Sulfate Affinity Research Articles

A Conformationally Flexible, Urea-Based Tripodal Anion Receptor: Solid-State, Solution, and Theoretical Studies

Tripodal tris(urea) cationic receptors 1 and 2 containing p-tolyl or octyl substituents, respectively, have been synthesized, and their association behavior with anionic guests has been studied via a variety of methods. The receptors are based around a hexasubstituted aryl core and contain both urea and pyridinium functionalities. For 1:1 complexes, anions reside within the central cavity of the host species, held by hydrogen bonds from both NH and CH donors. The following host-anion complexes have been characterized by X-ray crystallography: 1-(Br)3, 1-(PF6)3.2(CH3)2CO, and 1-(NO3)1.5(PF6)1.5. Each structure contains the receptor in a significantly different geometry, highlighting the anion-dependent conformational flexibility of 1. Solution 1H NMR spectroscopic titrations have shown the two host species to display significant affinity for both halides and hydrogen sulfate and strongly suggest the persistence of CH...X- interactions despite the presence of "stronger" NH donor groups. Variable-temperature 1H NMR studies on the more soluble octyl derivative 2 show that there is a distinct change in conformation associated with the formation of a 1:1 host/guest complex. Computations using density functional theory (with the B3LYP functional) have been employed to aid in understanding the geometry of the 1:1 host/chloride complexes of 1 and 2. These experiments suggest that the lowest energy conformation for 1-Cl is one in which the ureidopyridinium arms are orientated upward forming a cavity that is sealed by CH...pi interactions, effectively forming a unimolecular capsule, whereas for 2 a less symmetrical "2-up, 1-down" geometry is favored.

Inverting the Charges of Natural Nucleobase Quartets: A Planar Platinum–Purine Quartet with Pronounced Sulfate Affinity

Variations on nature: Coordinative metal–ligand bonds instead of cyclic hydrogen bonds, an anion (sulfate) in the center rather than a cation, and an overall positive charge instead of a negative one are the major differences between the artificial platinum–purine quartet 2 (left) and the naturally occurring guanine quartet G4 (right).

Antiviral effect of the heparan sulfate mimetic, PI-88, against dengue and encephalitic flaviviruses

Many viruses, including flaviviruses, display affinity for cell surface heparan sulfate (HS) proteoglycans with biological relevance in virus attachment/entry. This raises the possibility of the application of HS mimetics in antiviral therapy. We have evaluated the antiviral effect of the sulfated polysaccharides, suramin, pentosan polysulfate (PPS) and PI-88, which are currently approved or in trial for clinical use, against dengue virus (DEN) and the encephalitic flaviviruses, Japanese encephalitis virus, West Nile virus, and Murray Valley encephalitis virus. A flow cytometry-based method for the measurement of inhibition of virus infectivity was developed, which showed the in vitro antiviral activity of the three compounds, albeit with differences in efficiency which were virus-dependent. The 50% effective concentration (EC 50) values for DEN inhibition were in the order: PPS < suramin < PI-88, and for Japanese encephalitis virus, PPS < PI-88 ≤ suramin. Heparin inhibited the DEN infectivity 30-fold more efficiently than the best of the test compounds, which was not the case for encephalitic flaviviruses. The in vitro anti-flaviviral effectiveness of the HS mimetics did not reliably predict their in vivo therapeutic activity. In mouse models for DEN and flaviviral encephalitis, only PI-88 demonstrated a significant beneficial effect in disease outcome.

1085. Directed Evolution of AAV2 Leads to Improved Viral Function

Adeno-associated viral (AAV) vectors are extremely safe and efficient gene delivery vehicles. However, numerous challenges in vector design remain, including neutralizing antibody responses, tissue transport, and infection of resistant cell types. In each case, the AAV capsid structure determines how the vector performs when presented with these barriers. Nature has evolved multiple AAV serotypes using mutagenesis and recombination, indicating a natural level of plasticity in viral structure and function. We have employed analogous techniques to rapidly generate a large random mutant AAV2 library and have selected for mutant AAV virions that display novel phenotypes. Specifically, we have selected the AAV2 library for infectious particles with altered heparin sulfate (HS) affinity and for the ability to evade an AAV2 immune response. Characterization of the mutants with both lower and higher affinity to heparin led to the location of novel point mutations on the capsid structure. Several of these mutations lie inside the capsid while two exposed mutations (K258E and S261F) lie in the canyon region. Furthermore, we have evolved vector variants that have greater than 100-fold improved resistance to serum that neutralizes wild type AAV2, yet retain AAV2 gene delivery efficiency. Analysis of the point mutations revealed one change on the surface of the 3-fold spike (N587I) and another change on the 2-fold dimple region (T716A) were critical to the enhanced resistance. Testing these vectors in animal studies may indicate that they could mediate gene delivery even in patients with pre-existing immunity. Finally, our libraries can be further analyzed to isolate mutants that surpass other barriers to successful gene delivery.

