Heparin-binding Growth-associated Molecule Research Articles

HB-GAM/Pleiotrophin but Not RIHB/Midkine Enhances Chondrogenesis in Micromass Culture

The heparin-binding growth-associated molecule HB-GAM (also named pleiotrophin) and the retinoic acid-induced heparin-binding protein RIHB (chicken midkine) are developmentally regulated proteins forming a new family of heparin-binding molecules with putative functions during cell growth and differentiation. A direct involvement of these molecules during chondrogenesisin vivowas suggested by their patterns of expression. The putative chondrogenic activity of these molecules was investigatedin vitrousing micromass cultures from chicken limb bud mesenchymal cells. Exogenous HB-GAM, not RIHB, was found to enhance chondrogenesis in this system. These results provide a strong incentive for considering and further investigating the role of this protein in the control of limb cartilage differentiation.

Cortactin-Src Kinase Signaling Pathway Is Involved in N-syndecan-dependent Neurite Outgrowth

N-syndecan (syndecan-3) was previously isolated as a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and suggested to mediate the neurite growth-promoting signal from cell matrix-bound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. In the present study, we have transfected N18 neuroblastoma cells with a rat N-syndecan cDNA and show that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinase-active fractions that bind to the cytosolic moiety of N-syndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. We suggest that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway.

High Affinity Binding and Overlapping Localization of Neurocan and Phosphacan/Protein-tyrosine Phosphatase-ζ/β with Tenascin-R, Amphoterin, and the Heparin-binding Growth-associated Molecule

We have studied the interactions of the nervous tissue-specific chondroitin sulfate proteoglycans neurocan and phosphacan with the extracellular matrix protein tenascin-R and two heparin-binding proteins, amphoterin and the heparin-binding growth-associated molecule (HB-GAM), using a radioligand binding assay. Both proteoglycans show saturable, high affinity binding to tenascin-R with apparent dissociation constants in the 2-7 nM range. Binding is reversible, inhibited in the presence of unlabeled proteoglycan, and increased by approximately 60% following chondroitinase treatment of the proteoglycans, indicating that the interactions are mediated via the core (glyco)proteins rather than by the glycosaminoglycan chains, which may in fact partially shield the binding sites. In contrast to their interactions with tenascin-C, in which binding was decreased by approximately 75% in the absence of calcium, binding of phosphacan to tenascin-R was not affected by the absence of divalent cations in the binding buffer, although there was a small but significant decrease in the binding of neurocan. Neurocan and phosphacan are also high affinity ligands of amphoterin and HB-GAM (Kd = 0.3-8 nM), two heparin-binding proteins that are developmentally regulated in brain and functionally involved in neurite outgrowth. The chondroitin sulfate chains on neurocan and phosphacan account for at least 80% of their binding to amphoterin and HB-GAM. The presence of amphoterin also produces a 5-fold increase in phosphacan binding to the neural cell adhesion molecule contactin. Immunocytochemical studies showed an overlapping localization of the proteoglycans and their ligands in the embryonic and postnatal brain, retina, and spinal cord. These studies have therefore revealed differences in the interactions of neurocan and phosphacan with the two major members of the tenascin family of extracellular matrix proteins, and also suggest that chondroitin sulfate proteoglycans play an important role in the binding and/or presentation of differentiation factors in the developing central nervous system.

Morphologic changes suggesting abnormal renal differentiation in congenital nephrotic syndrome.

Retrograde differentiation (or dedifferentiation) has recently been proposed as a pathogenetic mechanism involved also in various renal diseases. Here we studied whether evidence of these mechanisms can be found in the kidneys of patients with congenital nephrotic syndrome of the Finnish type (CNF). These patients show isolated massive proteinuria but no primary symptoms from any other organ systems. For the analysis we used antibody markers of early (fibronectin, stem cell factor, Wilms' tumor gene product, cytokeratin) and later (laminin, midgestation and kidney, heparin binding growth-associated molecule) stages of nephron differentiation as well as for apoptosis (acridine orange staining), rescue from apoptosis (anti-Bcl-2 antibodies) and cell proliferation (antibodies to proliferating cell nuclear antigen). In the peritubular spaces atypically organized areas were found which appeared positive with markers of low stages of differentiation, but neither abnormal cell proliferation nor activation of the apoptotic pathway could be detected. As morphologic signs of abnormal tissue organization, we found clusters of tightly compacted and large glomeruli corresponding to the size of two to three normal glomeruli. However, all individual glomerular cell compartments (mesangial, endothelial, visceral epithelial cells) appeared balanced in relative cell numbers. Together these results may indicate abnormal early mesenchymoepithelial tissue interaction leading to excessive and poorly organized formation of glomeruli. This could be causally related also to the serious functional immaturity of CNF kidneys presented as isolated proteinuria.

