A mercury-thiol affinity system for rapid generation of overlapping labeled DNA fragments for DNA sequencing.

We describe an in vitro protocol for quickly generating overlapping terminal-labeled restriction fragments for DNA sequence analysis via the Maxam-Gilbert technique. The protocol involves introducing mercurated nucleotides into one end of a region to be sequenced, partial digestion with several restriction enzymes and terminal-labeling, separation of the mercurated restriction enzymes and terminal-labeling, separation of the mercurated restriction fragments from non-mercurated ones on a thiol column and resolution of the different mercurated fragments on one preparative agarose gel. The protocol was used to determine the nucleotide sequence of a 980 base pair cDNA that contains the coding region for a variable surface glycoprotein of Trypanosoma brucei. It could just as quickly and easily be used to obtain many terminal-labeled overlapping restriction fragments covering a region of several kilobases.     

Monoclonal-antibody studies of creatine kinase. The proteinase K-cleavage site.

Proteinase K cleaves a small peptide from native muscle-specific creatine kinase. We present evidence, from the binding of two monoclonal antibodies to formic acid-cleavage fragments and proteinase K-digest fragments of chick muscle creatine kinase, that the proteinase K-cleavage site is in the C-terminal region of the molecule. This specificity of proteinase K, which is not normally a highly specific enzyme, and the continued association of the two peptide fragments after cleavage suggest an unusual conformational feature in the cleavage-site region. By applying predictive methods for hydrophobicity and secondary structure to an amino acid sequence in this region, we suggest possible structural features at the cleavage site that are evidently conserved across avian and mammalian species. The most likely site is next to, or within, a beta-turn on the surface of the molecule.     

Binding and endocytosis of thrombospondin and thrombospondin fragments in endothelial cell cultures analyzed by cuprolinic blue staining, colloidal gold labeling, and silver enhancement techniques

We investigated the distribution of thrombospondin-specific binding sites and the uptake of thrombospondin-gold conjugates in cultured porcine endothelial cells by light and electron microscopy. Colloidal gold marker and silver enhancement techniques were applied for cytochemical detection of monomeric thrombospondin and fragments of thrombospondin. Thrombospondin binds to granular and fibrillar structures and to sites of cell-cell contact on the cell surface, as indicated by many proteoglycan-cuprolinic blue precipitates. Cell migration tracks on the culture dish bottom are most heavily stained. Labeling of intact thrombospondin and of proteolytic fragments of thrombospondin with colloidal gold followed by silver intensification enables one to detect its binding and uptake in endothelial cells. Binding to the cell surface and uptake of thrombospondin-gold particles was inhibited by heparin but not by hyaluronic acid or chondroitin sulfate. The heparin binding region at the N-terminal end of the thrombospondin molecule proved to be essential for cell surface binding. Gold-conjugated thrombospondin fragments devoid of the heparin binding region were not internalized. After 60 min incubation at 37 degrees C, thrombospondin-gold particles accumulated in the lysosomal compartment close to the nucleus. In the presence of monensin and ammonium chloride, vesicles in this area are swollen and the concentration of particulate marker is reduced. Binding and uptake of thrombospondin by vascular endothelial cells appears to require linkage of the heparin binding region of the thrombospondin molecule to coated pits and heparan sulfate-rich molecules as receptors. Colloidal gold conjugation of thrombospondin fragments proved to be useful for cytochemical characterization of molecular domains.     

The number of repeat sequences in microtubule-associated protein 4 affects the microtubule surface properties

The microtubule-binding domain of MAP4, a ubiquitous microtubule-associated protein, contains a Repeat region with tandemly organized repeat sequences. In this study, we focused on the variations of the Repeat region, and searched for MAP4 isoforms with diverse Repeat region organizations. We successfully isolated four types of MAP4 cDNAs, which differed from each other in both the number and the arrangement of the repeat sequences, from a single source (bovine adrenal gland). To examine the functional differences among the isoforms, we prepared the microtubule-binding domain polypeptides of three of the four isoforms, and examined their activities. The isoform fragments showed similar degrees of microtubule assembly promoting activity and microtubule binding affinity. This result suggested that the Repeat region variation is not important for the control of microtubule dynamics, which is believed to be the main function of MAPs. On the other hand, the microtubule bundle-forming activity differed among the isoform fragments. The bundle formation was augmented by increasing the number of repeat sequences in the fragments. Based on these results, we propose the hypothesis that the role of the MAP4 isoforms is to regulate the surface charge of microtubules.     

