3 School of Biological Sciences, Royal Holloway University of London, Egham Hill, Egham, Surrey TW20 0EX, United Kingdom
4 Laboratory for Molecular Structure, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters bar, Hertfordshire, EN6 3QC, United Kingdom
Received on August 21, 2002; revised on December 4, 2002; accepted on January 7, 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words:
GDNF
/
GFR1 receptor
/
glycosaminoglycan
/
heparan sulfate
/
heparin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is well established that GDNF exerts potent neuroprotective activity both in vitro and in vivo; indeed GDNF-expressing vectors have proven effective in protecting against neurodegeneration and promoting regeneration in murine (Choi-Lunberg et al., 1997) and primate (Kordower et al., 2000
) models of Parkinson's disease, and clinical trials of recombinant human (rh) GDNF infusion for this condition are now under way with promising initial results (Major, 2002
). However, the neuroprotective activity of GDNF is not limited to dopaminergic neurones, as numerous studies show beneficial effects in central and peripheral neurones and axons following challenge with various mechanical, ischemic, and neurotoxic insults. This raises the prospect of therapeutic applications of either gene- vectorexpressed GDNF or recombinant GDNF in a wide range of nervous system injury or disease.
Gene deletion studies in mice reveal an essential, nonredundant role for GDNF in the development of the enteric nervous system (Moore et al., 1996; Pichel et al., 1996
; Sanchez et al., 1996
). GDNF-/- mice also show other selective neurone deficits in the trigeminal motor nucleus, spinal chord, and sensory and sympathetic ganglia. However, the most striking feature of the GDNF-/- phenotype is the complete absence of kidneys, which is presumably the major contributor to the early postnatal mortality seen in this phenotype. Similar phenotypes, involving renal agenesis and the absence of enteric neurones, also result from targeted disruption of the genes encoding GFR
1 (Cacalano et al., 1998
; Tomac et al., 2000
) and Ret (Schuchardt et al., 1994
). These and other experimental observations suggest that high levels of GDNF expressed in the embryonic metanephric blastema serve to activate cells of the ureteric bud, which grows out from the preexisiting metanephric (Wolffian) duct. Cells at the tip of the bud express both GFR
1 and Ret, and contact with the blastema initiates subsequent events in kidney formation (reviewed by Schedl and Hastie, 2000
).
Heparan sulfate (HS) is an acidic polysaccharide that is nearly ubiquitous on cell surfaces and in the extracellular matrix. It has been shown to bind to an increasing number of diverse cytokines. The biosynthesis of HS involves postincorporation modifications of hexoses in the nascent polysaccharide chains. Such modifications include the epimerization of glucuronate residues to iduronate and their possible subsequent O-sulfation at the C-2 position (reviewed by Lindahl et al., 1994). This latter modification is catalyzed by a specific HS 2-O-sulfotransferase (HS2ST).
As part of a genetic screen approach to the study of embryogenesis, a mutation of the gene encoding HS2ST was recovered. Mice homozygous for disruption of the HS2ST gene exhibit a variety of developmental defects, but the most striking phenotype is absence of kidneys, recapitulating the phenotypes resulting from GDNF, GFR1, and cRet gene disruption (Bullock et al., 1998
). These observations could be accounted for by hypotheses in which GDNF and 2-O-sulfated HSs interact only via some indirect mechanism involving intermediary factors. However, the simplest and most direct hypothesis is that the high levels of GDNF expressed in the metanephric blastema bind to HS in the local extracellular matrix or on cell surfaces. This retention would serve in a critical manner to maintain a high localized concentration of the cytokine required to promote invasion by the extending uretric bud. Such a direct binding hypothesis predicts that 2-O-sulfate groups are essential for the binding of GDNF to HS, such that HS lacking 2-O-sulfate groups is substantially less active in this regard. It would also predict that HS-bound GDNF remains biologically active by being readily available to its cell surface receptors. GDNF is known to be a heparin-binding cytokine, because during purification, GDNF bound to a heparin-Sepharose column and was eluted between 0.5 and 1.5 M NaCl (Lin et al., 1994
). However, up to now there has been no detailed examination of the affinity and specificity of the binding of GDNF to sulfated glycosaminoglycans and how this might affect its receptor engagement and stability.