HB-GAM/pleiotrophin: localization of mRNA and protein in the chicken developing leg.

The heparin-binding growth-associated molecule HB-GAM (also named pleiotrophin) is a developmentally-regulated protein that belongs to a new family of heparin-binding molecules with putative functions during cell growth and differentiation. In order to study the localization of HB-GAM during chicken embryogenesis, we produced specific monoclonal antibodies to this factor. HB-GAM protein is first observed at stage 23 in the developing nervous system and later in the forming cartilage. We present an investigation of the HB-GAM mRNA expression and HB-GAM protein distribution in the developing leg by in situ hybridization and immunocytochemical studies. We focused our attention on the development of the tibia, where the HB-GAM protein appears at stage 27-28, i.e., just after the condensation of the mesodermal precursor cells of the chondrocytes. The protein then progressively accumulates in the central part of the embryonic cartilage (diaphysis). It persists until stage 42-44 in the regions where hypertrophic cartilage is being replaced by bone marrow. In contrast to the protein, the transcript is first detected at stage 26-27 and later expressed essentially in the epiphysis until stage 37. Therefore the localization of the mRNA does not parallel that of the protein and our data suggest a long half-life of the protein in the hypertrophic cartilage. In addition, the layer of stacked cells surrounding the cartilage core (usually considered as the osteoprogenitor cells) clearly expresses the HB-GAM message between stages 30-37 whereas differentiated osteoblasts do not. Furthermore, the distribution of HB-GAM protein in the osteoblast/osteoid layer suggests an involvement of this protein in early steps of osteogenesis. HB-GAM is absent from the newly formed bone.

N-syndecan and HB-GAM (heparin-binding growth-associated molecule) associate with early axonal tracts in the rat brain.

Heparin-Binding Growth-Associated Molecule (HB-GAM)/pleiotrophin is an 18 kDa extracellular matrix- and cell-surface-associated protein shown to enhance neurite outgrowth of perinatal forebrain neurones in vitro. The heparan sulphate proteoglycan N-syndecan (Raulo et al., 1994) has been isolated as a receptor/coreceptor for the HB-GAM. We have investigated, whether HB-GAM and N-syndecan could have a similar role in neurite outgrowth and axon guidance in early axonal tracts of brain. In the present study N-syndecan was found to be spatiotemporally associated with the developing axonal tracts already on embryonic day 9 in rat, as revealed by coexpression with class III beta-tubulin, which is one of the earliest neuronal markers (Easter et al., 1993; Brittis et al., 1995). Later, N-syndecan and HB-GAM were detected in the first afferent serotonergic projections arising from the pontine raphe nuclei. The expression pattern of HB-GAM peaked in the developing rhombencephalon at embryonic stage (E) 13-14. At the same time, N-syndecan was expressed in the developing raphe neurones growing neurites towards the diencephalon along HB-GAM immunoreactive pathways. When rhombencephalic neurones were cultured on decreasing concentrations of substrate-bound HB-GAM, E13 neurones showed a significantly better neurite outgrowth response than E11, E16 or E18 neurones. The neurite outgrowth of raphe neurones in vitro was inhibited by adding soluble heparin or N-syndecan into the culture medium, whereas addition of chondroitin sulphate had no effect. In a simple pathway assay, E13 raphe neurones selectively preferred attaching and growing neurites on pathways containing HB-GAM as compared with regions containing either laminin or fibronectin alone. Our results suggest that HB-GAM may function as a developmentally regulated cue for rhombencephalic neurones that possess N-syndecan on their cell membrane.

Regulation of mRNA localization by transmembrane signalling: local interaction of HB-GAM (heparin-binding growth-associated molecule) with the cell surface localizes beta-actin mRNA.

