Cytoplasmic retraction of the amino terminus of human multidrug resistance protein 1

Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) transport superfamily which also includes human multidrug resistance 1 (MDR1) gene product P-glycoprotein (Pgp). Overexpression of MRP1 or Pgp causes multidrug resistance in cancer cells. Different from Pgp, MRP1 contains an extra membrane-spanning domain (MSD1) with a putative extracellular amino terminus in addition to the core structure of two MSDs and two NBDs (nucleotide-binding domains). The structural and functional significance of the additional MSD1 in MRP1 remains elusive. In this study, we generated an IgG1 subclass monoclonal antibody, IU2H10, specific to the amino terminus of human MRP1 and mapped its epitope to 10 amino acids (S8ADGSDPLWD17). It can be used for Western blot, immunoprecipitation, and indirect immunofluorescence studies of human MRP1. However, surprisingly we found that IU2H10 cannot react with MRP1 unless cells are permeabilized. Furthermore, the IU2H10 epitope is exposed extracellularly when the carboxyl-terminal core domain of human MRP1 is deleted. Examination of the amino-terminal sequence of human MRP1 suggests that it consist of mainly coiled structures. These observations provide evidence for a model that is different from the prevailing extracellular location of the amino terminus of human MRP1. It is possible that part of the amino terminus of human MRP1, following exposure to the lumen of the endoplasmic reticulum, is retracted to the cytoplasm.     

The multidrug resistance protein is photoaffinity labeled by a quinoline-based drug at multiple sites

Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP has been shown to mediate the transport of cytotoxic natural products, in addition to glutathione-, glucuronidate-, and sulfate-conjugated cell metabolites. However, the mechanism of MRP drug binding and transport is at present not clear. In this study, we have used a photoreactive quinoline-based drug, N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide (IACI), to show the photoaffinity labeling of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein as the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1. Furthermore, a molar excess of leukotriene C(4), doxorubicin, colchicine, and other quinoline-based drugs, including MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI accumulation in MRP-expressing cells which was reversed by depleting ATP levels in H69/AR cells. Mild digestion of the purified IACI-photolabeled MRP with trypsin showed two large polypeptides ( approximately 111 and approximately 85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids approximately 900-1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1-900) containing the MSD1 and MSD2 plus the first nucleotide binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and approximately 6 plus 4 kDa photolabeled peptides, respectively. In addition, resolution of the exhaustively digested IACI-photolabeled MRP by HPLC showed two major and one minor radiolabeled peaks that eluted late in the gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying MRP-drug interactions.     

Mapping of the MRPm5 epitope to the cytosolic region between transmembrane helices 13 and 14 in the drug and organic anion transporter, MRP1 (ABCC1)

Multidrug resistance in human tumour cells is often associated with increased expression of the 190kDa multidrug resistance protein, MRP1, that belongs to the ATP-binding cassette superfamily of transport proteins. MRP1 is also an efficient transporter of many organic anions. In the present study, we have mapped the epitope of the MRP1-specific murine monoclonal antibody (MAb) MRPm5 to the decapeptide (1063)FFERTPSGNL(1072) located in the cytoplasmic loop (CL6) linking transmembrane helices 13 and 14 in the third membrane spanning domain of the protein. Several amino acids in the cytoplasmic loops of MRP1 have been reported to be important for its transport function; nevertheless, MAb MRPm5 does not inhibit vesicular uptake of the high affinity substrate leukotriene C(4). None of the other MRP1-reactive MAbs described to date map to CL6 of MRP1 which in turn enhances the utility of MAb MRPm5 for both clinical and experimental investigations of this transporter.     

A serotype-specific epitope of dengue virus 1 identified by phage displayed random peptide library

Abstract From a panel of monoclonal antibodies of dengue viruses, a serotype-specific epitope of dengue virus 1 was screened from a random peptide library displayed on phage. The epitope was the determinant reactive with monoclonal antibody 15F3-1 that was specific to dengue 1. The screening was monitored by a dot blotting procedure, and after three rounds of screening a consensus motif, HRYSWK, was found. This sequence matches the sequence HKYSWK, corresponding to the amino acid residues 885–890 of polyprotein or residues 111–116 of the non-structural protein 1 of dengue virus serotype 1. The linear epitope was confirmed by testing the antigenicity of chemically synthesized 8-branched peptide.     

