The structure and action of ricin, a cytotoxic N-glycosidase

The X-ray structure of the heterodimeric plant cytotoxin ricin has been elucidated. The A chain, known to be a specific N-glycosidase, has a prominent active site cleft. The B chain is a two domain lectin, which arose from the replication of a primitive sugar binding peptide. The molecular model reveals residues which are likely to be important in the action of the protein. Site directed mutagenesis reveals the relative importance of a number of these groups. A model for the mechanism of the A chain, including steric strain of the scissile bond and carboxonium ion transition state stabilization is proposed.     

The 2.8 A crystal structure of Gla-domainless activated protein C.

The structure of the Gla-domainless form of the human anticoagulant enzyme activated protein C has been solved at 2.8 A resolution. The light chain is composed of two domains: an epidermal growth factor (EGF)-like domain modified by a large insert containing an additional disulfide, followed by a typical EGF-like domain. The arrangement of the long axis of these domains describes an angle of approximately 80 degrees. Disulfide linked to the light chain is the catalytic domain, which is generally trypsin-like but contains a large insertion loop at the edge of the active site, a third helical segment, a prominent cationic patch analogous to the anion binding exosite I of thrombin and a trypsin-like Ca[II] binding site. The arrangement of loops around the active site partially restricts access to the cleft. The S2 and S4 subsites are much more polar than in factor Xa and thrombin, and the S2 site is unrestricted. While quite open and exposed, the active site contains a prominent groove, the surface of which is very polar with evidence for binding sites on the primed side, in addition to those typical of the trypsin class found on the non-primed side.     

Propagation of action potentials between parallel chains of cardiac muscle cells in PSpice simulation

Propagation of action potentials between parallel chains of cardiac muscle cells was simulated using the PSpice program. Excitation was transmitted from cell to cell along a strand of three or four cells not connected by low-resistance tunnels (gap-junction connexons) in parallel with one or two similar strands. Thus, two models were used: a 2 x 3 model (two parallel chains of three cells each) and a 3 x 4 model (three parallel chains of four cells each). The entire surface membrane of each cell fired nearly simultaneously, and nearly all the propagation time was spent at the cell junctions, thus giving a staircase-shaped propagation profile. The junctional delay time between contiguous cells in a chain was about 0.2-0.5 ms. A significant negative cleft potential develops in the narrow junctional clefts, whose magnitude depends on several factors, including the radial cleft resistance (Rjc). The cleft potential (Vjc) depolarizes the postjunctional membrane to threshold by a patch-clamp action. Therefore, one mechanism for the transfer of excitation from one cell to the next is by the electric field (EF) that is generated in the junctional cleft when the prejunctional membrane fires. Propagation velocity increased with elevation of Rjc. With electrical stimulation of the first cell of the first strand (cell A1), propagation rapidly spread down that chain and then jumped to the second strand (B chain), followed by jumping to the third strand (C chain) when present. The rapidity by which the parallel chains became activated depended on the longitudinal resistance of the narrow extracellular cleft between the parallel strands (Rol2). The higher the Rol2 resistance, the faster the propagation (lower propagation time) over the cardiac muscle sheet (2-dimensional). The transverse resistance of the cleft had no effect. When the first cell of the second strand (cell B1) was stimulated, propagation spread down the B chain and jumped to the other two strands (A and C) nearly simultaneously. When cell C1 was stimulated, propagation traveled down the C chain and jumped to the B chain, followed by excitation of the A chain. Thus, there was transverse propagation of excitation as longitudinal propagation was occurring. Therefore, transmission of excitation by the EF mechanism can occur between myocardial cells lying closely parallel to one another without the requirement of a specialized junction.     

Influence of imprinting with A and B chains of insulin on binding and functional changes in tetrahymena

Insulin and its A and B chain increased the quantity of intracellular PAS-positive material (glycogen) in tetrahymena, whereas the combined A+B chains decreased it. Imprinting—previous interaction—with insulin, its A and B chains in themselves and with the A+B chain increased the hormone binding capacity of tetrahymena, but the functional effect of imprinting (storage or breakdown of glycogen) showed a different tendency with insulin and A+B chain on the one hand, and A chain and B chain on the other. Since the imprinting potential of a molecule promotes the induction of receptor formation, the fact remains that both component chains of insulin were able to act as potential imprinters, although the A chain was superior to the B chain in this respect throughout, and combined treatment with the A+B chain ultimately induced the formation of a similar binding site as insulin itself.     