Localization of mRNAs is currently thought to be partially responsible for molecular sorting to specific compartments within the cell. In mammalian cells the best-studied example is the beta-actin mRNA that is localized to the cell processes, and its localization is necessary in migratory responses of cells. It is reasonable to assume that mRNA localization within cells is coupled to transmembrane signalling due to extracellular factors, but little is known about such putative mechanisms. We show here that HB-GAM, an extracellular matrix-associated factor that enhances migratory responses in cells, is able to localize beta-actin mRNA when locally applied to cells via microbeads. The HB-GAM-induced mRNA localization is specifically inhibited by low concentrations of heparin and by heparitinase treatment of cells, showing that cell-surface heparin-type glycans are required for the effect. The finding that soluble N-syndecan is also inhibitory suggests that the transmembrane proteoglycan N-syndecan, previously identified as an HB-GAM receptor, is involved in the mRNA-localizing effect of HB-GAM. Inhibition of the mRNA localization by the src-kinase inhibitor PP1 is compatible with an N-syndecan-mediated effect since the receptor function of N-syndecan has been recently found to depend on the src-kinase signalling pathway. The mRNA-localizing activity of N-syndecan is also suggested by the finding that affinity-purified anti-N-syndecan antibodies coated on microbeads are able to localize beta-actin mRNA.

Regulation of mRNA localization by transmembrane signalling: Local interaction of HB-GAM (heparin-binding growth-associated molecule) with the cell surface localizes β-actin mRNA

ABSTRACT Localization of mRNAs is currently thought to be partially responsible for molecular sorting to specific compartments within the cell. In mammalian cells the best-studied example is the β-actin mRNA that is localized to the cell processes, and its localization is necessary in migratory responses of cells. It is reasonable to assume that mRNA localization within cells is coupled to transmembrane signalling due to extracellular factors, but little is known about such putative mechanisms. We show here that HB- GAM, an extracellular matrix-associated factor that enhances migratory responses in cells, is able to localize β- actin mRNA when locally applied to cells via microbeads. The HB-GAM-induced mRNA localization is specifically inhibited by low concentrations of heparin and by heparitinase treatment of cells, showing that cell-surface heparin-type glycans are required for the effect. The finding that soluble N-syndecan is also inhibitory suggests that the transmembrane proteoglycan N-syndecan, previously identified as an HB-GAM receptor, is involved in the mRNA-localizing effect of HB-GAM. Inhibition of the mRNA localization by the src-kinase inhibitor PP1 is compatible with an N-syndecan-mediated effect since the receptor function of N-syndecan has been recently found to depend on the src-kinase signalling pathway. The mRNA- localizing activity of N-syndecan is also suggested by the finding that affinity-purified anti-N-syndecan antibodies coated on microbeads are able to localize β-actin mRNA.

Effect of heparin-binding growth-associated molecule (HB-GAM) on synaptic transmission and early LTP in rat hippocampal slices.

Heparin-binding growth-associated molecule (HB-GAM) is an 18-kDa developmentally regulated protein, which promotes neurite outgrowth, axonal guidance and synaptogenesis through interaction with cell-surface heparan-sulphate proteoglycans. We have studied the effect of HB-GAM on synaptic transmission and long-term potentiation (LTP) in the area CA1 of rat hippocampal slices, where HB-GAM mRNA is expressed in an activity-dependent manner. Injection of recombinant HB-GAM into the dendritic area inhibited tetanus-induced LTP without affecting baseline synaptic responses or the N-methyl-D-aspartate (NMDA)-receptor mediated transmission. HB-GAM did not depotentiate tetanus-induced LTP or prevent heterosynaptic LTP induced by application of tetraethylammonium (TEA), indicating that the effect was limited to early, synapse-specific stages of LTP induction. These results suggest that HB-GAM is involved in the regulation of synaptic plasticity in hippocampus.

Differential expression and biological activity of the heparin-binding growth-associated molecule (HB-GAM) in lung cancer cell lines.

