Immunochemical and physico-chemical characteristics of glycoprotein from retroplacental blood having several common antigenic determinants with trophoblastic beta 1-glycoprotein

A glycoprotein (TSG-fs) isolated from retroplacental blood is shown to have common antigenic determinants with trophoblast-specific beta 1-glycoprotein (TSG, SP-1). Antigenic activity of TSG-fs constitute 3.4% of TSG's activity. Like TSG, glycoprotein TSG-fs contains four intramolecular disulfide bonds and belongs to proteins with predominantly beta-structure. CD-spectroscopy data give evidence of high stability of the secondary structure of TSG-fs. Immunochemical identity of TSG-fs with the reduced and carboxymethylated derivative of TSG has been demonstrated. The above data allow to suggest that TSG-fs is a fragment of the TSG molecule.     

The role of tryptophan residues in the antigenic activity of carcinoembryonic antigen

Antigenic determinants of carcino-embryonic antigen (CEA) were spatially located using N-bromosuccinimide modification of tryptophan residues both in native (acetate buffer solution) and unfolded (guanidinium chloride solution) molecule of the antigen. Modification of exposed tryptophan residues failed to alter CEA antigenic activity and conformation of its protein portion as shown by CD spectroscopy. On the contrary, modification of buried tryptophan residues induced conformational changes of CEA protein portion connected with a considerable loss of its antigenic activity. It was shown that CEA antigenic activity depends on spatial structure of its protein moiety.     

Immunochemical characterization of a mycobacterial antigen designated β

Abstract A mycobacterial antigen called β, earlier demonstrated in ribosomal preparations, has been immunochemically characterized. Heat or alkali treatment destroyed the antigenic activity of β. At acidic pH β formed a precipitate with a protein/RNA ratio of 0.6. An anion exchanger resin adsorbed β at low but not at high ion strength. Trypsin diminished the antigenicity of β, while ribonuclease A seemed to split the β molecule. Dissociation of the protein from the RNA portion destroyed the antigenic activity of β. The results suggested that β is a ribonucleoprotein in which the antigen determinants are distributed on two or more peptides kept together by RNA.     

Relation between the spatial structure and antigenic activity of trophoblast-specific beta1-glycoprotein

Temperature- and pH-dependence of spatial structure of a native trophoblast-specific beta-glycoprotein (TSG), its desialated and deglycosylated derivatives, as well as of a fragment obtained by partial acid hydrolysis on the temperature and pH variations has been studied using CD and UV spectroscopy. Within the range 45-50 degrees C a conformational transition of the protein moiety of TSG, leading to partially reversible alterations in tertiary and secondary structures of this glycoprotein after cooling the solution to 20 degrees C has been found out. The results of spectral studies of the spatial structure of the TSG protein component have been compared with the data on antigen activity of native, temperature- and pH-denaturated, desialated, and deglycosilated TSG. It has been concluded that the protein moiety of TSG consists mainly of beta-structures; the greater part of antigen determinants of TSG is topographic and belongs to the protein component of TSG, and only 15% of antigen determinants are not connected with TSG's spatial structure.     

The ABO, Lewis and related blood group antigens; a review of structure and biosynthesis

Numerous studies have shown that the antigenic determinants of the ABO blood group system are closely related in biochemical terms to the antigenic determinants of the Hh, P, Lewis and Ii blood group systems. The blood group antigens of each of these systems are formed by the addition of specific sugars to an oligosaccharide precursor chain which may be bound through sphingosine to fatty acids (glycolipid) or through serine or threonine to a peptide chain (glycoproteins). The direct gene products of each of these blood group systems are the glycosyltransferase enzymes which catalyse the addition of the specific sugar thus conferring the specified blood group activity to the glycolipid or glycoprotein molecule. The antigenic determinants of the ABO and Lewis systems in addition to red cells also exist in the body secretions in soluble form when the relevant genes are expressed in the phenotype. The antigens expressed on both the red cells and in the secretions are determined by the interaction of Hh, Sese, ABO and Lele genes.     