Hydrozoan eggs can only be fertilized at the site of polar body formation

Hydrozoan eggs are normally fertilized at the site of polar body formation. The female pronucleus is just under the cell membrane at this site. Sperm are attracted to the eggs and aggregate at this site. This paper demonstrates that this site is the only region on the egg surface where the sperm can fuse with the egg. This has been done by cutting unfertilized eggs into fragments containing the site of polar body formation and fragments without this region. Sperm were added to the fragments and their ability to be fertilized was assayed by noting whether or not they cleaved. Only fragments containing the site of polar body formation cleaved. The absence of cleavage in fragments lacking the site of polar body formation cannot be attributed to the inability of these fragments to attract sperm. Such fragments attract sperm for several hours while fragments which contain the site of polar body formation stop attracting sperm a few minutes after fertilization. Cytological studies of egg fragments which do not contain the site of polar body formation show that they do not contain sperm nuclei. The lack of cleavage in these fragments cannot be attributed to the lack of a female pronucleus. By using centrifugation it is possible to move the female pronucleus away from the site of polar body formation. By cutting these centrifuged eggs in an appropriate way it is possible to create egg fragments with the site of polar body formation that lack the female pronucleus and egg fragments that lack the site of polar body formation but contain a female pronucleus. Only fragments which contain the site of polar body formation can be fertilized.     

Synapsin I is a highly surface-active molecule

Synapsin I is a neuron-specific phosphoprotein localized on the surface of small synaptic vesicles to which it binds with high affinity (Kd = 10 nM). Synapsin I exhibits a tendency to self-associate, suggesting that it might have amphiphilic properties. We have now found that synapsin I forms a stable monolayer at an air-water interface which can be compressed under a lateral force of up to 60 dynes/cm, indicating the presence of amphiphilic characteristics in its structure. This interpretation was also supported by circular dichroism spectra of synapsin I, which showed induction of secondary structure in the presence of trifluoroethanol. The various phosphorylated forms of synapsin I did not show any noticeable differences in the force-area isotherms. The monolayer properties of synapsin I fragments derived by cysteine-specific cleavage indicated the presence of amphiphilic characteristics throughout the entire sequence, although the C-terminal region showed less of such surfactant properties. Compositional studies of these fragments revealed that there is little interaction between the N-terminal and middle fragment regions, but that there may be some interaction between the C-terminal and middle fragment regions which affects the surface area occupied by these fragments. Based on this information, we propose a molecular topology for synapsin I consisting of amphiphilic regions and a hydrophilic region.     

S-layer anchoring and localization of an S-layer-associated protease in Caulobacter crescentus

The S-layer of the gram-negative bacterium Caulobacter crescentus is composed of a single protein, RsaA, that is secreted and assembled into a hexagonal crystalline array that covers the organism. Despite the widespread occurrence of comparable bacterial S-layers, little is known about S-layer attachment to cell surfaces, especially for gram-negative organisms. Having preliminary indications that the N terminus of RsaA anchors the monomer to the cell surface, we developed an assay to distinguish direct surface attachment from subunit-subunit interactions where small RsaA fragments are incubated with S-layer-negative cells to assess the ability of the fragments to reattach. In doing so, we found that the RsaA anchoring region lies in the first approximately 225 amino acids and that this RsaA anchoring region requires a smooth lipopolysaccharide species found in the outer membrane. By making mutations at six semirandom sites, we learned that relatively minor perturbations within the first approximately 225 amino acids of RsaA caused loss of anchoring. In other studies, we confirmed that only this N-terminal region has a direct role in S-layer anchoring. As a by-product of the anchoring studies, we discovered that Sap, the C. crescentus S-layer-associated protease, recognized a cleavage site in the truncated RsaA fragments that is not detected by Sap in full-length RsaA. This, in turn, led to the discovery that Sap was an extracellular membrane-bound protease, rather than intracellular, as previously proposed. Moreover, Sap was secreted to the cell surface primarily by the S-layer type I secretion apparatus.     

Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping

The binding domains of 28 monoclonal antibodies (mAbs) against the alpha, beta, and delta subunits of the Torpedo acetylcholine receptor were mapped on the primary sequences of these subunits. Small peptide fragments (2000-20,000 daltons) of the purified subunits were obtained by digestion with staphylococcal V8 protease and papain, separated on a discontinuous polyacrylamide gel electrophoretic system, and electroblotted onto diaminophenyl thioether paper. The blots were probed with the various monoclonal antibodies and also with antibodies against carboxy-terminal decapeptides of the alpha, beta, and delta subunits to identify the carboxy-terminal fragments. From inspection of the binding patterns of the various antibodies to the subunits fragments and the molecular weights of these fragments, and by using the carboxy termini of the subunits as reference points, it was possible to deduce the regions on the primary sequence of each subunit in which the antibodies bound and in some cases to order the binding sites within these sequences. mAb 148, which inhibits receptor function by cross-linking receptor molecules on the cytoplasmic side, was mapped to the sequence beta 368-406. The main immunogenic region of the native receptor, which is of pathological importance in the autoimmune disease myasthenia gravis, was mapped by using mAb 210 to within 80 amino acid residues (alpha 46-127). The overall antigenic structure of alpha subunits was examined. Synthetic peptides have been used to locate determinants responsible for 83% of the antibodies in antisera to denatured alpha subunits and 46% of the antibodies to denatured alpha subunits in antisera to intact receptor. Theoretical models of the transmembrane orientation of the subunit polypeptide chains were tested by determining whether mapped monoclonal antibodies bound to the extracellular or intracellular surface of receptor-rich membranes. Our results confirm previous reports that the carboxy termini of the subunits are exposed on the intracellular surface, as is part of the region between a putative channel-forming domain (M5) and a putative membrane-spanning region (M3). However, contrary to current theoretical models, the region between M5 and the putative membrane-spanning sequence M4 also appears to be on the intracellular surface, implying that M4 and M5 are not membrane-spanning domains.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells

New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 °C and retained their binding specificity. For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents. Received: 2 November 2000 / Accepted: 11 January 2001     

Zinc-induced dimerization of the amyloid-β metal-binding domain 1-16 is mediated by residues 11-14

Analysis of complex formation between amyloid-β fragments using surface plasmon resonance biosensing and electrospray mass spectrometry reveals that region 11-14 mediates zinc-induced dimerization of amyloid-β and may serve as a potential drug target for preventing development and progression of Alzheimer's disease.     

Expression of recombinant proteins on the surface of the coagulase-negative bacterium Staphylococcus xylosus.

An expression system to allow targeting of heterologous proteins to the cell surface of Staphylococcus xylosus, a coagulase-negative gram-positive bacterium, is described. The expression of recombinant gene fragments, fused between gene fragments encoding the signal peptide and the cell surface-binding regions of staphylococcal protein A, targets the resulting fusion proteins to the outer bacterial cell surface via the membrane-anchoring region and the highly charged cell wall-spanning region of staphylococcal protein A. The expression system was used to secrete fusion proteins containing sequences from a malaria blood-stage antigen and a streptococcal albumin-binding receptor to the cell surface of S. xylosus. Analysis of the recombinant cells by immunogold staining and immunofluorescence revealed that both the receptor and the malaria peptide were properly processed and exposed on the surface of the host cells. However, only approximately 40 to 50% of the recombinant cells were strongly stained with antiserum reactive with the albumin-binding receptor, while approximately 10 to 15% of the cells were stained with antiserum reactive with the malaria peptide. The incomplete staining of some of the cells suggests steric effects that make the recombinant fusion proteins inaccessible to the reactive antibodies because of variable cell wall structures. However, the results demonstrate for the first time that recombinant techniques can be used to express heterologous receptors and immunogens on the surface of gram-positive cells.     

Structural and functional studies of titin's fn3 modules reveal conserved surface patterns and binding to myosin S1--a possible role in the Frank-Starling mechanism of the heart.

The A-band part of titin, a striated-muscle specific protein spanning from the Z-line to the M-line, mainly consists of a well-ordered super-repeat array of immunoglobulin-like and fibronectin-type III (fn3)-like domains. Since it has been suspected that the fn3 domains might represent titin's binding sites to myosin, we have developed structural models for all of titin's 132 fn3-like domains. A subset of eight experimentally determined fn3 structures from a range of proteins, including titin itself, was used as homology templates. After grouping the models according to their position within the super-repeat segment of the central A-band titin region, we analyzed the models with respect to side-chain conservation. This showed that conserved residues form an extensive surface pattern predominantly at one side of the domains, whereas domains outside the central C-zone super-repeat region show generally less conserved surfaces. Since the conserved surface residues may function as protein-binding sites, we experimentally studied the binding properties of expressed multi-domain fn3 fragments. This revealed that fn3 fragments specifically bind to the sub-fragment 1 of myosin. We also measured the effect of fn3 fragments on the contractile properties of single cardiac myocytes. At sub-maximal Ca(2+) concentrations, fn3 fragments significantly enhance active tension. This effect is most pronounced at short sarcomere length, and as a result the length-dependence of Ca(2+) activation is reduced. A model of how titin's fn3-like domains may influence actomyosin interaction is proposed.     

