Identification and Cloning of Prs a 1, a 32-kDa Endochitinase and Major Allergen of Avocado, and Its Expression in the Yeast Pichia pastoris
1998; Elsevier BV; Volume: 273; Issue: 43 Linguagem: Inglês
10.1074/jbc.273.43.28091
ISSN1083-351X
AutoresSlawomir Sowka, Li-Shan Hsieh, Monika Krebitz, Akira Akasawa, Brian M. Martin, David A. Starrett, Clemens K. Peterbauer, Otto Scheiner, Heimo Breiteneder,
Tópico(s)Insect and Pesticide Research
ResumoAvocado, the fruit of the tropical treePersea americana, is a source of allergens that can elicit diverse IgE-mediated reactions including anaphylaxis in sensitized individuals. We characterized a 32-kDa major avocado allergen, Prs a 1, which is recognized by 15 out of 20 avocado- and/or latex-allergic patients. Natural Prs a 1 was purified, and its N-terminal and two tryptic peptide sequences were determined. We isolated the Prs a 1 encoding cDNA by PCR using degenerate primers and 5′-rapid amplification of cDNA ends. The Prs a 1 cDNA coded for an endochitinase of 326 amino acids with a leader peptide of 25 amino acids. We expressed Prs a 1 in the yeast Pichia pastoris at 50 mg/liter of culture medium. The recombinant Prs a 1 showed endochitinase activity, inhibited growth and branching of Fusarium oxysporum hyphae, and possessed IgE binding capacity. IgE cross-reactivity with latex proteins including a 20-kDa allergen, most likely prohevein, was demonstrated, providing an explanation for the commonly observed cross-sensitization between avocado and latex proteins. Sequence comparison showed that Prs a 1 and prohevein had 70% similarity in their chitin-binding domains. Characterization of chitinases as allergens has implications for engineering transgenic crops with increased levels of chitinases. Avocado, the fruit of the tropical treePersea americana, is a source of allergens that can elicit diverse IgE-mediated reactions including anaphylaxis in sensitized individuals. We characterized a 32-kDa major avocado allergen, Prs a 1, which is recognized by 15 out of 20 avocado- and/or latex-allergic patients. Natural Prs a 1 was purified, and its N-terminal and two tryptic peptide sequences were determined. We isolated the Prs a 1 encoding cDNA by PCR using degenerate primers and 5′-rapid amplification of cDNA ends. The Prs a 1 cDNA coded for an endochitinase of 326 amino acids with a leader peptide of 25 amino acids. We expressed Prs a 1 in the yeast Pichia pastoris at 50 mg/liter of culture medium. The recombinant Prs a 1 showed endochitinase activity, inhibited growth and branching of Fusarium oxysporum hyphae, and possessed IgE binding capacity. IgE cross-reactivity with latex proteins including a 20-kDa allergen, most likely prohevein, was demonstrated, providing an explanation for the commonly observed cross-sensitization between avocado and latex proteins. Sequence comparison showed that Prs a 1 and prohevein had 70% similarity in their chitin-binding domains. Characterization of chitinases as allergens has implications for engineering transgenic crops with increased levels of chitinases. recombinant Prs a 1 natural Prs a 1 radioallergosorbent test polyacrylamide gel electrophoresis high performance liquid chromatography polymerase chain reaction. Food allergy is a well known condition, afflicting a portion of the adult population that is hard to define. If one relies on self-perception, a prevalence of 15–20% of food allergic patients could be assumed (1European Commission Ortolani C. Pastorello E.A. Study of Nutritional Factors in Food Allergies and Food Intolerances. European Commission, Office for Official Publications of the European Communities, Luxembourg1997: 93-94Google Scholar, 2Young E. Stoneham M.D. Petruckevitch A. Barton J. Bona R. Lancet. 