This invention is related to the field of developmental biology. In particular it is related to the area of axon guidance cues.
The complex wiring of the adult nervous system is dependent upon the occurrence during neurodevelopment of an ordered series of axon guidance decisions that ultimately lead to the establishment of precise connections between neurons and their appropriate targets. These guidance events can act over long or short distances, and they can be either attractive or repulsive in nature (Tessier-Lavigne and Goodman, 1996). An important first step in elucidating the mechanisms by which long-distance clemotropic cues mediate axon guidance is identification of the receptors that bind these cues. Identification of two plylogenetically conserved gene families, the semaphorins and the netrins, has advanced our understanding of the cellular and molecular basis of long-range influences on axon guidance. Semaphorins and netrins function as chemotropic cues for specifac populations of neurons during development (Keynes and Cook, 1995). The netrins have been implicated in long-range attractive and repulsive guidance events in Caenorhabditis elegans (UNC-6), vertebrates (netrin-1 and netrin-2), and Drosopila (netrin-A and netrin-B) (Serafini et al., 1994; Varela-Eclavarria et al., 1997). Genetic studies in both invertebrates and vertebrates, and biochemical studies in vertebrates, show that two immunoglobulin (Ig) superfamily subgroups, one including the Deleted in Colorectal Cancer (DCC), UNC-40, and Frazzled proteins, and the other including the UNC-5, UNC5H1, UNC5H2, and RCM proteins, contain netrin receptors involved in mediating attractive and repulsive netrin functions (Tessier-Lavigne and Goodman, 1996; Ackerman et al., 1997; Fazeli et al., 1997; Leonardo et al., 1997). At present, however, semaphorin receptors have not been identified.
The semaphorins comprise a large family of both transmembrane and secreted glycoproteins, suggesting that some semaphorins act at a distance while others act locally (Kolodkin, 1996; Puschel, 1996). Semaphorpins are defined by a well-conserved extracellular semaphorin (sema) domain of approximately 500 amino acids. Secreted semaphorins contain an Ig domain that is C-terminal to the sema domain, while transmembrane semaphorins can have an Ig domain, type 1 thrombospondin repeat, or no obvious domain motif N-terminal to their transmembrane domain. Semaphorins are present in a variety of neuronal and non-neuronal tissues. Their function in neuronal growth cone guidance, however, has been addressed most extensively.
Two secreted semaphorins, vertebrate collapsin-1/Sema III/Sem D (species homologues) and Drosophia semaphorin II (D-sema II) (Matthes et al., 1995), have been shown to function selectively in repulsive growth cone guidance during development. Collapsin-1 (Coll-1) was identified in a search for growth cone collapsing factors from the membranes of adult click brain tissue (Luo et al., 1993). Acute application of recombinant Coll-1 induces the collapse of a subset of dorsal-root-ganglia (DRG) neuron growth cones at sub-nanomolar concentrations, but has no effect on chicken retinal ganglion cell growth cones. Brain-derived membrane extracts enriched for Coll-1 and immobilized to beads, however, provided sensory neurons in culture with a localized repulsive cue capable of steering growth cones away from beads rather than causing complete growth cone collapse (Fan and Raper, 1995).
Genes encoding human, rat and mouse Sema III/Sem D (referred to below as Sema III) were identified based on their similarity to other semaphorins (Giger et al., 1996; Kolodkin et al., 1993; Messersmith et al., 1995; Puschel et al., 1995). Sema III can act as a chemorepellent for NGF-dependent embryonic (E14) DRG sensory neurons. It has little effect, however, on neurotrophin-3 (NT-3)-responsive E14 DRG sensory afferents. The E14 ventral spinal cord secretes a chemorepellent activity selective for NGF-, not NT-3-, dependent E14 DRG sensory afferents (Fitzgerald et al., 1993; Messersmith et al., 1995; Puschel et al., 1996). This correlates well with the expression pattern of sema III in the ventral cord during the time of sensory afferent innervation, and the segregation of NT-3- and NGF-dependent sensory afferents, respectively, into ventral and dorsal targets in the spinal cord (Messersmith et al., 1995).
Indeed, antibody perturbation of Coll-1 at analogous stages in chick neurodevelopment supports the idea that Coll-1 is the ventral cord repellent (Shepherd et al., 1997). This is further supported by the observation that mice with a targeted deletion of the sema III gene exhibit defects in the trajectories of certain NGF-responsive sensory afferents (Behar et al., 1996). In addition, functional studies show that Sema III can act as a chemorepellent for spinal motor neurons and a subset of cranial motor neurons (Varela-Echavarria et al., 1997). Coupled with extensive analysis of sema III and Coll-1 expression (Giger et al., 1996; Shepherd et al., 1996; Wright et al., 1995), all of these data suggest that specific populations of embryonic and adult neurons require Sema III for establishment, and possibly maintenance, of their appropriate patterns of connections. The rapid response of DRG growth cones in culture to Coll-1 and Sema III, and the low concentrations of these factors needed to elicit a response, strongly suggest that a receptor-meldated signal transluction mechanism underlies the action of these proteins on the cytoskeletal reorganization events that ultimately influence growth cone guidance.
It is an object of the present invention to provide a method of monitoring the interaction of a semaphorin and a neuropilin.