Functional and structural consequences of cysteine substitutions in the NH2 proximal region of the human multidrug resistance protein 1 (MRP1/ABCC1)

The 190 kDa multidrug resistance protein 1 (MRP1; ABCC1) is comprised of three membrane spanning domains (MSDs) and two nucleotide binding domains (NBDs) configured MSD1-MSD2-NBD1-MSD3-NBD2. MRP1 overexpression in tumor cells results in an ATP-dependent efflux of many oncolytic agents and arsenic and antimony oxyanions. MRP1 also transports GSSG and GSH as well as conjugated organic anions, including leukotriene C(4) and 17beta-estradiol 17-(beta-D-glucuronide) and certain xenobiotics in association with GSH. Previous studies have shown that portions of MSD1 and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. In the present study, Cys residues at positions 43, 49, 85, 148, and 190 in MSD1 and positions 208 and 265 in CL3 were mutated to Ala and Ser, and the effects on protein expression, plasma membrane localization, trypsin sensitivity, organic anion transport, and drug resistance properties were investigated. Confocal microscopy showed that 11 of 14 mutants displayed significant levels of nonplasma membrane-associated MRP1. Most mutant proteins were also more resistant to trypsin proteolysis than wild-type MRP1. All Cys mutants transported organic anions (0.5-1.5-fold wild-type MRP1 activity), and cells expressing Ser-substituted but not Ala-substituted Cys43 and Cys265 MRP1 mutants exhibited a 2.5-fold decrease and a 3-fold increase in arsenite resistance, respectively; Cys43Ser MRP1 also conferred lower levels of vincristine resistance. These results indicate that certain Cys residues in the NH(2) proximal region of MRP1 can be important for its structure and selected transport activities.     

The leucotriene C4 binding sites in multidrug resistance protein 1 (ABCC1) include the first membrane multiple spanning domain

The multiple drug resistance protein 1 (MRP1 or ABCC1) transports anticancer drugs and normal cell metabolites. Leucotriene C(4) (LTC(4)) is one of the highest affinity substrates of MRP1. In this study, we have synthesized and characterized a novel photoreactive azido analogue of LTC(4) (AALTC(4)). The specificity of AALTC(4) binding to MRP1 was confirmed using an LTC(4)-specific monoclonal antibody. Moreover, binding with radioiodinated [(125)I]AALTC(4) (or IAALTC(4)) to MRP1 was dramatically competed with unmodified LTC(4) and to a lesser degree by glutathione (GSH). Oxidized glutathione (GSSG) slightly increased IAALTC(4) binding to MRP1, while MK571, verapamil, and vincristine inhibited IAALTC(4) binding to MRP1. Using AALTC(4) together with a panel of epitope-specific and LTC(4)-specific monoclonal antibodies, we identified LTC(4) binding sites in MRP1. Western blotting of large tryptic fragments of MRP1 with three well-characterized epitope-specific mAbs (MRPr1, QCRL1, and MRPm6) showed LTC(4) binding in both the N- and C-terminal halves of MRP1. Furthermore, a peptide corresponding to the N-terminal membrane-spanning domain of MRP1 (MSD0) was photoaffinity labeled by AALTC(4), indicating that MSD0 contains an LTC(4) binding site. Higher resolution mapping of additional LTC(4) binding sites was obtained using eight MRP1 variants with each containing hemaglutanin A (HA) epitopes at different sites (at amino acid 4, 163, 271, 574, 653, 938, 1001, or 1222). MRP1 variants were photoaffinity labeled with IAALTC(4) and digested with trypsin to isolate specific regions of MRP1 that interact with LTC(4). These results confirmed that sequences in MSD0 interact with IAALTC(4). Other regions that were photoaffinity labeled by IAALTC(4) include TM 10-11, TM 16-17, and TM 12, shown previously to encode MRP1 drug binding site(s). Together, our results show a high-resolution map of LTC(4) binding domains in MRP1 and provide the first direct evidence for LTC(4) binding within MSD0.     

