Purification of a meningeal cell-derived chondroitin sulphate proteoglycan with neurotrophic activity for brain neurons and its identification as biglycan.
Serum-free cultures of meningeal fibroblasts synthesize and release a chondroitin sulphate proteoglycan (CSPG) that markedly enhances survival but not adhesion of embryonic rat (embryonic day 15) neocortical neurons in vitro. The active molecule was purified from conditioned medium (meningeal cell-conditioned medium, MCM) in three steps by means of fast-performance liquid chromatography fractionation combined with a quantitative microphotometric bioassay: (i) preparative Q-Sepharose anion exchange chromatography under native conditions; (ii) rechromatography of biologically active Q-Sepharose fractions on a MonoQ column in the presence of 8 M urea; and (iii) final gel filtration of active MonoQ fractions on Superose 6 in the presence of 4 M guanidinium hydrochloride. Analytical sodium dodecyl sulphate-polyacrylamide gradient gel electrophoresis of active Superose 6 fractions revealed a single broad glycoprotein band with a molecular mass in the range of 220-340 kDa. Further characterization of the purified molecule with glycosaminoglycan:lyases revealed a core protein of 50 kDa and the nearly complete loss of neurotrophic activity after chondroitinase digestion, whereas heparitinase treatment changed neither electrophoretic mobility nor biological activity. Amino-terminal sequencing of the purified CSPG core protein revealed identity with the amino acid sequence of rat biglycan. Biglycan purified from bovine cartilage supported neuron survival with virtually the same activity as the CSPG purified from MCM (half-maximal activity approximate to 10(-8) M). In conclusion, we isolated a neurotrophic CSPG from meningeal cells with strong survival-enhancing activity for brain neurons that was identified as biglycan, a molecule not previously related to neural functions.
Junghans
U
U
Department of Neurology, University of Düsseldorf, Germany.
Koops
A
A
Westmeyer
A
A
Kappler
J
J
Meyer
H E
HE
Müller
H W
HW
D000818:Animals; D058578:Biglycan; D001921:Brain / Q000378:metabolism; D002470:Cell Survival; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D005347:Fibroblasts / Q000378:metabolism*; D007150:Immunohistochemistry; D008578:Meninges / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000378:metabolism; D011509:Proteoglycans / Q000032:analysis* / Q000378:metabolism*; D051381:Rats
C546886:Bgn protein, rat; D058578:Biglycan; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D011509:Proteoglycans; D011133:Polysaccharide-Lyases