We have established an enzyme-linked immunosorbent assay (ELISA) approach to investigate the binding of cytokines to glycosaminoglycans, involving capture on an immobilized heparinprotein conjugate. Using this approach we have found that interleukins (ILs) 2, 6, and 12 bind to heparin and HS (Najjam et al., 1997, 1998
; Hasan et al., 1999
; Mummery and Rider, 2000). In each case this binding is selective because it is undetectable with the cytokines TNF-
, IL-10, and IL-16 (unpublished data) and specific in that other sulfated glycosaminoglycans, particularly the chondroitin sulfates, are either poor or negligible competitors of heparin/HS binding (Najjam et al., 1997
; Hasan et al., 1999
; Mummery and Rider, 2000). Here we report the use of this ELISA approach to characterize the interaction between rhGDNF and heparin. We also investigate the effect of heparin binding on the interaction between GDNF and a soluble chimeric construct of its cognate receptor, GFR
1. These studies provide evidence for a major role for 2-O-sulfate groups in the binding of GDNF to heparin/HS and show that heparin-bound GDNF stills binds GFR
1 and is protected from proteolytic degradation.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Because soluble heparin is an effective competitor with the heparin-complex for GDNF binding, we examined the specificity of GDNFglycosaminoglycan interactions by employing a range of heparin-related preparations in this competitive variant of the ELISA. As may be seen in Figure 2A, 2.0 µg/ml heparin again gives essentially complete inhibition of binding. By comparison, bovine kidney HS, although used at a fivefold higher concentration, is a weak inhibitor, reducing GDNF binding only by some 30%. In contrast, fucoidan, a sulfated polyfucose from marine algae, appears to be a more potent inhibitor than heparin because it provides complete inhibition at a concentration that with heparin would only provide some 60% inhibition (see Figures 1B and 2B). The three forms of chondroitin sulfate, A, B, and C, are only weak inhibitors. Statistical analysis of two independent experiments by analysis of variance with post hoc analysis establishes that bovine kidney HS and all forms of chondroitin sulfate give significant inhibition at the p0.05 level but fails to establish any significant difference between the effects of all four of these glycosaminoglycans.
|
The structural characteristics of HSA and HSE were investigated by 500 mHz 1H nuclear magnetic resonance (NMR), and the ratios of various substituents, as determined by integration of specific peak signals (Mulloy et al., 1994; Yates et al., 1996
), are summarized in Table I. The 5th International Standard heparin (from porcine mucosa) was simultaneously analyzed as a control. As may be seen, HSE is extensively N-deacetylated and N-sulfated, to an extent approaching that seen in heparin. In contrast, in HSA only approximately half of the glucosamine residues remain in the unmodified N-acetyl form. All three preparations have similar ratios of 6-O-sulfate to N-sulfate. However the preparations differ markedly in the ratio of 2-O-sulfate to N-sulfate, that of HSA being considerably lower than that of heparin and less than 50% that seen with HSE. Given the markedly lower abundance of N-sulfate groups in HSA compared to the other preparations, it may be estimated that HSA has only around 25% of the 2-O-sulfate groups found in HSE.
|
|
|
We also sought to determine whether or not HS, like heparin, could potentiate the binding of rhGDNF to the GFR1 chimera. The results of a single experiment, representative of a series of three, are shown in Figure 4C. Here heparin at 1 µg/ml is seen to double the amount of rhGDNF captured by the immobilized GFR
1 chimera. This increase is entirely consistent with the data shown in Figure 4A and B and appears maximal under the experimental conditions because there is no further increase with 5 µg/ml heparin. Equivalent concentrations of HSE give essentially no increase in GDNFGFR
1 binding, however at 25 µg/ml HSE a 25% increase in binding is seen.
