RESUMEN
INTRODUCTION: Characterization of the complete IgE binding spectrum of cat allergens is important for the development of improved diagnosis and effective immunotherapeutics. While Fel d 1 remains unchallenged as the major cat allergen, we now report the isolation of two new allergens capable of binding similar concentrations of IgE in the allergic sera of some individuals. MATERIALS AND METHODS: Cat tongue and submandibular salivary gland cDNA libraries were screened by DNA hybridisation and IgE immunoassay. The isolated DNA fragments were sub-cloned into an E. coli expression system and the IgE reactivity was examined with human cat-allergic sera using a DELFIA IgE quantitation assay. RESULTS: Fel d 7, an 18 kDa von Ebner gland protein Can f 1 homologue, was isolated from the tongue library. Fel d 8, a 24-kDa latherin-like protein with homology to Equ c 5, was isolated from the submandibular library. The frequency of IgE binding of cat-allergic sera to recombinant Fel d 1, 7 and 8 was 60.5, 37.6 and 19.3%, respectively. Inhibition studies indicated some IgE binding cross-reactivity between Fel d 7 and dog dander extracts. DISCUSSION: The study reports the isolation and characterization of two new cat allergens. The isolation of these allergens provides the opportunity to determine the role that IgE binding proteins other than Fel d 1 play in cat-allergic disease. For cat-allergic individuals with moderate to mild rhinoconjunctivitis these allergens may play a more important role in the manifestation of their allergic disease.
Asunto(s)
Alérgenos/aislamiento & purificación , Gatos/inmunología , Lipocalina 1/aislamiento & purificación , Proteínas y Péptidos Salivales/aislamiento & purificación , Alérgenos/genética , Alérgenos/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Clonación Molecular , Inmunoglobulina E/sangre , Lipocalina 1/genética , Lipocalina 1/inmunología , Datos de Secuencia Molecular , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Proteínas y Péptidos Salivales/inmunología , Alineación de SecuenciaRESUMEN
Tear lipocalin and beta-lactoglobulin are members of the lipocalin superfamily. They have similar tertiary structures but unusually low overall sequence similarity. Non-native helical structures are formed during the early stage of beta-lactoglobulin folding. To address whether the non-native helix formation is found in the folding of other lipocalin superfamily proteins, the folding kinetics of a tear lipocalin variant were investigated by stopped-flow methods measuring the time-dependent changes in circular dichroism (CD) spectrum and small-angle X-ray scattering (SAXS). CD spectrum showed that extensive secondary structures are not formed during a burst-phase (within a measurement dead time). The SAXS data showed that the radius of gyration becomes much smaller than in the unfolded state during the burst-phase, indicating that the molecule is collapsed during an early stage of folding. Therefore, non-native helix formation is not general for folding of all lipocalin family members. The non-native helix content in the burst-phase folding appears to depend on helical propensities of the amino acid sequence.
Asunto(s)
Lactoglobulinas/metabolismo , Lipocalina 1/genética , Lipocalina 1/metabolismo , Secuencia de Aminoácidos , Dicroismo Circular , Escherichia coli/genética , Humanos , Cinética , Lactoglobulinas/química , Lipocalina 1/química , Lipocalina 1/aislamiento & purificación , Datos de Secuencia Molecular , Mutación Puntual , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Alineación de Secuencia , Urea/metabolismo , Difracción de Rayos XRESUMEN
PURPOSE: A number of studies have shown that the levels of some proteins in the aqueous humor (AH) are altered and correlate with the mechanisms or prognosis of many eye diseases. To identify the possible mechanisms that lead to the development of wet age-related macular degeneration (AMD), a proteomic analysis of the AH composition from wet AMD patients was performed and compared with that from non-AMD cataract patients. EXPERIMENTAL DESIGN: Six wet AMD and six non-AMD cataract patients were enrolled. A proteomic approach which included two-dimensional electrophoresis coupled with MS and bioinformatics methods were used to identify AH proteins with altered expression in wet AMD compared with non-AMD patients. An ELISA was used to validate the proteomic results. RESULTS: We separated 78 protein spots and identified 68 that were differently expressed in the wet AMD group and controls. Numerous proteins identified in this study are implicated in inflammation, apoptosis, angiogenesis, and oxidative stress. CONCLUSIONS AND CLINICAL RELEVANCE: The AH protein composition was significantly different between wet AMD and non-AMD patients. The proteins identified in this study may be potential biomarkers of wet AMD development and might play a role in the mechanisms of wet AMD.