1994; 343: 1127-1130Abstract PubMed Scopus (577) Google Scholar). However, on the basis of in vitroand in vivo (skin prick test) diagnosis, the percentage might be as low as 1.4–1.8% (1European Commission Ortolani C. Pastorello E.A. Study of Nutritional Factors in Food Allergies and Food Intolerances. European Commission, Office for Official Publications of the European Communities, Luxembourg1997: 93-94Google Scholar, 2Young E. Stoneham M.D. Petruckevitch A. Barton J. Bona R. Lancet. 1994; 343: 1127-1130Abstract PubMed Scopus (577) Google Scholar). Self-perception has the drawback of being based on imponderable psychological effects. On the other hand, extracts used for diagnostic procedures are, in particular in the case of food allergens, often of questionable quality. This is probably due to the varying composition and stability of the food extracts. For this reason, recombinant DNA techniques have significantly contributed to a reliable characterization of the responsible allergens from food extracts (3Vanek-Krebitz M. Hoffmann-Sommergruber K. Laimer-da-Camara-Machado M. Susani M. Ebner C. Kraft D. Scheiner O. Breiteneder H. Biochem. Biophys. Res. Commun. 1995; 214: 538-551Crossref PubMed Scopus (264) Google Scholar, 4Breiteneder H. Hoffmann-Sommergruber K. O'Riordain G. Susani M. Ahorn H. Ebner C. Kraft D. Scheiner O. Eur. J. Biochem. 1995; 233: 484-489Crossref PubMed Scopus (228) Google Scholar, 5Burks A.W. Cockrell G. Stanley J.S. Helm R.M. Bannon G.A. J. Clin. Invest. 1995; 96: 1715-1721Crossref PubMed Scopus (202) Google Scholar, 6Leung P.S. Chu K.H. Chow W.K. Ansari A. Bandea C.I. Kwan H.S. Nagy S.M. Gershwin M.E. J. Allergy Clin. Immunol. 1994; 94: 882-890Abstract Full Text PDF PubMed Scopus (213) Google Scholar). Allergy to avocado is of increasing importance, especially in Mexico and the United States, where consumption of avocado-based dishes is common. To judge from the few data available, the prevalence of avocado allergy in the general population could be estimated to be around 1% (8% in atopic individuals; Refs. 1European Commission Ortolani C. Pastorello E.A. Study of Nutritional Factors in Food Allergies and Food Intolerances. European Commission, Office for Official Publications of the European Communities, Luxembourg1997: 93-94Google Scholar and 7Telez-Diaz G. Ellis M.H. Morales-Russo F. Heiner D.C. Allergy Proc. 1995; 16: 241-243Crossref PubMed Scopus (2) Google Scholar). Avocado allergy is of particular relevance in the "latex-fruit syndrome" observed in at least 40% of latex-allergic individuals (8Blanco C. Carrillo T. Castillo R. Quiralte J. Cuevas M. Ann. Allergy. 1994; 73: 309-314PubMed Google Scholar, 9Brehler R. Theissen U. Mohr C. Luger T. Allergy. 1997; 52: 404-410Crossref PubMed Scopus (276) Google Scholar, 10Beezhold D.H. Sussman G.L. Liss G.M. Chang N.-S. Clin. Exp. Allergy. 1996; 26: 416-422Crossref PubMed Scopus (249) Google Scholar). Since 5–10% of health care workers are sensitized to latex, which is much more than the risk of latex allergy in the general population (11Slater J.E. Kay A.B. Allergy and Allergic Diseases. 2. Blackwell Science, Oxford1997: 981-993Google Scholar, 12Turjanmaa K. Alenius H. Makinen-Kiljunen S. Reunala T. Palosuo T. Allergy. 1996; 51: 593-602Crossref PubMed Scopus (239) Google Scholar), the percentage of health care workers affected with the latex-fruit syndrome may be as high as 2–4%. In this context, a precise characterization of the respective allergens is of special interest. Avocado can induce IgE-mediated reactions with different clinical manifestations including a high percentage of anaphylaxis (8Blanco C. Carrillo T. Castillo R. Quiralte J. Cuevas M. Ann. Allergy. 