It has been shown for several other cytokines that binding to heparin provides protection from proteolytic degradation. As shown in Figure 5, digestion of rhGDNF with LysC in vitro resulted in the clipping of the GDNF polypeptide; as on western blotting, the GDNF immunoreactive band migrates marginally faster. Under conditions that gave essentially complete clipping of the polypeptide, concentrations of heparin as low as 0.2 µg/ml completely protected the cytokine from this degradation. However, 2-O-desulfated heparin was less active in this regard; as with 0.2 µg/ml the product migrates as a smeared doublet with substantial immunoreactivity migrating faster, like the clipped polypeptide, albeit with some unprocessed polypeptide remaining. In addition to demonstrating a protective effect, these experiments provide a demonstration, independent of ELISA, that heparin binds with high affinity to GDNF.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As well as soluble heparin, several other glycosaminoglycans compete for the binding of rhGDNF to the immobilized heparin complex. As in our previous studies of IL-2 (Najjam et al., 1997) and IL-6 (Mummery and Rider, 2000
), but not in the case of IL-12 (Hasan et al., 1999
), fucoidan is a more potent inhibitor than heparin. Fucoidan is highly sulfated, with the major disaccharide repeat unit being a trisulfated difucose, in which both 2C positions are sulfated (Chevolot et al., 2001
). In contrast to the unbranched chains of mammalian glycosaminoglycans, it also has a branched, comb-like structure (Patankar et al., 1993
). Thus fucoidan offers a high density of sulfate groups with particularly diverse spatial organizations, enabling it to furnish potential binding sites for clusters of basic residues on the surfaces of polypeptides.
When comparing the results obtained here with rhGDNF for glycosaminoglycan inhibition of binding with those obtained previously by the same method with other cytokines, subtle differences in the patterns of inhibition are evident. For instance, in contrast to the results obtained here for GDNF where chondroitin sulfates A, B, and C are all significant inhibitors, albeit with modest potency, in the case of IL-6, only chondroitin sulfate A provided significant inhibition of binding (Mummery and Rider, 2000), and with IL-12 only chondroitin sulfate B was a significant inhibitor (Hasan et al., 1999
). These variations indicate that these different cytokines have differences in specificity for the glycosaminoglycan structures with which they interact.
The use of chemically modified heparins and HSs again reveals unique aspects in the binding of GDNF to heparin. In contrast to the results with rhGDNF here, equivalent studies of IL-6, using the same modified heparin preparations, showed that 2-O-sulfated heparin was the most active competitor, and moreover that the various HSs used were essentially indistinguishable from each other as partial inhibitors (Mummery and Rider, 2000). In addition to revealing an essential role for 2-O-sulfates in the binding of GDNF to heparin, the present findings also indicate some involvement of 6-O-sulfates and N-sulfates. In the case the N-desulfated heparin, the partial recovery of inhibitory activity on re-N-acetylation was also seen in our studies of the heparin-binding properties of the HIV-1 envelope glycoprotein gp120. It may be accounted for either by the blocking of the positively charged, which would otherwise occur on the unsubstituted amino group of the N-desulfated glucosamine, or by the requirement for a bulk derivative group on the amino group, whether charged (sulfate) or uncharged (acetyl), to maintain an appropriate conformation within the heparin chain for binding (Rider et al., 1994
).
The nitrous acid cleavage method used to generate the heparin oligosaccharides used in this study cleaves the chains preferentially at N-sulfated glucosamine residues (Shively and Conrad, 1976). The inhibitory activity here appears particularly sensitive to this treatment, compared to previous results using the same oligosaccharides in the study of rhIL-12 (Hasan et al., 1999
). Together with the sensitivity of inhibitory activity to chemical desulfation, particularly 2-O-desulfation, and the observation also that the most active HS is the highly sulfated HSE preparation, this all suggests that highly sulfated domains within heparin/HS chains (Gallagher et al., 1992
) are important in GDNF binding.