Rapid method for the affinity purification of thermostable α-amylase from Bacillus licheniformis

A rapid and efficient method the exploiting affinity of α-amylase for its substrate starch is described. α-amylase from Bacillus licheniformis was purified to homogeneity by ammonium sulphate precipitation and affinity chromatography with 230-fold purification. The α-amylase adsorption to various starches was examined in order to screen its ability for highest binding to starch. The α-amylase was bound to starch more tenaciously, hence various eluants like maltose, soluble starch and high salts could not elute the bound α-amylase. However, the bound α-amylase was instantly eluted using 2% (w/v) dextrin. The purified enzyme showed a single polypeptide on SDS-PAGE, with a molecular weight of 58 kD. Western blot analysis confirmed the specificity of antibody raised against purified α-amylase.

Evaluation of effect of some corticosteroids on glucose-6-phosphate dehydrogenase and comparative study of antioxidant enzyme activities

Corticosteroids are anti-inflammatory drugs that are similar to the natural corticosteroid hormones produced by the cortex of the adrenal glands. The objective of this study was to scrutinize effects of some corticosteroids on glucose-6-phosphate dehydrogenase (G6PD) and some antioxidant enzymes. Initially, G6PD was purified from human erythrocytes by using ammonium sulphate precipitation and affinity chromatography. The two drugs, dexamethasone phosphate and prednisolone, investigated on the purified enzyme inhibited the enzyme activity.Comparative in vivo studies were performed to determine the effects of dexamethasone phosphate on the antioxidant enzyme activities using Spraque-Dawley rats. G6PD and catalase (CAT) activities were found significantly lower than in the control, whereas glutathione peroxidase (GP) activity was significantly increased in the erythrocytes of rats the receiving drug; glutathione reductase (GR) activity was unaffected. The results imply that dexamethasone phosphate may affect oxidative stress by changing antioxidant enzyme activities.

Factor H Is a Dermatan Sulfate-Binding Protein: Identification of a Dermatan Sulfate-Mediated Protease That Cleaves Factor H

Dermatan sulfate mediates the blood coagulation cascade by binding to heparin cofactor II and potentiating the antithrombin activity. In order to explore another function of dermatan sulfate, a dermatan sulfate affinity column was prepared from biotinylated dermatan sulfate and Streptavidin Sepharose. When human plasma was applied on the dermatan sulfate column, factor H was bound and cleaved. The cleavage products, a 30-kDa N-terminal fragment and a 120-kDa fragment, were eluted from the column with 500 mM NaCl and detected after Western blotting with anti-factor H. The bond between the tandem arginine residues in the sixth domain of factor H was cleaved. When purified factor H was applied on the column, the factor H was not cleaved and was recovered from the column as an intact 150-kDa fraction. The finding that dermatan sulfate-mediated cleavage of factor H was inhibited by (p-amidinophenyl) methanesulfonyl fluoride, but not N-ethylmaleimide or EDTA, indicates that a serine protease in the plasma was activated on the dermatan sulfate column and factor H was cleaved without intervention of the plasma protease inhibitors. Amidase activity was detected in the effluent from the dermatan sulfate column but was abolished by pretreatment of the plasma with dermatan sulfate. Therefore, dermatan sulfate participates in the activation of a protease as well as having the protease inhibitory action.

