Comparative gene expression analysis of avian embryonic facial structures reveals new candidates for human craniofacial disorders.

Mammals and birds have common embryological facial structures, and appear to employ the same molecular genetic developmental toolkit. We utilized natural variation found in bird beaks to investigate what genes drive vertebrate facial morphogenesis. We employed cross-species microarrays to describe the molecular genetic signatures, developmental signaling pathways and the spectrum of transcription factor (TF) gene expression changes that differ between cranial neural crest cells in the developing beaks of ducks, quails and chickens. Surprisingly, we observed that the neural crest cells established a species-specific TF gene expression profile that predates morphological differences between the species. A total of 232 genes were differentially expressed between the three species. Twenty-two of these genes, including Fgfr2, Jagged2, Msx2, Satb2 and Tgfb3, have been previously implicated in a variety of mammalian craniofacial defects. Seventy-two of the differentially expressed genes overlap with un-cloned loci for human craniofacial disorders, suggesting that our data will provide a valuable candidate gene resource for human craniofacial genetics. The most dramatic changes between species were in the Wnt signaling pathway, including a 20-fold up-regulation of Dkk2, Fzd1 and Wnt1 in the duck compared with the other two species. We functionally validated these changes by demonstrating that spatial domains of Wnt activity differ in avian beaks, and that Wnt signals regulate Bmp pathway activity and promote regional growth in facial prominences. This study is the first of its kind, extending on previous work in Darwin's finches and provides the first large-scale insights into cross-species facial morphogenesis.

Anti-phosphocholine hybridoma antibodies. II. Functional analysis of binding sites within three antibody families.

The present investigation extends our immunochemical characterization of binding site heterogeneity among a large series of monoclonal anti-phosphocholine (PC) antibodies. Hybridoma proteins (HP) from eight genetically distinct strains are included in this study, yet no strain specific characteristics are observed. These HP, as previously shown (5), are divided into three well-defined families based on public or family-specific Id and L chain isotypes characteristic of three PC-binding myeloma proteins: T15, M603, and M511. All antibodies exhibited some degree of inter- or intra-family heterogeneity, or both. Some of this intra-family diversity was reflected by differential reactivity for PC when attached to three different carriers. In spite of this, the specificity profiles for hapten analogues of PC, as measured by hapten inhibition of binding, were the same for all members of the T15 family. Altering the carrier had no effect, thus suggesting that the binding site pocket for PC is essentially preserved, whereas that for carrier is variable. Similar conclusions were reached for most of the M603 HP, although the binding site is different from the T15 HP. The M511 HP stand in sharp contrast to the HP in the other two families because their binding sites exhibit extensive variability. The independence in reactivity for PC and PC plus carrier offers a rational explanation for idiotypic and/or structural heterogeneity within a family. More importantly it suggests interesting strategies for diversification within one group of antibodies.

The synthesis of sialylated oligosaccharides using a CMP-Neu5Ac synthetase/sialyltransferase fusion.

Large-scale enzymatic synthesis of oligosaccharides, which contain terminal N-acetyl-neuraminic acid residues requires large amounts of the sialyltransferase and the corresponding sugar-nucleotide synthetase, which is required for the synthesis of the sugar-nucleotide donor, CMP-Neu5Ac. Using genes cloned from Neisseria meningitidis, we constructed a fusion protein that has both CMP-Neu5Ac synthetase and alpha-2,3-sialyltransferase activities. The fusion protein was produced in high yields (over 1200 U/L, measured using an alpha-2,3-sialyltransferase assay) in Escherichia coli and functionally pure enzyme could be obtained using a simple protocol. In small-scale enzymatic syntheses, the fusion protein could sialylate various oligosaccharide acceptors (branched and linear) with N-acetyl-neuraminic acid as well as N-glycolyl- and N-propionyl-neuraminic acid in high conversion yield. The fusion protein was also used to produce alpha-2,3-sialyllactose at the 100 g scale using a sugar nucleotide cycle reaction, starting from lactose, sialic acid, phosphoenolpyruvate, and catalytic amounts of ATP and CMP.

Mechanisms of spectral shifts in lobster carotenoproteins. The resonance Raman spectra of ovoverdin and the crustacyanins.