1994; 73: 309-314PubMed Google Scholar, 13Blanco C. Carrillo T. Castillo R. Quiralte J. Cuervas M. Allergy. 1994; 49: 454-459Crossref PubMed Scopus (72) Google Scholar). For avocado pear extracts, immunoblotting studies revealed several antigenic constituents between 10 and 120 kDa (14Lavaud F. Prevost A. Cossart C. Guerin L. Bernard J. Kochman S. J. Allergy Clin. Immunol. 1995; 95: 557-564Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 15Ahlroth M. Alenius H. Turjanmaa K. Makinen-Kiljunen S. Reunala T. Palosuo T. J. Allergy Clin. Immunol. 1995; 96: 167-173Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), none of which have been characterized on a molecular level. Cross-reactivity of avocado and latex proteins has been reported (14Lavaud F. Prevost A. Cossart C. Guerin L. Bernard J. Kochman S. J. Allergy Clin. Immunol. 1995; 95: 557-564Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 15Ahlroth M. Alenius H. Turjanmaa K. Makinen-Kiljunen S. Reunala T. Palosuo T. J. Allergy Clin. Immunol. 1995; 96: 167-173Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The predominant allergen in avocado, shown to be cross-reactive among avocado, latex, and banana, is about 30 kDa (14Lavaud F. Prevost A. Cossart C. Guerin L. Bernard J. Kochman S. J. Allergy Clin. Immunol. 1995; 95: 557-564Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The cross-reactivity suggests that this avocado allergen might share antigenic determinants with some latex allergens, although Persea americana and Hevea brasiliensis are botanically unrelated. Here we report the cloning and expression of Prs a 1, a 32-kDa major allergen of avocado, cross-reactive with latex allergens. This cross-reactivity of the recombinant protein provides the first molecular basis of the association of type I allergic reactions to latex and avocado. rPrs a 11displayed endochitinase activity and inhibited the growth of Fusarium oxysporum in vitro. A total of 20 individual serum samples from patients with positive case histories, positive skin prick tests, positive RASTs (RAST classes higher than 4), and characteristic type I allergic reactions to latex was used in this study. Seven out of 20 patients (35%) reported symptoms after the ingestion of avocado. A serum pool from 22 healthy individuals with no histories of any type I allergy, negative skin prick tests, and negative RAST results to avocado and/or latex allergens was used as negative control. Ten grams of avocado pear mesocarp tissue (P. americana Miller cv. Haas) were homogenized in a Waring blender and mixed with 20 ml of extraction buffer consisting of 50 mm Tris-HCl, pH 8.0, 10 mm EDTA, 10 mm diethyldithiocarbamate, and 10 mm sodium sulfate. The mixture was then centrifuged at 40,000 ×g for 1 h. The supernatant was collected and filtered through a Millex-HV filter (Millipore Corp., Bedford, MA). Freshly harvested field latex was fractionated by ultracentrifugation (40,000 × g at 4 °C for 1 h) into (i) the rubber particles in the upper fraction, (ii) a translucent aqueous layer known as C-serum, and (iii) a pellet containing organelles collectively called "lutoids" (16d'Auzac J. Jacob J.-L. d'Auzac J. Jacob J.