Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

The autocrine/paracrine peptide signaling molecules such as growth factors have many promising biologic activities for clinical applications. However, one cannot expect specific therapeutic effects of the factors administered by ordinary drug delivery systems as they have limited target specificity and short half-lives in vivo. To overcome the difficulties in using growth factors as therapeutic agents, we have produced fusion proteins consisting of growth factor moieties and a collagen-binding domain (CBD) derived from Clostridium histolyticum collagenase. The fusion proteins carrying the epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) at the N terminal of CBD (CBEGF/CBFGF) tightly bound to insoluble collagen and stimulated the growth of BALB/c 3T3 fibroblasts as much as the unfused counterparts. CBEGF, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 days, whereas EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth-promoting effect in vivo, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 days and 7 days after injection. These results indicate that CBD may be used as an anchoring unit to produce fusion proteins nondiffusible and long-lasting in vivo.

Phage display of a catalytic antibody to optimize affinity for transition-state analog binding

Catalytic antibodies have shown great promise for catalyzing a tremendously diverse set of natural and unnatural chemical transformations. However, few catalytic antibodies have efficiencies that approach those of natural enzymes. In principle, random mutagenesis procedures such as phage display could be used to improve the catalytic activities of existing antibodies; however, these studies have been hampered by difficulties in the recombinant expression of antibodies. Here, we have grafted the antigen binding loops from a murine-derived catalytic antibody, 17E8, onto a human antibody framework in an effort to overcome difficulties associated with recombinant expression and phage display of this antibody. “Humanized” 17E8 retained similar catalytic and hapten binding properties as the murine antibody while levels of functional Fab displayed on phage were 200-fold higher than for a murine variable region/human constant region chimeric Fab. This construct was used to prepare combinatorial libraries. Affinity panning of these resulted in the selection of variants with 2- to 8-fold improvements in binding affinity for a phosphonate transition-state analog. Surprisingly, none of the affinity-matured variants was more catalytically active than the parent antibody and some were significantly less active. By contrast, a weaker binding variant was identified with 2-fold greater catalytic activity and incorporation of a single substitution (Tyr-100aH → Asn) from this variant into the parent antibody led to a 5-fold increase in catalytic efficiency. Thus, phage display methods can be readily used to optimize binding of catalytic antibodies to transition-state analogs, and when used in conjunction with limited screening for catalysis can identify variants with higher catalytic efficiencies.

The chitinase PfCHT1 from the human malaria parasite Plasmodium falciparum lacks proenzyme and chitin-binding domains and displays unique substrate preferences

Within hours after the ingestion of a blood meal, the mosquito midgut epithelium synthesizes a chitinous sac, the peritrophic matrix. Plasmodium ookinetes traverse the peritrophic matrix while escaping the mosquito midgut. Chitinases (EC 3.2.1.14) are critical for parasite invasion of the midgut: the presence of the chitinase inhibitor, allosamidin, in an infectious blood meal prevents oocyst development. A chitinase gene, PgCHT1, recently has been identified in the avian malaria parasite P. gallinaceum. We used the sequence of PgCHT1 to identify a P. falciparum chitinase gene, PfCHT1, in the P. falciparum genome database. PfCHT1 differs from PgCHT1 in that the P. falciparum gene lacks proenzyme and chitin-binding domains. PfCHT1 was expressed as an active recombinant enzyme in Escherichia coli. PfCHT1 shares with PgCHT1 a substrate preference unique to Plasmodium chitinases: the enzymes cleave tri- and tetramers of GlcNAc from penta- and hexameric oligomers and are unable to cleave smaller native chitin oligosaccharides. The pH activity profile of PfCHT1 and its IC50 (40 nM) to allosamidin are distinct from endochitinase activities secreted by P. gallinaceum ookinetes. Homology modeling predicts that PgCHT1 has a novel pocket in the catalytic active site that PfCHT1 lacks, which may explain the differential sensitivity of PfCHT1 and PgCHT1 to allosamidin. PfCHT1 may be the ortholog of a second, as yet unidentified, chitinase gene of P. gallinaceum. These results may allow us to develop novel strategies of blocking human malaria transmission based on interfering with P. falciparum chitinase.

