The Journal of Neuroscience, June 15, 2001, 21(12):4281–4289 Diversity and Specificity of Actions of Slit2 Proteolytic Fragments in Axon Guidance Kim T. Nguyen Ba-Charvet,1 Katja Brose,2 Le Ma,2 Kuan H. Wang,2 Vale´ rie Marillat,1 Constantino Sotelo,1 Marc Tessier-Lavigne,2 and Alain Che´ dotal1 1Institut National de la Sante´ et de la Recherche Me´ dicale U106, Baˆ timent de Pe´ diatrie, Hoˆ pital de la Salpeˆ trie` re, 75013 Paris, France, and 2Howard Hughes Medical Institute, Department of Anatomy and Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0452 The Slits are secreted proteins that bind to Robo receptors and olfactory bulb (OB) but not dorsal root ganglia (DRG) axons are play a role in axon guidance and neuronal migration. In verte- repelled by Slit2-N and Slit2-U. Moreover, only Slit2-N mem- brates, Slit2 is a major chemorepellent for developing axons branes or purified protein-induced OB growth cones collapse. and is involved in the control of midline crossing. In vivo, Slit2 Finally, we found that only recombinant Slit2-N could induce is cleaved into 140 kDa N-terminal (Slit2-N) and 55–60 kDa branching of DRG axons and that this effect was antagonized C-terminal (Slit2-C) fragments, although the uncleaved/full- by Slit2-U. Therefore, different axons have distinct responses to length form can also be isolated from brain extract. We ex- Slit2 fragments, and these proteins have different growth- plored the functional activities of Slit2 fragments by engineering promoting capacities. mutant and truncated versions of Slit2 representing the N-, C-, and full/uncleavable (Slit2-U) fragments. Only Slit2-N and Key words: repulsion; guidance; collapse; Robo; Slit2; Slit2-U bind the Robo proteins. We found that in collagen gel, branching Developing axons are guided by cues that can be either diffusible stretch of four leucine reach repeats, seven to nine EGF repeats, or bound to the extracellular matrix (ECM) or the cell mem- and a domain, named ALPS [for “agrin, laminin, perlecan, slit” brane. The Slits have been characterized recently as extracellular (Rothberg and Artavanis-Tsakonas, 1992)], LNS [for “laminin, ligands for the roundabout transmembrane receptors (Kidd et al., neurexin, slit” (Rudenko et al., 1999)], or laminin G-like (LG) 1999). Three distinct slit genes, named slit1, slit2, and slit3, have module (Hohenester et al., 1999). Full-length hSlit2 is proteolyti- been cloned in mammals (Holmes et al., 1998; Itoh et al., 1998; cally processed into 140 kDa N-terminal and 55–60 kDa Brose et al., 1999). In tissue culture, Slit1 and Slit2 proteins have C-terminal fragments in cell culture and in vivo. Drosophila Slit been shown to function as chemorepellents and collapsing factors appears to be similarly processed in vitro and in vivo, suggesting for olfactory, motor, hippocampal, and retinal axons (Li et al., conserved mechanisms for Slit proteolytic processing across spe- 1999; Nguyen Ba-Charvet et al., 1999; Erskine et al., 2000; Niclou cies (Brose et al., 1999; Wang et al., 1999). There is some et al., 2000; Ringstedt et al., 2000). Slit1 and Slit2 can also repel evidence that the different Slit2 fragments may have different tangentially migrating interneurons in the mouse telencephalon functional activities in vivo. The purification of a DRG axon (Hu, 1999; Zhu et al., 1999), and Drosophila Slit (dSlit) repels elongation- and branch-promoting activity suggested that the muscle precursors in the fly embryo (Kidd et al., 1999). However, N-terminal fragment of Slit2, but not the full-length protein, is there is also some positive effect associated with Slits, because capable of stimulating elongation and branching (Wang et al., Slit2 stimulates the formation of axon collateral branches by 1999). However, the purification of a repellent activity for mi- NGF-responsive neurons of the dorsal root ganglia (DRG) grating subventricular zone olfactory precursor interneurons and (Wang et al., 1999). retinal axons revealed that these activities also correspond to the ϳ Slits are large ECM glycoproteins of 200 kDa (Fig. 1A), full-length Slit2 protein (Hu, 1999; Niclou et al., 2000). It is not comprising, from their N terminus to their C terminus, a long clear which fragment or fragments are responsible for the other Slit2-associated activities, including the repellent effects of Slit2 Received Jan. 12, 2001; revised March 9, 2001; accepted March 21, 2001. on axons and cells in vertebrates and Drosophila (Brose et al., This work was supported by Institut National de la Sante´ et de la Recherche 1999; Kidd et al., 1999; Li et al., 1999; Nguyen Ba-Charvet et al., Me´dicale and Grant BIO4-CT960-774 to A.C., C.S., K.N.B.-C., and V.M., and by Action Concerte´e Incitative and Association pour la Recherche sur le Cancer 5249 1999; Wu et al., 1999; Zhu et al., 1999). It is also not known to A.C. K.N.B.