Resonance Raman data have been used to elucidate the mechanisms of the absorption spectral shifts occurring for astaxanthin upon binding to the carotenoproteins, ovoverdin and alpha-,beta- and gamma-crustacyanins, from the lobster Homarus americanus. Although distinguishable on the basis of small differences in their resonance Raman spectra the binding sites of the crustacyanins, giving rise to lambdamax at 605 +/- 25 nm, are essentially the same. The large red shift in lambdamax for the crustacyanins compared to free astaxanthin (lambdamax 480 nm), is accounted for by a charge-polarisation mechanism in which charged groups and possibly hydrogen bonds in the binding site set up pi electron polarisation in the ligand. Several alternate mechanisms can be eliminated. Ovoverdin is found to consist of three polypeptide chains of molecular weight 105,000, 95,000 and 78,000 which are not linked by disulfide bridges. The visible absorption peaks of ovoverdin at 460 and 640 nm are shown to arise from two astaxanthin molecules each bound at a different site. The spectral characteristics of the 460 nm site suggest a rigid hydrophobic environment for astaxanthin, in which no charge-ligand interactions occur. The mechanism of the spectral shift in the 640 nm site is the same as in the crustacyanins, i.e. a charge-polarisation effect. Resonance Raman spectra of ovoverdin and the crustacyanins could be obtained in situ; they were identical to the spectra of the purified proteins showing that the carotenoid sites were unperturbed by protein isolation.

The solution structure of concanavalin A probed by FT-IR spectroscopy.

The secondary structural properties of various forms of concanavalin A in solution were investigated by Fourier-transform infrared spectroscopy in the Amide I region. As in the crystal, the solution structure of the native protein consists mainly of antiparallel beta-sheet. Carbohydrate binding does not produce major changes in the overall secondary structure of concanavalin A, but affects infrared bands due to loops and beta-turns. Upon demetallization, the spectrum of concanavalin A shows only a small change in the Amide I band, indicating that whereas the beta-sheet structure is conserved, the tertiary properties may be altered. There are also changes in the bands from the tyrosine residues which are compatible with local changes in structure. Confirming tertiary structural differences, the cation-depleted apoprotein is much less stable, denaturing around 63 degrees C, while the native protein denatures only at temperatures around 85 degrees C. Tetramerization proceeds without significant secondary structural change. However, aggregation of the tetramers leads to a significant decrease of the bands corresponding to beta-sheet structure, and changes in the tyrosine bands.

Analysis of sequence variation among legume lectins. A ring of hypervariable residues forms the perimeter of the carbohydrate-binding site.

Twelve plant lectins from the Papilionoideae subfamily were selected to represent a range of carbohydrate specificities, and their sequences were aligned. Two variability indices were applied to the aligned sequences and the results were analysed using the three-dimensional structures of concanavalin A and the pea lectin. The areas of greatest variability were located in the carbohydrate-binding site region, forming a perimeter around a well-conserved core. These residues are inferred to be specificity determining, in the manner of antibodies, and the most variable position corresponded to Tyr100 in concanavalin A, a known ligand contact residue. In addition to the five peptide loops known to form the binding site from crystallographic studies, a sixth segment with variable residues was located in the binding-site region, and this may contribute to oligosaccharide specificity. In their overall composition, the lectin sites resemble those of the sugar-transport proteins rather than antibodies. The prospects for modelling lectin binding sites by the methods used for antibodies were also assessed.

Crystallization and preliminary X-ray diffraction studies of the complex of Maclura pomifera agglutinin with the disaccharide Gal beta 1-3GalNAc.

Single crystals of Maclura pomifera agglutinin, a seed lectin from the Moraceae family, complexed with the disaccharide Gal beta 1-3GalNAc have been obtained by the method of vapor diffusion with Li2SO4 as precipitant at pH 4.5. The crystals belong to the trigonal space group P3(1)21 or P3(2)21, with a = b = 67.4 A, c = 149.3 A. They contain two subunits per asymmetric unit and diffract beyond 2.7 A. This and other evidence indicate that both this lectin and the Artocarpus integrifolia lectin, jacalin, have dimeric structures rather than the tetrameric structures previously proposed.

Exploring the mimicry of polysaccharide antigens by anti-idiotypic antibodies. The crystallization, molecular replacement, and refinement to 2.8 A resolution of an idiotope-anti-idiotope Fab complex and of the unliganded anti-idiotope Fab.