The growth of human lung cancer cells is regulated positively and negatively by a variety of growth factors through autocrine as well as paracrine mechanisms. In the present report, we studied the differential role and expression of a neuropolypeptide growth factor in 26 lung cancer cell lines. Expression of the heparin-binding growth-associated molecule (HB-GAM) in 12 small cell lung cancer (SCLC) cell lines was compared to that in 14 non-small cell lung cancer (NSCLC) cell lines. HB-GAM mRNA was expressed in 9 of 12 SCLC and 3 of 14 NSCLC cell lines as determined by RT-PCR analyses. Normal human bronchial epithelial cells were used as negative controls. All cell lines which expressed HB-GAM mRNA produced HB-GAM protein as well. Western blot analysis showed that the tumor cells secreted HB-GAM into the media. HB-GAM, purified from lung cancer cell lines, exerted biological activity on fibroblasts, endothelial cells and SW13 cells as determined by thymidine incorporation and soft agar cloning assays. In addition, the biological activity of HB-GAM was blocked by a specific antibody in a dose-dependent way. Our findings suggest that HB-GAM may serve as a marker for SCLC cell lines and that it may function as a paracrine growth factor in human lung cancer. HB-GAM may be a further member of the network of growth factors involved in proliferation, angiogenesis and metastasis of lung tumors.

Differential expression and biological activity of the heparin‐binding growth‐associated molecule (HB‐GAM) in lung cancer cell lines

Differential expression and biological activity of the heparin‐binding growth‐associated molecule (HB‐GAM) in lung cancer cell lines

A Role of Midkine in the Development of the Neuromuscular Junction

Midkine (MK) is a member of a family of developmentally regulated neurotrophic and heparin-binding growth factors. It is expressed during the midgestation period in a retinoid-acid dependent manner during embryogenesis in the mouse.In vitro,it promotes neurite outgrowth from spinal cord neurons and cell migration. Its expression is strongest in the central nervous system, thus suggesting a function for this protein in neural development. In this study, the role of MK in synaptogenesis was examined in the Xenopus system. A Xenopus MK cDNA was cloned from an embryonic library encompassing neurulation and synaptogenesis stages. By Northern blot analysis, MK mRNA was detected from the onset of neurulation and throughout the stages of synaptogenesis in the Xenopus embryo. This suggests that MK is also an important growth regulator in Xenopus embryogenesis. To study the function of MK in the development of the neuromuscular junction (NMJ), fusion proteins were made and their ability to induce the formation of acetylcholine receptor (AChR) clusters in cultured muscle cells was studied. Beads coated with MK strongly induce AChR clustering. When nerve-muscle cocultures were labeled with antibodies made against the MK fusion protein, MK immunoreactivity was detected at the NMJ. Unlike heparin-binding growth-associated molecule (HB-GAM), another member of this growth factor family, MK expression cannot be detected in the muscle but is present in spinal cord neurites. Consistent with thesein vitrodata is the observation that MK mRNA is only localized in the central nervous system but the protein is deposited at the intersomitic junction where the NMJ is locatedin vivo.Exogenously applied MK does bind to the heparan sulfate proteoglycan on the surface of Xenopus muscle cells. Agrin, a heparan-sulfate proteoglycan that induces the formation of AChR clusters in cultured muscle cells, binds strongly to MK. Bath application of MK in conjunction with agrin results in a change in the pattern of AChR clustering induced by agrin alone. These data suggest that MK is a neuron-derived factor that participates in the signal transduction process during NMJ development.

Association of cortactin with developing neuromuscular specializations.

During the development of the neuromuscular junction (NMJ), motoneurons grow to the muscle cell and the nerve-muscle contact triggers the development of both presynaptic specialization, consisting of clusters of synaptic vesicles (SVs), and postsynaptic specialization, consisting of clusters of synaptic vesicles (SVs), and postsynaptic specialization, consisting of clusters of acetylcholine receptors (AChRs). Previous studies have shown that the activation of tyrosine kinases and the local assembly of an actin-based cytoskeletal specialization are involved in the development of both types of specializations. To understand the link between tyrosine phosphorylation and the assembly of the cytoskeleton, we examined the localization of cortactin in relationship to synaptic development. Cortactin is a 80/85 kD F-actin binding protein and is a substrate for tyrosine kinases. It contains a proline-rich motif and an SH3 domain and is localized at sites of active F-actin assembly. Using a monoclonal antibody against cortactin, its localization at developing NMJs in culture was observed. To understand the spatial and temporal relationship between cortactin and developing synaptic structures, cultured muscle cells and spinal neurons from Xenopus embryos were treated with beads coated with heparin-binding growth-associated molecule to induce the formation of AChR clusters and SV clusters and the localization of cortactin was followed by immunofluorescence. In untreated muscle cells, cortactin is often co-localized with spontaneously formed AChR clusters. After cells were treated with beads, cortactin became localized at bead-induced AChR clusters at their earliest appearance (1 h after the addition of beads). This association was most reliably detected at the early stage of the clustering process. On the presynaptic side, cortactin localization could be detected as early as 10 min after the bead-neurite contact was established. Cortactin-enriched contacts later showed concentration of F-actin (at 1 h) and clusters of SVs (at 24 h). These data suggest that cortactin mediates the local assembly of the cytoskeletal specialization triggered by the synaptogenic signal on both nerve and muscle.