Refinement of the structure of human basic fibroblast growth factor at 1.6 A resolution and analysis of presumed heparin binding sites by selenate substitution.

The three-dimensional structure of human basic fibroblast growth factor has been refined to a crystallographic residual of 16.1% at 1.6 A resolution. The structure has a Kunitz-type fold and is composed of 12 antiparallel beta-strands, 6 of which form a beta-barrel. One bound sulfate ion has been identified in the model, hydrogen bonded to the side chains of Asn 27, Arg 120, and Lys 125. The side chain of Arg 120 has two conformations, both of which permit hydrogen bonds to the sulfate. This sulfate binding site has been suggested as the binding site for heparin (Eriksson, A.E., Cousens, L.S., Weaver, L.H., & Matthews, B.W., 1991, Proc. Natl. Acad. Sci. USA 88, 3441-3445). Two beta-mercaptoethanol (BME) molecules are also included in the model, each forming a disulfide bond to the S gamma atoms of Cys 69 and Cys 92, respectively. The side chain of Cys 92 has two conformations of which only one can bind BME. Therefore the BME molecule is half occupied at this site. The locations of possible sulfate binding sites on the protein were examined by replacing the ammonium sulfate in the crystallization medium with ammonium selenate. Diffraction data were measured to 2.2 A resolution and the structure refined to an R-factor of 13.8%. The binding of the more electron-dense selenate ion was identified at two positions. One position was identical to the sulfate binding site identified previously. The second selenate binding site, which is of lower occupancy, is situated 5.6 A from the first. This ion is hydrogen bonded by the side chain of Lys 135 and Arg 120. Thus the side chain of Arg 120 binds two selenate ions simultaneously. It is suggested that the observed second selenate binding site should also be considered as a possible binding site for heparin, or that both selenate binding sites might simultaneously contribute to the binding of heparin.     

Interaction between the constituent A and B lens alpha-crystallin subunits leading to formation of alpha-neoprotein molecules

A and B constituent subunits associated in lens alpha-crystallin were found to interact with added B chains forming alpha-neoprotein molecules with lower A to B chain ratios than 2 A to 1 B in alpha-crystallin. Addition of 1% excess of B chains to the one in alpha-crystallin, which resulted in a ratio of 1.98 A to 1 B in the mixture, caused a change of quaternary structure in 30% of alpha-crystallin molecules within 18 h. At a ratio of 1.86 A to 1 B, all alpha-crystallin molecules were affected at this time. A maximum number of 495 B chains was found to form an association with 1 A chain, initially bound in alpha-crystallin. Such a high number may indicate that the reaction involves monomeric A chains binding aggregated macromolecules of B chains. It is in such form that B chains occur as macromolecules with an average molecular weight of 0.7 X 10(6) in aqueous solution. The alpha-neoprotein molecules selected for studies in this report had A to B chain ratios of 1.75:1, 1:1, and 0.2:1. Each behaved in immunodiffusion tests like single molecular entities. Antigenic determinants located on A as well as on B chains associated with each other in alpha-crystallin were found to be identical with determinants on the chains associated in the above alpha-neoprotein molecules. Determinants dependent on the quaternary structure of alpha-neoprotein and of alpha-crystallin molecules were completely different. Some of the quaternary determinants of various alpha-neoproteins were type specific and did not occur in molecules with different A to B chain ratios. Other quaternary determinants occurred in all alpha-neoproteins. An excess of A chains did not revert alpha-neoproteins to alpha-crystallin. However, alpha-neoprotein molecules did interact with added B chains forming neomolecules with lower A to B chain ratios.     

Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: insight into P450 conformational plasticity and membrane interaction

To better understand ligand-induced structural transitions in cytochrome P450 2B4, protein-ligand interactions were investigated using a bulky inhibitor. Bifonazole, a broad spectrum antifungal agent, inhibits monooxygenase activity and induces a type II binding spectrum in 2B4dH(H226Y), a modified enzyme previously crystallized in the presence of 4-(4-chlorophenyl)imidazole (CPI). Isothermal titration calorimetry and tryptophan fluorescence quenching indicate no significant burial of protein apolar surface nor altered accessibility of Trp-121 upon bifonazole binding, in contrast to recent results with CPI. A 2.3 A crystal structure of 2B4-bifonazole reveals a novel open conformation with ligand bound in the active site, which is significantly different from either the U-shaped cleft of ligand-free 2B4 or the small active site pocket of 2B4-CPI. The O-shaped active site cleft of 2B4-bifonazole is widely open in the middle but narrow at the top. A bifonazole molecule occupies the bottom of the active site cleft, where helix I is bent approximately 15 degrees to accommodate the bulky ligand. The structure also defines unanticipated interactions between helix C residues and bifonazole, suggesting an important role of helix C in azole recognition by mammalian P450s. Comparison of the ligand-free 2B4 structure, the 2B4-CPI structure, and the 2B4-bifonazole structure identifies structurally plastic regions that undergo correlated conformational changes in response to ligand binding. The most plastic regions are putative membrane-binding motifs involved in substrate access or substrate binding. The results allow us to model the membrane-associated state of P450 and provide insight into how lipophilic substrates access the buried active site.     

Three-dimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site.

The three-dimensional structure of endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 18.3% at 1.8 A resolution. The 190 amino acid length protein was found to exist as a single domain where the main chain folds to form two mostly antiparallel beta-sheets, which are packed against each other in parallel. The beta-sheet structure is twisted, forming a large cleft on one side of the molecule. The structure of XYNII resembles that of Bacillus 1,3-1,4-beta-glucanase. The cleft is an obvious suggestion for an active site, which has putative binding sites for at least four xylose residues. The catalytic residues are apparently the two glutamic acid residues (Glu86 and Glu177) in the middle of the cleft. One structure was determined at pH 5.0, corresponding to the pH optimum of XYNII. The second structure was determined at pH 6.5, where enzyme activity is reduced considerably. A clear structural change was observed, especially in the position of the side chain of Glu177. The observed conformational change is probably important for the mechanism of catalysis in XYNII.     

Contributions of amino acid side chains to the kinetics and thermodynamics of the bivalent binding of protein L to Ig kappa light chain

Protein L is a bacterial surface protein with 4-5 immunoglobulin (Ig)-binding domains (B1-B5), each of which appears to have two binding sites for Ig, corresponding to the two edges of its beta-sheet. To verify these sites biochemically and to probe their relative contributions to the protein L-Ig kappa light chain (kappa) interaction, we compared the binding of PLW (the Y47W mutant of the B1 domain) to that of mutants designed to disrupt binding to sites 1 and 2, using gel filtration, BIAcore surface plasmon resonance, fluorescence titration, and solid-phase radioimmunoassays. Gel filtration experiments show that PLW binds kappa both in 1:1 complexes and multivalently, consistent with two binding sites. Covalent dimers of the A20C and V51C mutants of PLW were prepared to eliminate site 1 and site 2 binding, respectively; both the A20C and V51C dimers bind kappa in 1:1 complexes and multivalently, indicating that neither site 1 nor site 2 is solely responsible for kappa binding. The A20R mutant was designed computationally to eliminate site 1 binding while preserving site 2 binding; consistent with this design, the A20R mutant binds kappa in 1:1 complexes but not multivalently. To probe the contributions of amino acid side chains to binding, we prepared 75 point mutants spanning nearly every residue of PLW; BIAcore studies of these mutants revealed two binding-energy "hot spots" consistent with sites 1 and 2. These data indicate that PLW binds kappa at both sites with similar affinities (high nanomolar), with the strongest contributions to the binding energy from Tyr34 (site 2) and Tyr36 (site 1). Compared to other protein-protein complexes, the binding is insensitive to amino acid substitutions at these sites, consistent with the large number of main chain interactions relative to side chain interactions. The strong binding of protein L to Ig kappa light chains of various species may result from the ambidextrous binding of the B1-B5 domains and the unimportance of specific side chain interactions.     

