Human neurochordins: identification of several P-epitope-bearing polypeptides and a study of their ontogenetic expression.

Neurochordins are a family of immunologically related high-Mr neural tissue glycoproteins. After SDS-agarose electrophoresis of human neural tissue extracts, two main neurochordins (A1 and B2) as well as several minor ones (O, A2, A3, B1, B3, C1, C2, D) were visualized on immunoblots stained with monoclonal antibody At5. Neurochordin expression starts in human embryos before 6 weeks of gestation. General antigenic activity of neurochordins increases between 6 and 24 weeks of gestation while its level does not alter from the second half of gestation up to the age of 11-13 years. Neurochordins extracted from large hemispheres of brain, from cerebellum and spinal cord of a 24-week embryo display a similar pattern after electrophoresis. Partially different pattern of neurochordins was observed with a brain tumor.

[The structure of chordin: characteristics of protein and carbohydrate components and their role in antigenicity]. / Struktura khordina: kharakteristika belkovogo i uglevodnogo komponentov i ikh rol' v antigennosti.

Using immobilized monoclonal antibodies, a tissue-specific antigen, chordin, was isolated from cell extracts of giant sturgeon (beluga) notochord. The antigen was further purified by gel filtration through SP-Sephadex (pH 2.1) and gel chromatography on TSK Toyopearl HW-60. Purified chordin preparations contained 40% of protein and 60% of carbohydrates. The predominant polar amino acids were threonine, serine, glycine, asparagine and glutamine (or aspartic and glutamic amino acids). The carbohydrate moiety comprised mannose, fucose, galactose, galactosamine and glucosamine. Treatment of chordin with three enroglycosidases specifically hydrolyzing the carbohydrate chains of proteoglycans did not affect the antigenic properties of chordin or its behaviour on gel filtration. These findings and the fact that 75% of galactosamine was converted to galactosaminite after treatment with alkaline NaBH4 permitted to relate chordin to glycoproteins carrying O-glycosidic carbohydrate-peptide bonds between the N-acetyl-galactosamine and beta-hydroxyamino acid residues. Besides, chordin seems to contain a N-glycosylamide carbohydrate-peptide bond as can be judged from glucosaminite formation after treatment of the antigen with alkaline LiHB4. The changes in the antigenic properties of chordin after its treatment with neuraminidase, pronase, sodium periodate, alkali, alkaline NaBH4 or LiBH4 suggest that the polypeptide moiety of the chordin molecule and, perhaps, the N-acetylgalactosamine within the composition of the carbohydrate-peptide bond are involved in the construction of its most immunogenic determinants (P-determinants).

P-epitope is characteristic for the neural tissue of vertebrates.

In a search for antigens immunologically related to chordin, a notochord-specific glycoprotein of sturgeneous fishes, extracts from 55 samples of human and rabbit tissues were tested for inhibition of [125I]chordin binding to rabbit polyclonal antibodies. The strongest inhibition was observed with brain extracts of both species. Human, chicken, rabbit, and newt brain extracts also inhibited chordin binding in liquid phase to monoclonal antibodies (MAbs) against the P-epitope, the most immunogenic epitope of this glycoprotein. Immunohistochemical studies done on human and chicken embryos, newt, sterlet, and sturgeon embryos, larvae, and juveniles revealed a strong immunoreactivity of the brain, spinal cord, and tissue of the peripheral nervous system with an anti-P MAb. Other tissues, with several exceptions, showed a negative reaction in immunohistochemical experiments. The authors found that the P-epitope is ontogenetically expressed in the neural tissue of chicken, newt, and sterlet at the period of cytodifferentiation. Gel chromatography of human, chicken, and newt brain extracts showed that in each case the P-epitope was associated with a polydisperse macromolecular material of similar size. These antigens were designated as neurochordins. Prolonged pronase digestion of human and chicken brain extracts resulted in fragments with M about 3 kDa (presumably glycopeptides), which reacted with anti-P MAbs. These fragments were of the same size as corresponding glycopeptides of the pronase digest of chordin. Thus, in the present study, the P-epitope has been shown to be characteristic for the neural tissue of several vertebrate species; in the brain, it has been found in association with neurochordins, macromolecular antigens that are presumably protein conjugates with carbohydrates.

[Tissue specificity of chordin]. / Izuchenie tkanevoi spetsifichnosti khordina.

Chordin is a tissue-specific protein antigen of notochord. Earlier this protein was discovered in the notochords of sturgeon (Acipenseridae) species; the notochord-specific antigenic determinants were detected in the notochord residues of teleost fish species and in notochord derivatives (nuclei pulposi) of mammals. Using the RIA technique, extracts from 35 samples of normal, fetal and tumour tissues of man were screened for chordin. Among other tissue samples tested, extracts from fetal brain and rectal adenocarcinoma exhibited marked cross-reactivity towards antibodies against chordin. Cross-reactivity towards chordin was observed in rabbit brain extract. This extract contained an antigen which was immunologically related (but not fully identical) to chordin. In total, in this and previous studies, 58 samples of fish and mammalian tissues were analyzed for chordin. However, antigenic determinants of chordin were identified only in extracts prepared from the notochords and nuclei pulposi as well as from brain and rectal adenocarcinoma. These findings suggest that chordin is an antigen with a restricted tissue specificity.

Monoclonal antibodies against chordin. Use in structural and immunohistochemical studies.

Eight MAbs have been developed against chordin and designated as At2-At9. It is shown that all antibodies are directed against identical, spatially overlapping or closely positioned epitopes of chordin. The chordin molecule has repetitive sites wherein epitopes for the eight MAbs are located. This site lies within a proteinase-resistant fragment of chordin, presumably a glycopeptide, of molecular mass between 2 and 10 kDa. Fluorescence staining of cryostat sections from stellate sturgeon with the use of At5 (indirect Coons' method) has revealed a positive reaction with notochord cells and sheath and with the spinal cord. No significant reaction with cartilage, muscle and kidney was detected.

[Glutaraldehyde and glyoxal fixation of ribonucleoproteins for their analysis by cesium chloride equilibrium gradient centrifugation]. / Fiksatsiia ribonukleoproteidov glutarovym al'degidom i glioksalem dlia ikh analiza ravnovesnym tsentriougirovaniem v gradiente plotnosti CsC1.

Conditions for fixation of different RNP (ribosomes, poliribosomes, informosomes) by glutaraldehyde and glyoxal for their subsequent analysis in CsCl density-gradient has been developed. Higher dialdehyde concentration and longer incubation time should be used for fixation of ribosomes and polyribosomes than for that of informosomes. For the fixation of all RNP studied their incubation with 0.01 M (0.1%) glutaraldehyde for several minutes is sufficient. Much higher concentration of the fixating agent (about 0.2-0.5 M i. e. 1-3%) and more prolonged time of incubation (in order of several 10 hours) are needed for the fixation of the RNP in the case of glyoxal. Conditions for selective aldehyde fixation of informosomes in the presence of ribosomes and polyribosomes has been developed.