs possess make them interesting as novel allergen carriers for SIT.

Isolated late asthmatic reactions provoked by inhalation of T-cell peptide epitopes are associated with bronchial mucosal infiltration of CD4+ T cells and increased airway hyperresponsiveness

Rationale We previously demonstrated the induction of IgE-independent, MHC-restricted, isolated late asthmatic reactions (LARs) by intradermal (i.d.) administration of T-cell peptide epitopes derived from the major cat allergen, Fel d 1 (J Exp Med 1999;189:1885-94). A bronchoscopy study investigating i.d. peptide-induced LARs found neither cellular infiltration nor elevated concentrations of inflammatory mediators. We subsequently observed that inhalation of Fel d 1 peptides induced isolated LARs with associated sputum eosinophilia, suggesting that local peptide delivery might lead to more superficial changes. Methods A randomized, placebo-controlled, crossover study was performed involving bronchial biopsies and bronchoalveolar lavage fluids from 12 subjects who developed LARs 6 hours after Fel d 1 peptides inhalation (responders) and 12 subjects who showed no clinical response (non-responders). Histamine PC 20 was performed at baseline and 1 week after each challenge. Results Peptide-induced LARs were associated with a significant elevation in bronchial biopsy CD4+ count (p=0.034), whereas there was no change in non-responders. A trend towards increased total CD3+ cells was also observed (p=0.052), that was significantly different (p=0.005) from the non-responder group. A trend for increased eosinophils was noted in responders with Congo Red stain (p=0.064) and anti-MBP (p=0.067). No differences in cell numbers in BALF were identified. A significant increase in airway hyperreponsiveness was identified in responders 7 days after peptide challenge compared with diluent (p=0.001002), that was not seen in nonresponders (p=0.85). Conclusion Thus, inhalation of T-cell epitope peptides induces isolated LARs, associated with bronchial infiltration of CD4+ T-cells, and increased airway hyperresponsiveness, but only modest eosinophilia.

Immunological characteristics of heterodimeric recombinant Fel d 1

Rationale Exposure and sensitization to domestic cat ( Felis domesticus) is a significant risk factor for developing allergic rhinitis and asthma. Earlier studies have shown that majority of the cat allergic patients develop allergic responses against major cat allergen Fel d 1. Several attempts have been made to produce of recombinant (r) Fel d 1 as heterodimer. We have recently shown that baculovirus expression system is able to produce rFel d 1 that has compatible characteristics to nFel d 1. Methods In the present study an immunological characterization of rFel d 1 in comparison to the purified nFel d 1 and cat allergen extract (IMP Fel d, ALK-Abello') was performed by use of IgE inhibition, lymphocyte proliferation and basophile histamine release assay(s). Results In inhibition assay against cat allergen extract, natural and rFel d 1 were able to inhibit 87% of the binding of cat allergen specific serum IgE antibodies. The basophile histamine release responses with rFel d 1 and nFel d 1 showed to be in line with the responses given by the cat allergen extract. Comparable T-cell responses towards natural and rFel d 1 was obtained in lymphocyte proliferation assay and when the response of Fel d 1-specific T-cell lines was investigated even though nFel d 1 seemed to be recognized more frequently. Conclusions These results show that heterodimeric rFel d 1 elicits immunological responses that are comparable to nFel d 1. The present study demonstrates that recombinant allergen(s) with well defined biochemical and immunological characteristics are feasible for development of therapeutic vaccines.

Allergen peptide immunotherapy in humans inhibits allergen-induced proliferation of CD4+ T cells but does not affect suppressive activity of CD4+CD25+ T cells

Rationale Our group has previously described immunotherapy using short peptides derived from the major cat allergen Fel d 1, to target CD4+ T cell activation without cross linking IgE. We previously reported that this treatment inhibited allergen late responses, and peripheral blood T cell activation, with increased IL-10 secretion in vitro, and infiltration of CD25+ T cells into allergen challenged skin. In addition we have demonstrated reduced suppressive activity of CD4+CD25+ regulatory T cells in atopic donors. Here, we examined the effect of peptide immunotherapy on regulation by CD4+CD25+ regulatory T cells. Methods 16 patients were entered into a double-blind placebo-controlled trial of an escalating dose regimen of intradermal mix peptides derived from Fel d 1 (total cumulative dose of 291 μg). Peripheral blood CD4+ and CD4+CD25+ T cells were isolated before and after treatment and proliferation to cat allergen extract was measured by thymidine incorporation at 7 days. Results Peptide immunotherapy resulted in significant reduction in the nasal response to allergen challenge (p<0.05) when compared to the placebo-treated group, and this was accompanied by a reduction in cat allergen-induced proliferation of CD4+ T cells (p<0.05). However, there was no change in suppression of cat allergen induced proliferation by CD4+CD25+ T cells. Conclusions Peptide immunotherapy reduces both clinical and CD4+ T cell responsiveness to allergen, but this is not due to increased suppression by CD4+CD25+ T cells.

