Probing the surface of eukaryotic cells using combinatorial toxin libraries.

The success of proteomics hinges in part on the development of approaches able to map receptors on the surface of cells. One strategy to probe a cell surface for the presence of internalized markers is to make use of Shiga-like toxin 1 (SLT-1), a ribosome-inactivating protein that kills eukaryotic cells [1, 2]. SLT-1 binds to the glycolipid globotriaosylceramide [3, 4], which acts as a shuttle, allowing the toxin to be imported and routed near ribosomes. We investigated the use of SLT-1 as a structural template to create combinatorial libraries of toxin variants with altered receptor specificity. Since all SLT-1 variants retain their toxic function, this property served as a search engine enabling us to identify mutants from these libraries able to kill target cells expressing internalizable receptors. Random mutations were introduced in two discontinuous loop regions of the SLT-1 receptor binding subunit. Minimal searches from screening 600 bacterial colonies randomly picked from an SLT-1 library identified toxin mutants able to kill cell lines resistant to the wild-type toxin. One such mutant toxin was shown to bind to a new receptor on these cell lines by flow cytometry. Toxin libraries provide a strategy to delineate the spectrum of receptors on eukaryotic cells.

Inhibition of the spindle assembly checkpoint kinase Mps-1 as a novel therapeutic strategy in malignant mesothelioma.

Malignant mesothelioma (MM) is an aggressive malignancy, highly resistant to current medical and surgical therapies, whose tumor cells characteristically show a high level of aneuploidy and genomic instability. We tested our hypothesis that targeting chromosomal instability in MM would improve response to therapy. Thr/Tyr kinase (TTK)/monopolar spindle 1 kinase (Mps-1) is a kinase of the spindle assembly checkpoint that controls cell division and cell fate. CFI-402257 is a novel, selective inhibitor of Mps-1 with antineoplastic activity. We found that CFI-402257 suppresses MM growth. We found that Mps-1 is overexpressed in MM and that its expression correlates with poor patients' outcome. In vitro, CFI-402257-mediated inhibition of Mps-1 resulted in abrogation of the mitotic checkpoint, premature progression through mitosis, marked aneuploidy and mitotic catastrophe. In vivo, CFI-402257 reduced MM growth in an orthotopic, syngeneic model, when used as a single agent, and more so when used in combination with cisplatin+pemetrexed, the current standard of care. Our preclinical findings indicate that CFI-402257 is a promising novel therapeutic agent to improve the efficacy of the current chemotherapeutic regimens for MM patients.

Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification.

The cellulose-binding domain (CBDCex) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the beta-barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 34:6993-7009). Although all five tryptophans can be oxidized by N-bromosuccinimide (NBS), stopped-flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDCex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDCex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDCex to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDCex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 7:604-607], each of the exposed tryptophans interacts reversibly with cellulose.

Essential carboxy groups in xylanase A.

An endo-1,4-beta-xylanase of Schizophyllum commune was purified to homogeneity through a modified procedure employing DEAE-Sepharose CL-6B and gel-filtration chromatography on Sephadex G-50. The role of carboxy groups in the catalytic mechanism was delineated through chemical modification studies. The water-soluble carbodi-imide 1-(4-azonia-4,4-dimethylpentyl)-3-ethylcarbodi-imide iodide (EAC) inactivated the xylanase rapidly and completely in a pseudo-first-order process. Other carbodi-imides and Woodward's Reagent K were less effective in decreasing enzymic activity. Significant protection of the enzyme against EAC inactivation was provided by a mixture of neutral xylo-oligomers. The pH-dependence of the EAC inactivation revealed the presence of a critical ionizable group with a pKa value of 6.6 in the active site of the xylanase. Treatment of the enzyme with diethyl pyrocarbonate resulted in modification of all three histidine residues in the enzyme with 100% retention of original enzymic activity. Titration of the enzyme with 5,5-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetimide and p-chloromercuribenzoate indicated the absence of free/reactive thiol groups. Reaction of the xylanase with tetranitromethane did not result in a significant activity loss as a result of modification of tyrosine residues.

