Localization of the antigenic determinants in carcinoembryonic antigen: the role of the carbohydrate component

The majority of topographic antigenic determinants of the carcinoembryonic antigen (CEA) representing a glycoprotein were shown to contain sugar residues of the CEA carbohydrate chains. Periodate oxidation of CEA followed by reduction afforded a corresponding CEA derivate (CEA-POR), which retained 3% antigenic activity of CEA. In secondary and tertiary structures CEA-POR was proved to be close to CEA pre heated to 80 degrees C. Formation of borate complexes with sugar residues of CEA decreased the CEA antigenic activity to 5% while a but did not affect the spatial structure of its protein core.     

The role of tryptophan residues in heparin-antithrombin interactions

A single tryptophan residue on antithrombin has been modified with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide. This alteration led to a 500-fold reduction in the heparin-dependent acceleration of thrombin-modified antithrombin interactions, as well as a 10-fold decrease in the avidity of the modified protease inhibitor for mucopolysaccharide. Preincubation of antithrombin with the octasaccharide binding domain of heparin prior to treatment with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide was able to suppress modification of the critical tryptophan and preserve the functional capacities of the protease inhibitor. Fluorescence quenching experiments indicated that the modifiable tryptophan groups of antithrombin were exposed to the solvent environment. Based upon these data, it was proposed that the loss of “heparin cofactor” activity of antithrombin must be predominantly due to an inability of the modified protease inhibitor to undergo a conformational transition required for mucopolysaccharide-dependent “activation” of the macromolecule.     

The role of tryptophan residues in the autoprocessing of prosubtilisin E

Subtilisin E, a serine protease from Bacillus subtilis, requires an N-terminal propeptide for its correct folding. The propeptide is autocleaved and digested by the subtilisin domain upon proper folding. Here we investigated the individual roles of the three Trp residues within the subtilisin domain (Trp106, Trp113 and Trp241) on propeptide processing, enzymatic activity and stability of subtilisin. When the propeptide processing was examined by SDS-PAGE after refolding by rapid dilution, the mutation at either position Trp106 or Trp113 was found to significantly delay the propeptide processing, while the mutation at Trp241 had no effect. Far-UV circular dichroism (CD) spectra of the mutants revealed that the mutations at the three positions did not affect appreciably the alpha-helix content of subtilisin. Secondary structure thermal unfolding monitored by CD spectroscopy revealed that none of the tryptophan residues had any significant effect on the stability of mature subtilisin. The enzymatic activity measurements showed that only Trp106 plays a major role in the enzymatic activity of subtilisin E. These results demonstrate that both Trp106 and Trp113 play a specific role in propeptide processing and enzymatic activity, while Trp241 plays no considerable role on any of these activities.     

Assessing the role of tryptophan residues in the binding site

Instead of looking at the interfacial area as a measure of the extent of a protein--protein recognition site, a new procedure has been developed to identify the importance of a specific residue, namely tryptophan, in the binding process. Trp residues which contribute more towards the free energy of binding have their accessible surface area reduced, on complex formation, for both the main-chain and side-chain atoms, whereas for the less important residues the reduction is restricted only to the aromatic ring of the side chain. The two categories of residues are also distinguished by the presence or absence of hydrogen bonds involving the Trp residue in the complex. A comparison of the observed change in the accessible surface area with the value calculated using an analytical expression provides another way of characterizing the Trp residues critical for binding and this has been used to identify such residues involved in binding non-proteinaceous molecules in protein structures.     