-L. Chrestin H. Physiology of Rubber Tree Latex. CRC Press, Inc., Boca Raton, FL1989: 59-96Google Scholar). The bottom fraction was resuspended in 50 mm Tris-HCl buffer, pH 8.0, containing 0.05% Triton X-100 and will hereafter be called the B fraction. C serum was again centrifuged for 1 h (40,000 × g, 4 °C) to remove residual latex particles. The pH of the extracts was adjusted with acetic acid to pH 7.0, dialyzed against water, lyophilized, and stored at −80 °C until use. For the experiments, the lyophilized protein extracts were resuspended in 200 mmNaCl. rPrs a 1, nPrs a 1, latex B fraction, C serum, and avocado extracts were analyzed by SDS-PAGE in 12% polyacrylamide gels under reducing conditions. For immunodetection, the separated proteins were transferred to ProBlott membranes (Applied Biosystems, Foster City, CA). Membrane strips were incubated with sera from allergic patients, and bound IgE was detected using phosphatase-labeled anti-human IgE goat serum (Kirkeggard and Perry Laboratories, Gaithersburg, MD) and the Chemiluminescence Horseradish Peroxidase system from Amersham Life Science (Little Chalfont, United Kingdom) according to the manufacturer's instructions (Fig. 1). Alternatively, bound IgE was detected using 125I-labeled rabbit anti-human IgE (RAST RIA, Pharmacia, Uppsala, Sweden) diluted 1:10 (Figs. Figure 5, Figure 6, Figure 7). Isoelectric focusing gel electrophoresis was performed with Ampholine® PAGplates, 3.5–9.5 (Pharmacia), at 500 V for 90 min.Figure 5Binding of human IgE to rPrs a 1. Lane 1, 20-μl supernatant (total protein amount 350 ng) of P. pastoris culture producing Prs a 1; lane 2, 20-μl supernatant (total protein amount <50 ng) of untransformed P. pastoris; lane 3, IgE immunoblot of the recombinant Prs a 1 (as in lane 1) with serum pool from patients allergic to avocado and/or latex; lane 4, IgE immunoblot of supernatant of untransformed P. pastoris (as in lane 2) probed with the same serum pool. Lanes 1 and 2, are Coomassie, stained SDS-PAGE gels.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Inhibition of patients' IgE binding to proteins in avocado extracts. A serum pool from patients allergic to avocado and/or latex was preincubated with 30 μg of recombinant Prs a 1 and then incubated with allergens blotted onto nitrocellulose strips. Lane 1, molecular mass standards; lane 2, 60-μg protein extracts from avocado fruit; lane 3, IgE binding of the serum pool to avocado proteins; lane 4, inhibition of IgE binding to avocado proteins by the addition of 30 μg of purified rPrs a 1. Lanes 1 and 2 are Coomassie-stained SDS-PAGE gels. The position of Prs a 1 is indicated. The same amount of protein was loaded in lanes 2–4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Inhibition of patients' IgE binding to proteins in latex B fraction with purified rPrs a 1. A serum pool from patients allergic to avocado and/or latex was preincubated with 30 μg of purified Prs a 1 and then incubated with nitrocellulose-blotted latex B fraction proteins. Lane 1, molecular weight standards; lane 2, 60 μg of latex B fraction proteins;lane 3, IgE binding of the serum pool to proteins from B fraction; lane 4, inhibition of IgE binding to B fraction proteins by 30 μg of purified rPrs a 1. The same amount of protein was loaded in lanes 2–4. The position of the 20-kDa allergen is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For the immunoblot inhibition experiments, a serum pool from 19 avocado and/or latex allergic patients (sera 2–20 in Fig. 1) was preincubated overnight at 4 °C with 30 μg of purified rPrs a 1. Preincubated sera were used to probe the ProBlott membrane strips containing total avocado extracts, latex B fraction, or latex C serum as described above. Twelve milliliters of avocado extracts were dialyzed against 20 mmcitric acid buffer, pH 3.8, and subjected to a HiTrapTM SP cation exchange column (Pharmacia) at a flow rate of 0.5 ml/min using a fast protein liquid chromatography system: buffer A, 20 mmcitric acid buffer, pH 3.8; buffer B, buffer A and 1 mNaCl. In a further step, the protein was