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Cycloproprene fatty acids in Gnetum gnemon (L.) seeds and leaves

Berry SK

J Sci Food Agric, 1980 Jul;31(7):657-62.
PMID: 6779057
Matched MeSH terms: Plant Proteins/analysis
Studies on the Indonesian Antiaris toxicaria sap

Fujimoto Y, Suzuki Y, Kanaiwa T, Amiya T, Hoshi K, Fujino S

J. Pharmacobio-dyn., 1983 Feb;6(2):128-35.
PMID: 6306201

The present research is on a milky sap obtained from the Antiaris toxicaria tree (Moraceae) which is called Upas or Ipoh in Indonesia. The crude sap was administered to anesthetized rats, and changes in electrocardiogram (ECG) and systemic blood pressure was observed. Biologically and pharmacologically active components were extracted from the crude sap by means of water-acetone solution. Based on the strength of chemical qualitative detection tests of the sap extract (SE), cardiac glycosides are supposed to be the main components. The SE inhibited the Na+-, K+-ATPase (EC 3.6.1.3) which was partially purified from guinea pig heart muscle. When the SE and, concurrently, authentic ouabain were applied to isolated frog heart muscles, the fall of twitch tension was observed after the increased tension on mechanograms. These facts suggest that the main components of the milky sap are cardiac glycosides, and glycosides affect Na+, K+-ATPase activity of muscle membrane and heart muscle contraction.

Matched MeSH terms: Plant Proteins/analysis
The rubber elongation factor of rubber trees (Hevea brasiliensis) is the major allergen in latex

Czuppon AB, Chen Z, Rennert S, Engelke T, Meyer HE, Heber M, et al.

J Allergy Clin Immunol, 1993 Nov;92(5):690-7.
PMID: 8227860

BACKGROUND: Allergy to latex-containing articles is becoming more and more important because it can result in unexpected life-threatening anaphylactic reactions in sensitized individuals.
METHODS: A protein of 58 kd with an isoelectric point of 8.45 was purified from raw latex and from latex gloves and identified as the major allergen, completely blocking specific IgE antibodies in the serum of latex-sensitized subjects. The allergen is a noncovalent homotetramer molecule, in which the 14.6 kd monomer was identified, by amino acid composition and sequence homologies of tryptic peptides, to be the rubber elongation factor found in natural latex of the Malaysian rubber tree.
RESULTS: Competitive immunoinhibition tests showed that the starch powder covering the finished gloves is the airborne carrier of the allergen, resulting in bronchial asthma on inhalation. The purified allergen can induce allergic reactions in the nanogram range.
CONCLUSION: The identification of the allergen (Hev b I) may help to eliminate it during the production of latex-based articles in the future.

Matched MeSH terms: Plant Proteins*
Characterization of a monoclonal antibody against latex protein associated with latex allergy

Kurup VP, Kumar A, Kelly KJ, Fink JN

J Allergy Clin Immunol, 1993 Nov;92(5):638-43.
PMID: 8227854 DOI: 10.1016/0091-6749(93)90005-z

Several hybridomas were produced against antigens extracted from the latex sap of Hevea brasiliensis. One of the monoclonal antibodies (LA-E3) reacted with antigens demonstrating binding to patient sera on crossed enzyme immunoelectrophoresis. This monoclonal antibody reacted with 2 of 10 glove extracts studied and with both Malaysian and Indian latex plant extracts. The antigens purified with monoclonal antibody affinity chromatography demonstrated specific IgE in the sera of patients with latex allergy as determined by ELISA. This monoclonal antibody can thus be utilized to obtain reliable antigens useful in the diagnosis of latex sensitivity, although additional antigens will likely be necessary to enhance sensitivity and specificity.