-C. is supported by the Fondation de France and a grant from the whether proteolytic processing is required for the repulsive ac- Institut International de Recherche en Paraple´gie, Geneva, on behalf of an anony- mous donation. V.M. is a recipient of a Ministe`re de l’Education Nationale de la tivity of Slit2. Recherche et de la Technologie fellowship. K.B. was supported by a National In this paper, we have begun to explore these questions by Institutes of Health (NIH) fellowship, K.H.W. was supported by a fellowship from engineering mutant versions of human Slit2 (hSlit2) representing the Howard Hughes Medical Institute (HHMI), and L.M. was supported by an NIH postdoctoral training grant. M.T.L. is an investigator of the HHMI. the two cleavage fragments, N- and C-, and the full/uncleavable Correspondence should be addressed to Alain Che´dotal, Institut National de la molecule and examining the activities of these mutants in binding Sante´ et de la Recherche Me´dicale U106, Baˆtiment de Pe´diatrie, Hoˆpital de la and functional assays. We show that different fragments have Salpeˆtrie`re, 47 Boulevard de l’Hoˆpital, 75013 Paris, France. E-mail: [email protected]. different activities but also that the response of an axon to Slit2 is Copyright © 2001 Society for Neuroscience 0270-6474/01/214281-09$15.00/0 not absolute but rather depends on the type of axon. 4282 J. Neurosci., June 15, 2001, 21(12):4281–4289 Nguyen Ba-Charvet et al. • Role of Slit2 Fragments in Axon Guidance Figure 1. A, Schematic representation of the various Slit2 constructs. Slit2-full represents the native full-length Slit2. The nine EGF repeats and the ALPS domain are represented, and the nine amino acids sequence deleted to generate Slit2-U is indicated (arrowhead). All proteins except Slit2-N are also myc tagged at their C termini, and Slit2-N and Slit2-C are tagged by the alkaline phosphatase at their N termini. B–D, Western blots using anti-alkaline phosphatase (B), 9E10 anti-myc (C), and anti-hSlit2 (D) antibodies. All Slit2 fragments can be detected in conditioned medium (sup) and after extraction in 1 M NaCl (salt). Slit2-NAP is recognized by anti-hSlit2 and anti-alkaline phosphatase (B, D), whereas Slit2-U is detected by anti-myc and anti-hSlit2 (C, D). Slit2-C can be detected by the anti-alkaline phosphatase and the anti-myc antibodies (B, C). All protein fragments migrate at the expected molecular weight, and Slit2-U migrates as a single band and therefore is not cleaved. MATERIALS AND METHODS cloned into the pSectagB. This mutation abolishes the cleavage of Slit2. Generation of Slit2 fragment expression constructs. The N-terminal cleav- The resulting protein was called Slit2-U (for uncleavable). age construct (Slit2-N) was made by cloning the N terminus of human Antibodies and Western analyses. To examine processing of hSlit2, Slit2, consisting of sequences encoding amino acids QACPAQ to FSP- conditioned media and high salt (1 M NaCl) extracts from cells trans- PMY, upstream of the putative cleavage site into the BamHI–XbaI sites fected with truncation constructs consisting of the N-terminal (tagged of pSectagB vector (Invitrogen, San Diego, CA) and thus contains both with AP, and detectable with the anti-hSlit2 and the anti-alkaline phos- myc and His epitope tags at its C terminus. A version of this construct phatase antibodies), the C-terminal (tagged with AP and myc) or the bearing an N-terminal alkaline phosphatase tag (Slit2-NAP) was con- uncleavable (myc tagged, and detectable with the anti-Slit2 antibody) structed by inserting coding sequences for human alkaline phosphatase fragments were collected. These extracts were TCA precipitated, solubi- into the HindIII site of Slit2-N. lized in SDS-PAGE sample buffer, run out on an SDS-PAGE (7.5%) gel, The C-terminal cleavage construct (Slit2-C) was made by cloning the and Western blotted by standard methods with a monoclonal 9E10 C terminus of human Slit2, consisting of sequences encoding amino acids anti-myc antibody (gift of J. M. Bishop, Stanford University), an anti- immediately downstream of the putative cleavage site beginning at alkaline phosphatase antibody (Dako, Glostrup, Denmark), or an anti- TSPCD to the stop into the XhoI–XbaI sites of pSectagB. A version of Slit2 antibody (see below). this construct bearing an N-terminal alkaline phosphatase tag (Slit2-N- Purification of Slit2-U, Slit2-N, and Slit2-C fragments. Slit2-N and AP) was constructed by inserting coding sequences for human alkaline Slit2-U proteins were purified on the basis of the association of the N phosphatase into the HindIII site of Slit2-C. terminus with wheat germ agglutinin (WGA). Salt extracts of cells Sequences encoding the amino acids SPPMVLPRT were deleted from expressing either Slit2-U or Slit2-N were prepared by incubating cells human Slit2 using PCR mutagenesis.