Showing papers by "University of Maryland Biotechnology Institute published in 1994"

Two crystal structures of the B1 immunoglobulin-binding domain of streptococcal protein G and comparison with NMR.

Abstract: The structure of the 56-residue B1 immunoglobulin-binding domain from streptococcal protein G has been determined in two different crystal forms. The crystal structures were deduced by molecular replacement, based on the structure of the B2 domain (Brookhaven accession code 1PGX). Final R values are 0.174 and 0.198 for orthorhombic and trigonal forms, for diffraction data from 6.0 to 2.07 A and from 6 to 1.92 A, respectively. The orthorhombic crystals have an unusually high packing density for protein crystals, with Vm = 1.66 and a solvent content of 26%. The protein structure is found to be very similar (rms deviation 0.25 A for 56 C alpha's) in the two crystal forms, with an efficiently packed hydrophobic core between a four-stranded beta-sheet and a four-turn alpha-helix. The B1 domain has the same fold and general structure as the B2 domain (rms deviations 0.36 and 0.39 A), despite the six residue differences between them. The crystallographic models differ from NMR-derived models in several local regions, primarily in the loop involving residues 46-51; other significant variations are observed in the helix and in the structure of bound water. The primary crystal contact is the same in both crystal forms, involving both sheet edges to form extended beta-sheets throughout the crystals.

Structure of S-lectin, a developmentally regulated vertebrate beta-galactoside-binding protein.

Abstract: The crystal structure of a 14-kDa bovine spleen S-lectin complexed with the disaccharide N-acetyllactosamine at 1.9-A resolution reveals a surprising structural relationship to legume lectins, despite the lack of sequence homology. Two monomers associate to form an extended beta-sandwich, each with the same jelly roll topology typical of legume lectins but with dramatically trimmed loops and with different dimer association. Each monomer binds one N-acetyllactosamine molecule in a topologically and spatially different site than that of legume lectins. The carbohydrate-binding site provides an unprecedented paradigm for carbohydrate binding, with a unique network of salt bridges. The specificity for beta-galactose arises from intricate interactions that constrain the position of the O4 atom.

Crosslinking of mammalian lectin (galectin-1) by complex biantennary saccharides.

Abstract: Galectins are beta-galactoside-binding proteins that occur intra- and extracellularly in many animal tissues. They have been proposed to form networks of glycoconjugates on the cell surface, where they may modulate various cell response pathways such as growth, activation and adhesion. The high resolution X-ray crystallographic analyses of three crystal forms of bovine galectin-1 in complex with biantennary saccharides of N-acetyllactosamine type reveal infinite chains of lectin dimers cross-linked through N-acetyllactosamine units located at the end of the oligosaccharide antenna. The oligosaccharide adopts a different low energy conformation in each of the three crystal forms.

Environmental Reservoir of Vibrio cholerae The Causative Agent of Cholera

Abstract: Until the late 1970s and early 1980s, Vibrio cholerae was believed to be highly host-adapted and incapable of surviving longer than a few hours or days outside the human intestine. This view, enunciated by Felsenfeld,’ was that “some authors claimed that cholera vibrios may survive in water, particularly seawater, for as long as 2 months. This is, however, scarcely possible under natural conditions, if reinfection of the water does not take place.” This perspective of cholera ecology dominated the literature since the organism was first identified by Robert Koch in 1884.2 For V. chohae the term ‘‘survival” had been viewed to reflect a high degree of host-adaptation, ea., “cholera vibrios” being able to exist for only very short periods of time outside the human intestine. But Koch’s speculation that multiplication takes place in river water, without any assistance, has proven to be prescient, since the most recent data show that toxigenic V. cholerae 01 exist for long periods in laboratory microcosm water.’ In fact, the evidence accumulated over the ast decade shows that V. chohae is an

Comparison of systematic search and database methods for constructing segments of protein structure

Abstract: Two principal methods of determining the conformation of short pieces of polypeptide backbone in proteins have been developed: using a database of known structures and systematically generating all conformations. In this paper, we compare the effectiveness of these two techniques. The completeness of the database for segments of different lengths is examined and it is found to contain most conformations for segments seven residues long, but to deteriorate rapidly for longer regions. When the database segment is to be incorporated into the rest of a structure, at least seven residues are required to build four new residues, because of the need to position the segment relative to the rest of the structure. It is found that such positioning using flanking residues results in large errors in the inserted region. We conclude that the database method is currently not effective for comparative modeling, even for short segments. The systematic search procedure is found to generate almost all structures of short segments found in proteins. In contrast to the database method, low root mean square error structures are obtained for a set of trial segments embedded in the rest of a protein structure. Thus, it should be considered the method of choice.