purified by a Vydac C-4 reverse phase column (Western Analytical Products, Temecula, CA) with a linear gradient of 0.12% trifluoroacetic acid in water (buffer C) and 0.12% trifluoroacetic acid in acetonitrile (buffer D). The rPrs a 1 was purified by one-step purification over the HiTrap 153TMSP column as described above. The protein concentration was assessed with the Protein Assay kit from Bio-Rad with bovine serum albumin as a standard. The amino acid sequences of the N terminus and two tryptic peptides of purified nPrs a 1 were determined by automated Edman degradation and analysis on a 477A gas phase microsequencer (Applied Biosystems) connected on-line to the phenylthiohydantoin analyzer, model 120A. A sample of the purified nPrs a 1 was desalted by extensive dialysis, dried down under a stream of nitrogen, and then dissolved in 50 μl of HPLC grade water. Neutral sugars were released by hydrolysis with trifluoroacetic acid added to a final concentration of 2 m in a sealed polypropylene microcentrifuge tube. After 6 h of hydrolysis at 100 °C, the sample was evaporated to dryness. The sample was redissolved in 55 μl of water and analyzed directly by high pH anion exchange chromatography with pulsed amperometric detection. The monosaccharides were separated on a Dionex Bio-LC system (Dionex, Sunnyvale, CA) equipped with the strong anion exchange column CarboPac PA1 (4 × 250 mm), a PA 1 guard column, and a PAD 2 detector. The elution was performed at 15 mm NaOH isocratic with a flow of 1 ml/min, 200 mm NaOH as eluent A and water as eluent B. RNA was prepared from avocado pear mesocarp tissue as described previously (17Starrett D.A. Laties G.G. Plant Physiol. 1993; 103: 227-234Crossref PubMed Scopus (26) Google Scholar). Samples corresponding to different stages of maturation were pooled and used for reverse transcription. The reverse transcription was performed with 1 μg of total RNA using an oligo(dT)16 primer and the GeneAmp RNA PCR kit (Perkin-Elmer). From the obtained N-terminal peptide sequence, two different degenerate sense oligonucleotides were designed for use in PCR: AVO1, 5′-TG(T/C)TG(T/C)AG(C/T)CA(A/G)TT(T/C)GG(A/G/T/C)TGGTG(T/C)GG-3′, and AVO2, 5′- TG(T/C)TG(T/C) TC(A/G/T/C)CA(A/G)TT(T/C)GG(A/G/T/C)TGGTG(T/C)GG-3′, both corresponding to CCSQFGWCG. The antisense oligonucleotide was the lock-docking oligo(dT) primer 5′-(T)30(G/A/C)(G/A/C/T)-3′. The PCR products were cloned into pCRII (TA Cloning kit,Invitrogen, San Diego, CA) and sequenced. To complete the cDNA, the fragment was extended by 5′-rapid amplification of cDNA ends using the AmpliFinder™ kit (CLONTECH, Palo Alto, CA). The full-length Prs a 1 cDNA sequence was analyzed for possible cleavage sites using the program SIGSEQ2 (18Filz R.J. Gordon J.I. Biochem. Biophys. Res. Commun. 1987; 146: 870-877Crossref PubMed Scopus (48) Google Scholar). Sequence analysis was performed using the Thermosequenase Fluorescent Labeled Primer Cycle Sequencing kit (Amersham Life Science) and the LI-COR DNA sequencer model 4000L (LI-COR, Lincoln, NE). Both strands of six different clones were analyzed to yield the final sequence of the amplified fragment. The FASTA program provided with the Wisconsin Package (Genetics Computer Group, Madison, WI) was used to search for protein sequence homologies of the 32-kDa avocado allergen to proteins in the SWISSPROT data base. The cDNA corresponding to the mature Prs a 1 protein was amplified by PCR using the phosphorothioate-modified primers: sense, 5′-TATCTCGAGAAAAGAGAACAATGTGGTAGACAAGCT-3′; antisense, 5′-TTAGCGGCCGC TCATTAGGATGAAGCAGCAAGG-3′ (priming regions underlined, XhoI and NotI sites in italics) and the Vent Polymerase (New England