Immunohistochemical distribution of the antigenic determinants detected by monoclonal antibodies to carcinoembryonic antigen

By using four distinct monoclonal antibodies to CEA, the molecular profile of which was clarified in our accompanying companion paper, immunohistochemical distribution of the antigenic determinants on both cancerous and noncancerous tissues as well as fetal tissues was studied with the use of the immunoperoxidase method. All of the monoclonal antibodies recognize different antigenic determinants on the tissue section. None of the antibodies stained granulocytes in the peripheral blood or in the normal liver tissues tested. Three of our monoclonal antibodies stained columnar epithelial cells in morphologically normal colonic mucosa; however, monoclonal antibody YK024 did not stain them. This antibody was also found to be unreactive with intestinal metaplasia lesions of the stomach, but reacted with a 16-wk-old fetal stomach as well as with cancerous parts of the colon and of the stomach. Moreover, it was found that this monoclonal antibody mainly reacted with moderately or poorly differentiated adenocarcinoma lesions of the colon and the stomach. Periodic acid treatment in this study, together with trypsin treatment on the antigen as described in our accompanying companion paper, may suggest that this antibody recognizes the carbohydrate antigenic determinant in nature.     

The role of tryptophan residues in an integral membrane protein: diacylglycerol kinase

Tryptophan residues are thought to play special roles in integral membrane proteins, anchoring transmembrane alpha-helices into the lipid bilayer. We have studied the effect of mutating the five Trp residues in the diacylglycerol kinase (DGK) of Escherichia coli to Leu residues. The fluorescence emission maxima for DGK and a variety of Trp mutants in bilayers of dioleoylphosphatidylcholine [di(C18:1)PC] are all centered at ca. 327 nm, suggesting that all five Trp residues are located close to the glycerol backbone region of the bilayer. This is also consistent with fluorescence quenching experiments, measuring the separation between the Trp residues and the bromine atoms in a bilayer of dibromostearoylphosphatidylcholine. Mutation of Trp residues in DGK was found to have significant effects on activity for DGK reconstituted into bilayers of di(C18:1)PC containing 30 mol % 1,2-dihexanoylglycerol (DHG). Of the mutants containing a single Trp residue, only that containing Trp-112 was found to give active protein. The presence of both Trp-25 and Trp-112 gave higher activity than Trp-112 alone. Trp-25 and Trp-112 are the most important Trp residues in DGK as far as activity is concerned. Effects of mutations on K(m) for DHG were generally greater than effects on v(max). The activity of wild-type and mutant DHGs reconstituted into bilayers of phosphatidylcholines was sensitive to the chain length of the phospholipid, with highest activities for chain lengths of C18 or C20 and lower activities in phosphatidylcholines with shorter or longer chains. Compared to wild-type DGK, the Trp mutants were less affected by long-chain phosphatidylcholines but more affected by short-chain phospholipids. In mutants lacking Trp-25, low activities in short-chain phospholipids followed from a decrease in v(max) compared to wild type, combined with an increase in K(m) value for DHG, as observed in the wild type. It is suggested that Trp-25 plays a role in maintaining the alignment of ATP and DHG at the active site. Fluorescence emission spectra for the Trp mutants do not change significantly with changing fatty acyl chain length from C14 to C24, showing efficient hydrophobic matching between DGK and the surrounding lipid bilayer. It is suggested that hydrophobic matching is achieved by tilting of the transmembrane alpha-helix or rotation of residues at the ends of the helices about the Calpha-Cbeta bond linking the residue to the helix backbone. As well as any structural effects, the presence of Trp residues in DGK has a clear effect on thermal stability.     

The role of tryptophan residues of NADPH-adrenodoxin reductase in the formation of complex with adrenodoxin

Chemical modification of tryptophan residues by N-bromosuccinimide was used to determine the role of these residues in the NADPH-adrenodoxin-catalyzed reduction of adrenodoxin, dichlorophenolindophenol and ferricyanide. It was shown that the rate of reduction of all electron acceptors diminishes with modification of tryptophan residues. The most significant decrease of the enzyme activity is observed in case of adrenodoxin-catalyzed reactions. It was suggested that tryptophan residues are responsible for the adrenodoxin reductase interaction with adrenodoxin.     

