Thomas Brümmendorf

Neural cell recognition molecule L1: from cell biology to human hereditary brain malformations

The neural cell recognition molecule L1 is a member of the immunoglobulin superfamily implicated in embryonic brain development. L1 is engaged in complex extracellular interactions, with multiple binding partners on cell surfaces and in the extracellular matrix. It is the founder of a neural group of related cell surface receptors that share with L1 a highly conserved cytoplasmic domain that associates with the cytoskeleton. Phenotypic analyses of human patients with mutations in the L1 gene and characterizations of L1-deficient mice suggest that L1 is important for embryonic brain histogenesis, in particular the development of axon tracts.

Neural cell recognition molecule F11: Homology with fibronectin type III and immunoglobulin type C domains

We report here the complete cDNA sequence of F11 130 kd polypeptide, a chick neural cell surface-associated glycoprotein implicated in neurite fasciculation and elongation. The predicted protein sequence of 1010 amino acids includes an amino-terminal signal peptide and a carboxy-terminal hydrophobic stretch, which is compatible with the consensus motif for covalent attachment of glycosyl-phosphatidylinositol. Accordingly, F11 lacks an intracellular domain, which is consistent with evidence obtained from protease protection experiments on isolated microsomes. In addition, the molecule comprises six domains related to the immunoglobulin domain type C and four resembling fibronectin repeat type III. Both types of repeats resemble those present in neural cell adhesion molecules L1 and N-CAM. The possible identity of F11 with the chick neural glycoprotein contactin is discussed.

Structure/function relationships of axon-associated adhesion receptors of the immunoglobulin superfamily

Evidence is accumulating that axonal members of the Ig superfamily (IgSF) interact in a complex manner with other axonal Ig-like proteins and with proteins of the extracellular matrix. Studies investigating the structure/function relationships of these proteins have highlighted the importance of Ig-like domains near the amino terminus (N-proximal) as both necessary and sufficient for homophilic and heterophilic binding. Although efforts have been made in the past year to correlate the structure and neurite-outgrowth-promoting ability of axonal IgSF members, this work is still at an early stage.

Axonal glycoproteins with immunoglobulin- and fibronectin type III-related domains in vertebrates : structural features, binding activities, and signal transduction

Abstract: The L1‐ and F11‐like axonal glycoproteins, implicated in neurite outgrowth and fasciculation, are members of the Ig superfamily comprising multiple fibronectin type III‐like domains. Their Ig‐like and fibronectin type III‐related domains are likely to be composed of seven β‐strands arranged in two opposing β‐sheets of highly similar topology. Whereas the F11‐like molecules lack a transmembrane sequence and are anchored in the plasma membrane by a glycosylphosphatidylinositol, the L1 ‐like molecules comprise cytoplasmic domains with highly conserved sequence motifs. Most of the latter proteins occur in different isoforms generated by alternative pre‐mRNA splicing, which has not been documented for molecules of the F11 subgroup. L1 ‐like proteins undergo heterophils as well as homophilic interactions, whereas only the former mode of binding was observed for F11 ‐like proteins. Evidence is accumulating that these Ig superfamily molecules with fibronectin type III‐like domains are interacting in a complex manner with each other and molecules of the extracellular matrix. Investigations assigning structure to function reveal that their individual extracellular domains serve distinct binding activities. Recent studies also suggest that L1 and NCAM are implicated in the transduction of transmembrane signals.

Pathological missense mutations of neural cell adhesion molecule L1 affect homophilic and heterophilic binding activities

Mutations in the gene for neural cell adhesion molecule L1 (L1CAM) result in a debilitating X‐linked congenital disorder of brain development. At the neuronal cell surface L1 may interact with a variety of different molecules including itself and two other CAMs of the immunoglobulin superfamily, axonin‐1 and F11. However, whether all of these interactions are relevant to normal or abnormal development has not been determined. Over one‐third of patient mutations are single amino acid changes distributed across 10 extracellular L1 domains. We have studied the effects of 12 missense mutations on binding to L1, axonin‐1 and F11 and shown for the first time that whereas many mutations affect all three interactions, others affect homophilic or heterophilic binding alone. Patient pathology is therefore due to different types of L1 malfunction. The nature and functional consequence of mutation is also reflected in the severity of the resultant phenotype with structural mutations likely to affect more than one binding activity and result in early mortality. Moreover, the data indicate that several extracellular domains of L1 are required for homophilic and heterophilic interactions.

