Assignment of the disulfide bonds of Ole e 1, a major allergen of olive tree pollen involved in fertilization.

The most prevalent allergen from olive tree pollen, Ole e 1, consists of a single polymorphic polypeptide chain of 145 amino acids which includes six cysteine residues at positions 19, 22, 43, 78, 90 and 131. By using an homogeneous form of the allergen expressed in Pichia pastoris, the array of the disulfide bridges has been elucidated. Specific proteolysis with thermolysin and reverse-phase HPLC separation of the peptides allowed the determination of the disulfide bond between Cys43 and Cys78. Another thermolytic product, which contained three peptides linked by the remaining four cysteines, was digested with Glu-specific staphylococcal V8 protease and the products isolated by reverse-phase HPLC. Amino acid compositions and Edman degradation of the peptide products indicated the presence of the disulfide bonds at Cys19-Cys90 and Cys22-Cys131. These data can help in the analysis of the three-dimensional structure of the protein as well as in studies of its allergenic determinants.     

Human acid sphingomyelinase.

Human acid sphingomyelinase (haSMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to ceramide and phosphorylcholine. An inherited haSMase deficiency leads to Niemann-Pick disease, a severe sphingolipid storage disorder. The enzyme was purified and cloned over 10 years ago. Since then, only a few structural properties of haSMase have been elucidated. For understanding of its complex functions including its role in certain signaling and apoptosis events, complete structural information about the enzyme is necessary. Here, the identification of the disulfide bond pattern of haSMase is reported for the first time. Functional recombinant enzyme expressed in SF21 cells using the baculovirus expression system was purified and digested by trypsin. MALDI-MS analysis of the resulting peptides revealed the four disulfide bonds Cys120-Cys131, Cys385-Cys431, Cys584-Cys588 and Cys594-Cys607. Two additional disulfide bonds (Cys221-Cys226 and Cys227-Cys250) which were not directly accessible by tryptic cleavage, were identified by a combination of a method of partial reduction and MALDI-PSD analysis. In the sphingolipid activator protein (SAP)-homologous N-terminal domain of haSMase, one disulfide bond was assigned as Cys120-Cys131. The existence of two additional disulfide bridges in this region was proved, as was expected for the known disulfide bond pattern of SAP-type domains. These results support the hypothesis that haSMase possesses an intramolecular SAP-type activator domain as predicted by sequence comparison [Ponting, C.P. (1994) Protein Sci., 3, 359-361]. An additional analysis of haSMase isolated from human placenta shows that the recombinant and the native human protein possess an identical disulfide structure.     

Determination of the disulfide bond arrangement of Newcastle disease virus hemagglutinin neuraminidase. Correlation with a beta-sheet propeller structural fold predicted for paramyxoviridae attachment proteins

Disulfide bonds stabilize the structure and functions of the hemagglutinin neuraminidase attachment glycoprotein (HN) of Newcastle disease virus. Until this study, the disulfide linkages of this HN and structurally similar attachment proteins of other members of the paramyxoviridae family were undefined. To define these linkages, disulfide-linked peptides were produced by peptic digestion of purified HN ectodomains of the Queensland strain of Newcastle disease virus, isolated by reverse phase high performance liquid chromatography, and analyzed by mass spectrometry. Analysis of peptides containing a single disulfide bond revealed Cys(531)-Cys(542) and Cys(172)-Cys(196) linkages and that HN ectodomains dimerize via Cys(123). Another peptide, with a chain containing Cys(186) linked to a chain containing Cys(238), Cys(247), and Cys(251), was cleaved at Met(249) with cyanogen bromide. Subsequent tandem mass spectrometry established Cys(186)-Cys(247) and Cys(238)-Cys(251) linkages. A glycopeptide with a chain containing Cys(344) linked to a chain containing Cys(455), Cys(461), and Cys(465) was treated sequentially with peptide-N-glycosidase F and trypsin. Further treatment of this peptide by one round of manual Edman degradation or tandem mass spectrometry established Cys(344)-Cys(461) and Cys(455)-Cys(465) linkages. These data, establishing the disulfide linkages of all thirteen cysteines of this protein, are consistent with published predictions that the paramyxoviridae HN forms a beta-propeller structural fold.     