Matched MeSH terms: Plant Proteins/immunology*
Precipitation of Hevea brasiliensis latex proteins with trichloroacetic acid and phosphotungstic acid in preparation for the Lowry protein assay

Yeang HY, Yusof F, Abdullah L

Anal Biochem, 1995 Mar 20;226(1):35-43.
PMID: 7785777

Many proteins derived from the latex of Hevea brasiliensis that remain soluble in trichloroacetic acid (TCA) can be precipitated by phosphotungstic acid (PTA). A combination of 5% TCA and 0.2% PTA precipitates a wide range of proteins effectively even when they are present in low concentrations (below 1 microgram ml-1). In addition to its protein purification function, acid precipitation also increases the sensitivity of the subsequent protein assay by allowing the test sample to be concentrated. Another advantage of protein precipitation by TCA and PTA is that very small amounts of protein (of the order of 10 micrograms) can be repeatably recovered without the use of precipitate-bulking agents such as sodium deoxycholate. This general procedure of protein purification and concentration is simple and rapid, but the use of PTA may not be fully compatible with the Bradford protein assay. A modified Lowry microassay is described which enables about 3 micrograms ml-1 to be quantitated at the photometric absorbance of 0.05. When used in conjunction with protein concentration by precipitating with TCA/PTA, approximately 0.4 microgram ml-1 protein present in 6 ml of solution can be assayed.

Matched MeSH terms: Plant Proteins/analysis*; Plant Proteins/isolation & purification
The 14.6 kd rubber elongation factor (Hev b 1) and 24 kd (Hev b 3) rubber particle proteins are recognized by IgE from patients with spina bifida and latex allergy

Yeang HY, Cheong KF, Sunderasan E, Hamzah S, Chew NP, Hamid S, et al.

J Allergy Clin Immunol, 1996 Sep;98(3):628-39.
PMID: 8828541 DOI: 10.1016/s0091-6749(96)70097-0

Two major water-insoluble proteins are located on the surface of rubber particles in Hevea brasiliensis latex. A 14.6 kd protein (Hev b 1), found mainly on large rubber particles (> 350 mm in diameter), and a 24 kd protein (Hev b 3), found mainly on small rubber particles (average diameter, 70 nm), are recognized by IgE from patients with spina bifida and latex allergy. Although Hev b 1 (also called the rubber elongation factor [REF]) has previously been reported as a major latex allergen, this conclusion has been disputed on the basis of results from other studies. The allergenicity of Hev b 1 is verified in this study by testing the recombinant protein generated from its gene. Because allergenicity is confined to patients with spina bifida and not observed in adults sensitive to latex, it is not a major latex allergen. The identification of Hev b 3 as another allergen originating from rubber particles is confirmed by immunogold labeling and electron microscopy. Observations with the monoclonal antibody USM/RC2 developed against Hev b 3 show that the protein has a tendency to fragment into several polypeptides of lower molecular weight (from 24 kd to about 5 kd) when stored at -20 degrees C. There is also indication of protein aggregation from the appearance of proteins with molecular weights greater than 24 kd. Fragmentation of Hev b 3 is induced immediately on he addition of latex B-serum, which is normally compartmentalized in the lutoids in fresh latex. In the preparation of ammoniated latex (used for the manufacture of latex products), the lutoids are ruptured, and the released B-serum reacts with Hev b 3 on the rubber particles to give rise to an array of low molecular weight polypeptides that are allergenic to patients with spina bifida.

Matched MeSH terms: Plant Proteins/adverse effects; Plant Proteins/immunology*; Plant Proteins/chemistry
Isolation of a mannose-binding and IgE- and IgM-reactive lectin from the seeds of Artocarpus integer

Lim SB, Chua CT, Hashim OH

J Immunol Methods, 1997 Dec 01;209(2):177-86.
PMID: 9461333

A mannose-binding lectin, termed champedak lectin-M, was isolated from an extract of the crude seeds of champedak (Artocarpus integer). On gel filtration chromatography, the lectin eluted in a single peak at elution volumes corresponding to 64 kDa. SDS-PAGE showed the mannose-binding lectin to be composed of 16.8 kDa polypeptides with some of the polypeptides being disulphide-linked to give dimers. When tested with all isotypes of immunoglobulins, champedak lectin-M demonstrated a selective strong interaction with human IgE and IgM, and a weak interaction with IgA2. The binding interactions of lectin-M were metal ion independent. The lectin was also shown to interact with horseradish peroxidase, ovalbumin, porcine thyroglobulin, human alpha1-acid glycoprotein, transferrin and alpha1-antitrypsin. It demonstrated a binding preference to Man alpha 1-3Man ligands in comparison to Man alpha 1-6Man or Man alpha 1-2Man.