Mutational analysis of the role of tryptophan residues in an antimicrobial peptide

Antimicrobial peptides belonging to the pediocin-like family of bacteriocins (class IIa bacteriocins) produced by lactic acid bacteria contain several tryptophan residues that are highly conserved. Since tryptophan residues in membrane proteins are often positioned in the membrane-water interface, we hypothesized that Trp residues in bacteriocins could be important determinants of the structure of membrane-bound peptides and of anti-microbial activity. To test this hypothesis, the effects of mutating each of the 3 tryptophan residues (Trp18, Trp33, and Trp41) in the 43-residue pediocin-like bacteriocin sakacin P were studied. Trp18 and Trp33 are located at each end of an amphihilic alpha-helix, whereas Trp41 is near the end of an unstructured C-terminal tail. Replacement of Trp33 with the hydrophobic residues Leu and Phe had marginal effects on activity, whereas replacement with the more polar Tyr and Arg reduced activity 10-20 and 500-1000 times, respectively, indicating that Trp33 and the C-terminal part of the helix interact with the hydrophobic core of the membrane. Any mutation of Trp18 and Trp41 reduced activity, indicating that these two residues play unique roles. Substitutions with other aromatic residues were the least deleterious, indicating that both Trp18 and Trp41 interact with the membrane-water interface. The suggested locations of the three Trp residues are compatible with a structural model in which the helix and the C-terminal tail form a hairpin-like structure, bringing Trp18 and Trp41 close to each other in the interface, and placing Trp33 in the hydrophobic core of the membrane. Indeed, the deleterious effect of the W18L and W41L mutations could be overcome by stabilizing the hairpin-like structure by introduction of a disulfide bridge between residues 24 and 44. These results provide a basis for a refined structural model of pediocin-like bacteriocins and highlight the unique role that tryptophan residues can play in membrane-interacting peptides.     

The specificity for the differentiation blocking activity of carcinoembryonic antigen resides in its glycophosphatidyl-inositol anchor

Ectopic expression of various members of the human carcinoembryonic antigen (CEA) family of intercellular adhesion molecules in murine myoblasts either blocks (CEA, CEACAM6) or allows (CEACAM1) myogenic differentiation. These surface glycoproteins form a subset of the immunoglobulin (Ig) superfamily and are very closely related, but differ in the precise sequence of their external domains and in their mode of anchorage to the cell membrane. CEA and CEACAM6 are glycophosphatidyl-inositol (GPI) anchored, whereas CEACAM1 is transmembrane (TM) anchored. Overexpression of GPI-linked neural cell adhesion molecule (NCAM) p125, also an adhesion molecule of the Ig superfamily, accelerates myogenic differentiation. The molecular requirements for the myogenic differentiation block were investigated using chimeric constructs in which the COOH-terminal hydrophobic domains of CEA, CEACAM1, and NCAM p125 were exchanged. The presence of the GPI signal sequence specifically from CEA in the chimeras was sufficient to convert both CEACAM1 and NCAM into differentiation-blocking proteins. Conversely, CEA could be converted into a neutral protein by exchanging its GPI anchor for the TM anchor of CEACAM1. Since the external domains of CEA, CEACAM1, and NCAM can all undergo homophilic interactions, and mutations in the self-adhesive domains of CEA abrogate its differentiation-blocking activity, the structural requirements for differentiation-inhibition are any self-adhesive domains attached to the specific GPI anchor derived from CEA. We therefore suggest that biologically significant functional information resides in the processed extreme COOH terminus of CEA and in the GPI anchor that it determines.     

The role of tryptophan residues in the hemolytic activity of stonustoxin,a lethal factor from stonefish (Synanceja horrida) venom

Stonustoxin (SNTX) is a pore-forming cytolytic lethal factor, isolated from the venom of the stonefish Synanceja horrida, that has potent hemolytic activity. The role of tryptophan residues in the hemolytic activity of SNTX was investigated. Oxidation of tryptophan residues of SNTX with N-bromosuccinimide (NBS) resulted in loss of hemolytic activity. Binding of 8-anilino-1-naphthalenesulphonate (ANS) to SNTX resulted in occlusion of tryptophan residues that resulted in loss of hemolytic activity. Circular dichroism and fluorescence studies indicated that ANS binding resulted in a conformational change of SNTX, in particular, a relocation of surface tryptophan residues to the hydrophobic interior. NBS-modification resulted in oxidised surface tryptophan residues that did not relocate to the hydrophobic interior. These results suggest that native surface tryptophan residues play a pivotal role in the hemolytic activity of STNX, possibly by being an essential component of a hydrophobic surface necessary for pore-formation. This study is the first report on the essentiality of tryptophan residues in the activity of a lytic and lethal factor from a fish venom.     