Dissociation of the Tubulin-sequestering and Microtubule Catastrophe-promoting Activities of Oncoprotein 18/Stathmin

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein that destabilizes microtubules, but the mechanism of destabilization is currently controversial. Based on in vitro microtubule assembly assays, evidence has been presented supporting conflicting destabilization models of either tubulin sequestration or promotion of microtubule catastrophes. We found that Op18 can destabilize microtubules by both of these mechanisms and that these activities can be dissociated by changing pH. At pH 6.8, Op18 slowed microtubule elongation and increased catastrophes at both plus and minus ends, consistent with a tubulin-sequestering activity. In contrast, at pH 7.5, Op18 promoted microtubule catastrophes, particularly at plus ends, with little effect on elongation rates at either microtubule end. Dissociation of tubulin-sequestering and catastrophe-promoting activities of Op18 was further demonstrated by analysis of truncated Op18 derivatives. Lack of a C-terminal region of Op18 (aa 100–147) resulted in a truncated protein that lost sequestering activity at pH 6.8 but retained catastrophe-promoting activity. In contrast, lack of an N-terminal region of Op18 (aa 5–25) resulted in a truncated protein that still sequestered tubulin at pH 6.8 but was unable to promote catastrophes at pH 7.5. At pH 6.8, both the full length and the N-terminal–truncated Op18 bound tubulin, whereas truncation at the C-terminus resulted in a pronounced decrease in tubulin binding. Based on these results, and a previous study documenting a pH-dependent change in binding affinity between Op18 and tubulin, it is likely that tubulin sequestering observed at lower pH resulted from the relatively tight interaction between Op18 and tubulin and that this tight binding requires the C-terminus of Op18; however, under conditions in which Op18 binds weakly to tubulin (pH 7.5), Op18 stimulated catastrophes without altering tubulin subunit association or dissociation rates, and Op18 did not depolymerize microtubules capped with guanylyl (α, β)-methylene diphosphonate–tubulin subunits. We hypothesize that weak binding between Op18 and tubulin results in free Op18, which is available to interact with microtubule ends and thereby promote catastrophes by a mechanism that likely involves GTP hydrolysis.

Chaperone activity and structure of monomeric polypeptide binding domains of GroEL

The chaperonin GroEL is a large complex composed of 14 identical 57-kDa subunits that requires ATP and GroES for some of its activities. We find that a monomeric polypeptide corresponding to residues 191 to 345 has the activity of the tetradecamer both in facilitating the refolding of rhodanese and cyclophilin A in the absence of ATP and in catalyzing the unfolding of native barnase. Its crystal structure, solved at 2.5 Å resolution, shows a well-ordered domain with the same fold as in intact GroEL. We have thus isolated the active site of the complex allosteric molecular chaperone, which functions as a “minichaperone.” This has mechanistic implications: the presence of a central cavity in the GroEL complex is not essential for those representative activities in vitro, and neither are the allosteric properties. The function of the allosteric behavior on the binding of GroES and ATP must be to regulate the affinity of the protein for its various substrates in vivo, where the cavity may also be required for special functions.

IFN consensus sequence binding protein potentiates STAT1-dependent activation of IFNγ-responsive promoters in macrophages

IFNγ, once called the macrophage-activating factor, stimulates many genes in macrophages, ultimately leading to the elicitation of innate immunity. IFNγ's functions depend on the activation of STAT1, which stimulates transcription of IFNγ-inducible genes through the GAS element. The IFN consensus sequence binding protein (icsbγ or IFN regulatory factor 8), encoding a transcription factor of the IFN regulatory factor family, is one of such IFNγ-inducible genes in macrophages. We found that macrophages from ICSBP−/− mice were defective in inducing some IFNγ-responsive genes, even though they were capable of activating STAT1 in response to IFNγ. Accordingly, IFNγ activation of luciferase reporters fused to the GAS element was severely impaired in ICSBP−/− macrophages, but transfection of ICSBP resulted in marked stimulation of these reporters. Consistent with its role in activating IFNγ-responsive promoters, ICSBP stimulated reporter activity in a GAS-specific manner, even in the absence of IFNγ treatment, and in STAT1 negative cells. Indicative of a mechanism for this stimulation, DNA affinity binding assays revealed that endogenous ICSBP was recruited to a multiprotein complex that bound to GAS. These results suggest that ICSBP, when induced by IFNγ through STAT1, in turn generates a second wave of transcription from GAS-containing promoters, thereby contributing to the elicitation of IFNγ's unique activities in immune cells.