The ABO, Lewis and related blood group antigens; a review of structure and biosynthesis

Abstract Numerous studies have shown that the antigenic determinants of the ABO blood group system are closely related in biochemical terms to the antigenic determinants of the Hh, P, Lewis and Ii blood group systems. The blood group antigens of each of these systems are formed by the addition of specific sugars to an oligosaccharide precursor chain which may be bound through sphingosine to fatty acids (glycolipid) or through serine or threonine to a peptide chain (glycoproteins). The direct gene products of each of these blood group systems are the glycosyltransferase enzymes which catalyse the addition of the specific sugar thus conferring the specified blood group activity to the glycolipid or glycoprotein molecule. The antigenic determinants of the ABO and Lewis systems in addition to red cells also exist in the body secretions in soluble form when the relevant genes are expressed in the phenotype. The antigens expressed on both the red cells and in the secretions are determined by the interaction of Hh, Sese, ABO and Lele genes.     

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.     

Role of carbohydrate in determining the immunochemical properties of the major glycoprotein (gp71) of Friend murine leukemia virus.

Treatment of Friend leukemia virus gp71 with protease-free glycosidase enzymes results in removal of the major portion of the carbohydrate without affecting the amount of protein present. The digested material migrates as protein of about 60,000 to 65,000 molecular weight on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Analyses of the serological properties of gp71 after enzyme treatment indicated that the type, group, and interspecies determinants were not destroyed. In contrast, treatment with proteolytic enzymes led to the complete destruction of the gp71 molecule, including the total elimination of its serological reactivity as measured by direct and competition radioimmunoassay and by a serum cytotoxicity assay. We conclude that the carbohydrate portion of gp71 is not of major significance in defining the antigenic determinants of this viral glycoprotein.     

Epitope mapping of the outer surface protein A (OspA) of the spirochete Borrelia burgdorferi using a panel of monoclonal antibodies and lanthanide competition fluoroimmunoassay

A panel of fourteen different monoclonal antibodies was used for detection and analysis of antigenic determinants located on the outer surface protein A (OspA) of the spirochete Borrelia burgdorferi, which is a causative agent of tick-borne borreliosis (Lyme disease). Two main and several minor partially overlapping antigenic determinants have been found on the surface of the OspA protein of Borrelia burgdorferi sensu stricto (strain 297) by lanthanide competition fluoroimmunoassay. One of the main antigenic determinants is located in the N- and the other in the C-half of the OspA molecule. The involvement of the OspA protein in intact Borrelia burgdorferi sensu stricto (four bacterial strains have been analyzed: 297, B31, FR90-594, and CA90-742) is associated with retention of the above-mentioned two major antigenic determinants, but unlike the case of the isolated OspA they are partially overlapping with each other and with other antigenic determinants. The protein of the spirochete Borrelia afzelii (two bacterial strains have been analyzed: Ip-21 and Pko) contains only one antigenic determinant, which is the same as the main determinant of the OspA protein of Borrelia burgdorferi sensu stricto located in the N-half of the OspA molecule.     

TSG101/mammalian VPS23 and mammalian VPS28 interact directly and are recruited to VPS4-induced endosomes

Class E vacuolar protein sorting (vps) proteins are required for appropriate sorting of receptors within the yeast endocytic pathway, and most probably function in the biogenesis of multivesicular bodies. We have identified the mammalian orthologue of Vps28p as a 221- amino acid cytosolic protein that interacts with TSG101/mammalian VPS23 to form part of a multiprotein complex. Co-immunoprecipitation and cross-linking experiments demonstrated that hVPS28 and TSG101 interact directly and that binding requires structural information within the conserved C-terminal portion of TSG101. TSG101 and hVPS28 are predominantly cytosolic. However, when endosomal vacuolization was induced by the expression of a dominant-negative mutant of another class E vps protein, human VPS4, a portion of both TSG101 and hVPS28 translocated to the surface of these vacuoles. We conclude that TSG101 and its interacting components are directly involved in endosomal sorting.     