Disulfide transfer between two conserved cysteine pairs imparts selectivity to protein oxidation by Ero1

The membrane-associated flavoprotein Ero1p promotes disulfide bond formation in the endoplasmic reticulum (ER) by selectively oxidizing the soluble oxidoreductase protein disulfide isomerase (Pdi1p), which in turn can directly oxidize secretory proteins. Two redox-active disulfide bonds are essential for Ero1p oxidase activity: Cys100-Cys105 and Cys352-Cys355. Genetic and structural data indicate a disulfide bond is transferred from Cys100-Cys105 directly to Pdi1p, whereas a Cys352-Cys355 disulfide bond is used to reoxidize the reduced Cys100-Cys105 pair through an internal thiol-transfer reaction. Electron transfer from Cys352-Cys355 to molecular oxygen, by way of a flavin cofactor, maintains Cys352-Cys355 in an oxidized form. Herein, we identify a mixed disulfide species that confirms the Ero1p intercysteine thiol-transfer relay in vivo and identify Cys105 and Cys352 as the cysteines that mediate thiol-disulfide exchange. Moreover, we describe Ero1p mutants that have the surprising ability to oxidize substrates in the absence of Cys100-Cys105. We show the oxidase activity of these mutants results from structural changes in Ero1p that allow substrates increased access to Cys352-Cys355, which are normally buried beneath the protein surface. The altered activity of these Ero1p mutants toward selected substrates leads us to propose the catalytic mechanism involving transfer between cysteine pairs evolved to impart substrate specificity to Ero1p.     

Pi7, an orphan peptide from the scorpion Pandinus imperator: a 1H-NMR analysis using a nano-NMR Probe

The three-dimensional solution structure of a novel peptide, Pi7, purified from the venom of the scorpion Pandinus imperator, and for which no specific receptor has been found yet, was determined by two-dimensional homonuclear proton NMR methods from a nanomole amount of compound using a nano-nmr probe. Pandinus imperator peptide 7 does not block voltage-dependent K(+)-channels and does not displace labeled noxiustoxin from rat brain synaptosomal membranes. The toxin has 38 amino acid residues and, similarly to Pi1, is stabilized by four disulfide bridges (Cys6-Cys27, Cys12-Cys32, Cys16-Cys34, and Cys22-Cys37). In addition, the lysine at position 26 crucial for potassium-channel blocking is replaced in Pi7 by an arginine. Tyrosine 34, equivalent to Tyr36 of ChTX is present, but the N-terminal positions 1 and 2 are occupied by two acidic residues Asp and Glu, respectively. The dihedral angles and distance restraints obtained from measured NMR parameters were used in structural calculations in order to determine the conformation of the peptide. The disulfide-bridge topology was established using distance restraints allowing ambiguous partners between S atoms combined with NMR-derived structural information. The structure is organized around a short alpha-helix spanning residues Thr9 to Thr20/Gly21 and a beta-sheet. These two elements of secondary structure are stabilized by two disulfide bridges, Cys12-Cys32 and Cys16-Cys34. The antiparallel beta-sheet is composed of two strands extending from Asn22 to Cys34 with a tight turn at Ile28-Asn29 in contact with the N-terminal fragment Ile4 to Cys6.     

Profile of the disulfide bonds in acetylcholinesterase

The inter- and intrasubunit disulfide bridges for the 11 S form of acetylcholinesterase isolated from Torpedo californica have been identified. Localized within the basal lamina of the synapse, the dimensionally asymmetric forms of acetylcholinesterase contain either two (13 S) or three (17 S) sets of catalytic subunits linked to collagenous and noncollagenous structural subunits. Limited proteolysis of these molecules yields a tetramer of catalytic subunits that sediments at 11 S. Each catalytic subunit contains 8 cysteine residues which were identified following tryptic digestion of the reduced, 14C-carboxymethylated protein. The tryptic peptides were purified by gel filtration followed by reverse-phase high performance liquid chromatography (HPLC) and then sequenced. The disulfide bonding profile was determined by treating the native, nonreduced 11 S form of acetylcholinesterase with a fluorescent, sulfhydryl-specific reagent, monobromobimane, prior to tryptic digestion. Peptides again were resolved by gel filtration and reverse-phase HPLC. One fluorescent cysteine-containing peptide was identified, indicating that a single sulfhydryl residue, Cys231, was present in its reduced form. Three pairs of disulfide-bonded peptides were identified. These were localized in the polypeptide chain based on the cDNA-deduced sequence of the protein and were identified as Cys67-Cys94, Cys254-Cys265, and Cys402-Cys521. Hence, three loops are found in the secondary structure. Cys572, located in the carboxyl-terminal tryptic peptide, was disulfide-bonded to an identical peptide and most likely forms an intersubunit cross-link. Since the 6 cysteine residues in acetylcholinesterase that are involved in intrachain disulfide bonds are conserved in the sequence of the homologous protein thyroglobulin, it is likely that both proteins share a common folding pattern in their respective tertiary structures. Cys231 and the carboxyl-terminal cysteine residue Cys572 are not conserved in thyroglobulin.     