Site-specific mutations in the mature form of human IL-18 with enhanced biological activity and decreased neutralization by IL-18 binding protein

IL-18 can be considered a proinflammatory cytokine mediating disease as well as an immunostimulatory cytokine that is important for host defense against infection and cancer. The high-affinity, constitutively expressed, and circulating IL-18 binding protein (IL-18BP), which competes with cell surface receptors for IL-18 and neutralizes IL-18 activity, may act as a natural antiinflammatory as well as immunosuppressive molecule. In the present studies, the IL-18 precursor caspase-1 cleavage site was changed to a factor Xa site, and, after expression in Escherichia coli, mature IL-18 was generated by factor Xa cleavage. Mature IL-18 generated by factor Xa cleavage was fully active. Single point mutations in the mature IL-18 peptide were made, and the biological activities of the wild-type (WT) IL-18 were compared with those of the mutants. Mutants E42A and K89A exhibited 2-fold increased activity compared with WT IL-18. A double mutant, E42A plus K89A, exhibited 4-fold greater activity. Unexpectedly, IL-18BP failed to neutralize the double mutant E42A plus K89A compared with WT IL-18. The K89A mutant was intermediate in being neutralized by IL-18BP, whereas neutralization of the E42A mutant was comparable to that in the WT IL-18. The identification of E42 and K89 in the mature IL-18 peptide is consistent with previous modeling studies of IL-18 binding to IL-18BP and explains the unusually high affinity of IL-18BP for IL-18.

Selective association of the methyl-CpG binding protein MBD2 with the silent p14/p16 locus in human neoplasia

DNA methylation of tumor suppressor genes is a common feature of human cancer. The cyclin-dependent kinase inhibitor gene p16/Ink4A is hypermethylated in a wide range of malignant tissues and the p14/ARF gene located 20 kb upstream on chromosome 9p21 is also methylated in carcinomas. p14/ARF (ARF, alternative reading frame) does not inhibit the activities of cyclins or cyclin-dependent kinase complexes; however, the importance of the two gene products in the etiology of cancer resides in their involvement in two major cell cycle regulatory pathways: p53 and the retinoblastoma protein, Rb, respectively. Distinct first exons driven from separate promoters are spliced onto the common exons 2 and 3 and the resulting proteins are translated in different reading frames. Both genes are expressed in normal cells but can be alternatively or coordinately silenced when their CpG islands are hypermethylated. Herein, we examined the presence of methyl-CpG binding proteins associated with aberrantly methylated promoters, the distribution of acetylated histones H3 and H4 by chromatin immunoprecipitation assays, and the effect of chemical treatment with 5-aza-2′-deoxycytidine (5aza-dC) and trichostatin A on gene induction in colon cell lines by quantitative reverse transcriptase–PCR. We observed that the methyl-CpG binding protein MBD2 is targeted to methylated regulatory regions and excludes the acetylated histones H3 and H4, resulting in a localized inactive chromatin configuration. When methylated, the genes can be induced by 5aza-dC but the combined action of 5aza-dC and trichostatin A results in robust gene expression. Thus, methyl-CpG binding proteins and histone deacetylases appear to cooperate in vivo, with a dominant effect of DNA methylation toward histone acetylation, and repress expression of tumor suppressor genes hypermethylated in cancers.

Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I

Cleavage and polyadenylation of mRNA 3′ ends in Saccharomyces cerevisiae requires several factors, one of which is cleavage factor I (CF I). Purification of CF I activity from yeast extract has implicated numerous proteins as functioning in both cleavage and/or polyadenylation. Through reconstitution of active CF I from separately expressed and purified proteins, we show that CF I contains five subunits, Rna14, Rna15, Pcf11, Clp1, and Hrp1. These five are necessary and sufficient for reconstitution of cleavage activity in vitro when mixed with CF II, and for specific polyadenylation when mixed with polyadenylation factor I, purified poly(A) polymerase, and poly(A) binding protein. Analysis of the individual protein–protein interactions supports an architectural model for CF I in which Pcf11 simultaneously interacts with Rna14, Rna15, and Clp1, whereas Rna14 bridges Rna15 and Hrp1.

Direct DNA binding by Brca1

The tumor suppressor Brca1 plays an important role in protecting mammalian cells against genomic instability, but little is known about its modes of action. In this work we demonstrate that recombinant human Brca1 protein binds strongly to DNA, an activity conferred by a domain in the center of the Brca1 polypeptide. As a result of this binding, Brca1 inhibits the nucleolytic activities of the Mre11/Rad50/Nbs1 complex, an enzyme implicated in numerous aspects of double-strand break repair. Brca1 displays a preference for branched DNA structures and forms protein–DNA complexes cooperatively between multiple DNA strands, but without DNA sequence specificity. This fundamental property of Brca1 may be an important part of its role in DNA repair and transcription.

Comparison of Binding Properties and Early Biological Effects of Elicitins in Tobacco Cells1

Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum). They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis. In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca2+ influx, extracellular medium alkalinization, and active oxygen species production). The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins. Furthermore, elicitins compete with one another for binding sites, suggesting that they interact with the same receptor. The four elicitins induced Ca2+ influx, extracellular medium alkalinization, and the production of active oxygen species in tobacco cell suspensions, but the intensity and kinetics of these effects were different from one elicitin to another. As a general observation the concentrations that induce similar levels of biological activities were lower for basic elicitins (with the exception of cinnamomin-induced Ca2+ uptake). The qualitative similarity of early events induced by elicitins indicates a common transduction scheme, whereas fine signal transduction tuning is different in each elicitin.