Biolabs, Beverly, MA). The sequence CTC GAG AAA AGA GAA, corresponding to the amino acid sequence LEKRE, was necessary to recreate the signal peptide cleavage site of the Saccharomyces cerevisiae α factor leader peptide present in the P. pastoris expression vector pPIC9 (Invitrogen). The signal peptidase cleavage in the above sequence lies between arginine and glutamic acid residues. Fortunately, glutamic acid is also the first amino acid of the mature Prs a 1, so no cloning artifacts were produced. The XhoI/NotI-digested PCR product was ligated to the respective sites of P. pastoris vector pPIC9 and sequenced to confirm the identity of the insert. The transformation of the P. pastoris strain GS115 (Invitrogen), screening for recombinant Prs a 1-producing clones, and extracellular expression were performed according to the instruction manual. The endochitinase assay with the purified rPrs a 1 was performed in 50 mm potassium phosphate, pH 8.0, according to Wirth et al. (19Wirth S.J. Wolf G.A. J. Microbiol. Methods. 1990; 12: 197-205Crossref Scopus (249) Google Scholar) using carboxymethyl-substituted soluble chitin labeled with remazol brilliant violet 5R (Loewe Biochemica, Otterfing, Germany). The exochitinase activity was measured using 4-nitrophenyl-N-acetyl-β-d-glucosaminide (simulates a dimer; Serva, Heidelberg, Germany) as described previously (20Berghem L.E.R. Pettersson L.G. Eur. J. Biochem. 1974; 46: 295-305Crossref PubMed Scopus (227) Google Scholar) and 4-nitrophenyl-N, N′-diacetyl chitobioside (simulates a trimer; Sigma) according to Ref. 21Roberts W.K. Selitrennikoff C.P. J. Gen. Microbiol. 1988; 134: 169-176Google Scholar. Lysozyme activity was determined by a modification of the method reported by Shugar et al. (22Shugar D. Biochim. Biophys. Acta. 1952; 8: 302-309Crossref PubMed Scopus (873) Google Scholar). Briefly, 0.2 mg of Micrococcus lysodeikticus cell walls (Sigma) in 900 μl of 100 mm potassium phosphate, pH 8.0, were mixed with 100 μl of enzyme solution, incubated at 37 °C, and the absorbance of the reaction mixture at 570 nm was measured every 10 min to determine the decrease in turbidity. Hen egg white lysozyme (Merck, Darmstadt, Germany) was assayed at 55 °C in 100 mm Tris-HCl, 100 mm NaCl, pH 9.0, as a positive control. For the growth inhibition assay, F. oxysporum spores were collected from 8-day-old cultures grown on potato dextrose agar plates (Difco). Assay mixtures contained 12 μl of 5× potato dextrose broth (Difco), 3000 spores of the test fungus in 10 μl of water, and 38 μl of the rPrs a 1 solution. The effect of rPrs a 1 was tested at concentrations of 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, and 100 μg/ml. In the controls, heat-denatured rPrs a 1 and sterile water were used instead of the solution containing the enzyme. After 40 h of incubation at 25 °C, portions of the samples were placed on microscope slides, and the lengths of the first 20 germ tubes were measured and averaged. Nineteen out of 20 serum samples from patients allergic to avocado and/or latex reacted with proteins from the avocado extract. Fifteen of them reacted with a 32-kDa protein, nine reacted with a 46-kDa protein, four reacted with a 28-kDa protein, and two reacted with a 14-kDa protein (Fig. 1). Both natural and recombinant Prs a 1 eluted at 0.26m NaCl in 20 mm citric acid buffer, pH 3.8, from the HiTrap 153TM SP column. Fractions containing natural Prs a 1 were collected and then further purified to homogeneity by HPLC reverse-phase chromatography (Fig. 2, lane 1), eluting at 38% buffer D. For the rPrs a 1, the first purification step was sufficient (Fig. 2, lane 2). The first 26 N-terminal amino acid