Induction of axonal growth by heterophilic interactions between the cell surface recognition proteins Fll and Nr-CAM/Bravo

F11 and Nr-CAM/Bravo are two axon-associated glycoproteins belonging to different subgroups of the immunoglobulin superfamily. In this report we have investigated the interaction of both proteins using neurite outgrowth and binding assays. Antibody blocking experiments demonstrate that neurite extension of tectal cells on immobilized F11 is mediated by Nr-CAM/Bravo. Binding studies further reveal a direct heterophilic interaction between F11 and Nr-CAM/Bravo. This activity can be mapped to the amino-terminal second or third immunoglobulin-like domain within F11 with domain-specific monoclonal antibodies and deletion mutant proteins expressed on COS cells. Furthermore, perturbation experiments with domain-specific monoclonal antibodies demonstrate that this region is required for adhesion and neurite extension.

Neurotractin/kilon promotes neurite outgrowth and is expressed on reactive astrocytes after entorhinal cortex lesion

The IgLON subgroup of the immunoglobulin superfamily consists of four members that are thought to be important in neural cell-cell recognition. Here, we cloned and characterized the murine IgLON subgroup member neurotractin/kilon, in the context of brain development and axonal regeneration. Neurotractin/kilon was found to be upregulated during brain development and is expressed on neurites of primary hippocampal neurons. To elucidate a potential role for neurotractin/kilon during regeneration in the CNS, we performed lesions in the entorhinal cortex, and showed that the expression of neurotractin/kilon is induced on reactive astrocytes. Notably, the expression on reactive astrocytes appears specifically in the denervated outer molecular layer of the dentate gyrus, where regenerative axon sprouting occurs. In vitro assays demonstrated that neurotractin/kilon attracts hippocampal axons in the stripe assay and that astroglial neurotractin/kilon promotes neurite outgrowth. These results suggest a function for neurotractin/kilon as a trans-neural growth-promoting factor for outgrowing axons following hippocampal denervation.

Neural cell recognition molecule F11: Membrane interaction by covalently attached phosphatidylinositol

The mode of membrane insertion of F11 130 kDa protein, a neural chick cell surface glycoprotein involved in neurite fasciculation, has been investigated. Up to 41% of total F11 130 kDa is released from adult chick brain plasma membranes by phosphatidylinositol specific phospholipase C (PI-PLC), whereas no release is mediated by lecithin/cephalin specific phospholipase C (PLC). PI-PLC dependent release of F11 is also observed from embryonal chick brain plasma membranes and from the surface of intact retinal cells. Biosynthetic labelling experiments demonstrate that F11 contains ethanolamine. Taken together, these results suggest that F11 interacts with the plasma membrane at least partially through covalently linked glycosyl-phosphatidylinositol (GPI) or a structurally similar lipid.

The Contactin-Related Protein FAR-2 Defines Purkinje Cell Clusters and Labels Subpopulations of Climbing Fibers in the Developing Cerebellum

FAR-2 is a novel neural member of the Ig superfamily, which is related to F11/F3/contactin and axonin-1/TAG-1. This protein is expressed by subpopulations of Purkinje cells in the chicken cerebellum and FAR-2-positive clusters of these neurons alternate with FAR-2-negative clusters in both tangential dimensions of the cerebellar cortex. Furthermore, FAR-2 is also expressed by one type of Purkinje cell afferents, namely, the climbing fibers, and different subpopulations of these axons show distinct levels of FAR-2 expression. Homology modeling using axonin-1 as a template reveals that the four aminoterminal Ig domains of FAR-2 form a compact U-shaped structure, which is likely to contain functionally important ligand-binding sites. FAR-2 is binding to the Ig superfamily protein NgCAM/L1, but not to the related receptor NrCAM, and it is also interacting with the modular ECM protein tenascin-R. These results suggest that FAR-2 may contribute to the formation of somatotopic maps of cerebellar afferents during the development of the nervous system.

The gene of the neural cell recognition molecule F11: conserved exon-intron arrangement in genes of neural members of the immunoglobulin superfamily

The chicken neural glycoprotein F11 is a cell recognition molecule implicated in neurohistogenesis, in particular in the context of neurite outgrowth and fasciculation. F11 is a glycosyl-phosphatidylinositol-linked member of the immunoglobulin superfamily that is also termed contactin or F3 in humans and rodents, respectively. In this study, we report the complete structure of the F11 gene. It is composed of 23 exons distributed over more than 100 kb of genomic DNA and each of the ten domains of the F11 protein is encoded by two exons. The sizes of the introns vary by two orders of magnitude ranging from 150 bp to more than 15 kb. All interdomain introns are in phase one, i.e. are inserted after the first nucleotide of a codon, being consistent with assembly of a F11 progenitor gene via exon shuffling. The intradomain introns are localized at variable sites within the domains and have different intron phases. This study reveals a remarkable similarity of the F11 gene with the gene of axonin-1, a related neural immunoglobulin superfamily member which is also implicated in neurite outgrowth and fasciculation. The intron positions with respect to the protein domain organization are found to be identical, strongly suggesting that both genes are derived from a common ancestor that already had this exon-intron structure.