Animal Lectins as Self/Non-Self Recognition Molecules.

Abstract: In recent years, the significant contributions from molecular research studies on animal lectins have elucidated structural aspects and provided clues not only to their evolution but also to their multiple biological functions. The experimental evidence has suggested that distinct, and probably unrelated, groups of molecules are included under the term "lectin." Within the invertebrate taxa, major groups of lectins can be identified: One group would include lectins that show significant homology to membrane-integrated or soluble vertebrate C-type lectins. The second would include those beta-galactosyl-specific lectins homologous to the S-type vertebrate lectins. The third group would be constituted by lectins that show homology to vertebrate pentraxins that exhibit lectin-like properties, such as C-reactive protein and serum amyloid P. Finally, there are examples that do not exhibit similarities to any of the aforementioned categories. Moreover, the vast majority of invertebrate lectins described so far cannot yet be placed in one or another group because of the lack of information regarding their primary structure. (See Table 1.) Animal lectins do not express a recombinatorial diversity like that of antibodies, but a limited diversity in recognition capabilities would be accomplished by the occurrence of multiple lectins with distinct specificities, the presence of more than one binding site, specific for different carbohydrates in a single molecule, and by certain "flexibility" of the binding sites that would allow the recognition of a range of structurally related carbohydrates. In order to identify the lectins' "natural" ligands, we have investigated the interactions between those proteins and the putative endogenous or exogenous glycosylated substances or cells that may be relevant to their biological function. Results from these studies, together with information on the biochemical properties of invertebrate and vertebrate lectins, including their structural relationships with other vertebrate recognition molecules, are discussed.

Role of P-450 aromatase in sex determination of the diamondback terrapin, malaclemys terrapin

Abstract: Sex determination in the diamondback terrapin, Malaclemys terrapin, is temperature-dependent. Eggs incubated at 31 degrees C, and above, hatch in approximately 45 days as females. Eggs incubated below 27 degrees C hatch in about 60 days as males. Sex is not reversible after hatching. Nest temperatures in the wild can be as low as 20 degrees C and as high as 37 degrees C with as much as a 10 degrees C diel cycle. The shortest incubation time measured in nature was 56 days and the longest approaching 120 days. Nests in our study site produced predominantly (> 95%) male hatchlings. Treatment of developing embryos with estrogen produces females at male producing temperatures while treatment with fadrozole (a nonsteroidal aromatase inhibitor) induces partial male-like gonads. Treatment with a steroidal aromatase inhibitor (4-hydroxyandrostenedione, 4-OHA) had no effect on sex determination. Both fadrozole and 4-OHA are potent competitive inhibitors (Ki approximately 40-50 nM) for terrapin in vitro aromatase activity. These findings are consistent with aromatase expression being a key step in sex determination of terrapins. We have cloned a partial single copy P-450 aromatase from the terrapin using a cDNA library constructed from ovarian mRNA. This partial clone is highly homologous to other vertebrate aromatases.

Cholera DFA: An improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration of Vibrio cholerae O1

Abstract: An improved fluorescent monoclonal antibody staining kit, Cholera DFA, for direct detection and enumeration of Vibrio cholerae O1 has been developed, employing a highly specific anti-A antigen monoclonal antibody, COLTA, labeled with fluorescein isothiocyanate (FITC). An optimized quantity of anti-photobleaching agent is used in a glycerol mounting medium to retard the rapid fading of immunofluorescent stained cells during fluorescent microscopy, thus enabling prolonged inspection of individual fields, as well asimproved photographic recording of results without loss of fluorescence intensity. When tested for specificity, all 30 strains of V. cholerae O1 reacted with Cholera DFA, whereas 100 heterologous species examined did not, yielding 100% specificity for all strains examined in this study. A field trial was conducted in Bangladesh, employing Cholera DFA and the results were compared with those obtained by conventional culture methods. Of 44 diarrheal stool specimens tested, Cholera DFA was positive for V. cholerae O1 in all culture-positive stool specimens and negative for all culture-negative stool specimens. The procedure is sensitive and highly specific, as well as simple, i.e., less complex than the indirect fluorescent assay, requiring only one reagent and less than 30 min to complete the staining process, while retarding rapid fading that often occurs with fluorescent microscopy.

