BIOLOGICAL OVERVIEW

Cadherin-N (CadN and also referred to as N-cadherin) is the fourth cadherin to be discovered in Drosophila. Shotgun, to date the best characterized Drosophila cadherin, is predominantly expressed in epithelial tissues in embryos and is critically required for dynamic epithelial rearrangements during embryogenesis. Two other proteins of the Drosophila cadherin superfamily have been characterized: Fat, a tumor suppressor, and Dachsous. None of these proteins are expressed primarily in the nervous system. While Shotgun is expressed in the embryonic nervous system, its expression is limited to midline glial cells of the central nervous system and to adherens junctions between sensory dendrites and their accessory cells in the periphery (Iwai, 1997).

Cadherin-N was discovered in an intensive search for novel Drosophila cadherin genes. CadN differs from Shotgun and from classic vertebrate cadherins by the presence of 15 cadherin repeats, versus the 6 found in Shotgun and the 4 found in all typical vertebrate cadherins. Drosophila Shotgun and Cadherin-N also share a similar complex region adjacent to the transmembrane domain. This region is not found in vertebrate cadherins. Nevertheless, the intracellular domains of both Cadherin-N and Shotgun bears a strong resemblence to the intracellular domains of vertebrate cadherins. It is apparent that the extracellular domains have evolved at a different (more conservative) rate than the extracellular domains (Iwai, 1997).

Fasciclin II-expressing axonal tracts are deformed in mutant flies. Anti-Fas II antibody labels four neurons that pioneer the first two longitudinal axon pathways: vMP2 and MP1. Loss of Cadherin-N does not appear to perturb the pathfinding of the pioneers at stage 12 and 13, since the pioneer growth cones normally navigate through the vMP2 pathway, and the pathway is generated as in the wild type. At stage 14, when follower neurons begin Fas II expression and join the pioneer tracts, three classes of pattern alteration are recognized. (1) In normal embryos, the vMP2 tract is constricted toward the midline in a segmentally repeated fashion, whereas the mutant route weaves to a lesser extent. Moreover, local associations between the vMP2 and MP1 fascicles are diminished in the mutants. (2) One commissural axon fascicle periodically detected in each segment stains more intensely in mutants than in wild type, implying that more axons join the mutant fascicle. (3) The MP1 pathway sometimes looks discontinuous. Irregular patterns become more conspicous at late stage 16 when Fas II-positive axons are assembling into three longitudinal bundles. Occasionally, the two more laterally located pathways are interupted, and the disconnected terminals are often swollen and/or have turned laterally. The bundles bifurcate locally, which represents either defasciculation or abnormal fusion (Iwai, 1997).

Apterous can be used as a marker for neuron growth directed by CadN. The LIM homeodomain transcription factor Apterous is expressed in only three interneurons per abdominal hemisegment; the fascicles of their ipsilaterally (same side) projecting axons can be monitored using DNA to encode an enzyme marker driven by the apterous promoter. The Apterous axons first extend medially, then their growth cones make right-angled turns and grow in an anterior direction. Subsequently, the transverse portion of each Apterous axon starts a medial shift, and its turning point reaches the most medial surface of the longitudinal axon tracts. In Cadherin-N mutants, the inital outgrowth is normal, however after the turn, mutant fascicles take oblique trajectories in contrast to the parallel pathways in normal embryos. Most of the mutant growth cones of the Apterous neurons reach adjacent anterior segments and navigate along the most medial path. However, they cannot immediately fasciculate with the misrouted axons of counterpart neurons in more anterior segments. Without fascicle formation, mutant growth cones appear to continue navigation, indicating that bundling is not an absolute prerequisite for further axon growth. Because of the broad neuronal distribution of Cadherin-N, it is supposed that abnormal patterning is not restricted to Apterous neurons (Iwai, 1997).

PROTEIN STRUCTURE

Amino Acids - 3097

Structural Domains

The enormous size of Cadherin-N is primarily due to the presence of 15 cadherin repeats in the extracellular region, presenting a contrast to the 4 repeats in all vertebrate classic cadherins and the 6 repeats in Shotgun, the Drosophila E-cadherin. Both Drosophila cadherins have insertions of similar sequences between the last extracellular cadherin repeat and the membrane-spanning segment. The insert contains a series of subdomains: an Fcc box (fly classic cadherin box), a cysteine-rich segment (C-rich 1), a laminin A (See Drosophila Laminin A) globular segment (LmA-G), and another cysteine-rich segment (C-rich 2). The Fcc box, comprising 170 amino acids is defined as such because database searches with its sequences identify only the comparable regions of Shotgun as a relative. Similar sequences are not found in vertebrate cadherins. The whole Cadherin-N LmA-G displays 25% sequence identity to mouse laminin A and to the presynaptic transmembrane protein Neurexin. The Cadherin-N cytoplasmic domain is much more similar to those domains of Shotgun and vertebrate classic cadherins with respect to both size and sequence. The intracellular domains of the two Drosophila cadherins and mouse N-cadherin range between 157 and 160 amino acids in length, and have 37% and 46% sequence identity in any combination among them, with the higher figure representing the Cadherin-N identity to murine N-cadherin, and the lower representing Shotgun identity to murine N-cadherin. The degree of sequence conservation between the two Drosophila cadherins (41% identity) is lower than the 63% identity between N- and E-cadherin in the same vertebrate species, as for example, in mice (Iwai, 1997).

Antibodies were raised to the extracellular domain and to the cytoplasmic region of Cadherin-N and were designated as DN-Ex and DN-In, respectively. Both antisera recognize a pair of bands of roughly 300 kDA, of which the upper band is always faint. When alpha-catenin is immunoprecipitated, the smaller band is easily detected, while the large is not. It is assumed that the larger band is a precursor, and that the smaller represents the mature form generated by proteolytic digestion. In addition to these high molecular weight forms, DN-Ex detects a major 200 kDa molecule, and DN-ln labels an intense 120 kDa band. The production of these two fragments can be explained by postulating that the mature form is cleaved into an N-terminal 200 kDa and a C-terminal 120 kDa fragment. It is likely that the 200 Da form is derived only from the extracellular domain: the 200 kDa band is still present in a Cadherin-N mutant strain at a proximal position in the extracellular domain. Both 120 and 200 kDa forms coprecipitate with alpha-catenin (Iwai, 1997).

date revised: 5 January 98

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