Taken overall, our findings show that GDNF binds strongly to heparin/HS and that in this interaction 2-O-sulfate groups play a particularly prominent role. This provides strong biochemical support for a hypothesis that the critical role for 2-O-sulfated HS in the developmental activity of GDNF, demonstrated in the HS2ST-/- mouse (Bullock et al., 1998), arises from the direct binding of GDNF to 2-O-sulfate-rich HS. How might such binding influence the bioactivity of GDNF? One important outcome of glycosaminoglycan binding, particularly for small proteins, is that the free diffusion of the protein away from sites of secretion will be slowed and restricted. Interestingly, in a study of the infusion of rhGDNF and other GFLs into rat brain, it was observed that coinfusion with heparin increased the volume of diffusion fivefold, and the authors speculated that this might arise through the blocking of the binding of the cytokine to sites in the extracellular matrix (Hamilton et al., 2001
). In kidney development, GDNF in the embryonic metanephric blastema functions as a chemoattractant factor (Schedl and Hastie, 2000
), and it is entirely plausible that in the absence of high-affinity binding to HS, GDNF diffuses away, and thus its concentration falls below a critical threshold. A second mechanism to explain the dependence of GDNF activity on HS, for which we provide some in vitro evidence, is that glycosaminoglycan protects the cytokine from proteolytic degradation. We show here that 2-O-desulfated heparin is less effective in this regard.
Finally, we provide further evidence in support of a direct binding hypothesis to explain the dependence of GDNF bioactivity on intact heparin-like glycosaminoglycan. This is that heparin-bound GDNF still engages its cognate receptor polypeptide, GFR1. Indeed, we report a modest potentiation of this first interaction in the GDNF signaling pathway. This effect is observed not only with heparin but also with a highly sulfated preparation of HS, albeit with the latter being less efficient in this regard.
The activity of GDNF in kidney development is especially sensitive to quantitative change, because unusually some 30% of mice heterozygotes for GDNF gene disruption show the absence of one or both kidneys (Sanchez et al., 1996; Pichel et al., 1996
; Moore et al., 1996
). It could therefore be argued that the combined effect of the mechanisms revealed thus far for the involvement of HS in the biological functioning of GDNF might be adequate to explain the phenotype of the HS2ST-/- mouse. Nonetheless, it remains likely that there are additional mechanisms to be elucidated by further studies of effects of heparin and HS on GDNF signaling.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NMR spectroscopy
1H NMR spectroscopy was performed using a Varian Unity 500 MHz spectrometer, with deuterated trimethylsilylpropionic acid as an external reference. The resulting spectra were all in agreement with those reported by Mulloy et al. (1994) and Yates et al. (1996)
.
Preparation and characterization of chemically modified heparins
The parent material for all modified heparins was bovine lung heparin from the 2nd International Standard heparin. N-desulfation of heparin and its subsequent re-N-acetylation were performed as described by Mulloy et al. (1994). Selective 2-O-desulfation and 6-O-desulfation, including re-N-sulfation of the latter to correct for a degree of N-desulfation, were performed as fully described elsewhere (Ostrovsky et al., 2002
). The extent of desulfation at specific positions was assessed by 1H NMR spectroscopy. The intensity of the characteristic signal for H2 of N-sulfated glucosamine in the spectra of N-desulfated, re-N-acetylated heparin was below the limit of detection, indicating that the N-desulfation was at least close to completion. Likewise, the signal for H2 of 2-O-sulfated iduronate, which is prominent in the 1H spectrum of the parent heparin, was undetectable in the spectrum of the 2-O-desulfated heparin. Similarly, the signals for H6 of the 6-O-sulfated glucosamine were undetectable in the 6-O-desulfated material. In the case of each of the eventual selectively desulfated products, the NMR spectra showed no detectable nonspecific desulfation.
