Cloning of Cockroach Allergen, Bla g 4, Identifies Ligand Binding Proteins (or Calycins) as a Cause of IgE Antibody Responses

Abstract

An allergen cloned from a Blattella germanica (German cockroach) cDNA library, encoded a 182-amino acid protein of 20,904 Da. This protein, designated B. germanica allergen 4 (Bla g 4), was expressed as a glutathione S-transferase fusion protein in Escherichia coli and purified by affinity chromatography and high-performance liquid chromatography. The prevalence of serum IgE antibody to recombinant Bla g 4 in 73 cockroach allergic patients with asthma ranged from 40% (antigen binding radioimmunoassay) to 60% (plaque immunoassay). Cockroach allergic patients gave positive intradermal skin tests to recombinant Bla g 4 at concentrations of 10 ؊3 -10 ؊5 g/ml, whereas non-allergic controls, or cockroach allergic patients with no detectable serum IgE antibody to Bla g 4, gave negative skin tests to 1 g/ml. Polymerase chain reaction and Southern analysis identified a 523-base pair DNA encoding Bla g 4 in both B. germanica and Periplaneta americana (American cockroach). However, Northern analysis showed that mRNA encoding Bla g 4 was transcribed in B. germanica but not in P. americana, suggesting that allergen expression was species specific. Sequence similarity searches showed that Bla g 4 was a ligand binding protein or calycin and unexpectedly revealed that this family contained several important allergens: ␤-lactoglobulin, from cow milk, and rat and mouse urinary proteins. Although the overall sequence homology between these proteins was low (ϳ20%), macromolecular modeling techniques were used to generate two models of the tertiary structure of Bla g 4, based on comparisons with the x-ray crystal coordinates of bilin binding protein and rodent urinary proteins. The results show that members of the calycin protein family can cause IgE antibody responses by inhalation or ingestion and are associated with asthma and food hypersensitivity.

The lipocalin protein family: structure and function

“…To what extent are the apparent biological properties of individual lipocalins mediated directly by the protein structure itself (ligand binding [35,83,84], complexation [35,44,50] and receptor interaction [61,136]), by post-translational modifications (glycosylation state [99,109], chromophores [51], or membrane anchors [5]), or by combination of the two, such as the formation of disulphide linked complexes [41,44,49] ? Are the allergenic properties of several, evolutionarily distinct lipocalins [6] merely a co-incidental property of their individual structures, or do they result from conserved structural features, such as the ability to interact with cellular receptors, common to the family ? Can the immunosuppressive and cell-regulation functions of the family be linked to macromolecular complexation, perhaps through the modulation of proteinase-mediated cell signalling [137], either by the down-regulation of protease inhibitors [50] or by the direct inhibition of proteases themselves [42] ?…”

“…This includes several examples from arthropods : butterfly insecticyanin, grasshopper lazarillo [5], cockroach Bla g 4 protein [6] and lobster crustacyanin [7] ; and there is evidence to suggest that carotenoprotein lipocalins may also be present in species from the phylum Coelenterata [8]. Other calycins, FABPs and avidins are also present in both vertebrates and invertebrates, including Arthropoda and Coelentarata, but thus far there is only one example from prokaryotes, namely streptavidin.…”

“…However, evidence linking the observation of immunosuppression in the test tube and effects in the normal and pathological functioning of the immune system is lacking for the lipocalins ; further work is need to establish this link and demonstrate their role in i o. In passing, it is worth noting that several lipocalins have been shown to give rise to inflammation [6] : bovine β-lactoglobulin, rodent urinary proteins and cockroach Bla g 4 protein are all allergens, the latter specifically associated with bronchial asthma.…”

IgE reactivity of tandem repeats derived from cockroach allergen, Bla g 1

“…The results suggest that rBla g 1 is a good candidate for studies of allergy diagnosis, when used together with other recombinant cockroach allergens, such as Bla g 2, Bla g 4 and Bla g 5. We have estimated that a cocktail of these four allergens could diagnose > 95% of cockroach allergic patients [9]. Natural cockroach allergenic products contain large amounts of nonallergenic proteins, are prone to form precipitates and may contain proteolytic enzymes.…”

“…In the US, this problem is particularly important in inner city areas where infestation by the German cockroach (Blattella germanica) is common [2][3][4][5]. Several German cockroach allergens have been cloned including Bla g 1, Bla g 2, Bla g 4, Bla g 5 and Bla g 6 [6][7][8][9][10]. Bla g 1 is the only German cockroach allergen that shows antigenic cross-reactivity with an American cockroach (Periplaneta americana) allergen, Per a 1 [7,[11][12][13][14].…”