Animal models of allergic and inflammatory conjunctivitis.

Allergic eye diseases are complex inflammatory conditions of the conjunctiva with an increasing prevalence and incidence. The diseases are often concomitant with other allergic diseases such as allergic rhinitis, atopic dermatitis and allergic asthma. Despite the disabling and prominent symptoms of ocular allergies, they are less well studied and further insights into the molecular basics are still required. To establish new therapeutic approaches and assess immunological mechanisms, animal models of ocular allergies have been developed in the past years. The major forms of allergic ocular diseases, seasonal and perennial allergic conjunctivitis, vernal and atopic keratoconjunctivitis and giant papillary conjunctivitis, each have different pathophysiological and immunological components. In contrast to these distinct entities, the current animal models are based on the sensitization against a small number of allergens such as ovalbumin, ragweed pollen or major cat allergens and consecutive challenge. Different animal species have been used so far. Starting with guinea-pig models of allergic conjunctivitis to assess pharmacological aspects, new models including rats and mice have been developed which mimic major features of ocular allergy. The presently preferred species for the investigation of the immunological basis of the disease is represented by murine models of allergic conjunctivitis. In the future, combined ocular, nasal and aerosolic challenges with allergens may provide a model of allergy that encompasses simultaneously the target organs eye, nose and airways with conjunctivitis, rhinitis and asthma.

Formation of Disulfide Bonds and Homodimers of the Major Cat Allergen Fel d 1 Equivalent to the Natural Allergen by Expression in Escherichia coli

Dander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy. Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful. In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced. A synthetic gene coding for direct fusion of the Fel d 1 chain 2 N-terminally to chain 1 was constructed by overlapping oligonucleotides in PCR. Escherichia coli expression resulted in a non-covalently associated homodimer with an apparent molecular mass of 30 kDa defined by size exclusion chromatography. Furthermore, each 19,177-Da subunit displayed a disulfide pattern identical to that found in the natural Fel d 1, i.e. Cys3(1) Cys73(2), Cys44(1)-Cys48(2), Cys70(1)-Cys7(2), as determined by electrospray mass spectrometry after tryptic digestion. Circular dichroism analysis showed identical folds of natural and recombinant Fel d 1. Furthermore, recombinant Fel d l reacted specifically with serum IgE, inducing expression of CD203c on basophils and lymphoproliferative responses in cat-allergic patients. The results show that the overall fold and immunological properties of the recombinant Fel d 1 are very similar to those of natural Fel d 1. Moreover, the recombinant Fel d 1 construct provides a tool for defining the three-dimensional structure of Fel d 1 and represents a reagent for diagnosis and allergen-specific immunotherapy of cat allergy.

The Crystal Structure of the Major Cat Allergen Fel d 1, a Member of the Secretoglobin Family

The domestic cat (Felis domesticus) is one of the most important causes of allergic asthma worldwide. The dominating cat allergen, Fel d 1, is composed of two heterodimers. Recently, it has been shown that recombinant Fel d 1, consisting of chain 2 and chain 1 fused together without additional linker, has immunological properties indistinguishable from the natural heterodimeric protein. Herein, we report the crystal structure of recombinant monomeric Fel d 1 at 1.85-A resolution, determined by multi-wavelength anomalous diffraction using selenomethionine substituted protein. Fel d 1 is an all-helical protein and consists of eight helices. The two halves of the recombinant Fel d 1 molecule, corresponding to the wild-type Fel d 1 chains, are very similar in three-dimensional structure, despite the lack of significant sequence identity. The structure of the Fel d 1 presents a striking similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties. An internal, asymmetric cavity is formed in the Fel d 1 that could bind an endogenous ligand. The distribution of residues lining this cavity suggests that such a ligand must be amphipathic. The structure of Fel d 1 displays the localization of three previously defined Fel d 1 IgE epitopes on the surface of the protein. The three-dimensional structure provides a framework for rational design of hypoallergenic mutants aimed for treatment of cat allergy.