The monoclonal antibody YsT9.1 is specific for the lipopolysaccharide A antigen of Brucella abortus. A complex formed between the Fab of YsT9.1 (Ab1) and the Fab of its antidiotopic monoclonal antibody T91AJ5 (Ab2) has been crystallized, and data have been collected to 2.8 A resolution. The space group is monoclinic P2(1), with one molecule per asymmetric unit. The structure was solved using a limited Patterson-correlation search over the asymmetric-unit of rotation space, and has been refined to an R-factor of 0.174. The complex is a head-to-head dimer, with the contact between the two Fabs almost completely restricted to their hypervariable loops. The interactions between the two Fabs in the complex are dominated by tyrosine residues, not only in the formation of hydrogen bonds, but in their participation in an aromatic ring network that spans the two Fv domains. The anti-idiotope was found to be unable to carry an internal image of the antigen and induce polysaccharide-specific "anti-anti-idiotopes" (Ab3) because the polysaccharide binding cleft on the Ab1 is too narrow and deep to allow comprehensive contact with the binding site of Ab2. The antibody T91AJ5 therefore is a class gamma anti-idiotope. The contact surfaces of the two Fabs are highly complementary; however, they are distinctly different in character. Each Fab has two separate binding surfaces of approximately equal size, but while the two binding surfaces of Ab1 are partitioned between the light and heavy chains, the two binding surfaces of Ab2 are shared between the chains, with the heavy chain responsible for about 60% of the total binding area. The structure of the unliganded Fab of Ab2 has also been solved by molecular replacement, and refined to an R-factor of 0.152 at 2.8 A resolution. This Fab crystallizes in the orthorhombic space group P2(1)2(1)2(1), with one molecule per asymmetric unit. The second hypervariable loop of the heavy chain of the Ab2 Fab is observed to undergo a significant and necessary conformational rearrangement in going from the unliganded to complexed form, and thus complex formation is an example of "induced fit" of antigen to antibody. The unliganded Ab1 Fab packs in the standard head-to-tail fashion observed for other Fabs. This mode of packing is precluded in the complex, by its head-to-head nature, and it is found to pack in tilted layers with most intermolecular contacts made between adjacent Ab2 Fab molecules.(ABSTRACT TRUNCATED AT 400 WORDS)

The glycopeptides of the mouse immunoglobulin A T15.

Cleavage of mouse IgA T15 with papain yielded (a) a glycosylated Fab fragment, (b) a non-glycosylated Fc fragment and (c) a glycosylated C-terminal peptide. The cleavage sites at the hinge and at the end of the C alpha 3 domain were located by sequencing. The two glycopeptides were prepared from the Fab and C-terminal fragments by pronase digestion. The C alpha 1 glycopeptide at Asn 155 was complex type with alpha (1-3)galactose terminal groups, and closely resembled the Asn 171 glycopeptide of mouse IgM (Anderson et al. (1985) Arch. Biochem. Biophys. 243, 605-618). In contrast, the C-terminal glycopeptide at Asn 446 was entirely different from the corresponding IgM glycopeptide, being complex rather than high-mannose type.

The circular dichroism of phosphocholine-specific mouse hybridoma and myeloma proteins: unusual properties of the hybridoma protein 101.6G6.

The circular dichroism (CD) spectra of five myeloma and six hybridoma proteins specific for phosphocholine were measured in the 250-310-nm range. The effect on the CD spectra of adding phosphocholine was also examined. The five myeloma proteins all had distinctive native spectra and, except for M603 and W3207, unique changes occurred on ligand binding. The hybridomas were chosen as pairs from each of the three known families of phosphocholine-specific immunoglobulins. Those from the T15 or M603 families resembled the appropriate prototype. However, the proteins from the M167 family were all distinctively different in their CD properties. In particular, the hybridoma protein 101.6G6 showed large CD changes on hapten binding and values for the association constant for phosphocholine of 1.1 X 10(5) M-1 and of 5.8 X 10(2) M-1 for acetylcholine were obtained by CD spectrophotometric titration. The CD properties of the proteins are interpreted in the light of the sequence data so far available, including the possible role of the D-segment.