Heparin-binding ELISA
A covalent heparinBSA conjugate was synthesized by coupling the reducing ends of the heparin chains to the protein using sodium cyanoborohydride, as fully described elsewhere (Najjam et al., 1997), except that 34 mg BSA (equivalent to 0.5 µmol) and 910 mg heparin (
75 µmol) were added to the reaction mixture. The resulting complex was separated from the unconjugated reactants by gel filtration on Sepharose 4B (Amersham Pharmacia Biotech, Little Chalfont, UK) (Najjam et al., 1997
). Mock-treated BSA was prepared with cyanoborohydride in the same way but in the absence of heparin with subsequent gel filtration.
ELISA wells were coated with either 5 ng heparinBSA complex (as measured by BSA content) or mock-treated BSA and washed as described previously (Mummery and Rider, 2000). After blocking for 5 min with phosphate buffered saline (PBS) containing 1% (w/v) BSA, wells were washed three times with PBS, and rhGDNF diluted in PBS-BSA was added for 2 h at room temperature. In competitive experiments, rhGDNF was preincubated with glycosaminoglycans in PBS-BSA for 30 min before addition to the plate. Wells were washed three times with PBS containing 0.05% (v/v) Tween 20; anti-GDNF, diluted 1/200 in PBS-BSA, was added for 30 min. Following an additional three washes with PBS-Tween, alkaline phosphatase second antibody (Sigma) diluted 1/1000 in PBS-BSA was added. After 30 min, wells were washed three times with PBS-Tween and twice more in PBS. Substrate solution, 100 µl/well was added and after a suitable color development, typically around 30 min, the plate was read at 405 nm in an Emax plate reader (Molecular Devices, Sunnyvale, CA). All plate incubations were carried out at room temperature with gentle shaking, except for the final incubation with the substrate, which was carried out at 37°C with rapid shaking in microtitreplate incubator (Wellwarm 1, Labsystems, Cambridge, UK).
GDNFGFR1 binding ELISA
ELISA plate wells were coated overnight at 4°C with 100 µl mouse anti-human IgG ascites fluid diluted 1/200 in 30 mM sodium bicarbonate buffer, pH 8.5. After three washes in PBS, wells were blocked for 30 min with PBS-BSA at room temperature. After three washes with PBS, GFR1 chimera, 20 ng/well, was added in PBS-BSA for 1 h before washing again in PBS. rhGDNF, preincubated in PBS-BSA in the absence or presence of the appropriate glycosaminoglycan, was then added and incubated for 90 min before more PBS washes. Anti-GDNF diluted 1/300 in PBS-BSA was added, and the remainder of the assay was conducted as described, except the alkaline phosphatase second antibody was from Jackson Immunoresearch and diluted 1/250.
LysC digestion of GDNF
Endoproteinase LysC was stored frozen at 0.1 µg/ml enzyme in 20 mM TrisHCl buffer, pH 8.0, containing 0.05% 2-mercaptoethanol. Aliquots of 65 µl containing 100 ng GDNF and 5 µg BSA carrier protein in the same buffer but without mercaptoethanol were preincubated in the absence or presence of varying concentrations of bovine lung heparin or its 2-O-desulfated derivative for 30 min at lab temperature; then 5 µl of 15-fold diluted LysC stock solution was added. After incubation for 75 min at 37°C, reactions were stopped by the addition of concentrated sodium dodecyl sulfatepolyarcrylamide gel electrophoresis sample buffer, followed immediately by boiling for 3 min. Samples containing 15 ng GDNF were then separated on 14% acrylamide gels, which were overrun by 50% of the time needed for the tracker dye to reach the bottom of the gel.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
2 To whom correspondence should be addressed; e-mail: c.rider{at}rhul.ac.uk
![]() |
Abbreviations |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baloh, R., Enomoto, H., Johnson, E., and Milbrandt, J. (2000) The GDNF family ligands and receptorsimplications for neural development. Curr. Opin. Neurobiol., 10, 103110.[CrossRef][ISI][Medline]
Bullock, S., Fletcher, J., Beddington, R., and Wilson, V. (1998) Renal agenesis in mice homozygous for a gene trap mutation in the gene encoding heparan sulfate 2-sulfotransferase. Genes Dev., 12, 18941906.