Carbonic anhydrase inhibitors. Inhibition of isozymes I, II, IV, V, and IX with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate

The inhibition of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes; the cytosolic hCA I and II, the membrane-bound hCA IV, the mitochondrial hCA V, and the tumor-associated, transmembrane hCA IX, with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate; such as chlorate, perchlorate, bromate, iodate, periodate, silicate, bismuthate, vanadate, molybdate, and wolframate is reported. Apparently, the geometry of the inhibitor (tetrahedral or trigonal) does not influence its binding to the Zn(II) ion of the enzyme active site, but the nature of the central element is the most important factor influencing potency. Isozymes hCA I and II are best inhibited by chlorate, perchlorate, and silicate, together with the anions structurally related to sulfate, sulfamate, and sulfamidate, but sulfate itself is a weak inhibitor (inhibition constant of 74 mM against hCA I and 183 mM against hCA II). Molybdate is a very weak hCA I inhibitor ( K I of 914 mM) but it interacts with hCA II ( K I of 27.5 mM). Isozyme IV is well inhibited by sulfate ( K I of 9 mM), sulfamate, and sulfamidate (in the low micromolar range), but not by perchlorate ( K I of 767 mM). The mitochondrial isozyme V has the lowest affinity for sulfate ( K I of 680 mM) and carbonate ( K I of 95 mM) among all the investigated isozymes, suggesting on one hand its possible participation in metabolon(s) with sulfate anion exchanger(s), and on the other hand an evolutionary adaptation to working at higher pH values (around 8.5 in mitochondria) where rather high amounts of carbonate in equilibrium with bicarbonate may be present. Metasilicate, isosteric to carbonate, is also about a 10 times weaker inhibitor of this isozyme as compared to other CAs investigated here ( K I of 28.2 mM). Surprisingly, the tumor-associated isozyme IX is resistant to sulfate inhibition ( K I of 154 mM) but has affinity in the low micromolar range for carbonate, sulfamate, and sulfamidate ( K I in the range of 8.6–9.6 μM). This constitutes another proof that this isozyme best works at acidic pH values present in tumors, being inhibited substantially at higher pH values when more carbonate may be present. Bromate and chlorate are quite weak CA IX inhibitors ( K I s of 147–274 mM).

Functional characterization and genomic organization of the human Na +-sulfate cotransporter hNaS2 gene ( SLC13A4)

Sulfate plays an essential role in human growth and development. Here, we characterized the functional properties of the human Na +-sulfate cotransporter (hNaS2), determined its tissue distribution, and identified its gene ( SLC13A4) structure. Expression of hNaS2 protein in Xenopus oocytes led to a Na +-dependent transport of sulfate that was inhibited by thiosulfate, phosphate, molybdate, selenate and tungstate, but not by oxalate, citrate, succinate, phenol red or DIDS. Transport kinetics of hNaS2 determined a K m for sulfate of 0.38 mM, suggestive of a high affinity sulfate transporter. Na + kinetics determined a Hill coefficient of n = 1.6 ± 0.6, suggesting a Na: SO 4 2 - stoichiometry of 2:1. hNaS2 mRNA was highly expressed in placenta and testis, with intermediate levels in brain and lower levels found in the heart, thymus, and liver. The SLC13A4 gene contains 16 exons, spanning over 47 kb in length. Its 5′-flanking region contains CAAT- and GC-box motifs, and a number of putative transcription factor binding sites, including GATA-1, AP-1, and AP-2 consensus sequences. This is the first study to characterize hNaS2 transport kinetics, define its tissue distribution, and resolve its gene ( SLC13A4) structure and 5′ flanking region.

Suppression of the Biological Activities of the Epidermal Growth Factor (EGF)-like Domain by the Heparin-binding Domain of Heparin-binding EGF-like Growth Factor

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that has a high affinity for heparin and heparan sulfate. While interactions with heparin are thought to modulate the biological activity of HB-EGF, the precise role of the heparin-binding domain has remained unclear. We analyzed the activity of wild-type HB-EGF and a mutant form lacking the heparin-binding domain (DeltaHB) in the presence or absence of heparin. The activity of the EGF-like domain of HB-EGF was determined by measuring binding to diphtheria toxin (DT) as well as the growth factor activity in EGF receptor-expressing cells. The binding affinity of DeltaHB for DT was much higher than that of wild-type HB-EGF in the absence of heparin. The binding affinity of HB-EGF for DT was increased by addition of exogenous heparin and reached the level close to the affinity of DeltaHB, whereas that of DeltaHB was not affected. Moreover, the growth factor activity of DeltaHB was much higher than that of wild-type HB-EGF in the absence of heparin but was not affected by addition of exogenous heparin, whereas HB-EGF had increased growth factor activity with added heparin. These results indicate that the heparin-binding domain suppresses the activity of the EGF-like domain of HB-EGF and that association of heparin with HB-EGF via this domain removes the suppressive effect. Thus, we conclude that the heparin-binding domain serves as a negative regulator of this growth factor.

Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit

Recognition of the large secreted protein Slit by receptors of the Robo family provides fundamental signals in axon guidance and other developmental processes. In Drosophila, Slit-Robo signalling regulates midline crossing and the lateral position of longitudinal axon tracts. We report the functional dissection of Drosophila Slit, using structure analysis, site-directed mutagenesis and in vitro assays. The N-terminal region of Slit consists of a tandem array of four independently folded leucine-rich repeat (LRR) domains, connected by disulphide-tethered linkers. All three Drosophila Robos were found to compete for a single highly conserved site on the concave face of the second LRR domain of Slit. We also found that this domain is sufficient for biological activity in a chemotaxis assay. Other Slit activities may require Slit dimerisation mediated by the fourth LRR domain. Our results show that a small portion of Slit is able to induce Robo signalling and indicate that the distinct functions of Drosophila Robos are encoded in their divergent cytosolic domains.

A wide range of medium-sized, highly cationic, α-helical peptides show antiviral activity against herpes simplex virus

Ten highly cationic, α-helical peptides were synthesized and tested for antiviral activity against herpes simplex virus 1 and 2 (HSV-1 and HSV-2). Several of the peptides were found to exhibit antiviral activity. The peptides affinity for heparan sulfate (HS) increased with the number of cationic residues. Net charge could be decisive for the anti-HSV-1 activity, while secondary structure of the peptides seems more important for the anti-HSV-2 activity. The peptides were able to inhibit the entry of HSV-1 into the host cell, probably by blocking HS at the cell surface. HSV plaque formation was inhibited in a dose-dependent manner when cells were exposed to the peptides prior to the addition of virus. Lower inhibition activity was observed when the virus was allowed to attach to the cell surface before the addition of peptide. However, the plaque size was smaller compared to the untreated control, indicating that the peptides may also interfere with cell-to-cell spread of the virus. The two most potent antiviral peptides exhibited synergy with acyclovir against HSV.

Modelling of anti‐HSV activity of lactoferricin analogues using amino acid descriptors

Herpes simplex virus (HSV) causes a number of diseases and new therapies are being pursued vigorously. Earlier studies have shown that modified peptides based on lactoferricins reduce HSV-1 and HSV-2 infection, and structure-activity studies indicate that the anti-viral activity correlates with the binding affinity for heparan sulphate and chondroitin sulphate. In this study it is shown that theoretically derived amino acid descriptors can be used to model the anti-viral activity of peptides, as well as other peptide properties, even more accurately.

Determinants of virulence of classical swine fever virus strain Brescia.

Two related classical swine fever virus (CSFV) strain Brescia clones were isolated from blood samples from an infected pig. Virus C1.1.1 is a cell-adapted avirulent variant, whereas CoBrB is a virulent variant. Sequence analysis revealed 29 nucleic acid mutations in C1.1.1, resulting in 9 amino acid substitutions compared to the sequence of CoBrB (476)R. Using reverse genetics, parts of the genomes of these viruses, which contain differences that lead to amino acid changes, were exchanged. Animal experiments with chimeric viruses derived from C1.1.1 and CoBrB (476)R showed that a combination of amino acid changes in the structural and nonstructural regions reduced the virulence of CSFV in pigs. Moreover, the presence of a Leu at position 710 in structural envelope protein E2 seemed to be an important factor in the virulence of the virus. Changing the Leu at position 710 in the CoBrB (476)S variant into a His residue did not affect virulence. However, the (710)His in the C1.1.1/CoBrB virus, together with adaptive mutations (276)R, (476)R, and (477)I in E(rns), resulted in reduced virulence in pigs. These results indicated that mutations in E(rns) and E2 alone do not determine virulence in pigs. The results of in vitro experiments suggested that a high affinity for heparan sulfate of C1.1.1 E(rns) may reduce the spread of the C1.1.1/CoBrB virus in pigs and together with the altered surface structure of E2 caused by the (710)L-->H mutation may result in a less efficient infection of specific target cells in pigs. Both these features contributed to the attenuation of the C1.1.1/CoBrB virus in vivo.