Matched MeSH terms: Plant Proteins/metabolism*
Amino acid sequence similarity of Hev b 3 to two previously reported 27- and 23-kDa latex proteins allergenic to spina bifida patients

Yeang HY, Ward MA, Zamri AS, Dennis MS, Light DR

Allergy, 1998 May;53(5):513-9.
PMID: 9636811

Separate studies have reported spina bifida patients to be especially allergic to proteins of 27 and 23 kDa found in the serum of centrifuged natural rubber latex. An insoluble latex protein located on the surface of small rubber particles, Hev b 3, has similarly been found to be allergenic to spina bifida patients. In this study, internal amino acid sequences of Hev b 3 showed similarity to the published sequences for the 27- and 23-kDa latex proteins. The latter allergens are hence identified as Hev b 3. Determination of the molecular weight of Hev b 3 revealed various species of 22-23 kDa. The consistent gaps of about 266 Da observed between various forms of the intact protein suggest that the protein undergoes post-translational modification. To determine whether Hev b 3 also occurs in a soluble form in the latex serum, its presence in molecular-filtered serum was checked by ELISA and Western blot. The results showed Hev b 3 to be largely absent in the C-serum from fresh latex. The protein is therefore insoluble in its native state. However, a small amount of the solubilized protein was detected in ammonia-stabilized latex (commonly used in the manufacture of latex products).

Matched MeSH terms: Plant Proteins*
Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida

Wagner B, Krebitz M, Buck D, Niggemann B, Yeang HY, Han KH, et al.

J Allergy Clin Immunol, 1999 Nov;104(5):1084-92.
PMID: 10550757

BACKGROUND: Two natural rubber latex proteins, Hev b 1 and Hev b 3, have been described in spina bifida (SB)-associated latex allergy.
OBJECTIVE: The aim of this study was to clone and express Hev b 3 and to obtain the immunologic active and soluble recombinant allergen for diagnosis of SB-associated latex allergy.
METHODS: A complementary DNA (cDNA) coding for Hev b 3 was amplified from RNA of fresh latex collected from Malaysian rubber trees (Hevea brasiliensis). PCR primers were designed according to sequences of internal peptide fragments of natural (n) Hev b 3. The 5'-end sequence was obtained by specific amplification of cDNA ends. The recombinant (r) Hev b 3 was produced in Escherichia coli as a 6xHis tagged protein. Immunoblotting and inhibition assays were performed to characterize the recombinant allergen.
RESULTS: An Hev b 3 cDNA clone of 922 bp encoding a protein of 204 amino acid residues corresponding to a molecular weight of 22.3 kd was obtained. In immunoblots 29/35, latex-allergic patients with SB revealed IgE binding to rHev b 3, as did 4 of 15 of the latex-sensitized group. The presence of all IgE epitopes on rHev b 3 was shown by its ability to abolish all IgE binding to nHev b 3. Hev b 3 is related to Hev b 1 by a sequence identity of 47%. Cross-reactivity between these 2 latex allergens was illustrated by the large extent of inhibition of IgE binding to nHev b 1 by rHev b 3.
CONCLUSION: rHev b 3 constitutes a suitable in vitro reagent for the diagnosis of latex allergy in patients with SB. The determination of the full sequence of Hev b 3 and the production of the recombinant allergen will allow the epitope mapping and improve diagnostic reagents for latex allergy.