Molecular basis for the lack of mimicry of Brucella polysaccharide antigens by Ab2gamma antibodies.

The crystal structure of the complex of an anti-Id Fab with an Fab specific for a Brucella polysaccharide antigen has previously been reported (Evans et al., 1994, J. Mol. Biol. 241, 691-705). To complement this study, the binding characteristics and immunological properties of this Ab2 and two others raised with a second anti-Brucella antibody were investigated, including quantitative kinetic measurements by surface plasmon resonance. The affinities of the Fabs from the Ab2s for the Ab1s were three orders of magnitude greater than those estimated for the antigen, but the Ab2s failed to induce antigen-binding Ab3s, that is, they were of the Ab2gamma type. The avidities of the Ab1s for antigen were however within one order of magnitude of their avidities for Ab2. Tests of 16 other anti-Brucella polysaccharide antibodies showed that the two idiotopes were not present in them, and in confirmation of the lack of a dominant idiotope, N-terminal sequencing of their H and L chains showed a wide variety of V genes were employed in the immune response to the Brucella polysaccharides. The failure of the Ab2 to induce antigen-reactive Ab3 thus appears to be due to neither intrinsic affinity nor idiotope frequency, but arises instead from structural reasons, for example, the incomplete penetration of the Ab2 into the binding-site cleft of the Ab1. The surface topography of polysaccharide antigens and their binding-sites thus appears to be especially difficult for Ab2s to mimic and will restrict their routine use as surrogates for T-cell independent polysaccharide antigens.

Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen.

The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.

Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen.

A 1460-bp DNA encoding the two chains of the antigen-binding fragment (Fab) portion of a monoclonal antibody have been chemically synthesized and expressed in Escherichia coli. The antibody, Se155-4, is specific for a Salmonella serogroup B O-antigen and its crystal structure is under investigation. The genes were synthesized according to a strategy that allows for easy manipulation in genetic engineering studies of the Fab-binding site. Each gene is preceded by the ompA secretory signal and a ribosome-binding site, and has been expressed from the two-cistron DNA under the control of the lac promoter. Active Fab of 50 kDa with an inter-chain disulfide bond has been isolated from the periplasm of E. coli in a one-step affinity purification in high yield (2 micrograms/ml of cells). The bacterially produced Fab is as active as purified mouse Fab in antigen-binding and competitive immunoassays. This is the first example of a completely synthetic Fab gene and provides an ideal system to probe the nature of antigen binding by anti-carbohydrate antibodies.

Magnesium as a natural substitute for manganese in concanavalin A and other lectins.

Addition of magnesium to apo-concanavalin A in the presence of calcium was shown by ultraviolet difference spectroscopy to generate a holoprotein spectroscopically identical to the MnCa-holoprotein. The MgCa- and MnCa-forms bound equally strongly to Sephadex G-75. In kinetic experiments, the binding of Mg2+ was much slower than Mn2+ binding; Kd for Mg2+ was estimated as 7.4 mM. The combined Mg2+ and Mn2+ contents of 10 lectins specific for D-galactose or N-acetyl-D-galactosamine were each close to one atom per subunit, suggesting occupancy of the Mn2+ site by Mg2+ is common in plant lectins.

The amino acid sequences of jacalin and the Maclura pomifera agglutinin.

Amino acid sequences for the alpha-chains of the Moraceae lectins, jacalin and Maclura pomifera agglutinin, were determined by protein sequencing. Both are 133 residues long and contain several genetically variant positions; the overall homology is 85%. A possible site for the known glycopeptide of jacalin was located. The alpha-chains have a conserved tryptophan residue that may be part of the binding-site.

Post-translational processing of two alpha-amylase inhibitors and an arcelin from the common bean, Phaseolus vulgaris.

Mass spectrometric methods were used to investigate the proteolytic processing and glycopeptide structures of three seed defensive proteins from Phaseolus vulgaris. The proteins were the alpha-amylase inhibitors alphaAI-1 and alphaAI-2 and arcelin-5, all of which are related to the seed lectins, PHA-E and PHA-L. The mass data showed that the proteolytic cleavage required for activation of the amylase inhibitors is followed by loss of the terminal Asn residue in alphaAI-1, and in all three proteins, seven or more residues were clipped from the C-termini, in the manner of the seed lectins. In most instances, individual glycoforms could be assigned at each Asn site, due to the unique masses of the plant glycopeptides. It was found that alphaAI-1 and alphaAI-2 differed significantly in their glycosylation patterns, despite their high sequence homology. These data complement the previous X-ray studies of the alpha1-amylase inhibitor and arcelin, where many of the C-terminal residues and glycopeptide residues could not be observed.