The Role of an Agrin–Growth Factor Interaction in ACh Receptor Clustering

The clustering of acetylcholine receptors (AChRs) at the neuromuscular junction is mediated in part by the heparan-sulfate proteoglycan agrin. However, our previous studies have also suggested the role of heparin-binding growth-associated molecule (HB-GAM) in AChR clustering. Here the role of an agrin–HB-GAM interaction in this process was examined using culturedXenopusmuscle cells. Agrin-coated beads were found to be weakly effective while agrin-coated beads further treated with HB-GAM were highly effective in AChR cluster induction. Protein overlay assays showed specific binding of HB-GAM to agrin. In addition, agrin-enriched neuritic tracks bound HB-GAM in a manner that showed a high degree of colocalization between the neural agrin and the applied factor. Finally, the introduction of exogenous HB-GAM together with soluble agrin resulted in the appearance of AChR clusters on the dorsal surface of cells in an agrin isoform-dependent manner; a dramatic change from the characteristic ventral AChR clustering seen in response to agrin alone. These results suggest that agrin may mediate AChR clustering by interacting with muscle-bound heparin-binding growth factors such as HB-GAM.

Role of HB-GAM (Heparin-Binding Growth-Associated Molecule) in Proliferation Arrest in Cells of the Developing Rat Limb and Its Expression in the Differentiating Neuromuscular System

HB-GAM (heparin-binding growth-associated molecule) is a secretory, extracellular matrix-associated protein that was isolated by screening for proteins that enhance neurite outgrowth in perinatal rat brain neurons. In the present study we have investigated the possible role of HB-GAM in cell proliferation in the developing rat limb. Exogenously added recombinant HB-GAM was found to inhibit the proliferation of mesenchymal and epithelial cells in cultured limb buds, as demonstrated by bromodeoxyuridine incorporation and by staining for PCNA (proliferating cell nuclear antigen). The inhibitory effect of HB-GAM on cell proliferation was reversed by heparin, suggesting that HB-GAM may bind to a heparin-type carbohydrate epitope that is required for cell proliferation in the developing limb. Endogenous HB-GAM of the developing limb was found to be expressed in a proximal-to-distal pattern, in agreement with the putative role in proliferation arrest and cell differentiation. In addition, double immunostaining of HB-GAM with PCNA showed that in early (Embryonic Day 12) limb mesenchyme HB-GAM was associated mainly with the surface of growth-arrested cells. Furthermore, HB-GAM was associated with the muscle surface, as demonstrated in double immunostaining of HB-GAM with desmin and myosin heavy chain proteins. Coinciding with the onset of synapse formation (Embryonic Day 16), HB-GAM was found in patches on the muscle cell surface in close proximity to nicotinic acetylcholine receptor clusters. This finding is in agreement with a previous study that has suggested a role for HB-GAM in the differentiation of the neuromuscular junction in Xenopus muscle.

Developmentally Regulated Neurite Outgrowth Response from Dorsal Root Ganglion Neurons to Heparin‐binding Growth‐associated Molecule (HB‐GAM) and the Expression of HB‐GAM in the Targets of the Developing Dorsal Root Ganglion Neurites

Heparin-binding growth-associated molecule (HB-GAM) is a highly conserved cell surface- and extracellular matrix-associated protein that enhances neurite outgrowth in brain neurons in vitro. To study the possible response of peripheral neurons, we cultured chicken dorsal root ganglion neurons from different developmental stages from embryonic day 4.5 (E4.5; St 25) to E9 (St 35) on recombinant HB-GAM. We discovered that the neurite outgrowth response to HB-GAM is maximal at E5.5-6.5 (St 28-30). In order to correlate this in vitro phenomenon with in vivo phenomena, immunohistochemical staining and in situ hybridization were performed on cryosections. The protein expression of HB-GAM peaked at E6 (St 29) and was most extensive on the dorsal spinal cord and dorsal roots. Using Dil labelling, we confirmed that at the time when sensory afferents travel longitudinally in the bundle of His of the spinal cord, HB-GAM protein expression there is at its peak. Though HB-GAM is a secreted protein, at the RNA level the timing of HB-GAM appearance and existence in the spinal cord and sensory ganglia is in accordance with its protein expression. Our results demonstrate that peripheral neurons are responsive to substrate-bound HB-GAM in a developmentally regulated manner, and that the expression of both HB-GAM mRNA and protein in vivo is spatially and temporally matched to this in vitro phenomenon. HB-GAM is therefore a putative cue for the growth of sensory afferents to and within the dorsal spinal cord.