Characterization of Zinc Uptake, Binding, and Translocation in Intact Seedlings of Bread and Durum Wheat Cultivars

Durum wheat (Triticum turgidum L. var durum) cultivars exhibit lower Zn efficiency than comparable bread wheat (Triticum aestivum L.) cultivars. To understand the physiological mechanism(s) that confers Zn efficiency, this study used 65Zn to investigate ionic Zn2+ root uptake, binding, and translocation to shoots in seedlings of bread and durum wheat cultivars. Time-dependent Zn2+ accumulation during 90 min was greater in roots of the bread wheat cultivar. Zn2+ cell wall binding was not different in the two cultivars. In each cultivar, concentration-dependent Zn2+ influx was characterized by a smooth, saturating curve, suggesting a carrier-mediated uptake system. At very low solution Zn2+ activities, Zn2+ uptake rates were higher in the bread wheat cultivar. As a result, the Michaelis constant for Zn2+ uptake was lower in the bread wheat cultivar (2.3 μm) than in the durum wheat cultivar (3.9 μm). Low temperature decreased the rate of Zn2+ influx, suggesting that metabolism plays a role in Zn2+ uptake. Ca inhibited Zn2+ uptake equally in both cultivars. Translocation of Zn to shoots was greater in the bread wheat cultivar, reflecting the higher root uptake rates. The study suggests that lower root Zn2+ uptake rates may contribute to reduced Zn efficiency in durum wheat varieties under Zn-limiting conditions.

Binding and disruption of phospholipid bilayers by supramolecular RNA complexes

In an RNA world, RNAs would have regulated traffic through normally impermeable bilayer membranes. Using selection-amplification we previously found RNAs that bind stably and increase the ionic conductance of phospholipid membranes at high Mg2+ and Ca2+ concentrations. Now selection in reduced divalents yields RNAs that bind phosphatidylcholine liposomes under conditions closer to physiological. Such affinity for phospholipid membranes requires interactions between RNAs. In fact, we detected no functional monomeric membrane-binding RNAs. A membrane-active end-to-end heterotrimer consisting of 2 RNA 9 and 1 RNA 10 is defined by nucleotide protection, oligonucleotide competition, and mutant analysis. Oligomers of the heterotrimer bind stably, cause release of liposome-encapsulated solutes, and disrupt model black membranes. Individual RNA molecules do not show any of these activities. This novel mechanism of RNA binding to lipid membranes may not only regulate membrane permeability, but suggests that arrays of catalytic or structural RNAs on membranes are plausible. Finally, a selection met only by RNA complexes evokes new possibilities for selection-amplification itself.

Binding to the naturally occurring double p53 binding site of the Mdm2 promoter alleviates the requirement for p53 C-terminal activation

Genotoxic stress activation of the tumor suppressor transcription factor p53 involves post-translational C-terminal modifications that increase both protein stability and DNA binding activity. We compared the requirement for p53 protein activation of p53 target sequences in two major p53-regulated genes, p21/WAF1 (encoding a cell cycle inhibitory protein) and Mdm2 (encoding a ubiquitin ligase that targets p53 for proteolytic degradation). The p53 binding site in the proximal p21/WAF1 promoter contains a single p53 binding consensus sequence, while the p53 binding site in the Mdm2 promoter contains two consensus sequences linked by a 17 bp spacer. Binding of recombinant p53 protein to the p21/WAF1 binding site required monoclonal antibody PAb421, which can mimic activating phosphorylation and/or acetylation events at the C-terminus. In contrast, recombinant p53 bound strongly to the Mdm2 binding site in the absence of PAb421 antibody. Separate binding to each consensus sequence of the Mdm2 binding site still required PAb421, indicating that p53 binding was not simply due to greater affinity to the Mdm2 consensus sequences. Linking two p21/WAF1 binding sites with the 17 bp spacer region from the Mdm2 gene eliminated the PAb421 requirement for p53 binding to the p21/WAF1 site. These results suggest a mechanism for regulation of Mdm2 gene transcription that differs from that other p53-induced genes by its lack of a requirement for C-terminal activation of p53 protein. A steady induction of Mdm2 protein would maintain p53 protein at low levels until post-translational modifications following DNA damage increased p53 activity towards other genes, mediating p53 growth inhibitory and apoptotic activities.

A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.