Probing the conformational and dynamical effects of O-glycosylation within the immunodominant region of a MUC1 peptide tumor antigen.

MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is under-glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc) and TF (Gal beta1-3 GalNAc) carbohydrates. Here, we report the results of 1H NMR structural studies, natural abundance 13C NMR relaxation measurements and distance-restrained MD simulations designed to probe the structural and dynamical effects of Tn-glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine-residue MUC1 peptide of the sequence, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6-Arg7-Pro8-Ala9, and a Tn-glycosylated version of this peptide, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6(alphaGalNAc)-Arg7-Pro8-Ala9. The results of these studies show that a type I beta-turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar beta-turn within the PDTRP core peptide epitope of the under-glycosylated cancer-associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I beta-turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.     

Human T cells recognize polymorphic and non-polymorphic regions of the Plasmodium falciparum circumsporozoite protein.

In order to characterize T cell epitopes in the Plasmodium falciparum circumsporozoite (CS) protein sequence, we isolated T cell clones, from non-immune donors, which reacted with synthetic peptides corresponding to two predicted CS protein T cell epitopes. Peptide CS.T3 (corresponding to a non-polymorphic region of the CS protein, residues 378-398) was recognized in association with either DR2 or DRw9 restriction elements. T cell clones recognizing CS.T3 also reacted with the sporozoite-derived CS protein. Peptide CS.T2 corresponds to a polymorphic region (residues 325-341) of the CS protein. Unlike the CS.T3-specific clones, the CS.T2-specific clones did not recognize the CS protein. Since the CS.T2 peptide includes residues which are polymorphic in different P. falciparum isolates, we investigated whether these residues were critical for recognition of the peptide. We show here that a single amino acid substitution at a position of the CS protein which shows genetic polymorphism affects recognition of the sequence by human T cells. The implications of these data for malaria vaccine development are discussed.     

Purification of the human apical conjugate export pump MRP2 reconstitution and functional characterization as substrate-stimulated ATPase.

The multidrug resistance protein MRP2 (ABCC2) acts as an ATP-dependent conjugate export pump in apical membranes of polarized cells and confers multidrug resistance. Purified MRP2 is essential for the detailed functional characterization of this member of the family of ATP-binding cassette (ABC) transporter proteins. In human embryonic kidney cells (HEK293), we have permanently expressed MRP2 containing an additional C-terminal (His)6-tag. Immunoblot and immunofluorescence analyses detected the MRP2-(His)6 overexpressing clones. Isolated membrane vesicles from the MRP2-(His)6-expressing cells were active in ATP-dependent transport of the glutathione S-conjugate leukotriene C4 and were photoaffinity-labelled with 8-azido-[alpha-32P]ATP. MRP2-(His)6 was solubilized from membranes of MRP2-(His)6-cells and purified to homogeneity in a three-step procedure using immobilized metal affinity chromatography, desalting, and immunoaffinity chromatography. The identity of the pure MRP2-(His)6 was verified by MS analysis of tryptic peptides. The purified MRP2-(His)6 glycoprotein was reconstituted into proteoliposomes and showed functional activity as ATPase in a protein-dependent manner with a Km for ATP of 2.1 mM and a Vmax of 25 nmol ADP x mg MRP2-1 x min-1. This ATPase activity was substrate-stimulated by oxidized and reduced glutathione and by S-decyl-glutathione. Future studies using pure MRP2 reconstituted in proteoliposomes should allow further insight into the molecular parameters contributing to MRP2 transport function and to define its intracellular partners for transport and multidrug resistance.     