Interplasmid transposition of Drosophila hobo elements in non-drosophilid insects.

Abstract: A modified hobo element from Drosophila melanogaster was introduced into embryos of the housefly, Musca domestica (family Muscidae) and the Queensland fruitfly, Bactrocera tryoni (family Tephritidae) to assess its ability to transpose. Hobo was capable of transposition in these species and transposition products had all of the hallmarks of hobo transposition products recovered from D. melanogaster, including the movement only of sequences precisely delimited by the inverted terminal repeats of hobo, the creation of an 8 bp duplication of the insertion site and an absolute requirement for hobo-encoded transposase. Transposition of hobo into the target gene resulted in a non-random distribution of insertion sites, with 10 of 38 independent insertions into the same nucleotide position. The results indicate that hobo can transpose in heterologous species, further demonstrating the similarity of hobo to Ac (Activator) of Zea mays and Tam3 of Antirrhinum majus. Hobo has excellent potential to act as a gene vector or gene tagging agent in nondrosophilid insects.

Expression of multiple flagellin-encoding genes of Proteus mirabilis.

Abstract: The overproduction of flagella is a distinguishing characteristic of Proteus mirabilis swarmer cell differentiation. The synthesis of flagellin, the principal protein composing the flagellar filament, is coordinately regulated as part of a larger regulon of genes whose expression is a prerequisite in urinary pathogenesis. In this report, the regulation of expression of the flaA locus, comprising flaA and flaB, two tandemly linked and nearly identical copies of flagellin-encoding genes, is examined. Transcriptional expression studies reveal that flaA, but not flaB, is expressed by wild-type cells, and flaA transcription increases eightfold during differentiation. The flaA transcriptional start site for both swimmer and swarmer cells was determined to be located at a guanine, 8 bases downstream of the flaA sigma 28 promoter. FlaA- mutants are nonmotile and undifferentiated and do not synthesize flagellin, while FlaB- mutants are wild type, thus verifying that FlaA is the sole flagellin produced by wild-type cells and that flaB is silent. FlaA- mutants frequently revert to a Mot+ phenotype that is antigenically distinct from that of wild-type cells. Southern blot analysis of the flaA Mot+ revertants reveals a deletion of between 2 and 7kb in the flaA locus. Biochemical analyses of revertant flagellin indicate major changes in protein size and composition but conservation of the first 28 N-terminal residues. The result of this process is to produce an antigenically distinct flagellum that may be significant in ensuring the survival of P. mirabilis during pathogenesis.

Hydrogen-deuterium exchange in the free and immunoglobulin G-bound protein G B-domain.

Abstract: Hydrogen-deuterium exchange experiements have been used to measure backbone amide proton (NH) exchange rates in the free and IgG-bound protein G B2-domain (GB2). Exchange rates were analyzed in terms of the free energy required for transient opening of an H-bonded NH (delta Gop), and exchange mechanisms were interpreted in the context of local and global opening motions. In free GB2 at 22 degrees C, 28 detectable NHs have delta Gop values which approximate the free energy of thermal unfolding (delta Gu) obtained from calorimetry. This indicates that the majority of detectable NHs exchange through a global unfolding mechanism, reflecting the cooperative two-state unfolding behavior observed thermodynamically [Alexander et al. (1992) Biochemistry 31, 3597-3603]. IgG binding results in a broadening of exchange rates and delta Gop values, consistent with a less cooperative exchange mechanism than in free GB2. The large range of protection factors (1.3 to > 210) also indicates that exchange does not occur cooperatively for all detectable NHs in bound GB2. Nineteen of the detectable NHs have significantly slowed exchange rates in the complex with protection factors > 5. Residues with protection factors of the order of 100 or more occur in both the helix region (F30, K31, A34) and in the central core of the beta-sheet (V6, F52, V54). The highest protection factors are consistent with a binding constant of approximately 10(8) M-1. The pattern of high protection observed in the helix overlaps with the putative binding site suggested from previous studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of in vitro replication of the oyster parasite Perkinsus marinus by the natural iron chelators transferrin, lactoferrin, and desferrioxamine.