Structural characterization and independent folding of a chimeric glycoprotein comprising granulocyte-macrophage colony stimulating factor and erythropoietin sequences

MEN 11300 is a hybrid glycoprotein of 297 amino acids obtained by fusion of the cDNA encoding GM-CSF with the cDNA encoding EPO followed by transfection of the hybrid gene into CHO cells. The oligonucleotide construct incorporated a spacing sequence between the two individual cDNAs which encodes eight amino acids constituting a linker peptide intended to separate the GM-CSF and EPO moieties. The recombinant MEN 11300 protein was submitted to a detailed structural characterization including the verification of the entire amino acid sequence, the assignment of the disulfide bridges pattern, the identification of the glycosylation sites and the definition of the glycosidic moiety, including site-specificity. Partial processing of the C-terminal Arg residue and the occurrence of N-glycosylation sites at Asn27, Asn155, Asn169, Asn214 were established. Moreover, O-glycosylation at Ser257 and at the N-terminal region was also detected. A large heterogeneity was observed in the N-glycans due to the presence of differently sialylated and fucosylated branched complex type oligosaccharides whereas O-linked glycans were constituted by GalGalNAc chains with a different number of sialic acids. The disulfide bridges pattern was established by direct FABMS analysis of the proteolytic digests or by ESMS analysis of HPLC purified fractions. Pairing of the eight cysteine residues resulted in Cys54-Cys96, Cys88-Cys121, Cys138-Cys292, and Cys160-Cys164. This S-S bridges pattern is identical to that occurring in the individual natural GM-CSF and EPO, thus showing that the two protein moieties in MEN 11300 can independently acquire their native three-dimensional structure.      

Oxidative modification of aldose reductase induced by copper ion. Definition of the metal-protein interaction mechanism

Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys(303) appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys(298)-Cys(303). However, a Cys(80)-Cys(303) disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys(80)-Cys(303) disulfide to the more stable product Cys(298)-Cys(303) is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys(80)-Cys(303) disulfide. However, no evidence of conditions favoring the formation of the Cys(298)-Cys(303) disulfide was observed. Our proposal is that the generation of the Cys(298)-Cys(303) disulfide, either directly or by rearrangement of the Cys(80)-Cys(303) disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms.     

Size distribution of allergenic Cry j 2 released from airborne <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> pollen grains during the pollen scattering seasons

Japanese cedar (Cryptomeria japonica) pollinosis is one of seasonal allergic rhinitis that mainly occurs in Japan. The pollinosis is caused by two main kinds of allergenic proteins called Cry j 1 and Cry j 2 which exist in Cryptomeria japonica pollen. In our previous study, we reported that the size-segregated of airborne fine allergenic Cry j 1 and morphological change of Cry j 1 due to the contact with rainfall. However, the study on airborne allergenic Cry j 2 in different particle sizes has not been identified until now. Therefore, the main aim of this study is to investigate the size distribution and scattering behavior of allergenic Cry j 2. The Cry j 2 particles were collected and determined in different particle sizes at the urban sampling points during the most severe pollination season of 2012 in Saitama, Japan. After the size-segregated Cry j 2 allergenic particles were collected using an Andersen high-volume (AHV) atmospheric sample, the airborne Cry j 2 concentrations were determined with a surface plasmon resonance (SPR) method. At the same time, the airborne Cryptomeria japonica pollens were also counted by the Durham pollen sampler. The higher concentrations of the allergenic Cry j 2 were detected even in particle sizes equal to or less than 1.1 μm (PM_1.1) than other particle sizes. The airborne particles ranges from 0.06 to 11 μm were also collected by a low-pressure impactor (LPI) atmospheric sampler. After that, the concentrations of Cry j 2 allergenic particles in fine particle sizes were measured by the SPR method either. With the help of this study, we have confirmed the existence of fine daughter allergenic particles, which clearly differ from the parent pollen grains in size, especially after the rainy days. It is possible that the daughter allergenic species will be released from the fractions of cell wall and burst pollen grains. We concluded that rainwater was one of the important factors that affects the release of pollen allergenic proteins of both Cry j 1 and Cry j 2 from the parent pollen grains.     