Matched MeSH terms: Plant Proteins/genetics; Plant Proteins/immunology*; Plant Proteins/isolation & purification
Detection of immunoglobulin antibodies in the sera of patients using purified latex allergens

Kurup VP, Yeang HY, Sussman GL, Bansal NK, Beezhold DH, Kelly KJ, et al.

Clin Exp Allergy, 2000 Mar;30(3):359-69.
PMID: 10691894

BACKGROUND: Latex allergy is largely an occupational allergy due to sensitization to natural rubber latex allergens present in a number of health care and household products. Although several purified allergens are currently available for study, information on the usefulness of these purified, native or recombinant allergens in the demonstration of specific immunoglobulin (Ig) E in the sera of patients is lacking.
OBJECTIVE: To evaluate the purified latex allergens and to demonstrate specific IgE antibody in the sera of health care workers and spina bifida patients with clinical latex allergy.
METHODS: Two radioallergosorbent and an enzyme-linked immunosorbent assay (ELISA) using latex proteins Hev b 1, 2, 3, 4, 6 and 7 along with two glove extracts and Malaysian nonammoniated latex (MNA) were evaluated to demonstrate IgE in the sera of health care workers and spina bifida with latex allergy and controls with no history of latex allergy.
RESULTS: ELISA using the purified latex allergens demonstrated specific IgE in 32-65% health care workers and 54-100% of spina bifida patients with latex allergy. The corresponding figures for RAST were 13-48 and 23-85 for RAST-1 and 19-61 and 36-57 for RAST-2. These results were comparable with the results obtained with glove extracts and crude rubber latex proteins.
CONCLUSIONS: When used simultaneously, latex proteins Hev b 2 and Hev b 7 reacted significantly with specific serum IgE in 80% of health care workers and 92% of spina bifida patients with latex allergy by ELISA technique, while this combination gave lower positivity when the RASTs were used. By the addition of Hev b 3, specific IgE was detected in all spina bifida patients with latex allergy. Both RASTs failed to show specific IgE in the control subjects, while the ELISA showed significant latex-specific IgE in 22% of controls.

Matched MeSH terms: Plant Proteins/immunology
Appraisal of latex glove proteins in the induction of sensitivity to multiple latex allergens

Yeang HY, Chow KS, Yusof F, Arif SA, Chew NP, Loke YH

J Investig Allergol Clin Immunol, 2000 Jul-Aug;10(4):215-22.
PMID: 11039838

Six Hevea brasiliensis latex protein allergens, Hevb 1, Hev b 2, Hev b 3, Hev b 4, and two variants of Hev b 7 (7b and 7c), were purified from Hevea latex, while a seventh protein, Hev b 5, was prepared in recombinant form. The presence of these proteins in glove extracts was indicated by their respective antibodies in the serum of rabbits immunized against the extracts. The relative propensities of IgE binding to the individual latex allergens were compared using sera from latex-allergic patients. IgE recognition of Hev b 4, Hev b 7b, Hev b 5 and Hev b 2 was most frequently encountered, with 75, 61, 31 and 28%, respectively, of the patient sera reacting. Sensitivity to multiple latex proteins was common, and out of the 31 seropositive patients, 23 (74%/ ) had IgE against at least two latex allergens, while 12 (39%) had IgE specific for at least three allergens. Statistical analysis of the data suggested that many patients might have acquired sensitivity to Hev b 2, Hev b 4 and Hev b 7b from a common source. (e.g., from latex products). On the other hand, sensitivity to Hev b 5 and to Hev b 7c were interrelated. It is plausible that sensitivity to these two proteins might have been acquired from sources other than latex products (e.g., from certain foods).

Matched MeSH terms: Plant Proteins/adverse effects*; Plant Proteins/genetics; Plant Proteins/immunology
Purification and characterization of 31-kDa palm pollen glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients

Kimura Y, Maeda M, Kimupa M, Lai OM, Tan SH, Hon SM, et al.