Identification of an essential tyrosyl residue in the binding site of Schizophyllum commune xylanase A.

Ultraviolet difference spectroscopy studies with the Schizophyllum commune xylanase in the presence of inhibitors and substrates indicated the participation of one or more tyrosyl residues in the binding of substrates to xylanase. Chemical modification experiments with group-specific reagents in the absence and presence of substrates confirmed the essential role of a tyrosyl residue in substrate binding while discounting the participation of tryptophan. A fourth-derivative absorbance spectroscopic method was developed to facilitate the quantitation of modified tyrosyl and tryptophanyl residues. This analysis showed that two tyrosyl residues of the xylanase are modified by tetranitromethane in the absence of substrate with the concomitant loss of catalytic activity. Protection of the xylanase with xylooligosaccharides resulted in the nitration of only one residue, and such enzyme derivatives retained 94% catalytic activity. Differential modification of the xylanase with tetranitromethane generated an enzyme derivative with the characteristic absorbance at 428 nm of 3-nitrotyrosine. Amino acid analysis and N-terminal sequencing of peptides with strong absorbance at 428 nm isolated from the protease-digested modified enzyme by reverse-phase HPLC identified the essential residue as Tyr97. Alignment of the S. commune xylanase amino acid sequence with those of the 18 other known family G xylanases revealed that Tyr97 is a conserved aromatic residue, further suggesting its essential role in substrate binding.

Identification of a glutamate residue at the active site of xylanase A from Schizophyllum commune.

The xylanase A (endo-1,4-beta-D-xylan xylanhydrolase) of the basidiomycete Schizophyllum commune was treated with the powerful carboxylate-modifying reagent 1-(4-azonia-4,4-dimethyl-pentyl)-3-ethylcarbodiimide iodide (EAC) in the presence of substrate. This treatment was followed by complete inactivation of the enzyme with [14c]EAC after the removal of excess reagent and protecting ligand. The inactivated enzyme was digested with endoproteinase Arg-C or trypsin, and peptides were separated and purified using reverse-phase high-performance liquid chromatography. Following sub-digestion of individual radioactive peptides with staphylococcal V8 protease and endoproteinase Lys-C, amino acid composition analysis and sequencing analysis revealed that the [14C]EAC label was bound exclusively to Glu87. Comparison of the primary sequences of related xylanase with that of xylanase A revealed that Glu87 is a highly conserved residue. Based on this similarity and the mechanism of carbodiimide action, Glu87 is proposed to act as the nucleophile in the catalytic mechanism of xylanase A. The possible environment of the putative catalytic glutamate residue was explored using hydrophobic-cluster analysis and secondary-structure prediction based on the primary sequence of xylanase.

Action pattern of xylo-oligosaccharide hydrolysis by Schizophyllum commune xylanase A.

The endo-1,4-beta-xylanase of the basidiomycete Schizophyllum commune, designated xylanase A, was studied to determine its action pattern, rates of reaction and bond-cleavage frequencies on xylo-oligomer and xylo-alditol substrates ranging in degree of polymerization (Dp) from xylotriose (X3) to xyloheptaose (X7). An HPLC method using a Dionex HPLC and Carbopac PA1 ion-exchange column with pulsed amperometric detection was developed to quantify both substrate loss and increase of products. Xylanase A had no detectable activity on xylobiose (X2) and low activity on xylotriose and xylotetraose (X4) but cleaved X5-X7 rapidly with X2 and X3 as major products. Initial rate data from hydrolyses of individual oligomers at 25 degrees C and pH 5.81 indicated that the Michaelis constant (Km) decreased with increasing chain length (n) of oligomer. Turnover number (kcat) increased with chain length up to n = 7 suggesting that the specificity region of xylanase A spans about seven xylose units. Bond-cleavage frequencies obtained from xylanase A hydrolysis of xylo-alditols indicated a strong preference for internal linkages of the xylose chain. The action pattern of xylanase A on reduced substrates suggests that the catalytic site is located assymetrically within the binding cleft of the enzyme.

