Heinz Ulrich Barnikol

The immunoglobulin-like genetic predetermination of the brain: the protocadherins, blueprint of the neuronal network.

Abstract. The morphogenesis of the brain is governed by synaptogenesis. Synaptogenesis in turn is determined by cell adhesion molecules, which bridge the synaptic cleft and, by homophilic contact, decide which neurons are connected and which are not. Because of their enormous diversification in specificities, protocadherins (pcdhα, pcdhβ, pcdhγ), a new class of cadherins, play a decisive role. Surprisingly, the genetic control of the protocadherins is very similar to that of the immunoglobulins. There are three sets of variable (V) genes followed by a corresponding constant (C) gene. Applying the rules of the immunoglobulin genes to the protocadherin genes leads, despite of this similarity, to quite different results in the central nervous system. The lymphocyte expresses one single receptor molecule specifically directed against an outside stimulus. In contrast, there are three specific recognition sites in each neuron, each expressing a different protocadherin. In this way, 4,950 different neurons arising from one stem cell form a neuronal network, in which homophilic contacts can be formed in 52 layers, permitting an enormous number of different connections and restraints between neurons. This network is one module of the central computer of the brain. Since the V-genes are generated during evolution and V-gene translocation during embryogenesis, outside stimuli have no influence on this network. The network is an inborn property of the protocadherin genes. Every circuit produced, as well as learning and memory, has to be based on this genetically predetermined network. This network is so universal that it can cope with everything, even the unexpected. In this respect the neuronal network resembles the recognition sites of the immunoglobulins.

Golgi retention of human protein NEFA is mediated by its N-terminal Leu/Ile-rich region

The subcellular localization of the human Ca2+‐binding EF‐hand/leucine zipper protein NEFA was studied in HeLa cells by immunofluorescence microscopy. Double immunostaining using mouse anti‐NEFA monoclonal antibody 1H8D12 and rabbit anti‐ERD2 polyclonal antibody proved that NEFA is localized in the Golgi apparatus. The result was confirmed by the expression of NEFA–green fluorescent protein (GFP) fusion protein in the Golgi in the same cell line. Cycloheximide treatment proved NEFA to be a Golgi‐resident protein. Seven NEFA deletion mutants were constructed to ascertain the peptide region relevant for Golgi retention. The expression of each NEFA–GFP variant was detected by fluorescence microscopy and immunoblotting. Only the ΔN mutant, lacking the N‐terminal Leu/Ile‐rich region, failed to be retained in the Golgi after cycloheximide treatment. The other six deletion mutants in which either the basic region, the complete EF‐hand pair domain, the two EF‐hand motifs separately, the leucine zipper and the leucine zipper plus the C‐terminal region is deleted, were localized to the Golgi. The peptide sequence within the Leu/Ile‐rich region is discussed as a novel Golgi retention motif.

Genetic determination of antibody specificity

The best system for the study of cell differentiation is a cell which in its differentiated state differs only by one product. This is the case in the immune system. The undifferentiated, but omnipotent stem cell differentiates into a committed B cell which produces only one type of specific antibody out of a million different, genetically fixed possibilities. Gene translocation and fusion is the basis of this differentiation process.

The Role of Gene Translocation in the Determination of Specificity and Class of the Antibody Molecule

Structural studies of monoclonal immunoglobulins demonstrated that immunoglobulin chains are organized in the antigen-binding variable (V) part and the constant (C) part. The variability pattern indicated the separate genetic control of V and C parts. The decisive steps in B cell differentiation are gene translocation and fusion events which assemble V and C genes to the actual one active H chain gene and the one active L chain gene in a B cell or plasma cell. Further gene translocations, now solely on the H chain C genes, effect the H chain class switch. By DNA structure analysis of immunoglobulin genes from stem cells and plasma cells, the details of the internal organization of the genes and of the gene translocations were recognized. RNA processing of H chain precursor mRNA decides whether the immunoglobulin molecule is produced as an antigen receptor or an antibody molecule. It also enables the simultaneous expression of antigen receptors as IgM and IgD molecules on the B cell membrane.

The Genetic Control of Antibody Variability

Publisher Summary This chapter reviews the genetic control of antibody variability. There is a conclusive evidence that variable and constant parts are under different genetic control and that the number of genes involved in the synthesis of the variable, and constant parts must be different. In accordance with the one-gene-one-protein dogma, it has to be assumed that the C-terminal part of one chain type is controlled by one gene. This assumption is confirmed by the localization of genetic factors or allotypes on the C-terminal part of κ type proteins. The factors that are alleles segregate in a simple Mendelian manner. This means that the gene controlling the C-terminal part occurs only once in the germ line. Tandemly duplicated C-genes would imply that evolution, which results in different but for every species constant C-terminal parts in the immunoglobulins of man and mouse, works in a parallel but identical way, which seems to be very unlikely. Most of the exchanges between proteins within one subgroup can be explained by single base mutation in the triplet coding for that particular exchanged amino acid

Immune Receptors and Cell Differentiation

Cell differentiation is a very complex phenomenon, because the products of differentiation are usually manyfold and heterogeneous. The best system for the study of this phenomenon would therefore be a cell which in its differentiated state differs only by one product. This is the case in the immune system. The stem cell, from which all antibody producing cells derive, is an undifferentiated cell. It still contains the potential to produce all antibodies. The differentiated B-cell has lost this omnipotency, it produces only one type of specific antibody which is chemically homogeneous and therefore can be analysed by chemical means. Sequence studies with such monoclonal immunoglobulins have revealed that the stem cell does not only have the potential, but also contains the information for the synthesis of about 1 Million different antibody molecules. This information is laid down in the form of Thousands of different V-(specificity) genes, it is genetically fixed and passed from generation to generation. These V-genes are, however, only partial genes, they are inactive in the stem cell. Activation occurs, when one of these partial genes undergoes translocation and fusion with a second gene, the C-gene, during differentiation. Now this gene is complete and can be transcribed and translated. At the same time this cell, in which the V-gene translocation and fusion with the C-gene occurs, becomes unipotent, it produces only one type of specific antibody. This antibody molecule is incorporated into the cell wall as an receptor molecule where it waits for an appropriate antigen. This differentiation process is irreversible.

Molekulare Grundlagen der Antikörperbildung

Die Frage nach der Herkunft der Antikorperspezifitat ist so alt wie die Immunologie selbst. Zur Debatte stehen im wesentlichen zwei Moglichkeiten: entweder ist die Information fur die verschiedenen moglichen Immunantworten genetisch fixiert, oder aber sie entsteht in jeder Antikorper bildenden Zelle durch somatische Mutation neu. Ein Weg, diese Frage zu klaren, ist die Strukturanalyse monoklonaler Immunglobuline.