Identification of the disulfide bonds in human plasma protein SP-40,40 (apolipoprotein-J).

SP-40,40, a human plasma protein, is a modulator of the membrane attack complex formation of the complement system as well as a subcomponent of high-density lipoproteins. In the present study, the positions of the disulfide bonds in SP-40,40 were determined. SP-40,40 was purified from human seminal plasma by affinity chromatography using an anti-SP-40,40 monoclonal antibody and reversed-phase, high-performance liquid chromatography (HPLC). The protein was digested with trypsin and the fragments were separated by reversed-phase HPLC. The peptides containing disulfide bonds were fluorophotometrically detected with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The peptides containing more than two disulfide bonds were further digested with Staphylococcus aureus V8 protease and lysylendopeptidase, and the fragments were isolated by HPLC. The amino acid compositions and the amino acid sequences of the peptides containing only a disulfide bond were determined. Disulfide bonds thus determined were between Cys58(alpha)-Cys107(beta), Cys68(alpha)-Cys99(beta), Cys75(alpha)-Cys94(beta), and Cys86(alpha)-Cys80(beta). Since there was no free sulfhydryl groups in the SP-40,40 molecule, Cys78(alpha) and Cys91(beta) should also be linked by a disulfide bond. It is notable that all of the disulfide bonds in SP-40,40 are not only formed by inter-chain pairing, but also appear to form an antiparallel ladder-like structure between the two chains. The unique structure could be related to the functions of SP-40,40.     

Functional structure of the somatomedin B domain of vitronectin

The N-terminal somatomedin B domain (SMB) of vitronectin binds PAI-1 and the urokinase receptor with high affinity and regulates tumor cell adhesion and migration. We have shown previously in the crystal structure of the PAI-1/SMB complex that SMB, a peptide of 51 residues, is folded as a compact cysteine knot of four pairs of crossed disulfide bonds. However, the physiological significance of this structure was questioned by other groups, who disputed the disulfide bonding shown in the crystal structure (Cys5-Cys21, Cys9-Cys39, Cys19-Cys32, Cys25-Cys31), notably claiming that the first disulfide is Cys5-Cys9 rather than the Cys5-Cys21 bonding shown in the structure. To test if the claimed Cys5-Cys9 bond does exist in the SMB domain of plasma vitronectin, we purified mouse and rat plasma vitronectin that have a Met (hence cleavable by cyanogen bromide) at residue 14, and also prepared recombinant human SMB variants from insect cells with residues Asn14 or Leu24 mutated to Met. HPLC and mass spectrometry analysis showed that, after cyanogen bromide digestion, all the fragments of the SMB derived from mouse or rat vitronectin or the recombinant SMB mutants are still linked together by disulfides, and the N-terminal peptide (residue 1-14 or 1-24) can only be released when the disulfide bonds are broken. This clearly demonstrates that Cys5 and Cys9 of SMB do not form a disulfide bond in vivo, and together with other structural evidence confirms that the only functional structure of the SMB domain of plasma vitronectin is that seen in its crystallographic complex with PAI-1.     

Disulfide pairing and secondary structure of ASP1, an olfactory-binding protein from honeybee (Apis mellifera L).

In insects, the transport of airborne, hydrophobic odorants and pheromones through the sensillum lymph is accomplished by olfactory-binding proteins (CBPs). We report the structural characterization of a honeybee OBP called ASP1 found in workers and drones, previously observed to bind queen pheromone components. A novel method based on ion-spray mass spectrometry analysis of cyanylation-induced cleavage products of partially reduced protein with Tris(2-carboxyethyl)phosphine was needed to determine the recombinant ASP1 disulfide bond pairing. It was observed to be Cys(I)-Cys(III), Cys(II)-Cys(V), Cys(IV)-Cys(VI), similar to those already described for other OBPs from honeybee and Bombyx mori suggesting that this pattern occurs commonly throughout the diverse family of insect OBPs. Circular dichroism revealed that ASP1 is an all-alpha protein in accordance with NMR preliminary data, but unlike lipocalin-like vertebrate OBPs.     