Quantitation of tryptophan and tyrosine residues in proteins by fourth-derivative spectroscopy.

A method for quantitation of tryptophan and tyrosine residues in proteins by fourth-derivative ultraviolet spectroscopy is described. The direct quantitation of tryptophan is based on measurement of a tryptophan-specific trough at 292 nm in the fourth derivative of a protein's ultraviolet absorption spectrum. A peak overlapping the tryptophan and tyrosine signatures at A282 is used to quantify tyrosine content. The procedure is accomplished by adding back known quantities of tyrosine to the sample and subtracting the contribution of tryptophan to the A282 peak to obtain an internal calibration curve. This curve is linear, with the ordinate axis intercept relating the quantity (residues/mole) of tyrosine present in the protein. This nondestructive and facile method was used to successfully predict the tryptophan and tyrosine content of a variety of well-characterized proteins. The utility of this method was further demonstrated by resolving the number of tryptophan and tyrosine residues in proteins oxidized by N-bromosuccinimide.

Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues.

The family 2a carbohydrate-binding module (CBM2a) of xylanase 10A from Cellulomonas fimi binds to the crystalline regions of cellulose. It does not share binding sites with the N-terminal family 4 binding module (CBM4-1) from the cellulase 9B from C.fimi, a module that binds strictly to soluble sugars and amorphous cellulose. The binding of CBM2a to crystalline matrices is mediated by several residues on the binding face, including three prominent, solvent-exposed tryptophan residues. Binding to crystalline cellulose was analyzed by making a series of conservative (phenylalanine and tyrosine) and non-conservative substitutions (alanine) of each solvent-exposed tryptophan (W17, W54 and W72). Other residues on the binding face with hydrogen bonding potential were substituted with alanine. Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72. Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity. Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, we propose a model for the interaction between the polypeptide and the crystalline surface.

Shiga-like toxin-1 receptor on human breast cancer, lymphoma, and myeloma and absence from CD34(+) hematopoietic stem cells: implications for ex vivo tumor purging and autologous stem cell transplantation.

The ribosome-inactivating protein, Shiga-like toxin-1 (SLT-1), targets cells that express the glycolipid globotriaosylceramide (CD77) on their surface. CD77 and/or SLT-1 binding was detected by flow cytometry and immunocytochemistry on lymphoma and breast cancer cells recovered from biopsies of primary human cancers as well as on B cells or plasma cells present in blood/bone marrow samples of multiple myeloma patients. Breast cancer cell lines also expressed receptors for the toxin and were sensitive to SLT-1. Treatment of primary B lymphoma, B-cell chronic lymphocytic leukemia, and myeloma B or plasma cells with SLT-1-depleted malignant B cells by 3- to 28-fold, as measured by flow cytometry. Depletion of myeloma plasma cells was confirmed using a cellular limiting dilution assay followed by reverse transcriptase-polymerase chain reaction analysis of clonotypic IgH transcripts, which showed a greater than 3 log reduction in clonotypic myeloma cells after SLT-1 treatment. Receptors for the toxin were not detected on human CD34(+) hematopoietic progenitor cells (HPC). HPC were pretreated with a concentration of SLT-1 known to purge primary malignant B cells and cultured for 6 days. The number of HPC was comparable in toxin-treated and untreated cultures. HPC were functionally intact as well. Colony-forming units (CFU) were present at an identical frequency in untreated and SLT-1 pretreated cultures, confirming that CFU escape SLT-1 toxicity. The results suggest the ex vivo use of SLT-1 in purging SLT-1 receptor-expressing malignant cells from autologous stem cell grafts of breast cancer, lymphoma, and myeloma patients.