Biosci Biotechnol Biochem, 2002 Apr;66(4):820-7.
PMID: 12036055

A basic glycoprotein, which was recognized by IgE from oil palm pollinosis patients, has been purified from oil palm pollen (Elaeis guineensis Jacq.), which is a strong allergen and causes severe pollinosis in Malaysia and Singapore. Soluble proteins were extracted from defatted palm pollen with both Tris-HCl buffer (pH 7.8) and Na-acetate buffer (pH 4.0). The allergenic glycoprotein was purified from the total extract to homogeneity with 0.4% yield by a combination of DEAE- and CM-cellulose, SP-HPLC, and gel filtration. The purified oil palm pollen glycoprotein with molecular mass of 31 kDa was recognized by the beta1-2 xylose specific antibody, suggesting this basic glycoprotein bears plant complex type N-glycan(s). The palm pollen basic glycoprotein, designated Ela g Bd 31 K, was recognized by IgE of palm pollinosis patients, suggesting Ela g Bd 31 K should be one of the palm pollen allergens. The preliminary structural analysis of N-glycans linked to glycoproteins of palm pollens showed that the antigenic N-glycans having alpha1-3 fucose and alpha1-2 xylose residues (GlcNAc(2 to approximately 0)Man3Xyl1Fuc(1 to approximately 0)GlcNAc2) actually occur on the palm pollen glycoproteins, in addition to the high-mannose type structures (Man(9 to approximately 5)GlcNAc2).

Matched MeSH terms: Plant Proteins/isolation & purification; Plant Proteins/chemistry*
Allergenic proteins of natural rubber latex

Yeang HY, Arif SA, Yusof F, Sunderasan E

Methods, 2002 May;27(1):32-45.
PMID: 12079415 DOI: 10.1016/S1046-2023(02)00049-X

As the living cytoplasm of laticiferous cells, Hevea brasiliensis latex is a rich blend of organic substances that include a mélange of proteins. A small number of these proteins have given rise to the problem of latex allergy. The salient characteristics of H. brasiliensis latex allergens that are recognized by the International Union of Immunological Societies (IUIS) are reviewed. These are the proteins associated with the rubber particles, the cytosolic C-serum proteins and the B-serum proteins that originate mainly from the lutoids. Procedures for the isolation and purification of latex allergens are discussed, from latex collection in the field to various preparative approaches adopted in the laboratory. As interest in recombinant latex allergens increases, there is a need to validate recombinant proteins to ascertain equivalence with their native counterparts when used in immunological studies, diagnostics, and immunotherapy.

Matched MeSH terms: Plant Proteins/immunology
The manufacture of gloves from natural rubber latex

Yip E, Cacioli P

J Allergy Clin Immunol, 2002 Aug;110(2 Suppl):S3-14.
PMID: 12170237 DOI: 10.1067/mai.2002.124499

Gloves that will provide a barrier of protection from infectious organisms are an essential feature of medical practice for the protection of both patients and medical personnel. Natural rubber latex has consistently been the most satisfactory raw material for the manufacture of gloves. Certain latex proteins, carried over into the finished product by inadequate manufacturing processes, may pose a risk of provoking allergic reactions in some patients and medical workers. As with any allergy, the risk depends on the route of exposure and dose. Hence, the method of manufacture, including the means used to coat gloves to make donning easy, can influence the eventual exposure of sensitive people to latex allergens. In this article, we describe the several processes in use and their effects on latex protein content.

Matched MeSH terms: Plant Proteins/immunology; Plant Proteins/isolation & purification
Neoculin as a new taste-modifying protein occurring in the fruit of Curculigo latifolia

Shirasuka Y, Nakajima K, Asakura T, Yamashita H, Yamamoto A, Hata S, et al.

Biosci Biotechnol Biochem, 2004 Jun;68(6):1403-7.
PMID: 15215616

A unique taste-modifying activity that converts the sense of sourness to the sense of sweetness occurs in the fruit of the plant Curculigo latifolia, intrinsic to West Malaysia. The active component, known as curculin, is a protein consisting of two identical subunits. We have found a new taste-modifying protein, named neoculin, of the same origin. Both chemical analysis and cDNA cloning characterized neoculin as a heterodimeric protein consisting of an acidic, glycosylated subunit of 113 amino acid residues and a basic subunit that is the monomeric curculin itself.