Study of the structural characteristics of the regulatory subunit of cAMP-dependent protein kinase type II using monoclonal antibodies

Monoclonal antibodies to the regulatory subunit of cAMP-dependent protein kinase type II from porcine brain were used to study the antigenic properties of the enzyme regulatory subunit (RII). The monoclonal antibodies were bound to linear antigenic determinants on the protein molecule surface. The cAMP binding to RII interfered with the interaction between monoclonal antibodies and the protein. The use of different proteolytic fragments of RII allowed for the localization of antigenic determinants in the N-terminal moiety of RII.     

ABH-blood-group antigens and glycolipids of human saliva

The nature of ABH-blood-group antigens in saliva was investigated. Human saliva was examined serologically for ABH-blood-group activity in its native form and after various treatments. The activity of the native form persisted in the delipidated samples, but was entirely lost after alkaline degradation. The lipid portion of saliva was completely inactive in the ABH hemagglutination inhibition system. The same results were obtained when purified glycolipid fraction of saliva was used instead of whole lipid extract. Neither alkaline treatment nor excessive amounts of salivary lipids effected antigenic activity of A-active glycosphingolipids of hog gastric mucosa admixed to saliva samples before alkaline degradation and/or in presence of large amounts of salivary lipids. The isolated glycolipid fractions contained at least eight glycolipids, each of which was composed of glucose, glyceryl ethers and fatty acids and differed from others with respect to number of glucose residues. Sphingosine and sugar residues involved in formation of ABH antigenic determinants were not detected. These findings together with data on stomach secretion [1,2] led us to the conclusion that ABH-blood-group antigens of saliva are exclusively of glycoprotein nature.     

Nature of the antigenic determinants of T locus antigens

The nature of the antigenic specificities of several antigens associated with the T/t complex in the mouse were analyzed by means of glycosidase and haptene inhibition studies. Results indicate that on testicular cells sugar residues are involved in at least six different T/t antigenic determinants. The immunodominant sugar appears to be different for each of the specificities. The specificity for the following T/t antigens resides predominantly in the sugars indicated: T:sialic acid; t12:beta-D-galactose; tw32:beta-D-galactose; t0:L-fucose; tw1:N-acetyl-D-galactosamine; tw18:L-fucose. It seems probable that these sugars are found at the terminal reducing ends of the carbohydrate portion of T/t-bearing moleculse. These studies imply that at least some of the genes in the T locus code for glycosyltransferases or regulators of glycosyltransferases which modigy oligosaccharide structures and impart specificity to the T/t antigens by alteration of their terminal sugar residues.     

The 120 kilodalton outer membrane protein (rOmp B) of Rickettsia rickettsii is encoded by an unusually long open reading frame: evidence for protein processing from a large precursor

A Rickettsia rickettsii outer surface membrane protein (rOmp B), of an apparent molecular mass of 120 kilodaltons, is a major surface antigen of the Rickettsiae that displays genus, species, and sub-species specific antigenic determinants. The 5' portion of this gene was found to be unstable in plasmids, but was stably cloned in a lambda vector. The nucleotide sequence of the 5' terminus has been determined, thus completing the DNA sequence of the entire gene. Genetic analysis revealed an unusually large open reading frame with the capacity to encode a product much larger than the mature protein. A 32 kilodalton peptide from purified rickettsiae was isolated and the amino terminus was sequenced, which revealed that the peptide is encoded by the 3' portion of this large open reading frame. This suggests a role for post-translational processing of rOmp B from a large precursor molecule.     

Immunochemical characterization of human plasma fibronectin.