Evidence for the role of glycosylation in accessibility of the extracellular domains of human MRP1 (ABCC1)

To enable cell surface localization of the human multidrug resistance protein (MRP1, ABCC1) and to assess the role of the extracellular domains of this transporter, the FLAG epitope tag was introduced into different extracellular loops of the three membrane-spanning domains (MSDs) of the transporter. We constructed and expressed various partially and fully glycosylation-deficient, FLAG-tagged MRP1 proteins in a Vaccinia virus-based transient expression system, and the cell surface expression level of MRP1 on intact cells was followed by flow cytometry, using the FLAG tag specific monoclonal antibody M2. We also expressed the wild-type MRP1 protein and some of the FLAG-tagged mutants in stably transfected HEK293 cells, and followed the cell surface expression and the transport function of MRP1 both by monitoring the efflux of fluorescent substrate and by their ability to confer resistance to HEK293 transfectants to anticancer agents such as daunorubicin and etoposide. When we inserted the FLAG epitope in extracellular loops of the MSD1 or MSD3, the tag was accessible upon removal of N-glycosylation sites (N --> Q at positions 17, 23, and 1006, respectively), whereas the FLAG epitope placed in the MSD2 was not accessible even after removal of all three N-glycosylation sites, indicating that MSD2 region is deeply buried in the plasma membrane. However, all FLAG tagged MRP1 mutants were expressed at the cell surface to the same extent as the wild-type protein and also exhibited normal transport function. Our results demonstrate that the accessibility of the external FLAG epitope is strongly dependent on the position of the tag and the glycosylation state of the different FLAG-tagged MRP1s, and the conformation of extracellular loops in MSD1 and MDS3 does not appear to contribute to the functional status of MRP1.     

A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida

In this study, we have mapped and characterized a B cell epitope of sulfated glycoprotein ZP2 (ZP2) as a step toward the development of a multi-epitope zona pellucida (ZP) vaccine. Recombinant polypeptides expressed by random deoxyribonuclease-digested fragments of ZP2 cDNA were screened for binding to IE-3, a monoclonal antibody to murine ZP2. Positive clones contained cDNA inserts encoding polypeptide corresponding to ZP2(103-134). When normal or ovariectomized female mice were immunized with three overlapping peptides that span this region of ZP2 (101-120, 111-130, 121-140), only ZP2(121-140) elicited IgG antibodies that reacted with mouse ovarian ZP, indicative of the presence of native B epitope and helper T cell epitope in ZP2(121-140). To more finely map the ZP2 B cell epitope, a random peptide display library was screened with the IE-3 antibody, and a consensus tetramer sequence VxYK that matched the ZP2(123-126) sequence VRYK was located. Competitive immunofluorescence analysis with single alanine-substituted VxYK peptides ranked the relative contribution of the three critical B cell epitope residues as Y > V > K. A chimeric peptide was constructed that contained the YRYK motif of ZP2 and a bovine RNase T cell epitope. Although (C57BL/6xA/J) F1 (B6AF1) female mice immunized with the chimeric peptide developed ZP antibody response, this peptide elicited antibody only in mice of the histocompatibility complex (MHC) H-2(k or b) haplotype. In contrast, ZP2(121-140) peptide elicited antibody in inbred mice with three additional mouse MHC haplotypes. Moreover, although ZP2(121-140) contained a T cell epitope, no oophoritis was observed after immunization of B6AF1 mice with ZP2(121-140) in complete Freund's adjuvant (CFA). In a preliminary trial, female B6AF1 mice immunized with ZP2(121-140) in CFA had reduced litter sizes as compared with mice injected with CFA alone.     