Structural analysis and disulfide-bridge pairing of two odorant-binding proteins from Bombyx mori

Pheromone-binding protein (PBP) and general odorant-binding proteins (GOBPs) were purified from the antennae of Bombyx mori and structurally characterised. The amino acid sequence of GOBP-2 has been corrected. The disulphide arrangements of PBP and GOBP-2 have been determined by a combined mass spectrometric/Edman degradation approach. The same cysteine pairings, Cys19-Cys54, Cys50-Cys108, and Cys97-Cys117, were found in both proteins, suggesting that such patterns occur commonly throughout this family of molecules. This arrangement of disulphide bonds indicates that the three-dimensional structure of insect OBPs is defined by three loops, rich in helical content, which can vary in size and charge distribution from one protein to another.     

Convergent solid-phase synthesis of hirudin.

Hirudin variant 1 (HV1), a small protein consisting of 65 amino acids and three disulfide bonds, was synthesized by using Fmoc-based convergent methods on 2-chlorotrityl resin (CLTR). The linear sequence was assembled by the sequential condensation of 7 protected fragments, on the resin-bound 55-65 fragment. The conditions of fragment assembly were carefully studied to determine the most efficient synthetic protocol. Crude reduced [Cys(16, 28)(Acm)]-HV1 thus obtained was easily purified to homogeneity by RP-HPLC. Disulfide bridges were successfully formed by a two-step procedure, involving an oxidative folding step to form Cys(6)-Cys(14) and Cys(22)-Cys(39) linkages, followed by iodine oxidation to form the Cys(16)-Cys(28) bond. The correct disulfide bond alignment was established by peptide mapping using Staphylococcus aureus V8 protease at pH 4.5.     

cDNA cloning and expression of acutin

Acutin, a thrombin-like enzyme was purified from Agkistrodon acutus venom in three steps by DEAE-Sepharose CL-6B, Superose 12 column on FPLC and Mono-Q column chromatographies. Its first 15 N-terminal amino acid residues sequence was then determined and the acutin cDNA was isolated from venom gland total RNA using RT-PCR. Determination of its nucleotide sequence allowed elucidation of the amino acid sequence of mature peptide for the first time. The mature acutin has 233 amino acids and its amino acid sequence exhibits significant homology with those of thrombin-like enzymes from crotaline snakes venoms. Based on the homology, the catalytic residues and disulfide bridges of acutin were deduced to be as follows: catalytic residues, His41, Asp84 and Ser179; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys231, Cys118-Cys185, Cys150-Cys164, Cys175-Cys200. The recombinant acutin has been expressed in E. coli and purified by affinity column. The renatured recombinant acutin is reported for the first time to have the activity of clotting fibrinogen and arginine-esterase.     

Primary structure of the major pepsin inhibitor from the intestinal parasitic nematode Ascaris suum

The major pepsin inhibitor from Ascaris suum was isolated by affinity chromatography and chromatofocusing. Its amino acid sequence was determined by automated Edman degradation of peptide fragments. Peptides were produced by chemical and enzymatic cleavage of pyridylethylated protein and were purified by reverse-phase high-performance liquid chromatography. The inhibitor consists of 149 residues with the following sequence: QFLFSMSTGP10FICTVKDNQV20FVANLPWTML30EGDDIQVGKE40 FAARVEDCTN50VKHDMAPTCT60KPPPFCGPQD70MKMFNFVGCS80VLGNKLFIDQ90KYVRDLTAK D100 HAEVQTFREK110IAAFEEQQEN120QPPSSGMPHG130AVPAGGLSPP140PPPSFCTVQ149. It has a molecular weight of 16,396. All cysteines are engaged as disulfide bonds: Cys(13)-Cys(59), Cys(48)-Cys(66), and Cys(79)-Cys(146). The protein is probably composed of two domains connected by a short hydrophobic region. This is the first aspartyl protease inhibitor of animal origin that has been sequenced. The sequence has no significant homology with any other known protein.     

Oxidized galectin-1 promotes axonal regeneration in peripheral nerves but does not possess lectin properties.