The critical role of residues 43R and 44Q of carcinoembryonic antigen cell adhesion molecules-1 in the protection from killing by human NK cells

The multifunctional carcinoembryonic Ag cell adhesion molecule (CEACAM)1 protein has recently become the focus of intense immunological research. We have previously shown that the CEACAM1 homophilic interactions inhibit the killing activity of NK cells. This novel inhibitory mechanism plays a key role in melanoma immune evasion, inhibition of decidual immune response, and controlling NK autoreactivity in TAP2-deficient patients. These roles are mediated mainly by homophilic interactions, which are mediated through the N-domain of the CEACAM1. The N-domain of the various members of the CEACAM family shares a high degree of similarity. However, it is still unclear which of the CEACAM family members is able to interact with CEACAM1 and what are the amino acid residues that control this interaction. In this study we demonstrate that CEACAM1 interacts with CEACAM5, but not with CEACAM6. Importantly, we provide the molecular basis for CEACAM1 recognition of various CEACAM family members. Sequence alignment reveals a dichotomy among the CEACAM family members: both CEACAM1 and CEACAM5 contain the R and Q residues in positions 43 and 44, respectively, whereas CEACAM3 and CEACAM6 contain the S and L residues, respectively. Mutational analysis revealed that both 43R and 44Q residues are necessary for CEACAM1 interactions. Implications for differential expression of CEACAM family members in tumors are discussed.     

The role of tryptophan residues in the 5-Hydroxytryptamine(3) receptor ligand binding domain

Aromatic amino acids are important components of the ligand binding site in the Cys loop family of ligand-gated ion channels. To examine the role of tryptophan residues in the ligand binding domain of the 5-hydroxytryptamine(3) (5-HT(3)) receptor, we used site-directed mutagenesis to change each of the eight N-terminal tryptophan residues in the 5-HT(3A) receptor subunit to tyrosine or serine. The mutants were expressed as homomeric 5-HT(3A) receptors in HEK293 cells and analyzed with radioligand binding, electrophysiology, and immunocytochemistry. Mutation of Trp(90), Trp(183), and Trp(195) to tyrosine resulted in functional receptors, although with increased EC(50) values (2-92-fold) to 5-HT(3) receptor agonists. Changing these residues to serine either ablated function (Trp(90) and Trp(183)) or resulted in a further increase in EC(50) (Trp(195)). Mutation of residue Trp(60) had no effect on ligand binding or receptor function, whereas mutation of Trp(95), Trp(102), Trp(121), and Trp(214) ablated ligand binding and receptor function, and all but one of the receptors containing these mutations were not expressed at the plasma membrane. We propose that Trp(90), Trp(183), and Trp(195) are intimately involved in ligand binding, whereas Trp(95), Trp(102), Trp(121), and Trp(214) have a critical role in receptor structure or assembly.     

The role of tryptophan residues and hydrophobic interaction in the binding of riboflavin in egg-yolk flavoprotein.

Egg-yolk flavoprotein has 7.2 tryptophan residues exposed, while the apoprotein shows an apparent exposure of 80 percent of these (5.7 residues) with dimethylsulphoxide as the perturbant. In the apoprotein at pH 6.9 only 4 groups are perturbed to ethylene glycol, 3.2 to glycerol and 1.4 to sucrose. Diminishing estimates of exposure obtained with increasing molecular diameter of the perturbant suggests that part of indole chromophores of apoprotein are located in "crevices" of the protein molecule. The apoprotein was treated with 2-hydroxy-5-nitrobenzyl bromide, H2O2 and N-bromosuccinimide under conditions designed to accomplish modification of tryptophan residues. Five to six of the eight tryptophans present in the protein were modified. Under these conditions the apoprotein completely looses its capacity for binding riboflavin and the fluorescent intensity of the protein at 360 nm is quenched at the same time to about 80 percent of its initial value. The presence of nonpolar amino acid residues on the surface of the apoprotein suggested the importance of hydrophobic interactions as the dominant factor controlling the binding of riboflavin. The hydrophobic probes Indocyanine green and 4-benzoylamide-4-aminostilbene-2,2-disulphonic acid bound to the apoprotein giving equimolar complexes with dissocation constants, KD 6.5-10(-7) M and 1.8-10(-6) M, respectively, Addition of an equimolar amount of riboflavin quantitatively displaced these dyes from their complexes with apoprotein as shown by spectrophotometric and spectrofluorometric studies.     