Identification and characterization of functionally important elements in the multidrug resistance protein 1 COOH-terminal region

The ATP binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), transports a broad spectrum of conjugated and unconjugated compounds, including natural product chemotherapeutic agents. In this study, we have investigated the importance of the COOH-terminal region of MRP1 for transport activity and basolateral plasma membrane trafficking. The COOH-terminal regions of some ABCC proteins have been implicated in protein trafficking, but the function of this region of MRP1 has not been defined. In contrast to results obtained with other ABCC proteins, we found that the COOH-proximal 30 amino acids of MRP1 can be removed without affecting trafficking to basolateral membranes. However, the truncated protein is inactive. Furthermore, removal of as few as 4 COOH-terminal amino acids profoundly decreases transport activity. Although amino acid sequence conservation of the COOH-terminal regions of ABC proteins is low, secondary structure predictions indicate that they consist of a broadly conserved helix-sheet-sheet-helix-helix structure. Consistent with a conservation of secondary and tertiary structure, MRP1 hybrids containing the COOH-terminal regions of either the homologous MRP2 or the distantly related P-glycoprotein were fully active and trafficked normally. Using mutated proteins, we have identified structural elements containing five conserved hydrophobic amino acids that are required for activity. We show that these are important for binding and hydrolysis of ATP by nucleotide binding domain 2. Based on crystal structures of several ABC proteins, we suggest that the conserved amino acids may stabilize a helical bundle formed by the COOH-terminal three helices and may contribute to interactions between the COOH-terminal region and the protein's two nucleotide binding domains.     

A monomeric 3(10)-helix is formed in water by a 13-residue peptide representing the neutralizing determinant of HIV-1 on gp41

The peptide gp41(659-671) (ELLELDKWASLWN) comprises the entire epitope for one of the three known antibodies capable of neutralizing a broad spectrum of primary HIV-1 isolates and is the only such epitope that is sequential. Here we present the NMR structure of gp41(659-671) in water. This peptide forms a monomeric 3(10)-helix stabilized by i,i+3 side chain-side chain interactions favored by its primary sequence. In this conformation the peptide presents an exposed surface, which is mostly hydrophobic and consists of conserved HIV-1 residues. The presence of the 3(10)-helix is confirmed by its characteristic CD pattern. Studies of the 3(10)-helix have been hampered by the absence of a model peptide adopting this conformation. gp41(659-671) can serve as such a model to investigate the spectral characteristics of the 3(10)-helix, the factors that influence its stability, and the propensity of different amino acids to form a 3(10)-helix. The observation that the 3(10)-helical conformation is highly populated in the peptide gp41(659-671) indicates that the corresponding segment in the cognate protein is an autonomous folding unit. As such, it is very likely that the helical conformation is maintained in gp41 throughout the different tertiary structures of the envelope protein that form during the process of viral fusion. However, the exposure of the gp41(659-671) segment may vary, leading to changes in the reactivity of anti-gp41 antibodies in the different stages of viral fusion. Since gp41(659-671) is an autonomous folding unit, peptide immunogens consisting of the complete gp41(659-671) sequence are likely to induce antibodies highly cross-reactive with HIV-1.     

Structural and functional consequences of mutating cysteine residues in the amino terminus of human multidrug resistance-associated protein 1

Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette membrane transport superfamily and is responsible for multidrug resistance in cancer cells. Currently, there are nine known human MRPs. Distinct from many other members of the ATP-binding cassette superfamily, human MRP1 and four other MRPs have an additional membrane-spanning domain (MSD) with a putative extracellular amino terminus. The functional significance of this additional MSD (MSD1) is currently unknown. To understand the role of MSD1 in human MRP1 structure and function, we studied the amino-terminal 33 amino acids. We found that the amino terminus of human MRP1 has two cysteine residues (Cys(7) and Cys(32)) that are conserved among the five human MRPs that have MSD1. Mutation analyses of the two cysteines in human MRP1 revealed that the Cys(7) residue is critical for the MRP1-mediated drug resistance and leukotriene C(4) transport activity. On the other hand, mutation of Cys(32) reduced only moderately the MRP1 function. The effect of Cys(7) mutation on MRP1 activity appears to be due to the 5-7-fold decrease in the maximal transport rate V(max). We also found that mutation of Cys(7) changed the amino-terminal conformation of MRP1. This conformational change is likely responsible for the decrease in V(max) of LTC(4) transport mediated by the mutant MRP1. Based on these studies, we conclude that the amino terminus of human MRP1 is important and that the Cys(7) residue plays a critical role in maintaining the proper structure and function of human MRP1.     