Matched MeSH terms: Plant Proteins/isolation & purification*; Plant Proteins/pharmacology; Plant Proteins/chemistry
Isolation and characterization of the early nodule-specific protein homologue (Hev b 13), an allergenic lipolytic esterase from Hevea brasiliensis latex

Arif SA, Hamilton RG, Yusof F, Chew NP, Loke YH, Nimkar S, et al.

J Biol Chem, 2004 Jun 04;279(23):23933-41.
PMID: 15024009

Recurring reports of a highly allergenic 42-46-kDa protein in Hevea brasiliensis latex appeared to have been resolved with the discovery of a 43-kDa allergenic latex protein that was a homologue to patatin. However, the low to moderate prevalence of sensitization to the protein, designated Hev b 7, among latex-allergic patients could not adequately explain the frequent observations of the 42-46-kDa allergen. This led to the hypothesis that another, more allergenic protein of a similar molecular mass existed in Hevea latex. We report the isolation and purification of a 42.98-kDa latex glycoprotein showing homology to the early nodule-specific protein (ENSP) of the legumes Medicago sativa, Medicago truncatula, and Glycine max. The protein is allergenic, being recognized by immunoglobulin E (IgE) in sera from latex-allergic patients. The IgE epitope resides on the carbohydrate moiety of the protein, and the presence of a similar carbohydrate component on potato tuber patatin enables the latter to inhibit IgE binding to the ENSP homologue. The cDNA encoding the ENSP homologue was isolated by reverse transcription-PCR and cloned. The protein predicted from the cDNA sequence has 391 amino acids, the first 26 of which constitute a putative signal peptide. The deduced molecular mass of the mature protein is 40.40 kDa, while its isoelectric point is estimated at 5.0. The discrepancy between the predicted and observed molecular mass might be due to glycosylation, for which three N-sites on the protein are predicted. The purified protein showed lipase and esterase activities and may be involved in plant defense.

Matched MeSH terms: Plant Proteins
Hev b 5 and Hev b 13 as allergen markers to estimate the allergenic potency of latex gloves

Yeang HY, Arif SA, Raulf-Heimsoth M, Loke YH, Sander I, Sulong SH, et al.

J Allergy Clin Immunol, 2004 Sep;114(3):593-8.
PMID: 15356563 DOI: 10.1016/j.jaci.2004.05.039

BACKGROUND:
Sensitization to natural rubber latex has been linked to proteins from medical latex gloves. Various assays to estimate the amount of residual allergenic proteins extractable from latex gloves to assess their potential exposure hazard have inherent weaknesses.

OBJECTIVE:
This investigation was aimed at developing 2-site immunoenzymetric assays and identifying appropriate protein markers to assess the allergenic potential of latex gloves.

METHODS:
The presence of 6 latex allergens--Hev b 1, 2, 3, 5, 6, and 13--was measured in a cross-section of commercial latex medical gloves by using monoclonal and polyclonal antibody-based 2-site immunoenzymetric assays. The overall allergenic potential of these gloves was assessed by IgE-inhibition assay. Stepwise multiple regression analyses were performed to identify marker allergens that best explained the variation in latex glove allergenicity.

RESULTS:
All 6 latex allergens were detected in at least some of the glove samples. Hev b 5 and Hev b 13 were identified as the marker allergens that combined best to explain the variation in the glove allergenicity. The significant multiple correlation (R=0.855) between these 2 markers and glove allergenic potency forms the basis of an assay to gauge latex glove allergenicity.

CONCLUSION:
The overall allergenic potential of latex gloves can be estimated by using Hev b 5 and Hev b 13 as indicator allergens. The correlation between glove allergenicity and the level of these allergens was maintained for low-protein gloves (<200 microg/g). This estimation of glove allergenicity was superior to that obtained by using total protein readings.

