Monocyte chemotactic protein-1 receptor CCR2B is a glycoprotein that has tyrosine sulfation in a conserved extracellular N-terminal region.

Monocyte chemotactic protein-1 (MCP-1) binding to its receptor, CCR2B, plays an important role in a variety of diseases involving infection, inflammation, and/or injury. In our effort to understand the molecular basis of this interaction and its biological consequences, we recognized a conserved hexad of amino acids at the N-terminal extracellular domain of several chemokine receptors, including CCR2B. Human embryonic kidney 293 cells expressing Flag-tagged CCR2B containing site-directed mutations in this region, 21-26, including a consensus tyrosine sulfation site were used to determine MCP-1 binding and its biological consequences. The results showed that several of these amino acids are important for MCP-1 binding and consequent lamellipodium formation, chemotaxis, and signal transduction involving adenylate cyclase inhibition and Ca(2+) influx into cytoplasm. Mutations that prevented adenylate cyclase inhibition and Ca(2+) influx did not significantly inhibit lamellipodium formation and chemotaxis, suggesting that these signaling events are not involved in chemotaxis. CCR2B was found to be sulfated at Tyr(26); this sulfation was abolished by the substitution of Tyr with Ala and severely reduced by substitution of Asp(25), a part of the consensus sulfation site. The expressed CCR2B was found to be N:-glycosylated, as N:-glycosidase F treatment of the receptor or growth of the cells in tunicamycin reduced the receptor size to the same level, from 50 to 45 kDa. Thus, CCR2B is the first member of the CC chemokine receptor family shown to be a glycoprotein that is sulfated at the N-terminal Tyr. These post-translational modifications probably have significant biological functions.

Identification of monoclonal antibody At5 as a new member of HNK-1 antibody family: the reactivity with myelin-associated glycoprotein and with two brain-specific proteoglycans, phosphacan and neurocan.

Monoclonal antibody At5 was primarily developed against chordin, a notochord-specific antigen of Acipenseridae (sturgeon fishes). In higher vertebrates the antibody reacted mainly with neural tissue antigens. In this study we have shown that the specificity of monoclonal antibody At5 is similar to that of antibodies of HNK-1 family which react with two glycolipids and with several high molecular weight glycoconjugates of neural tissue. We have demonstrated by protein sequencing and immunoblotting that one of At5 target antigens of human brain is dMAG, a derivative of myelin-associated glycoprotein. In the preparations of At5 antigens proteoglycans phosphacan and neurocan were identified by immunoblotting with specific monoclonal antibodies 6B4 and 1G2, respectively. The distribution of At5 and 6B4 immunoreactivity was studied on sections of mixed oligoastrocytoma. Oligodendroglioma area of this tumor was intensely stained with both antibodies, whereas astrocytoma area did not exhibit any At5 or 6B4 immunoreactivity.

Developmental expression of glial fibrillary acidic protein gene in human embryos.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein of astrocytes. We have shown by Northern blot analysis that GFAP mRNA first appears in the spinal cord at the age of 8 weeks and in the brain at the age of 9 weeks of embryogenesis, and its relative contents increases at later stages. A plasmid selected from a cDNA expression library with an insert encoding for almost full length of human GFAP was used in this study.

Phosphorylation of two human neurochordins by mammalian casein kinase 1.

Neurochordins are a family of immunologically interrelated high-M(r) neural tissue glycoproteins which includes four major molecular species and several minor ones. Incubation of the total preparation of immunoaffinity-isolated neurochordins with ATP and rabbit casein kinase 1 resulted in phosphorylation of two neurochordin polypeptides, A and B3. The maximum levels of modification were 7-8 and 2 mol of 32P incorporated per 1 mol of polypeptide, respectively. Phosphoamino acid analysis of the phosphorylated neurochordins showed that casein kinase 1 modified exclusively serine residues in both polypeptides. Casein kinase 2 was not effective in phosphorylating neurochordins in vitro.

Electrophoresis of neurochordins, a family of high molecular weight neural tissue glycoproteins, on horizontal submerged agarose gels in the presence of dodecyl sulfate.

We have developed a procedure for the use of agarose gels to separate electrophoretically neurochordins, a family of high molecular weight neural tissue glycoproteins. This procedure is a modification of the method of horizontal submerged electrophoresis on agarose gels routinely applied for the separation of DNA restrictions. For protein analysis we prepared gels of higher agarose concentration (3%), and 0.1% of sodium dodecyl sulfate was present in the buffer used both for gel formation and as an electrode buffer. After electrophoresis agarose gels can be directly stained with Coomassie blue, or proteins can be transferred from the gel to nitrocellulose or nylon filters for immunostaining. The main advantage of the proposed method compared to the method of electrophoresis on large-pore, composite agarose/polyacrylamide gels is its simplicity. Other proteins of very high molecular weight can also be successfully separated by this technique.

Neurochordins of higher vertebrates: similarities in P-epitope-bearing polypeptide composition and a study of P-epitope expression in cultured neural tissue cells.

Neurochordins are a family of high Mr P-epitope-bearing neural tissue glycoproteins. Comparison of SDS-agarose electrophoretic patterns of extracts from human, rat, mouse and chicken brain after immunostaining of blots with an anti-P-epitope MAb At5 demonstrated that in each case neurochordins from the A, B and C group were present. A similar result was obtained when polyclonal serum against total human neurochordin was used. The data favours the idea of high evolutional conservatism of neurochordins of higher vertebrate species. Expression of the P-epitope on glial cells in culture was studied. Strong immunostaining of oligodendrocytes with MAb At5 and the absence of this staining in astrocytes and Schwann cells make it possible to use At5 for identification of cell types in tissue culture experiments as well as for differential diagnostics of brain tumours.

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.

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.

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.