Mutational analysis of the Saccharomyces cerevisiae ATP-binding cassette transporter protein Ycf1p

Ycf1p is a member of the ATP-binding cassette transporter family of membrane proteins. Strong sequence similarity has been observed between Ycf1p, the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance protein (MRP). In this work, we have examined the functional significance of several of the conserved amino acid residues and the genetic requirements for Ycf1p subcellular localization. Biochemical fractionation experiments have established that Ycf1p, expressed at single-copy gene levels, co-fractionates with the vacuolar membrane and that this co-fractionation is independent of vps15 , vps34 or end3 gene function. Several cystic fibrosis-associated alleles of the CFTR were introduced into Ycf1p and found to elicit defects analogous to those seen in the CFTR. An amino-terminal extension shared between Ycf1p and MRP, but absent from CFTR, was found to be required for Ycf1p function, but not its subcellular localization. Mutant forms of Ycf1p were also identified that exhibited enhanced biological function relative to the wild-type protein. These studies indicate that Ycf1p will provide a simple, genetically tractable model system for the study of the trafficking and function of ATP-binding cassette transporter proteins, such as the CFTR and MRP.     

Binding of a photoaffinity analogue of glutathione to MRP1 (ABCC1) within two cytoplasmic regions (L0 and L1) as well as transmembrane domains 10-11 and 16-17

MRP1 (or ABCC1) is an ABC membrane protein that transports a wide range of natural products as well as glutathione (GSH)-, glucuronate-, and sulfate-conjugated metabolites. In addition, free GSH is required for MRP1 to transport several chemotherapeutic drugs. However, the mechanisms regulating the influence of GSH on MRP1 is poorly understood, and the location of GSH binding site(s) within MRP1 have yet to be determined. To address these issues, we have synthesized a [(125)I] labeled azido-derivative of GSH (IAAGSH) to photoaffinity label MRP1. Our results revealed that IAAGSH labeled MRP1 with high specificity, and binding was inhibited by MRP1 substrates leukotriene C(4) and MK571. Interestingly, verapamil and vincristine enhanced IAAGSH photolabeling of MRP1, in agreement with observations that both drugs enhance GSH transport. We observed GSH to be the best inhibitor of photoaffinity labeling, as compared to oxidized glutathione (GSSG) and four different GSH alkyl derivatives. These observations indicate that IAAGSH interacted with MRP1 in a similar manner as unmodified GSH. Moreover, using eight MRP1-HA variants, each containing hemagglutinin A (HA) epitopes inserted at different sites in MRP1, we mapped the GSH binding sites in MRP1. Our GSH analogue photoaffinity labeled four MRP1 polypeptides that were located within two cytoplasmic domains in linker sequences (L0 and L1) as well as transmembrane domains 10-11 and 16-17. The photoaffinity labeling of polypeptides within L0 and L1 domains is further confirmed using two MRP1-specific monoclonal antibodies (MRPr1 and QCRL1) with epitopes within the linker domains. Taken together, this study provides the most precise information to date on the location of GSH binding sites in MRP1.     

Protection against experimental encephalomyelitis. Idiotypic autoregulation induced by a nonencephalitogenic T cell clone expressing a cross-reactive T cell receptor V gene.

The recovery process in experimental autoimmune encephalomyelitis (EAE) in Lewis rats is characterized by an increasing diversity of T cell clones directed at secondary epitopes of myelin basic protein. Of particular interest, residues 55 to 69 of guinea pig basic protein could induce protection against EAE. A nonencephalitogenic T cell clone, C455-69, that was specific for this epitope transferred protection against both active and passive EAE. Clone C4 was found to express V beta 8.6 in its Ag receptor, and residues 39 to 59 of the TCR V beta 8.6 sequence were found to be highly crossreactive with the corresponding residues 39 to 59 of TCR V beta 8.2, which is known to induce protective anti-idiotypic T cells and antibodies. Like the TCR V beta 8.2 peptide, the V beta 8.6 sequence induced autoregulation and provided effective treatment of established EAE. Thus, the EAE-protective effect of the guinea pig basic protein 55-69 sequence was most likely mediated by T cell clones such as C4 that could efficiently induce anti-TCR immunity directed at a cross-reactive regulatory idiotope.     

