6B4 proteoglycan/phosphacan, an extracellular variant of receptor-like protein-tyrosine phosphatase zeta/RPTPbeta, binds pleiotrophin/heparin-binding growth-associated molecule (HB-GAM).
A major chondroitin sulfate proteoglycan in the brain, 6B4 proteoglycan/phosphacan, corresponds to the extracellular region of a receptor-like protein-tyrosine phosphatase, PTPzeta/RPTPbeta. Here, we purified and characterized 6B4 proteoglycan-binding proteins from rat brain. From the CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) extract of brain microsomal fractions, 18-, 28-, and 40-kDa proteins were specifically isolated using 6B4 proteoglycan-Sepharose. N-terminal amino acid sequencing identified the 18-kDa protein as pleiotrophin/heparin-binding growth-associated molecule (HB-GAM). Scatchard analysis of 6B4 proteoglycan-pleiotrophin binding revealed low (Kd = 3 nM) and high (Kd = 0.25 nM) affinity binding sites. Chondroitinase ABC digestion of the proteoglycan decreased the binding affinities to a single value (Kd = 13 nM) without changing the number of binding sites. This suggested the presence of two subpopulations of the proteoglycan with different chondroitin sulfate structures. Heparin potently inhibited binding of 6B4 proteoglycan to pleiotrophin (IC50 = 3.5 ng/ml). Heparan sulfate and chondroitin sulfate C inhibited moderately (IC50 = 150 and 400 ng/ml, respectively), but, in contrast, chondroitin sulfate A and keratan sulfate were poor inhibitors (IC50 > 100 microg/ml). Immunofluorescence and immunoblotting analyses indicated that both 6B4 proteoglycan and PTPzeta are located on cortical neurons. Anti-6B4 proteoglycan antibody added to the culture medium suppressed pleiotrophin-induced neurite outgrowth of cortical neurons. These results suggested that interaction between 6B4 proteoglycan and pleiotrophin is required for the action of pleiotrophin, and chondroitin sulfate chains on 6B4 proteoglycan play regulatory roles in its binding.
Maeda
N
N
Division of Molecular Neurobiology, National Institute for Basic Biology, and the Department of Molecular Biomechanics, The Graduate University for Advanced Studies, Okazaki 444, Japan.
Nishiwaki
T
T
Shintani
T
T
Hamanaka
H
H
Noda
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016501:Neurites; D009474:Neurons / Q000201:enzymology / Q000378:metabolism; D011485:Protein Binding; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011509:Proteoglycans / Q000276:immunology / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D011508:Chondroitin Sulfate Proteoglycans; D016207:Cytokines; D011509:Proteoglycans; C066845:pleiotrophin; C516895:PTPRZ1 protein, human; D017027:Protein Tyrosine Phosphatases; C516897:Ptprz1 protein, rat; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5