The family 6 carbohydrate binding module CmCBM6-2 contains two ligand-binding sites with distinct specificities

The microbial degradation of the plant cell wall is an important biological process, representing a major component of the carbon cycle. Enzymes that mediate the hydrolysis of this composite structure are modular proteins that contain non-catalytic carbohydrate binding modules (CBMs) that enhance catalytic activity. CBMs are grouped into sequence-based families, and in a previous study we showed that a family 6 CBM (CBM6) that interacts with xylan contains two potential ligand binding clefts, designated cleft A and cleft B. Mutagenesis and NMR studies showed that only cleft A in this protein binds to xylan. Family 6 CBMs bind to a range of polysaccharides, and it was proposed that the variation in ligand specificity observed in these proteins reflects the specific cleft that interacts with the target carbohydrate. Here the biochemical properties of the C-terminal cellulose binding CBM6 (CmCBM6-2) from Cellvibrio mixtus endoglucanase 5A were investigated. The CBM binds to the beta1,4-beta1,3-mixed linked glucans lichenan and barley beta-glucan, cello-oligosaccharides, insoluble forms of cellulose, the beta1,3-glucan laminarin, and xylooligosaccharides. Mutagenesis studies, informed by the crystal structure of the protein (presented in the accompanying paper, Pires, V. M. R., Henshaw, J. L., Prates, J. A. M., Bolam, D., Ferreira, L. M. A. Fontes, C. M. G. A., Henrissat, B., Planas, A., Gilbert, H. J., Czjzek, M. (2004) J. Biol. Chem. 279, 21560-21568), show that both cleft A and B can accommodate cello-oligosaccharides and laminarin displays a preference for cleft A, whereas xylooligosaccharides exhibit absolute specificity for this site, and the beta1,4,-beta1,3-mixed linked glucans interact only with cleft B. The binding of CmCBM6-2 to insoluble cellulose involves synergistic interactions between cleft A and cleft B. These data show that CmCBM6-2 contains two binding sites that display differences in ligand specificity, supporting the view that distinct binding clefts with different specificities can contribute to the variation in ligand recognition displayed by family 6 CBMs. This is in sharp contrast to other CBM families, where variation in ligand binding is a result of changes in the topology of a single carbohydrate-binding site.     

Laminin, a multidomain protein. The A chain has a unique globular domain and homology with the basement membrane proteoglycan and the laminin B chains

Laminin (Mr = 800,000) is a glycoprotein consisting of three chains, A, B1, and B2, and has diverse biological activities. Previously we reported the complete primary structure of the B1 and B2 chains of mouse laminin deduced from cDNA sequence (Sasaki, M., Kohno, K., Kato, S., Martin, G. R., and Yamada, Y. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 935-939; Sasaki, M., and Yamada, Y. (1988) J. Biol. Chem. 262, 17111-17117). Here we describe the isolation, characterization, and sequence of cDNA clones spanning 9,520 bases which encode the entire A chain of mouse laminin. The nucleotide sequence of the clones contains an open reading frame of 3,084 amino acids including 24 amino acids of a signal peptide. The A chain contains some eight distinct domains including alpha-helices, cysteine-rich repeats and globules. There is considerable sequence and structural homology between the A chain and the B1 and B2 chains. However, the A chain has a unique globular structure containing homologous repeats at the carboxyl terminus and constituting one third of the molecular mass of the chain. Furthermore, the A chain contains three globules and three cysteine-rich domains at the amino terminus, whereas the B1 and B2 chains have only two each of such domains. The A chain shows homology to the basement membrane heparan sulfate proteoglycan core protein and the extracellular domain of the Drosophila neurogenic protein Notch. There is an RGD (Arg-Gly-Asp) sequence in one of the cysteine-rich domains of the A chain. This potential cell binding sequence could be active as another adhesion signal in addition to the previously identified cell binding sequence YIGSR (Tyr-Ile-Gly-Ser-Arg) of the B1 chain.     