Trypanosoma brucei sspp.: cleavage of variant specific and common glycoproteins during exposure of live cells to trypsin

Intact bloodstream forms of Trypanosoma brucei brucei, T.b. gambiense, and T.b. rhodesiense and procyclic forms of T.b. brucei and T.b. gambiense were incubated in trypsin, solubilized for gel electrophoresis, and analyzed for removal of surface molecules. Silver-stained gels and transfer blots probed with horseradish peroxidase-conjugated or radiolabeled lectins revealed that only three glycoproteins, Gp120p, Gp91p, and Gp23p, were removed from the surface of procyclic forms by trypsin. The variant specific glycoproteins, Gp23b, Gp120b, and in some clones Gp91b were surface molecules cleaved from bloodstream forms. Greater than 90% of the variant specific glycoprotein (VSG) was removed from the surface of all clones studied within 1 hr following the addition of trypsin. The removal of VSG was coincident with appearance of 37 to 50 kDa glycopeptide fragments of VSG with different clones yielding different sized fragments. Detailed kinetic analysis of proteins from whole cell extracts and supernatants of the DuTat 1.1 clone of T.b. rhodesiense using concanavalin A (Con A) and polyclonal antibodies revealed that three major VSG fragments were released during trypsinization. The electrophoretic mobility of the three VSG fragments of DuTat 1.1 was not altered when samples were boiled in sodium dodecyl sulfate to inhibit the endogenous phospholipase C. Antiserum to the cross-reactive determinant bound to intact VSG, but did not bind VSG fragments. Thus, the major Con A binding fragments of DuTat 1.1 VSG and perhaps those of the other clones we studied were probably derived from the N-terminal domain of the molecule. The data suggest that VSG is cleaved by trypsin in situ at the hinge region, but remains attached to the cell surface via weak interaction with neighboring molecules.     

Boar sperm surface glycoproteins: Isolation,localization, and temporal expression during spermatogenesis

Boar sperm glycoprotein fractions were isolated by Lens culinaris hemagglutinin affinity chromatography of detergent-solubilized ejaculated spermatozoa, followed by preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In order to develop methods for further investigations of the sperm proteins, we proceeded with two of the isolated glycoproteins. Antibodies were raised in female rabbits against each of the two sperm glycoproteins. By a combination of immunosorbent chromatography, using the antibodies obtained, and preparative SDS polyacrylamide gel electrophoresis, highly purified sperm proteins were isolated. The sperm proteins were immobilized on Sepharose gel columns and specific immunoglobulin Fab fragments were enriched by affinity chromatography. The specificity of the Fab fragments was ascertained by immunoprecipitation analysis. The Fab fragments were used in indirect immunofluorescence analysis to localize the corresponding antigens on the surface of boar spermatozoa. Both antigens were exclusively confined to the postacrosomal region. Immunohistochemical staining of boar testis sections revealed that both antigens are expressed from the spermatid stage. This technique also revealed that one of the antigens congregated at the Golgi complex-acrosome region during spermatogenesis.     

NH2-terminal localization of that part of the staphylococcal enterotoxins polypeptide chain responsible for binding with membrane receptor and mitogenic effect

It is established that the part of the SEA and SEC2 polypeptide chain responsible for the binding of these toxin proteins with the membrane receptor on the surface of rabbit thymocytes and mitogenic effect is localised in the NH2-terminal region of the molecule. The SEC2 splits in two fragments T1 (17 kdalton) and T2 (12.5 kdalton) under limited proteolysis by trypsin in the presence of 2-ME. The amino acid terminal residues of SEA, SEC2 and their proteolytic fragments are also studied.     

Bacterial expression and purification of recombinant bovine Fab fragments.