Thermal stabilization of a single-chain Fv antibody fragment by introduction of a disulphide bond.

A disulphide bond was introduced into a single-chain Fv form of the anticarbohydrate antibody, Se155-4 by replacing Ala-L57 of the light chain and Asp-H106 of the heavy chain with cysteines, by site-directed mutagenesis. To maintain the salt-bridge from the latter residue to Arg-H98, Tyr-107 was also altered to Asp. The resulting ds-scFv was shown to retain full antigen-binding activity, by enzyme immunoassay and surface plasmon resonance analysis of binding kinetics. Compared with the parent scFv, the disulphide bonded form was shown to have enhanced thermal stability, by Fourier transform IR spectroscopy. The Tm was raised from 60 degrees C to 69 degrees C. The ds-scFv form thus combines the stable monomeric form of the disulphide form with the expression advantages of the scFv.

Characterisation of residues in antibody binding sites by chemical modification of surface-adsorbed protein combined with enzyme immunoassay.

Specific functional group modification of an antibody adsorbed to microtitre plates has been used to probe the binding site residues that determine antigen specificity. Chemical modification of adsorbed protein in tandem with enzyme immunoassay (termed CMAP-EIA) consumes only modest amounts of antibody, while allowing a variety of reagents to be rapidly screened in situ. Modification of tyrosine and arginine residues with 1-fluoro-2,4-dinitrobenzene, and p-hydroxyphenylglyoxal resulted in reduced binding of polysaccharide antigen from Yersinia enterocolitica O-polysaccharide to its homologous monoclonal antibody, YsT9-1. Modification with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide under various conditions indicated that carboxylate groups may also be involved. Parallel experiments with diethylpyrocarbonate and acetic anhydride were used to rule out the involvement of histidine and lysine residues respectively. In all cases, binding of an anti-idiotypic antibody, AJ5, could only be reduced at concentrations of modifying reagent substantially higher than those required to reduce polysaccharide antigen binding to YsT9-1. The results are discussed with regard to the structure of the combining site of YsT9-1 as determined by X ray crystallography and by modelling, and the role of particular residues in complex formation with antigen and in the idiotope.

O-antigen biotin conjugates. Preparation and use in direct competitive enzyme immunoassays.

Bacterial O-antigens coupled to biotin were shown to function well as labelled antigens in direct enzyme immunoassay. O-polysaccharides released from lipopolysaccharides by mild acid hydrolysis were oxidized by sodium periodate at sites located within the lipopolysaccharide inner core region and the generated aldehyde groups were subjected to reductive amination with 1,3-diaminopropane to yield per-aminated O-polysaccharide derivatives. Biotin was coupled to the introduced amino groups by way of a N-hydroxy-succinimide ester derivative. Biotinylated polysaccharides were used in direct enzyme immunoassays, that employed monoclonal antibody coated microtitre plates and detection of bound biotinylated antigen by streptavidin/horseradish peroxidase reagent. The assay format was designed for rapid estimation of the affinity constants of monoclonal antibodies for small oligosaccharide inhibitors, but the assay is also well suited to fast and sensitive detection of bacterial O-polysaccharides at concentrations within the range 1-10 ng/ml.

Surgical treatment of vertigo with retrolabyrinthine vestibular neurectomy.

Results for control of vertigo and preservation of hearing in patients who have had a retrolabyrinthine vestibular neurectomy (RVN) by our group were analyzed retrospectively. This procedure consists of selective section of the vestibular nerve in the posterior cranial fossa. Vertigo was completely controlled in all but two of 31 patients, one of whom required revision surgery to control attacks. Analysis of these two cases suggests that the cause of persistent vertigo is incomplete neurectomy. With our current surgical technique in patients with Meniere's disease, hearing results were not statistically different from our results with surgery of the endolymphatic sac. Control of vertigo was much more successful with the RVN than endolymphatic sac surgery.