Matched MeSH terms: Plant Proteins/adverse effects; Plant Proteins/chemistry
Low prevalence of latex sensitivity in South African spina bifida children in Cape Town

Johar A, Lim DL, Arif SA, Hawarden D, Toit GD, Weinberg EG, et al.

Pediatr Allergy Immunol, 2005 Mar;16(2):165-70.
PMID: 15787875

Spina bifida children have a high prevalence of latex allergy in studies reported from Europe and the USA. This study investigated the prevalence of latex allergy in a cohort of 24 spina bifida children at the Red Cross Children's Hospital from Cape Town, South Africa. The children were investigated using a detailed questionnaire, skin prick tests (ALK-Abello), ImmunoCap RASTs, Western blotting and ELISA, using the purified latex proteins Hev b1 and Hev b3 and whole latex preparation. A low overall prevalence of latex sensitization of 16.7% was found in the children. Children who were sensitive reacted to water insoluble to Hev b1 and Hev b3 proteins. The low prevalence of latex sensitization in the South African children may not be entirely explained by stringent latex avoidance. The children were from a low socioeconomic social status and 'hygiene' and other factors should be considered.

Matched MeSH terms: Plant Proteins/immunology
Molecular cloning and immunoglobulin E reactivity of a natural rubber latex lecithinase homologue, the major allergenic component of Hev b 4

Sunderasan E, Bahari A, Arif SA, Zainal Z, Hamilton RG, Yeang HY

Clin Exp Allergy, 2005 Nov;35(11):1490-5.
PMID: 16297147 DOI: 10.1111/j.1365-2222.2005.02371.x

BACKGROUND:
Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53-55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53-55 kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information.

OBJECTIVE:
We sought to clone the transcript encoding the Hev b 4 major protein, and characterize the native protein and its recombinant form in relation to IgE binding.

METHODS:
The 5'/3' rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients.

RESULTS:
The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue.

CONCLUSION:
The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular weight of the protein and the value calculated from its cDNA.

Matched MeSH terms: Plant Proteins/immunology
Allergen concentration in natural rubber latex

Yeang HY, Hamilton RG, Bernstein DI, Arif SA, Chow KS, Loke YH, et al.

Clin Exp Allergy, 2006 Aug;36(8):1078-86.
PMID: 16911364 DOI: 10.1111/j.1365-2222.2006.02531.x

BACKGROUND:
Hevea brasiliensis latex serum is commonly used as the in vivo and in vitro reference antigen for latex allergy diagnosis as it contains the full complement of latex allergens.

OBJECTIVE:
This study quantifies the concentrations of the significant allergens in latex serum and examines its suitability as an antigen source in latex allergy diagnosis and immunotherapy.

METHODS:
The serum phase was extracted from centrifuged latex that was repeatedly freeze-thawed or glycerinated. Quantitation of latex allergens was performed by two-site immunoenzymetric assays. The abundance of RNA transcripts of the latex allergens was estimated from the number of their clones in an Expressed Sequence Tags library.

RESULTS:
The latex allergens, Hev b 1, 2, 3, 4, 5, 6, 7 and 13, were detected in freeze-thawed and glycerinated latex serum at levels ranging from 75 (Hev b 6) to 0.06 nmol/mg total proteins (Hev b 4). Hev b 6 content in the latex was up to a thousand times higher than the other seven latex allergens, depending on source and/or preparation procedure. Allergen concentration was reflected in the abundance of mRNA transcripts. When used as the antigen, latex serum may bias the outcome of latex allergy diagnostic tests towards sensitization to Hev b 6. Tests that make use of latex serum may fail to detect latex-specific IgE reactivity in subjects who are sensitized only to allergens that are present at low concentrations.

CONCLUSION:
Latex allergy diagnostics and immunotherapy that use whole latex serum as the antigen source may not be optimal because of the marked imbalance of its constituent allergens.

Matched MeSH terms: Plant Proteins/analysis; Plant Proteins/genetics; Plant Proteins/immunology*

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