20. Directed Evolution of AAV2 Yields Novel Capsid Variants

Adeno-associated virus (AAV) is a highly promising gene delivery vehicle, though gene transfer barriers can in cases limit its efficiency. These barriers arise at every step of delivery: the transit of the vector from injection to a cell surface, receptor binding and uptake, intracellular trafficking, and nuclear steps. The AAV gene transfer properties at each of these steps are determined by its capsid structure. Previous capsid modifications that alter AAV tropism, as well as the existence of multiple AAV serotypes, suggest that the structure-function relationships of the AAV capsid are reasonably plastic. We have taken advantage of this remarkable capsid plasticity to generate a large random mutant AAV library (1e6) and select for mutant AAV virions that can overcome several barriers to infection. Specifically, we have selected the AAV2 library for infectious particles with altered heparan sulfate (HS) affinity and for the ability to evade an AAV2 immune response. First, we have generated mutants with both lower and higher affinity to heparin, which could prove valuable in controlling the therapeutic zone of an AAV vector in tissues where ECM HS hinders AAV2 diffusion. Furthermore, we have generated vector variants that have greater than 100-fold improved resistance to human serum that neutralizes wild type AAV2, yet retain AAV2 gene delivery efficiency. These vectors may enable high gene delivery efficiency even in patients with pre-existing immunity, and the locations of point mutations on the capsid surface suggest new regions of functional importance to the virus. These AAV libraries therefore both provide useful variants for gene therapy applications as well as offer new avenues to dissect basic AAV biology.

Crystal Structure of the Human RORα Ligand Binding Domain in Complex with Cholesterol Sulfate at 2.2 Å

The retinoic acid-related orphan receptor alpha (RORalpha) is an orphan member of the subfamily 1 of nuclear hormone receptors. Our recent structural and functional studies have led to the hypothesis that cholesterol or a cholesterol derivative is the natural ligand of RORalpha. We have now solved the x-ray crystal structure of the ligand binding domain of RORalpha in complex with cholesterol-3-O-sulfate following a ligand exchange experiment. In contrast to the 3-hydroxyl of cholesterol, the 3-O-sulfate group makes additional direct hydrogen bonds with three residues of the RORalpha ligand binding domain, namely NH-Gln(289), NH-Tyr(290), and NH1-Arg(370). When compared with the complex with cholesterol, seven well ordered water molecules have been displaced, and the ligand is slightly shifted toward the hydrophilic part of the ligand binding pocket, which is ideally suited for interactions with a sulfate group. These additional ligand-protein interactions result in an increased affinity of cholesterol sulfate when compared with cholesterol, as shown by mass spectrometry analysis done under native conditions and differential scanning calorimetry. Moreover, mutational studies show that the higher binding affinity of cholesterol sulfate translates into an increased transcriptional activity of RORalpha. Our findings suggest that cholesterol sulfate could play a crucial role in the regulation of RORalpha in vivo.

Anti-HSV activity of lactoferricin analogues is only partly related to their affinity for heparan sulfate

Earlier studies have shown that the heparan sulfate (HS) on the cell surface acts as a receptor for herpes simplex virus (HSV). We have recently shown that bovine lactoferricin (LfcinB), a small part of the milk protein lactoferrin, inhibits HSV-1 and HSV-2 infection, probably by blocking the entry of the virus. The human homologue (18–42), which shares 36% sequence similarity with LfcinB (17–41), displayed much lower antiviral activity. In the present study, a set of cyclic and linear human and bovine Lfcin derivatives were constructed to investigate the relation between their affinity to HS and chondroitin sulfate (CS) and their antiviral activity against HSV-1 and HSV-2. The lactoferrin (LF) proteins and several of the Lfcin derivatives exhibited similar affinity for HS, but the LF proteins possess a much higher antiviral activity than the smaller peptides. Our structure–activity relationship studies on the Lfcin derivates confirmed that affinity for HS, that was correlated to the net positive charge, is an important factor, but does not well predict the antiviral activity. Structural parameters such as hydrophobicity, molecular size, spatial distribution of charged and lipophilic amino acids, and the cyclic structure of Lfcin also seem to be important factors to govern antiviral activity against HSV.