Activity-induced enhancement of HB-GAM expression in rat hippocampal slices.

Heparin-binding growth-associated molecule (HB-GAM) is a developmentally regulated secretory protein with neurite outgrowth-promoting activity. High-frequency stimulation leading to induction of long-term potentiation (LTP) resulted in increased expression of HB-GAM in rat hippocampal area CA1. When tetanization was given in the presence of antagonists of the N-methyl-D-aspartate (NMDA) receptor and postsynaptic voltage-gated calcium channels, the mRNA level was comparable to control levels. The results indicate that high frequency stimulation inducing LTP results in calcium-dependent enhancement in HB-GAM expression, and imply a role for this extracellular protein in the modulation of synaptic function in the hippocampus.

155 HB-GAM (heparin-binding growth associated molecule) and n-syndecan play a role in the formation of thalamocortical connections ?

155 HB-GAM (heparin-binding growth associated molecule) and n-syndecan play a role in the formation of thalamocortical connections ?

HB-GAM is a cytokine present in Alzheimer's and Down's syndrome lesions.

The distribution of heparin binding growth associated molecule (HB-GAM) in the cerebral amyloidoses of Alzheimer's disease (AD) and Down's syndrome (DS), conditions characterized by the deposition of amyloid beta (A beta), was investigated immunohistochemically. Antibodies to HB-GAM, a cytokine which plays an important role in brain development and maturation, showed strong immunoreactivity with senile plaques in both AD and DS. Anti-HB-GAM reacted with pre-amyloid lesions, but only when markers of dystrophic neurites were present. The presence of HB-GAM in AD brains, but not in control brains, was confirmed by Western blotting. We suggest that the presence of HB-GAM in A beta lesions is a marker of neuronal injury.

Neurite Outgrowth in Brain Neurons Induced by Heparin-binding Growth-associated Molecule (HB-GAM) Depends on the Specific Interaction of HB-GAM with Heparan Sulfate at the Cell Surface

Heparin-binding growth-associated molecule (HB-GAM) is a cell-surface- and extracellular matrix-associated protein that lines developing axons in vivo and promotes neurite outgrowth in vitro. Because N-syndecan (syndecan-3) was found to function as a receptor in HB-GAM-induced neurite outgrowth, we have now studied whether the heparan sulfate side chains of N-syndecan play a role in HB-GAM-neuron interactions. N-Syndecan from postnatal rat brain was found to inhibit HB-GAM-induced but not laminin-induced neurite outgrowth when added to the assay media. The inhibitory activity was abolished by treating N-syndecan with heparitinase, but it was retained in N-syndecan-derived free glycosaminoglycan chains, suggesting that N-syndecan heparan sulfate at the cell surface is involved in HB-GAM-induced neurite outgrowth. Binding to HB-GAM and inhibition of neurite outgrowth was observed with heparin-related polysaccharides only; galactosaminoglycans were inactive. Significant inhibition of neurite outgrowth was induced by heparin and by N-syndecan heparan sulfate but not by heparan sulfates from other sources. A minimum of 10 monosaccharide residues were required for HB-GAM-induced neurite outgrowth. Experiments with selectively desulfated heparins indicated that 2-O-sulfated iduronic acid units, in particular, are of importance to the interaction with HB-GAM, were implicated to a lesser extent. Structural analysis of N-syndecan from 6-day-old rat brain indicated that the heparan sulfate chains contain sequences of contiguous, N-sulfated disaccharide units with an unusually high proportion (82%) of 2-O-sulfated iduronic acid residues. We suggest that this property of N-syndecan heparan sulfate is essential for HB-GAM binding and induction of neurite outgrowth.