6B4 Proteoglycan/Phosphacan, an Extracellular Variant of Receptor-like Protein-tyrosine Phosphatase ζ/RPTPβ, Binds Pleiotrophin/Heparin-binding Growth-associated Molecule (HB-GAM)

doi:10.1074/jbc.271.35.21446

6B4 Proteoglycan/Phosphacan, an Extracellular Variant of Receptor-like Protein-tyrosine Phosphatase ζ/RPTPβ, Binds Pleiotrophin/Heparin-binding Growth-associated Molecule (HB-GAM)*

From the Division of Molecular Neurobiology, National Institute for Basic Biology, and the Department of Molecular Biomechanics, The Graduate University for Advanced Studies, Okazaki 444, Japan

^‡ To whom correspondence should be addressed:
Division of Molecular Neurobiology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji-cho, Okazaki 444, Japan
. Tel.: 81-564-55-7590; Fax: 81-564-55-7595.

Abstract

A major chondroitin sulfate proteoglycan in the brain, 6B4 proteoglycan/phosphacan, corresponds to the extracellular region of a receptor-like protein-tyrosine phosphatase, PTPζ/RPTPβ. 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 (K_d = 3 n) and high (K_d = 0.25 n) affinity binding sites. Chondroitinase ABC digestion of the proteoglycan decreased the binding affinities to a single value (K_d = 13 n) 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 μg/ml). Immunofluorescence and immunoblotting analyses indicated that both 6B4 proteoglycan and PTPζ 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.

Footnotes

↵* This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan and from Mizutani Foundation for Glycoscience. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PBS

phosphate-buffered saline

PTP

protein-tyrosine phosphatase

CAH

carbonic anhydrase

HB-GAM

heparin-binding growth-associated molecule

BSA

bovine serum albumin

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

PMSF

phenylmethylsulfonyl fluoride

PAGE

polyacrylamide gel electrophoresis

ELISA

enzyme-linked immunosorbent assay.
↵² H. Hamanaka, N. Maeda, and M. Noda, unpublished observation.
↵³ T. Nishiwaki, N. Maeda, and M. Noda, unpublished observation.
- Received March 8, 1996.
- Revision received May 20, 1996.