Polymorphic residues on the I-A beta chain modulate the stimulation of T cell clones specific for the N-terminal peptide of the autoantigen myelin basic protein

The effect of polymorphic residues on the A alpha A beta molecule on T cell recognition of the N-terminal nonapeptide of myelin basic protein (R1-9) was determined. Ak-restricted T cell clones recognizing R1-9 were isolated. The peptide-Ia specificities of these clones were determined by testing the response to 1) a panel of peptide analogs of R1-11, 2) splenic APC from mice expressing MHC molecules from serologically distinct haplotypes, and 3) L cell transfectants expressing mutant/recombinant A beta cDNA containing combinations of polymorphic nucleotide sequences from the k and u alleles. Comparisons were made between the Ak-restricted clones and a previously characterized panel of Au-restricted clones. Certain Ak-restricted clones were able to recognize MBP peptide analogs that were not recognized by any of the Au-restricted clones. The Au-restricted T cell clones did not cross-react with R1-9 presented in the context of Ak, whereas the majority of the Ak-restricted clones responded to R1-9 presented in the context of Au. This nonreciprocal cross-reactivity was also reflected in the relative responses of the two sets of T cell clones to the interchange of u- and k-derived residues in the A beta chain. Residues in regions corresponding both the alpha-helical or beta-sheet portions of the hypothetical Ia three-dimensional structure were involved. The results suggest that overall specificity of the T cell clones is the summation of numerous distinct subspecificities for different regions of the peptide-Ia ligand. These results indicate that there can be striking differences in T cell specificity for an autoantigenic epitope, even in the context of A alpha A beta molecules from very closely related haplotypes.     

Human immunodeficiency virus type 1-neutralizing monoclonal antibody 2F5 is multispecific for sequences flanking the DKW core epitope

Human monoclonal antibody 2F5 is one of a few human antibodies that neutralize a broad range of HIV-1 primary isolates. The 2F5 epitope on gp41 includes the sequence ELDKWA, with the core residues, DKW, being critical for antibody binding. HIV-neutralizing antibodies have never been elicited by immunization with peptides bearing ELDKWA, suggesting that important part(s) of the 2F5 paratope remain unidentified. The use of longer peptides extending beyond ELDKWA has resulted in increased epitope antigenicity, but neutralizing antibodies have not been generated. We sought to develop peptides that bind to 2F5, and that function as specific probes of the 2F5 paratope. Thus, we used 2F5 to screen a set of phage-displayed, random peptide libraries. Tight-binding clones from the random peptide libraries displayed sequence variability in the regions flanking the DKW motif. To further reveal flanking regions involved in 2F5 binding, two semi-defined libraries were constructed having 12 variegated residues either N-terminal or C-terminal to the DKW core (X(12)-AADKW and AADKW-X(12), respectively). Three clones isolated from the AADKW-X(12) library had similar high affinities, despite a lack of sequence homology among them, or with gp41. The contribution of each residue of these clones to 2F5 binding was evaluated by Ala substitution and amino acid deletion studies, and revealed that each clone bound 2F5 by a different mechanism. These results suggest that the 2F5 paratope is formed by at least two functionally distinct regions: one that displays specificity for the DKW core epitope, and another that is multispecific for sequences C-terminal to the core epitope. The implications of this second, multispecific region of the 2F5 paratope for its unique biological function are discussed.     

The multidrug resistance protein family

The human multidrug resistance protein (MRP) family contains at least six members: MRP1, the godfather of the family and well known as the multidrug resistance protein, and five homologs, called MRP2-6. In this review, we summarize what is known about the protein structure, the expression in tissues, the routing in cells, the physiological functions, the substrate specificity, and the role in multidrug resistance of the individual members of the MRP family.