Experimental reactivity and antigenic activity of the bacterial-fungal antigen complexes

The immunogenic and reactogenic properties of monovaccines prepared from Staphylococcus aureus, S. epidermidis and Candida albicans, as well as those of a associated polyvaccine prepared from these infective agents, were experimentally studied on rabbits. The monovaccines and the associated bacterial-fungal vaccines were found to be safe and faintly reactogenic; in the blood serum of vaccinated rabbits a growth in the titer of agglutinins and its preservation at a high level for 4 months were noted.     

The role of plasmin on the double antibody radioimmunoassay of carcinoembryonic antigen in human blood samples.

Double antibody radioimmunoassay (RIA) of carcinoembryonic antigen (CEA) on a series of freshly drawn out concurrent plasma and serum samples of normal human blood donors showed that the serum CEA values were invariably higher than the corresponding plasma CEA values. Extraneous addition of fibrinogen brought down the serum-CEA level to a value comparable to or less than the corresponding plasma value. The effect of certain factors associated with blood clotting, particularly Ca++, fibrinogen, and the fibrinolytic enzyme plasmin, was investigated. Ca++ was shown to play no role whereas the effect of fibrinogen was shown to be indirect in that it served as a specific substrate for plasmin, thereby preventing the plasmin degradation of the primary antibody used in the RIA. The finding stresses the role of enzymes like plasmin in double antibody RIA in general and may explain some of the anomalous results obtained when testing biologic material containing plasmin-like substances.     

The role of phosphoric acid residues in the formation of antigenic determinants of the structural components of DNA

The inhibitory activity of thymidine, thymidine triphosphate and thymidyl oligonucleotides was studied in thymidine-antithymidine antibodies reaction. Thymidine was shown to have the greatest inhibitory effect, with thymidine triphosphate and thymidyl oligonucleotide inhibitory activity less expressed and reducing with the increase in oligonucleotide length. The effect of thymidine, thymidine triphosphate and thymidyl oligonucleotides on the interaction of antisera and SLE patients' sera with denatured DNA was studied. It was shown that thymidine triphosphate and particularly thymidyl oligonucleotides are characterized by greater inhibitory capacity, as compared to thymidine. It was found that only thymine dimers bound by phosphate groups can inhibit the interaction of UV-irradiated DNA with antiserum specific for UV-modified DNA. The data obtained suggest that the charge determined by phosphoric acid residues plays an essential role in the interaction of antibodies induced to charged structural DNA components.     

Assessment of the role of tryptophan residues in phospholipase A2 by fluorescence quenching

Tryptophan (Trp) fluorescence of two phospholipases A2 (PLA2) from Naja naja atra and Naja nigricollis snake venoms was quenched by acrylamide and iodide. Trp residues in N. naja atra PLA2 were equally accessible to acrylamide and iodide. Iodide quenching studies indicate that there are two classes of Trp fluorophores in N. nigricollis CMS-9. The accessible class consists of Trp-18 and Trp-19. Removal of the N-terminal octapeptide caused a perturbation of the micro-environment of the Trp residues in the PLA2 enzymes. The presence of a substrate lowers the susceptibility of the Trp residues to iodide quenching in N. naja atra PLA2, suggesting that all three Trp residues are at the substrate binding site, but in N. nigricollis CMS-9 Trp-18 and Trp-19 are related to substrate binding.