Cacalano, G., Farinas, I., Wang, L., Hagler, K., Forgie, A., Moore, M., Armanini, M., Phillips, H., Ryan, A., Reichardt, L., and others. (1998) GFRalpha1 is an essential receptor for GDNF in the developing nervous system and kidney. Neuron, 21, 5362.[ISI][Medline]
Chevolot, L., Mulloy, B., Ratiskol, J., Foucault, A., and Colliec-Jouault, S. (2001) A disaccharide repeat unit is the major structure of fucoidans from two species of brown algae. Carbohyd. Res., 330, 529535.[CrossRef][ISI][Medline]
Choi-Lundberg, D., Lin, Q., Chang, Y.-N., Chaing, Y., Hay, C., Mohajeri, H., Davidson, B., and Bohn, M. (1997) Dopaminergic neurons protected from degeneration by GDNF gene therapy. Science, 275, 838841.
Gallagher, J., Turnbull, J., and Lyon, M. (1992) Patterns of sulphation in heparan sulphate: polymorphism based on a common theme. Int. J. Biochem., 24, 553560.[CrossRef][ISI][Medline]
Hamilton, J., Morrison P., Chen, M., Harvey-White, J., Pernaute, R., Phillips, H., Oldfield, E., and Bankiewicz, K. (2001) Heparin coinfusion during convection-enhanced delivery (CED) increases the distribution of the glial-derived neurotrophic factor (GDNF) ligand family in rat striatum and enhances the pharmacological activity of neutrin. Exp. Neurol., 168, 155161.[CrossRef][ISI][Medline]
Hasan, M., Najjam, S., Gordon, M., Gibbs, R., and Rider, C. (1999) IL-12 is a heparin-binding cytokine. J. Immunol., 162, 10641070.
Kordower, J., Emborg, M., Bloch, J., Ma, S., Chu, Y., Leventhal, L., McBride, J., Chen, E., Palfi, S., Roitberg, B., and others. (2000) Neurodegeneration prevented by lentiviral delivery of GDNF in primate models of Parkinson's disease. Science, 290, 767773.
Kotzbauer, P., Lampe, P., Heuckeroth, R., Golden, J., Creedon, D., Johnson, E., and Milbrandt, J. (1996) Neurturin, a relative of glial- cell-line-derived neurotrophic factor. Nature, 384, 467470.[CrossRef][ISI][Medline]
Lin, L.-F., Doherty, D., Lile, J., Bektesh, S., and Collins, F. (1993) GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science, 260, 11301132.[ISI][Medline]
Lin L.-F., Zhang, T., Collins, F., and Armes, L. (1994) Purification and initial characterization of rat B49 glial line-derived neurotrophic factor. J. Neurochem., 63, 758768.[ISI][Medline]
Lindalhl, U., Lidholt, K., Spillmann, D., and Kjellen, L. (1994) More to "heparin" than anticoagulation. Thromb. Res., 75, 132.[ISI][Medline]
Major, S. (2002) New treatment improves symptoms of Parkinson's disease. Brit. Med. J., 324, 997.