It is another object of the present invention to provide an isolated and purified subgenomic nucleic acid molecule which encodes a new mammalian neuropilin.
It is an object of the present invention to provide a method of identifying axon guidance cues.
Another object of the invention is to provide a method for monitoring the interaction between a semaphorin and a neuropilin so that agonists and antagonists can be identified.
Another object of the invention is to provide a polypeptide useful for antagonizing the interaction between a semaphorin and a neuropilin.
Another object of the invention is to provide a polypeptide useful for antagonizing the interaction between a semaphorin and a neuropilin.
It is still another embodiment of the invention to provide an antibody preparation useful for isolating and detecting a neuropilin protein.
It is yet another object of the invention to provide an isolated and purified mammalian neuropilin protein.
These and other objects of the invention are achieved by one or more of the embodiments described below. In one embodiment a method of monitoring the interaction of a semaphorin and a neuropilin is provided. The method comprises the steps of:
contacting a first protein comprising an extracellular domain of a neuropilin with a second protein which comprises an extracellular domain of a semaphorin under conditions where the extracellular domain of the neuropilin binds to the extracellular domain of the semaphorin;
determining the binding of the first protein to the second protein or second protein to the first protein.
According to yet another embodiment an isolated and purified subgenomic nucleic acid molecule is provided. The molecule encodes a mammalian neuropilin-2 and has at least 90% sequence identity to SEQ ID NO: 1.
In yet another embodiment of the invention a method of identifying axon guidance cues is provided. The method comprises the steps of:
contacting a detectably labeled mammalian neuropilin protein or semaphorin-binding portion thereof with a mixture of proteins secreted by neuronal cells;
removing proteins of the mixture which do not bind to the detectably labeled mammalian neuropilin; wherein a protein of the mixture which binds to the detectably labeled mammalian neuropilin or said portion is identified as a candidate axon guidance cue.
According to another aspect of the invention a method is provided for monitoring the interaction between a semaphorin and a neuropilin. The method comprises the steps of:
contacting a fusion protein comprising a semaphorin sema or Ig basic domain with cells which express a neuropilin;
detecting the fusion protein comprising the semaphorin sema or Ig basic domain which binds to the cells.
As another aspect of the invention a method is provided for monitoring the interaction between a semaphorin and a neuropilin. The method comprises the steps of:
contacting a protein comprising a semaphorin sema or Ig basic domain with cells which express a polypeptide comprising an extracellular domain of a neuropilin;
detecting the protein comprising the semaphorin sema or Ig basic domain which binds to the cells.
As still another aspect of the invention a polypeptide portion of neuropilin useful for antagonizing the interaction between a semaphorin and a neuropilin is provided. The polypeptidecomprises the extracellular domain of a neuropilin.
In yet another embodiment of the invention an isolated dand purified protein which is neuropilin-2 is provided. The amino acid sequence of neuropilin-2 is shown in SEQ ID NO: 2. Other neuropilins having at least 90% amino acid identity with SEQ ID NO: 2 are also provided.
According to still another aspect of the invention a method of monitoring the interaction between a semaphorin and a neuropilin is provided. The method comprises the steps of:
coculturing in a matrix (a) embryonic nerve cells with (b) cells which have been transfected with an expression construct encoding a semaphorin and which express the semaphorin;
adding to the cells an inhibitor of binding of the semaphorin and the neuropilin;
determining the inhibition of embryonic nerve cell axon outgrowth adjacent to the cells which express the semaphorin in the presence and absence of inhibitor.
As another aspect of the invention a method is provided for monitoring the interaction between a semaphorin and a neuropilin. The method comprises the steps of:
culturing embryonic nerve cells under conditions in which they display growth cones;
contacting the embryonic nerve cells with a semaphorin and an anti-neuropilin antibody;
observing the effect of the antibody on the collapse of the growth cones.
Yet another aspect of the invention is provided by an antibody preparation which specifically binds to a neuropilin protein. The antibody does not specifically bind to neuropilin-1. Particular antibodies bind exclusively to the extracellular domain.
An additional embodiment of the invention is an isolated and purified mammalian neuropilin protein which comprises a signal sequence, two complement binding domains, two coagulation factor domains, a MAM domain, a transmembrane domain and a cytoplasmic domain, with the proviso that the neuropilin protein is not neuropilin-1.
The invention thus provides the art with potent modulators of nerve cell growth, immune responsiveness, and viral pathogenesis, which can be used in the treatment and diagnosis of neurological disease and neuro-regeneration, immune modulation including hypersensitivity and graft-rejection, and diagnosis and treatment of viral and oncological infection/diseases.
The neuropilins, neuropilin-encoding nucleic acids, and unique portions thereof also are useful in screening chemical libraries for regulators of semaphorin-mediated cell activity, in genetic mapping, as probes for related genes, as diagnostic reagents for genetic, neurological, immunological, and oncological disease and in the production of specific cellular and animal systems for the development of neurological, immunological, oncological and viral disease therapy.
The medical applications of such compounds, their agonists, and their antagonists are enormous and include modulating neuronal growth regenerative capacity, treating neurodegenerative disease, and mapping (e.g., diagnosing) genetic neurological defects.