Pharmacokinetics and tissue distribution of uraemic indoxyl sulphate in rats.

The purpose of the present study was to examine the pharmacokinetic properties of indoxyl sulphate, a harmful uraemic toxin that accumulates during chronic renal failure. The pharmacokinetics and tissue distribution of indoxyl sulphate were examined in normal and 5/6 nephrectomized (CRF) rats. The uptake process of indoxyl sulphate by rat renal cortical slices in vitro was also investigated. Endogenous indoxyl sulphate was found to be mainly distributed in the kidney. The rate of elimination of indoxyl sulphate from plasma was lower in CRF rats compared with sham-operated rats. The majority of intact indoxyl sulphate was excreted in the urine. In renal cortical slice experiments, uptake of indoxyl sulphate was a saturable process with a K(m) of 43.0 microm. Furthermore, sulphate conjugates, such as oestrone sulphate and dehydroepiandrosterone sulphate, inhibited the uptake of indoxyl sulphate to a greater extent than PAH. Thus, indoxyl sulphate is primarily eliminated from the plasma via the kidney by active tubular secretion, and renal uptake of indoxyl sulphate appears to be mediated by an organic anion transport system with a high affinity for oestrone sulphate and dehydroepiandrosterone sulphate.

Cloning of two contrasting high-affinity sulfate transporters from tomato induced by low sulfate and infection by the vascular pathogen Verticillium dahliae.

Two cDNAs, LeST1-1 (AF347613) and LeST1-2 (AF347614), encoding sulfate transporters have been cloned from tomato (Lycopersicon esculentum Mill.) by reverse transcription-polymerase chain reaction and their expression characterised. Sharing 76% identity at the amino acid level, the transporters are phylogenetically associated with the Group-1, high-affinity plant sulfate transporters. Both were shown to have high affinity for sulfate by uptake kinetic analysis using a yeast (Saccharomyces cerevisiae) sulfate-transporter mutant. Km values of 11.5 microM and 9.8 microM were calculated for LeST1-1 and LeST1-2, respectively, the same order of magnitude as those previously reported for several other Group-1 high-affinity sulfate transporters. In situ hybridisation to S-deficient tomato roots showed LeST1-1 to be expressed in the epidermis and pericycle, whereas LeST1-2 expression was located to the epidermis only. Northern analysis shows that the mRNA abundances of both LeST1-1 and LeST1-2 are upregulated in the root in response to sulfate deprivation. LeST1-1 is specifically expressed in root tissue, a characteristic of Group-1 sulfate transporters. LeST1-2, however, was also detected in tomato leaves and stems and is upregulated and expressed to a similar extent in these tissues under conditions of sulfate deprivation. Induction of LeST1-2 expression was also observed in the vascular tissues of a resistant line of tomato infected with the vascular wilt pathogen Verticillium dahliae.

Collapsin Response Mediator Proteins of Neonatal Rat Brain Interact with Chondroitin Sulfate

Chondroitin sulfate proteoglycans are structurally and functionally important components of the extracellular matrix of the central nervous system. Their expression in the developing mammalian brain is precisely regulated, and cell culture experiments implicate these proteoglycans in the control of cell adhesion, neuron migration, neurite formation, neuronal polarization, and neuron survival. Here, we report that a monoclonal antibody against chondroitin sulfate-binding proteins from neonatal rat brain recognizes collapsin response mediator protein-4 (CRMP-4), which belongs to a family of proteins involved in collapsin/semaphorin 3A signaling. Soluble CRMPs from neonatal rat brain bound to chondroitin sulfate affinity columns, and CRMP-specific antisera co-precipitated chondroitin sulfate. Moreover, chondroitin sulfate and CRMP-4 were found to be localized immuno-histochemically in overlapping distributions in the marginal zone and the subplate of the cerebral cortex. CRMPs are released to culture supernatants of NTera-2 precursor cells and of neocortical neurons after cell death, and CRMP-4 is strongly expressed in the upper cortical plate of neonatal rat where cell death is abundant. Therefore, naturally occurring cell death is a plausible mechanism that targets CRMPs to the extracellular matrix at certain stages of development. In summary, our data indicate that CRMPs, in addition to their role as cytosolic signal transduction molecules, may subserve as yet unknown functions in the developing brain as ligands of the extracellular matrix.