26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis

To explore the structure essential for the catalysis in 26 kDa endochitinase from barley seeds, we calculated theoretical pKa values of the ionizable groups based on the crystal structure, and then the roles of ionizable side chains located near the catalytic residue were examined by site-directed mutagenesis. The pKa value calculated for Arg215, which is located at the bottom of the catalytic cleft, is abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. No enzymatic activity was found in the Arg215-mutated chitinase (R215A) produced by the Escherichia coli expression system. The transition temperature of thermal unfolding (T(m)) of R215A was lower than that of the wild type protein by about 6.2 degrees C. In the crystal structure, the Arg215 side chain is in close proximity to the Glu203 side chain, whose theoretical pKa value was found to be abnormally low (-2.4), suggesting that these side chains may interact with each other. Mutation of Glu203 to alanine (E203A) completely eliminated the enzymatic activity and impaired the thermal stability (deltaT(m) = 6.4 degrees C) of the enzyme. Substrate binding ability was also affected by the Glu203 mutation. These data clearly demonstrate that the Arg215 side chain interacts with the Glu203 side chain to stabilize the conformation of the catalytic cleft. A similar interaction network was previously found in chitosanase from Streptomyces sp. N174 [Fukamizo et al. (2000) J. Biol. Chem. 275, 25633-25640]; hence, this type of interaction seems to be at least partly conserved in the catalytic cleft of other glycosyl hydrolases.     

Fine mapping of the antigen–antibody interaction of scFv215, a recombinant antibody inhibiting RNA polymerase II from Drosophila melanogaster

A bacterially expressed single chain antibody (scFv215) directed against the largest subunit of drosophila RNA polymerase II was analysed. Structure and function of the antigen binding site in scFv215 were probed by chain shuffling and by site‐specific mutagenesis. The entire variable region of either the heavy or light chain was replaced by an unrelated heavy or light chain. Both replacements resulted in a total loss of binding activity suggesting that the antigen binding site is contributed by both chains. The functional contributions of each complementarity determining region (CDR) were investigated by site specific mutagenesis of each CDR separately. Mutations in two of the CDRs, CDR1 of light chain and CDR2 of heavy chain, reduced the binding activity significantly. Each of the amino acids in these two CDRs was replaced individually by alanine (alanine walking). Seven amino acid substitutions in the two CDRs were found to reduce the binding activity by more than 50%. The data support a computer model of scFv215 which fits an epitope model based on a mutational analysis of the epitope suggesting an alpha‐helical structure for the main contact area. Copyright © 1999 John Wiley & Sons, Ltd.     

Crystal structure of the rigor-like human non-muscle myosin-2 motor domain

We determined the crystal structure of the motor domain of human non-muscle myosin 2B (NM-2B) in a nucleotide-free state and at a resolution of 2.8 Å. The structure shows the motor domain with an open active site and the large cleft that divides the 50kDa domain in a closed state. Compared to other rigor-like myosin motor domain structures, our structure shows subtle but significant conformational changes in regions important for actin binding and mechanochemical coupling. Moreover, our crystal structure helps to rationalize the impact of myosin, heavy chain 9 (MYH9)-related disease mutations Arg709Cys and Arg709His on the kinetic and functional properties of NM-2B and of the closely related non-muscle myosin 2A (NM-2A).     

Cell and heparin binding in the distal long arm of laminin: identification of active and cryptic sites with recombinant and hybrid glycoprotein [published erratum appears in J Cell Biol 1993 Dec;123(6 Pt 1):1623]

The long arm of laminin, which binds heparin and cells, consists of three polypeptides (A, B1, and B2) joined in a coiled-coil rod attached to a terminal A chain globule (G). Previously, we found that recombinant globular domain (rG) supported heparin and myoblast binding (Yurchenco, P. D., U. Sung, M. D. Ward, Y. Yamada, and J. J. O'Rear. 1993. J. Biol. Chem. 268:8356-8365). To further analyze long arm functions, we expressed the distal moiety of the mouse laminin A chain extending from the middle of the rod to the carboxyl terminus (rAiG). This larger glycoprotein, secreted by Sf9 insect cells infected with recombinant baculovirus, was intercalated in vitro into the corresponding disulfide-linked B chain segments of laminin fragment E8 (distal long arm rod and proximal globule). The hybrid molecule (B- rAiG) possessed a structure similar to laminin long arm as judged by electron microscopy and limited proteolysis. By joining rAiG with E8-B chains, the affinity of G domain for heparin decreased from that observed with rAiG and rG to one similar to native protein. HT1080 cells adhered to E8, rAiG, and B-rAiG, less well to rG, and not to denatured E8/B-rAiG, the A and B chain moieties of E8, or to a mixture of rG and E8-B chains. Cell adhesion to E8 and B-rAiG, in contrast to rAiG, was inhibited with antibodies specific for alpha 6 and beta 1 integrin chains. Since intercalation (a) restored a conformationally dependent alpha 6 beta 1 integrin recognition site present in native protein, (b) inactivated a cryptic cell binding activity in the A chain, and (c) inhibited a heparin binding site present in proximal G domain, we conclude that biological activities of laminin are different from that of its isolated subunits.     