We have previously described a recombinant phagemid expression vector, pComBov, designed for the production of native sequence bovine monoclonal antibodies (mAb) generated by antibody phage display. Bovine mAb Fab fragments isolated from libraries constructed using pComBov in Escherichia coli strain XL1-Blue, which is routinely used for antibodies expressed on the surface of phage, were expressed at very low yields. Therefore, a study was undertaken to determine optimal growth conditions for maximal expression of bovine Fab fragments in E. coli. By varying the E. coli strain, and the temperature and length of the culture growth, we were able to substantially increase the yield of soluble Fab fragments. A high yield of Fab fragments was found in the culture growth medium, which enabled us to devise a rapid and simple single-step method for the purification of native (nondenatured) Fabs based on immobilized metal affinity chromatography against a six-histidine amino acid carboxyl-terminal extension of the heavy-chain constant region. Using these methods we were able to express and purify antigen-specific bovine Fab fragments from E. coli.     

Identification and Therapeutic Potential of a Vitronectin Binding Region of Meningococcal Msf

The human pathogen Neisseria meningitides (Nm) attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf), a trimeric autotransporter, binds to the activated form of vitronectin (aVn) to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.     

Absence of furrowing activity following regional cortical tension reduction in sand dollar blastomere and fertilized egg fragment surfaces

The purpose of the present investigation was to test experimentally the possibility that division mechanism establishment at the equator of sand dollar eggs may be a consequence of cortical tension gradients between the equator and the poles. Cytochalasin has been shown to decrease tension at the sea urchin egg surface. The concave ends of cytochalasin D-containing agarose cylinders were held against regions of the surface of Echinarachnius parma blastomeres and enucleated fertilized egg fragments. The ability to interfere with normal furrowing activity was used as a biological indicator of the effectiveness of cytochalasin. When agarose containing 2 microg/mL cytochalasin contacted the equatorial region of the blastomeres resulting from the first cleavage, or the equatorial surfaces of nucleated fertilized egg halves, furrowing was blocked, stalled or delayed, indicating that the concentration of cytochalasin was effective. When the same concentration of cytochalasin was applied to the poles, the cells and nucleated fertilized egg fragments divided in the same way as the controls, indicating that the effectiveness of the cytochalasin did not spread from the poles to the equator and that bisection did not interfere with the division of nucleated fertilized egg fragments. When the same concentration of cytochalasin was applied to diametrically opposed surfaces of enucleated, spherical egg fragments, there was no evidence of furrowing activity between the areas that contacted the cytochalasin or in any other part of the surface. Because of the tension-reducing effect of cytochalasin, a tension gradient existed between the regions affected and unaffected by cytochalasin. The results strongly suggest that establishment of the division mechanism by simple gradients of tension at the surface is unlikely.     

Following single antibody binding to purple membranes in real time

Antibody binding to surface antigens in membranes is the primary event in the specific immune defence of vertebrates. Here we used force microscopy to study the dynamics of antibody recognition of mutant purple membranes from Halobacterium salinarum containing a genetically appended anti-Sendai recognition epitope. Ligation of individual anti-Sendai antibodies to their antigenic epitopes was observed over time. Their increase in number within a small selected area revealed an apparent kinetic on-rate. The membrane-bound antibodies showed many different conformations that ranged from globular to V- and Y-like shapes. The maximum distance of two Fab fragments of the same antibody was observed to be approximately 18 nm, indicating an overall strong intrinsic flexibility of the antibody hinge region. Fab fragments of bound anti-Sendai antibodies were allocated to antigenic sites of the purple membrane, allowing the identification and localization of individual recognition epitopes on the surface of purple membranes.     

Grafting of material-binding function into antibodies Functionalization by peptide grafting

Quite recently, a few antibodies against bulk material surface have been selected from a human repertoire antibody library, and they are attracting immense interest in the bottom-up integration of nanomaterials. Here, we constructed antibody fragments with binding affinity and specificity for nonbiological inorganic material surfaces by grafting material-binding peptides into loops of the complementarity determining region (CDR) of antibodies. Loops were replaced by peptides with affinity for zinc oxide and silver material surfaces. Selection of CDR loop for replacement was critical to the functionalization of the grafted fragments; the grafting of material-binding peptide into the CDR2 loop functionalized the antibody fragments with the same affinity and selectivity as the peptides used. Structural insight on the scaffold fragment used implies that material-binding peptide should be grafted onto the most exposed CDR loop on scaffold fragment. We show that the CDR-grafting technique leads to a build-up creation of the antibody with affinity for nonbiological materials.