PTP is a receptor-type protein-tyrosine phosphatase that is synthesized as a chondroitin sulfate proteoglycan and uses pleiotrophin as a ligand. The chondroitin sulfate portion of this receptor is essential for high affinity binding to pleiotrophin. Here, we purified phosphacan, which corresponds to the extracellular domain of PTP, from postnatal day 7 (P7) and P12 rat cerebral cortex (PG-P7 and PG-P12, respectively) and from P20 rat whole brain (PG-P20). The chondroitin sulfate of these preparations displayed immunologically and compositionally different structures. In particular, only PG-P20 reacted with the monoclonal antibody MO-225, which recognizes chondroitin sulfate containing the GlcA(2S)␤1-3GalNAc(6S) disaccharide unit (D unit). Analysis of the chondroitinase digestion products revealed that GlcA␤1-3GalNAc(4S) disaccharide unit (A unit) was the major component in these preparations and that PG-P20 contained 1.3% D unit, which was not detected in PG-P7 and PG-P12. Interaction analysis using a surface plasmon resonance biosensor indicated that PG-P20 had ϳ5-fold stronger affinity for pleiotrophin (dissociation constant (K D ) ‫؍‬ 0.14 nM) than PG-P7 and PG-P12, although all these preparations showed similar low affinity binding to pleiotrophin after chondroitinase ABC digestion (K D ‫؍‬ 1.4 ϳ 1.6 nM). We also found that shark cartilage chondroitin sulfate D containing ϳ20% D unit bound to pleiotrophin with moderate affinity (K D ‫؍‬ 2.7 nM), whereas whale cartilage chondroitin sulfate A showed no binding to this growth factor. These results suggest that variation of chondroitin sulfate plays important roles in the regulation of signal transduction in the brain.

Heparan sulfate (HS) binds with various proteins including growth factors, morphogens, and extracellular matrix molecules to regulate their biological functions. These regulatory interactions are considered to be dependent on the structure of HS, which is determined by HS sulfotransferases. To gain insights into the functions of HS sulfotransferases in the development of the nervous system, we examined the expression of these enzymes (3-O-sulfotransferase-1 [3-OST-1], -2, -4; 6-OST-1, -2, -3; and N-deacetylase /N-sulfotransferase-1 [NDST-1], -2, -3) by in situ hybridization and real-time reverse transcription-polymerase chain reaction (RT-PCR). The expression of these genes was spatiotemporally regulated. In the E16 cerebrum, the expression of these genes showed two patterns: (1) selective expression at cortical plate (CP) and ventricular zone (VZ) and (2) wider expression by the cells in the marginal zone (MZ), CP, subplate (SP), and VZ. At P1, most genes showed similar expression patterns, but after P7, these genes were expressed differentially in a layer-specific manner. In the P1 cerebellum, the external granule cell layer (EGL) expressed most genes, the expressions of which were down-regulated at P7. In contrast, Purkinje cells began to express many of these genes after P7. These complex expression patterns suggest that the structure of HS is altered spatiotemporally for regulating various biological activities in the developing brain including the proliferation of neuronal progenitors, extension of axons, and formation of dendrites. We discuss possible functional roles of these sulfotransferases in the signaling of several HS-binding proteins such as fibroblast growth factors, slit, netrin, and sonic hedgehog.