Abstract: The mammalian iron-binding proteins transferrin and lactoferrin, the bactericidal peptide lactoferricin B, and the bacterial siderophore desferrioxamine were tested for their ability to inhibit the in vitro replication of the oyster parasite Perkinsus marinus. All three chelators were effective in reducing the parasite proliferation in a dose-dependent manner. Lactoferricin B, a peptide of lactoferrin that exhibits bactericidal properties unrelated to iron chelation, had no inhibitory activity on the parasite. When the chelators were partially or completely saturated with the appropriate iron equivalents, their inhibitory effects on the parasite proliferation were diminished or abolished accordingly, confirming that this activity was related to the chelator's capacity for iron sequestration. Our results indicate that the parasite has a strong requirement for soluble iron and its growth rates are correlated with iron availability. We propose that excess iron accumulation in the host Crassostrea virginica promotes parasite proliferation. P. marinus may avoid oxidative damage that would compromise its intracellular survival by exhaustion the host's intracellular selected iron pools required for superoxide and hydroxyl radical production.

Determinants for intracellular sorting of cytoplasmic and nuclear intermediate filaments.

Abstract: The mechanism by which nuclear and cytoplasmic filaments are sorted in vivo was studied by examining which lamin sequences are required to target an otherwise cytoplasmic IF protein, the small neurofilament subunit (NF-L), to the nuclear lamina. By swapping corresponding domains between NF-L and lamin A, nuclear envelope targeting of NF-L was shown to require the presence of the "head" domain, a 42-amino acid sequence unique to lamin rod domains, a nuclear localization signal and the CAAX motif. Replacement of the entire COOH-terminal tail of lamin A with that of NF-L had no discernible effect on nuclear localization of lamin A, provided the substituted NF-L tail contained a NLS and a CAAX motif. This chimeric protein exhibited characteristics more typical of lamin B than that of the parental lamin A. With regard to cytoplasmic assembly properties, substitution of the head domain of lamin A for that of NF-L did not substantially affect the ability of NF-L to coassemble with vimentin in the cytoplasm. In contrast, insertion of a 42-amino acid sequence unique to lamin rod domains into NF-L profoundly affected NF-L coassembly with vimentin indicating that the 42-amino acid insertion in lamins may be important for sorting lamins from cytoplasmic IF proteins.

A gene for the enterotoxin zonula occludens toxin is present in Vibrio mimicus and Vibrio cholerae O139

Abstract: The presence of the zonula occludens toxin (ZOT) gene, which encodes an enterotoxin produced by serotype O1 strains of the pathogenic bacterium, Vibrio cholerae , in addition to cholera toxin, was investigated in selected strains of V. mimicus and the new pandemic V. cholerae non-O1 serotype O139. The zot gene was detected by polymerase chain reaction (PCR) amplification, using sets of primers based on the sequence of the V. cholerae O1 zot sequence. PCR amplification of genomic DNAs of both cholera toxin gene ( ctx ) positive and ctx − strains of V. mimicus detected the presence of zot gene. An Acc -I- Eco RV V. cholerae zot gene fragment designed to overlap PCR products was used as a probe. Southern hybridization studies confirmed that the PCR fragments from V. mimicus and V. cholerae O139 were strongly homologous to the V. cholerae O1 zot gene. The zot gene was found with 3 to 5 strains of V. mimicus of which only one strain harbored the ctx gene. The presence of a zot gene in ctx − toxigenic V. mimicus indicates a possible role of ZOT in the toxigenicity of this species. We conclude that, in addition to ctx, V. mimicus and V. cholerae O139 have the potential to produce ZOT.

Isolation of a Vibrio cholerae transposon-mutant with an altered viable but nonculturable response.

Abstract: We have isolated more than 2500 mutants of Vibrio cholerae by using transposon mutagenesis. Mutants were screened under low nutrient conditions in artificial seawater for an altered viable but nonculturable response, compared to the wild-type. Mutant JR09H1 entered the viable but nonculturable state more rapidly than the wild-type at both 25°C and 4°C.