Milbrandt, J., de Sauvage, F., Fahrner, T., Baloh, R., Leitner, M., Tansey, M., Lampe, P., Heuckeroth, R., Kotzbauer, P., Simburger, K., and others. (1998) Persephin, a novel neurotrophic factor related to GDNF and neuturin. Neuron, 20, 245253.[ISI][Medline]
Moore, M., Klein, R., Farinas, I., Sauer, H., Armanini, M., Phillips, H., Reichardt, L., Ryan, A., Carver-Moore, K., and Rosenthal, A. (1996) Renal and neuronal abnormalities in mice lacking GDNF. Nature, 382, 7679.[CrossRef][ISI][Medline]
Mulloy, B., Forster, M., Jones, C., Drake, A., Johnson E., and Davies D. (1994) The effect of substitution on the solution conformation of heparin: a spectroscopic and molecular modelling study. Carbohyd. Res., 255, 126.[CrossRef][ISI][Medline]
Mummery, R. and Rider, C. (2000) Characterization of the heparin-binding properties of IL-6. J. Immunol., 165, 56715679.
Najjam, S., Gibbs, R., Gordon, M., and Rider, C. (1997) Characterisation of human recombinant interleukin 2 binding to heparin and heparan sulphate using an ELISA approach. Cytokine, 9, 10131022.[CrossRef][ISI][Medline]
Najjam, S., Mulloy, B., Theze, J., Gordon, M., Gibbs, R., and Rider, C. (1998) Further characterisation of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites. Glycobiology, 8, 509516.
Ostrovsky, O., Berman, B., Gallagher, J., Mulloy, B., Fernig, D., Delehedde, M., and Ron, D. (2002) Differential effects of heparin saccharides on the formation of specific fibroblast growth factor (FGF) and FGF receptor complexes. J. Biol. Chem., 277, 24442453.
Patankar, M., Oehninger, S., Barnett, T., Williams, R., and Clark, G. (1993) A revised structure for fucoidan may explain some of its biological activities. J. Biol. Chem., 267, 2177021776.
Pichel, J., Shen, L., Sheng, H., Granholm, A.-C., Drago, J., Grinberg, A., Lee, E., Huang, S., Saarma, M., Hoffer, B., and others. (1996) Defects in enteric innervation and kidney development in mice lacking GDNF. Nature, 382, 7376.[CrossRef][ISI][Medline]
Rider, C., Coombe, D., Harrop, H., Hounsell, E., Bauer, C., Feeney, J., Mulloy, B., Mahmood, N., Hay, A., and Parish, C. (1994) Anti-HIV-1 activity of chemically modified heparins: correlation between binding to the V3 loop of gp120 and inhibition of cellular HIV-1 infection in vitro. Biochemistry, 33, 69746980.[ISI][Medline]
Sanchez, M., Silos-Santiago, I., Frisen, J., He, B., Lira, S., and Barbacid, M. (1996) Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature, 382, 7073.[CrossRef][ISI][Medline]
Schedl, A. and Hastie, N. (2000) Cross-talk in kidney development. Curr.t Opin. Genet. Dev., 10, 534549.
Schuchardt, A., D'Agati, V., Larsson-Blomberg, L., Costantini, F., and Pachnis, V. (1994) Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature, 367, 380383.[CrossRef][ISI][Medline]
Shively J. and Conrad, J. (1976) Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry, 15, 39323942.[ISI][Medline]
Tomac, A., Grinberg, A., Huang, S., Nosrat, C., Wang, Y., Borlongan, C., Lin, S.-Z., Chiang, Y.-H., Olson, L., Westphal, H., and Hoffer, B. (2000) Glial cell-line derived neurotrophic factor receptor alpha1 availability regulates glial cell-line derived neurotrophic factor signalling: evidence from mice carrying one or two mutated alleles. Neuroscience, 95, 10111023.[CrossRef][ISI][Medline]
Yates, E., Santini, F., Guerrini, M., Naggi, A., Torri, G., and Casu, B. (1996) 1H and 13C NMR spectral assignments of the major sequences of twelve systematically modified heparin derivatives. Carbohyd. Res., 294, 1527.[CrossRef][ISI][Medline]