residues determined by protein microsequencing were EQCGRQAGGALCPGGLCCSQFGWCGS. To confirm that this sequence was the N terminus of the mature peptide, an additional cleavage site analysis was performed using the SIGSEQ2 software. The cleavage site indicated by this program matched exactly the experimental data. The amino acid sequences of the two tryptic peptides derived from nPrs a 1 were GPIQISYNYNYGPAGA and TALWFWMTPQSPK. All three peptide sequences matched exactly the deduced amino acid sequence of Prs a 1 (Fig. 3).Figure 3Nucleotide and deduced amino acid sequence of Prs a 1. The deduced amino acid sequence is indicatedbelow the encoding nucleotides. The leader peptide is shown in boldface type, and the chitin-binding motif characteristic for class I chitinases (Glu26–Cys64), and two additional motifs of family 19 chitinases as described in the PROSITE data base (Cys96–Ala118 and Ile222–Met232) are in italic type. The peptide sequences derived from nPrs a 1 and a putative polyadenylation site are underlined. The sequence data are available from the EMBL data base under the accession numberZ78202.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The de-acetylated monosaccharides found by high pH anion exchange chromatography with pulsed amperometric detection were GalNH2 and Gal. The lack of Man and GlcNH2 is consistent with the absence of potentialN-glycosylation sites in the Prs a 1 sequence. The molar ratio of the O-linked carbohydrates GalNH2 and Gal to each other was 3.5:1. Fig. 3 depicts the Prs a 1 sequence with the identified motifs as analyzed by DNA sequencing of six independent cDNA clones. The cDNA codes for a polypeptide of 326 amino acids including a leader peptide of 25 residues as determined by N-terminal amino acid microsequencing and software analysis. The cleavage of the leader sequence results in the mature protein with a calculated molecular mass of 32.0 kDa. No potentialN-glycosylation sites were detected. A chitin recognition and/or binding motif corresponding to the consensus pattern CX 4,5CCSX 2GXCGX 4(F/Y/W)C (Fig. 3) was detected by the program MOTIFS (Wisconsin Package). Two additional consensus patterns, CX 4,5FY(S/T)X 3(F/Y)(L/I/V/M/F)XAX 3(Y/F)X 2F(G/S/A) and (L/I/V/M)(G/S/A)FX(S/T/A/G)2(L/I/V/M/F/Y)W(F/Y)W(L/I/V/M) (Fig. 3), which are characteristic for chitinases from family 19 of glycosyl hydrolases according to the classification by Henrissatet al. (23Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar), were identified without mismatches. Chitinases from family 19 belong to endochitinases (EC 3.2.1.14), enzymes that catalyze the hydrolysis of β-1,4-N-acetyl-d-glucosamine linkages in chitin polymers. Enzymes from family 19 are also known as class I A (another notation for class I A is I) and class I B (II) endochitinases. Class I A and I B endochitinases differ in the presence (I A) or absence (I B) of an N-terminal chitin-binding domain. The catalytic domain of these enzymes consists of about 220–240 amino acid residues. In this nomenclature, Prs a 1 is a class I A (class I) chitinase. In the Prs a 1 sequence, a glycine- and serine-rich hinge region of about 20 amino acids connects the chitin-binding domain (amino acid residues 1–39 in Fig. 3) with the C-terminal catalytic domain of about 240 amino acid residues. Prs a 1 shares substantial sequence similarities with endochitinases from plants. Only similarities to chitinases present in plant-derived foods were taken into account, since they may play important roles in the context of plant food allergies. The Prs a 1 endochitinase from avocado shares 77.0% identity with a chitinase from Triticum aestivum (in a 300-amino acid