The pairing of the separated A and B chains of insulin and its derivatives, FTIR studies.

From the amide I bands of their deconvolved FTIR spectra, the S-thiomethyl derivatives of the insulin A, B, despentapeptide(26-30) B and desoctapeptide(23-30) B chains all contain significant amounts of ordered secondary structure. The intact B chain is considerably more ordered than either the A or the truncated B chains. Comparison of the spectra of the separated and mixed intact chains of insulin suggests further folding upon mixing of the chains leading to significant increases in ordered secondary structures, presumably because of stabilization by interaction of the chains. The interactions of the A chain with the DPI B chain appear to be weaker as compared to that with the intact B chain. The above results suggest that only the intact A and B chains contain sufficient structural information to recognize each other and interact to form a native-like structure which make the correct formation of the disulfide linkages possible.     

V region sequences of anti-DNA and anti-RNA autoantibodies from NZB/NZW F1 mice

The V region sequences of two anti-DNA (A52, D42) and two anti-RNA (D44, D444) autoantibodies, derived from lupus prone NZB/NZW F1 female mice, were determined by mRNA sequencing. The sequences had the following features: 1) there was no clear sequence relationship between anti-DNA and anti-RNA antibodies; 2) there were no major similarities between any of the L chain sequences and each VL gene segment belonged to a different mouse VK subgroup; 3) the H chains of the two anti-RNA antibodies showed closely related sequences of VH gene segments and very similar third complementarity determining regions (CDR3); 4) the H chains of the two anti-DNA antibodies had VH segments belonging to different VH gene families but had a unique and similar combination of D segments and junctional sequences, suggesting a common recognition element for Ag and/or for idiotypic regulation in the H chain CDR3; and 5) the VH gene segment of one anti-DNA antibody (D42) was found to be very similar to the VH gene segment of a CBA mouse hybridoma antibody (6G6) which binds to the environmental Ag phosphocholine. The three-dimensional structure of the Fv-region of the anti-DNA antibody (D42) was modeled by computer and a stretch of poly(dT), ssDNA was docked to a cleft in the antibody combining site, formed by the three H chain CDR and by CDR1 and CDR3 of the L chain. The cleft is characterized by a preponderance of arginine and tyrosine residues, lining both the walls and base of the cleft.     

Cell surface and intracellular functions for ricin galactose binding.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.     

Extended repertoire of permissible peptide ligands for HLA-B*2702.

Recognition of self peptides bound to the class I major histocompatibility complex molecule HLA-B27 is thought to trigger proliferation of autoreactive T cells and result in autoimmune arthritic diseases. Previous work from other laboratories established that a predominant feature of endogenous peptides eluted from purified B27 is an arginine at position 2. We studied the binding of peptides containing both natural and unnatural amino acids by the subtype HLA-B*2702, with the goal of gaining insight into peptide binding by this B27 subtype that is associated with susceptibility to arthritic disease. A soluble from of B*2702 was depleted of endogenous peptides. We tested the binding of peptides substituted with cysteine, homocysteine, or an alpha-amino-epsilon-mercapto hexanoic acid side chain (Amh) instead of the naturally occurring arginine at position 2, to determine whether the peptide sulfhydryl residue could be covalently linked to cysteine 67 in the B*2702 binding cleft. Although none of the altered peptide sequences bound covalently to B*2702, the affinities of the homocysteine- and Amh-substituted peptides were close to that of the native peptide sequence. Substitutions at position 2 with other side chains, such as glutamine and methionine, also resulted in peptides that bound with only slightly reduced affinity. These results demonstrate that peptide side chains other than arginine at position 2 can be accomodated within the B*2702 peptide binding site with only minor reductions in affinity. This extended repertoire of permissible B27-binding peptides should be taken into account for a consideration of disease-associated peptide sequences.