Galectin-1 has recently been identified as a factor that regulates initial axonal growth in peripheral nerves after axotomy. Although galectin-1 is a well-known beta-galactoside-binding lectin, its potential to promote axonal regeneration as a lectin has not been reported. It is essential that the process of initial repair in peripheral nerves after axotomy is well clarified. We therefore undertook to investigate the relation between the structure and axonal regeneration-promoting activity of galectin-1. Recombinant human galectin-1 secreted into the culture supernatant of transfected COS1 cells (rhGAL-1/COS1) was purified under nonreducing conditions and subjected to structural analysis. Mass spectrometric analysis of peptide fragments from rhGAL-1/COS1 revealed that the secreted protein exists as an oxidized form containing three intramolecular disulfide bonds (Cys2-Cys130, Cys16-Cys88 and Cys42-Cys60). Recombinant human galectin-1 (rhGAL-1) and a galectin-1 mutant in which all six cysteine residues were replaced by serine (CSGAL-1) were expressed in and purified from Escherichia coli for further analysis; the purified rhGAL-1 was subjected to oxidation, which induced the same pattern of disulfide linkages as that observed in rhGAL-1/COS1. Oxidized rhGAL-1 enhanced axonal regeneration from the transected nerve sites of adult rat dorsal root ganglion explants with associated nerve stumps (5.0-5000 pg. mL-1), but it lacked lectin activity. In contrast, CSGAL-1 induced hemagglutination of rabbit erythrocytes but lacked axonal regeneration-promoting activity. These results indicate that galectin-1 promotes axonal regeneration only in the oxidized form containing three intramolecular disulfide bonds, not in the reduced form which exhibits lectin activity.     

Purification, identification, and cDNA cloning of Jun a 2, the second major allergen of mountain cedar pollen

The second major allergen of Juniperus ashei (mountain cedar) pollen, Jun a 2, has been purified and its cDNA cloned. The purified protein has a molecular mass of 43 kDa and its N-terminal 9-residue amino acid sequence is highly homologous to those of Cry j 2 and Cha o 2, the second major allergen of Cryptomeria japonica and Chamaecyparis obtusa pollen, respectively. cDNA clones encoding Jun a 2 were isolated after PCR based amplification, and their nucleotide sequences were determined. The cDNA contains an open reading frame of 507 amino acid residues, and encodes a putative 54-residue signal sequence and a 453-residue intermediate, which releases a C-terminal fragment upon maturation. Three possible N-linked glycosylation sites and 20 cystein-residues are found in the deduced amino acid sequence. The amino acid sequence of Jun a 2 shows 70.7 and 82.0% identity with those of Cry j 2 and Cha o 2, respectively. Immunological observations that IgE antibodies in sera of Japanese pollinosis patients bind not only to Cry j 2 and Cha o 2 but also to Jun a 2 strongly suggest that Jun a 2 is an allergen of mountain cedar pollen, and that allergenic epitopes of these three allergens are similar.     

Cysteine cross-linking defines part of the dimer and B/C domain interface of the Escherichia coli mannitol permease

Part of the dimer and B/C domain interface of the Escherichia coli mannitol permease (EII(mtl)) has been identified by the generation of disulfide bridges in a single-cysteine EII(mtl), with only the activity linked Cys(384) in the B domain, and in a double-cysteine EII(mtl) with cysteines at positions 384 and 124 in the first cytoplasmic loop of the C domain. The disulfide bridges were formed in the enzyme in inside-out membrane vesicles and in the purified enzyme by oxidation with Cu(II)-(1,10-phenanthroline)(3), and they were visualized by SDS-polyacrylamide gel electrophoresis. Discrimination between possible disulfide bridges in the dimeric double-cysteine EII(mtl) was done by partial digestion of the protein and the formation of heterodimers, in which the cysteines were located either on different subunits or on one subunit. The disulfide bridges that were identified are an intersubunit Cys(384)-Cys(384), an intersubunit Cys(124)-Cys(124), an intersubunit Cys(384)-Cys(124), and an intrasubunit Cys(384)-Cys(124). The disulfide bridges between the B and C domain were observed with purified enzyme and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mannitol did not influence the formation of the disulfide between Cys(384) and Cys(124). The close proximity of the two cysteines 124 was further confirmed with a separate C domain by oxidation with Cu(II)-(1,10-phenanthroline)(3) or by reactions with dimaleimides of different length. The data in combination with other work show that the first cytoplasmic loop around residue 124 is located at the dimer interface and involved in the interaction between the B and C domain.