Human plasma fibronectin has been purified by a non-denaturing affinity chromatography procedure [Vuento & Vaheri, (1979) Biochem.J. 183, 331--337], and antisera have been raised by immunizing rabbits with the native protein. The antisera reacted strongly with native fibronectin, but only weakly with reduced and alkylated fibronectin or with heat-denaturated fibronectin. Denaturation also affected the haemagglutinating and gelatin-binding activities of fibronectin and increased its susceptibility to proteolytic degradation. The antisera reacted with fragments of fibronectin obtained by proteolysis with plasmin. Large fragments (mol.wt. 180000--200000), lacking the region harbouring the interchain disulphide bridges but containing the sites responsible for gelatin-binding and haemagglutinating activity, showed as intense a reaction with the antisera as intact fibronectin. Smaller peptides showed a weaker reaction. All fragments tested showed sensitivity to denaturation in their reaction with the antisera. The results were interpreted as showing that: (1) native fibronectin has an ordered conformation that is easily perturbed by denaturation; (2) most of the antigenic determinants of the protein are dependent on conformation; (3) the region of the fibronectin molecule containing the interchain disulphide bridges has only few antigenic determinants; and (4) covalent interaction of the two subunits does not contribute to the antigenic structure recognized by rabbit antisera. The observed correlation between the antigenic activity and a structural and functional intactness of fibronectin suggests that the antibodies to native fibronectin could be used as a conformational probe in studies on this protein.     

Cleavage of bacterial flagellin with cyanogen bromide. Antigenic properties of the protein fragments

1. Four polypeptide fragments, obtained by cyanogen bromide treatment of the protein flagellin from Salmonella adelaide, were tested for their antigenic activity by using them as inhibitors in three different assays: bacterial immobilization, haemagglutination of sensitized erythrocytes and quantitative micro precipitation. Immunodiffusion studies were also performed on the protein fragments. 2. Cleavage of the flagellin molecule in this way gave no detectable loss of antigenic determinants. Fragment A (mol.wt. 18000), the largest of the polypeptides, contained all the antigenic specificities present on flagellin that were recognized by the antisera used. In one test, fragment B (mol.wt. 12000) also contained antigenic activity to an extent not easily explainable by contamination with fragment A. Fragments C (mol.wt. 5500) and D (mol.wt. 4500) appeared to be antigenically inactive.     

Genetic mapping of antigenic determinants on a membrane protein

The antigenic determinants recognized by two monoclonal antibodies were mapped on LamB, an outer membrane protein of Escherichia coli. The procedure consisted of performing immunoprecipitation experiments with extracts of strains which produced truncated fragments of LamB, either in a free form (deletion and nonsense mutants) or fused to another polypeptide (malK-lamB and lamB-lacZ fusion strains). The conclusion is that the two antigenic determinants are located within 70 residues from the COOH-terminal end of LamB, which contains a total of 421 amino acids. Since these two antigenic sites were previously demonstrated to be exposed at the cell surface, it follows that a COOH-terminal portion of LamB must be located on the outer surface of the outer membrane.     

Studies on Tryptophane Residues in CF1-ATPase and the Enzymatic Property of CF1-ATPase Modified by NBS and Photooxidation

The numbers of tryptophane residues in spinach CF1-ATPase were measured by means of chemical modification with N-bromosuccinimide (NBS) and photooxidation. There are 3.5 tryptophane residues in CF1-ATPase, among which two are essential for the enzyme activity. Photooxidation of CF1-ATPase led to increased O2 uptake of the reaction system and loss in activity of CF1-ATPase . Immunological property of CF1-ATPase has been altered by chemical modification with NBS and photooxidation. The resuits show that tryptophane residues seen to be essential for activity and antigenic properties of CF1-ATPase.     

Conformational integrity of myoglobin after immunization with Freund's adjuvant.

The use of Freund's complete adjuvant for immunizing goats with myoglobin produces mainly antibodies directed against antigenic determinants present in the native protein. Only about 9% of the total antibodies produced are directed toward determinants not expressed in tha native molecule. This shows that neither emulsification nor the subsequent in vivo events leading up to the immune response appreciably perturb the conformation of the protein surface.