overlap), 73.8% with Oryza sativa (in 301 amino acids), 73.5% with Solanum tuberosum (in 294 amino acids), 72.9% with Brassica napus (in 292 amino acids), and 71.0% with Cucumis sativa (in 300 amino acids) endochitinases. Another similarity to a known major latex allergen was revealed by the sequence comparison of Prs a 1 with prohevein (Fig. 4 A). The similarity between the two proteins is confined to their chitin-binding domains. The degree of identity over this region of 43 amino acid residues is 70%. Two other similarities of interest in the context of the latex-fruit syndrome, to a 33-kDa banana allergen (24Mikkola J. Alenius H. Turjanmaa K. Palosuo T. Reunala T. J. Allergy Clin. Immunol. 1997; 99 (abstr.): 342Google Scholar) and a latex 29-kDa allergen (25Posch A. Chen Z. Wheeler C. Dunn M. Raulf-Heimsoth M. Baur X. J. Allergy Clin. Immunol. 1997; 99: 385-395Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) were found (Fig. 4, B and C). The extracellular expression using the pPIC9 vector yielded a prominent band of 32 kDa (Fig. 5, lane 1). The yield, estimated by the method of Bradford was approximately 50 mg/liter culture supernatant. The rPrs a 1 protein could be separated from low molecular weight degradation products by one-step purification over a HiTrapTM SP column (Fig. 2, lane 2). Under standard SDS-PAGE conditions, the protein migrated exactly the same as natural Prs a 1 (Fig. 2). The experimental pI of the rPrs a 1 was determined to be 8.8 (data not shown). rPrs a 1 exhibited endochitinase activity but no exochitinase activity (Table I). In addition, as some plant chitinases also display the activity defined in EC 3.2.1.14 (lysozyme), we tested rPrs a 1 for lysozyme activity with hen egg white lysozyme as a control (Table I). Interestingly, hen egg white lysozyme showed a weak endochitinase activity, but Prs a 1 showed no lysozyme activity.Table IChitinase and lysozyme activities of rPrs a 1 and hen egg white lysozymeEnzyme activityEC numberSubstraterPrs a 1HEWL1-aHEWL, hen egg white lysozyme.EndochitinaseEC3.2.1.14CM-chitin-RBV1-bCM-chitin-RBV, carboxymethyl chitin labeled with remazol brilliant violet 5R.1411.7 ± 0.05 OD units/mg1.6 ± 0.05 OD units/mgLysozymeEC3.2.1.17M. lysodeikticus cell wallsNM1-cNM, not measurable.1.413 ± 0.08 × 105 units/mgExochitinaseEC 3.2.1.14(formerly 3.2.1.29)4-Nitrophenyl-N, N-diacetyl-β-d-chitobiosideNM1-cNM, not measurable.ND1-dND, not determined.ExochitinaseEC 3.2.1.14 (formerly 3.2.1.30)4-Nitrophenyl-N-acetyl-β-d-glucosaminideNM1-cNM, not measurable.ND1-dND, not determined.1-a HEWL, hen egg white lysozyme.1-b CM-chitin-RBV, carboxymethyl chitin labeled with remazol brilliant violet 5R.1-c NM, not measurable.1-d ND, not determined. Open table in a new tab Growth of F. oxysporum was inhibited by 95% at a concentration of 35 ± 3 μg/ml purified rPrs a 1 and 33 ± 3 μg/ml purified nPrs a 1. The inhibition curve of the purified nPrs a 1 was equivalent to the recombinant product within the S.D. values. As a control, heat-denatured rPrsa 1 did not inhibit the growth of the test fungus. In addition, an altered morphology was observed in samples treated with rPrs a 1. Only 15% of the germ tubes incubated with 35 μg of rPrs a 1/ml were branched, all of them having only a single branch. In contrast, most of the control mycelia were highly branched. The IgE binding capacity of rPrs a 1 was assessed by direct binding of IgE from a serum pool of avocado- and/or latex-allergic patients to solid phase bound rPrs a 1. The 32-kDa rPrs a 1 was the only component of P. pastorisculture supernatants, which bound serum IgE (Fig. 5, lane 3). No IgE
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