Sensitization to cat allergens in non–cat owner patients with respiratory allergy

Cats represent one of the most important sources of indoor allergens. The sensitization rate can reach up to 60% in western countries. Keeping cats indoors is uncommon in big cities in Turkey, but cats living in the streets are common. To investigate the prevalence of sensitization to cats in patients with respiratory allergy from Izmir, Turkey, and its relationship to home cat allergen levels. A total of 387 patients (70.8% female; mean age, 34.3 years) with respiratory allergic diseases (rhinitis and/or asthma) were included in this study. Skin prick test to cat was performed. House dust samples were collected from the living room of 25 patients and 14 healthy subjects. The major cat allergen (Fel d 1) levels were measured by Dustscreen. Fel d 1 levels given by the manufacturer were as follows: 0.05, 0.13, 0.40, 1.1, and 6.2 mU/mL. The prevalence of cat sensitivity was 44.7% (n = 173). Only 6 patients (1.6%) had a history of feeding a cat in their houses. Thirty-six (92%) of 39 houses had detectable levels of cat allergen (mean Fel d 1 level, 2.24 +/- 2.69 mU/mL). The mean Fel d 1 levels were 1.58 +/- 2.51 mU/mL in the healthy group, 1.91 +/- 2.61 mU/mL in the asthmatic group, and 3.26 +/- 2.85 mU/mL in the group with allergic rhinitis (P = 0.12). The prevalence of cat sensitivity in patients who had 1.1 mU/mL of Fel d 1 in their homes was 57.1%. This rate was five times lower (11.1%) in patients who had the highest Fel d 1 level (6.2 mU/mL) in their homes. The prevalence of cat sensitivity in Izmir, where cats are generally not kept within homes, is as high as in western countries. The sampled houses have measurable levels of Fel d 1 even in the absence of indoor cats. High prevalence of cat sensitivity in Izmir is probably due to indirect exposure.

Production, crystallization and preliminary crystallographic study of the major cat allergen Fel d 1.

The domestic cat (Felis domesticus) is an important cause of allergic disease worldwide. The major cat allergen 1 (Fel d 1) has been expressed in Escherichia coli, purified and refolded in a soluble form. Crystals of Fel d 1 were obtained in 13% 2-methyl-2,4-pentanediol, 0.1 M sodium acetate pH 4.8. The Fel d 1 crystals belong to space group P2(1), with unit-cell parameters a = 43.3, b = 51.5, c = 67.7 A, and diffract to 1.9 A resolution.

Characterization of two forms of mouse salivary androgen-binding protein (ABP): implications for evolutionary relationships and ligand-binding function.

Mouse salivary androgen-binding protein (ABP) is a member of the secretoglobin family produced in the submaxillary glands of house mice (Mus musculus). We report the cDNA sequences and amino acid sequences of the beta and gamma subunits of ABP from a mouse cDNA library, identifying the two subunits by their pIs and molecular weights. An anomalously high molecular weight of the alpha subunit is likely due to glycosylation at a single site. A phylogenetic comparison of the three subunits of ABP with the chains of other mammalian secretoglobins shows that ABP is most closely related to mouse lachrymal protein and to the major cat allergen Fel dI. An evaluation of the most conserved residues in ABP and the other secretoglobins, in light of structural data reported by others [Callebaut, I., Poupon, A., Bally, R., Demaret, J.-P., Housset, D., Delettre, J., Hossenlopp, P., and Mornon, J.-P. (2000) Ann. N.Y. Acad. Sci. 923, 90-112; Pattabiraman, N., Matthews, J., Ward, K., Mantile-Selvaggi, G., Miele, L., and Mukherjee, A. (2000) Ann. N.Y. Acad. Sci. 923, 113-127], allows us to draw conclusions about the critical residues important in ligand binding by the two different ABP dimers and to assess the importance of ligand binding in the function of the molecule. In addition to the cDNAs, which represent those of the musculus subspecies of Mus musculus, we also report the coding regions of the beta and gamma subunit cDNAs from two other mouse inbred strains which represent the other two subspecies: M. musculus domesticus and M. musculus castaneus. The high nonsynonymous/synonymous substitution rate ratios (K(a)/K(s)) for both the beta and gamma subunits suggest that these two proteins are evolving under strong directional selection, as has been reported for the alpha subunit [Hwang, J., Hofstetter, J., Bonhomme, F., and Karn, R. (1997) J. Hered. 88, 93-97; Karn, R., and Clements, M. (1999) Biochem. Genet. 37, 187-199].