Elsevier

Molecular Immunology

Volume 37, Issues 12–13, 1 September 2000, Pages 789-798
Molecular Immunology

Unique and shared IgE epitopes of Hev b 1 and Hev b 3 in latex allergy

https://doi.org/10.1016/S0161-5890(00)00095-XGet rights and content

Abstract

Of the several latex proteins cloned and expressed, the rubber elongation factor, Hev b 1, and the closely related Hev b 3, represent two major allergens associated with latex allergy. Although both allergens demonstrated IgE binding with sera from latex allergic patients, it was not known whether these two molecules shared any epitopes. Hence, in the present study using health care workers (HCW) and spina bifida (SB) patients with latex allergy, we investigated the IgE binding epitopes in Hev b 1 and Hev b 3. Recombinant Hev b 1 and Hev b 3 were expressed in a prokaryotic expression system, while overlapping decapeptides of Hev b 1 and Hev b 3 were synthesized on derivatized cellulose membrane. Eight IgE binding epitopes for Hev b 1 and eleven for Hev b 3 were identified using sera from latex allergic patients with SB. On further analysis of synthetic peptides encompassing these epitopes, similar IgE antibody reactivity was demonstrated with three Hev b 1 epitopes b1E3, b1E5, b1E6 and two Hev b 3 epitopes; b3E10 and b3E 11. For Hev b 1, a unique IgE binding epitope was identified in the region of amino acid residues 16–25. In competitive ELISA, peptides bIE2 and bIE4 together inhibited 58% of IgE binding of Hev b 1, while b3E5 showed 22% inhibition in the IgE binding of Hev b 3. The results of the present study suggest that the understanding of linear and conformational IgE epitopes in the major latex allergens may provide better insight into the structure–function relationship of the allergens, and may lead to the development of better patient care and management strategies in latex allergy.

Introduction

Natural rubber latex (NRL) is widely used in the manufacturing of medical devices and a number of domestic and industrial utility articles. Since the first clinical report of latex allergy, the IgE mediated type 1 allergy to NRL proteins has become an important medical and occupational problem (Nutter, 1979, Kelly et al., 1994, Kurup et al., 1995, Turjanmaa et al., 1996). Severe allergic reactions are frequently reported to occur perioperatively in children with spina bifida (SB). Recent studies have shown that as many as 8% of health care workers (HCW) and 28–67% of SB patients are sensitized to latex with an anaphylactic risk of over 500-fold compared to the general populations (Kelly et al., 1994, Slater, 1994, Turjanmaa et al., 1996).

Latex hypersensitivity reactions occur shortly after exposure to the allergen. The specific IgE antibodies bound to the high affinity FcεRI receptors on mast cells interact with allergens (through IgE binding epitopes) resulting in activation of mast cells and release of various pharmacological mediators (Abbas et al., 1997). These mediators, such as histamine, leukotrienes and prostaglandins could be responsible for the manifestation of the clinical symptoms of latex allergy (Breiteneder and Scheiner, 1998). Thus, the IgE binding epitopes of major latex allergens may have an important role in the immune mechanism of latex hypersensitivity (Chen et al., 1996). Some of these epitopes may also have potential possibilities in controlling the disease by immunotherapy (Kraft et al., 1998, Posch et al., 1998, Breiteneder and Scheiner, 1998). However, at the present time, our understanding of the structure–function relationship of the major latex allergens is not complete, although recent studies using overlapping synthetic peptides have provided information on the major B-cell epitopes for Hev b 1, Hev b 5 and Hev b 6 (Chen et al., 1996, Banerjee et al., 1997, Beezhold et al., 1997, Slater et al., 1999).

Hev b 1, identified as a 14.6 kD water insoluble protein, is associated mainly with large rubber particles (>350 nm diameter) and is involved in rubber biosynthesis (Czuppon et al., 1993). On the other hand, Hev b 3, isolated from small rubber particles (>70 nm), is highly unstable and frequently degrades into several polypeptides of lower molecular weight (from 24 to about 5 kD) even when stored at −20°C (Yeang et al., 1996). Previous studies have emphasized the involvement of these two latex proteins in both humoral and cell mediated immune responses in latex sensitized patients (Alenius et al., 1995, Alenius et al., 1996, Lu et al., 1995, Chen et al., 1997, Yeang et al., 1998, Wagner et al., 1999, Johnson et al., 2000).

In view of the prevalence and severity of latex hypersensitivity reactions in both children and adults and involvement of B cell epitopes in the clinical manifestation of the disease, we focused our attention on defining the specific structure–function relationship of Hev b 1 and Hev b 3. Recombinant allergens were overexpressed and their structural and functional similarities were compared. Overlapping peptides were synthesized on derivatized cellulose membranes and the IgE binding epitopes of Hev b 1 and Hev b 3 were studied using sera from patients with SB. The identified SB serum reacting IgE binding epitopes were further evaluated using sera from HCW. The results indicate that the common IgE binding epitopes are located near the C-terminal regions of Hev b 3, while for Hev b 1 the specific epitope binding to SB patients were seen at the amino acid residues 16–25. Several shared and unique epitopes in Hev b 1 and Hev b 3 were identified and such information may be of value in the reliable diagnosis of latex hypersensitivity and in understanding the pathophysiology of the disease.

Section snippets

Subjects

Serum samples from 20 subjects with latex allergy, ten HCW and ten SB patients, were used for the study. All of the latex allergic subjects were diagnosed by clinical history, immediate wheal and flare skin test reactivity to a latex glove extract, or to an allergen extract prepared from natural rubber latex (NRL) (Lu et al., 1995). A control group of ten subjects with no clinical evidence of latex allergy or reactivity to Hev b 1 and Hev b 3 from HCW and SB groups were also included in the

Recombinant Hev b l and Hev b 3 share similar functional properties

Purified recombinant Hev b 1 and Hev b 3 overexpressed in pET system demonstrated proteins at the molecular weight regions of 14–15 kD and 23–25 kD respectively (Fig. 1). In ELISA, both proteins showed distinct IgE and IgG binding with sera from latex allergy patients with SB and HCW (data not shown). The competitive ELISA studies using individual sera from SB and HCW groups are shown in Fig. 2A and B. Three out of four sera preincubated with rHev b 3 at a concentration of 20 μg could inhibit

Discussion

The development of clinical latex allergy in individuals involves a series of interactions between T and B-lymphocytes. The mediators released from activated T lymphocytes and cognate interaction between B cells bearing appropriate antigen specific surface immunoglobulins and allergen specific T-cells lead to isotype switching and generation of allergen specific IgE (Abbas et al., 1997). The specific IgE then binds to the high affinity FcεR1 on mast cells and basophils and finally the

Acknowledgements

This study was supported in part by Veterans Affairs Medical Research, by the Ansell Corporation, Children's Hospital Foundation, by Regent Medical. The technical assistance of Laura Castillo is gratefully acknowledged.

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