title: Composition of perineuronal net extracellular matrix in rat brain: a different disaccharide composition for the net-associated proteoglycans.
abstract: We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs). Adult rat brain was sequentially extracted with Tris-buffered saline (TBS), TBS-containing detergent, 1 m NaCl, and 6 m urea. Extracting tissue sections with these buffers showed that the diffuse and membrane-bound CSPGs were extracted in the first three buffers, but PNN-associated CSPGs remained and were only removed by 6 m urea. Most of the CSPGs were extracted to some degree with all the buffers, with neurocan, brevican, aggrecan, and versican particularly associated with the stable urea-extractable PNNs. The CSPGs in stable complexes only extractable in urea buffer are found from postnatal day 7-14 coinciding with PNN formation. Disaccharide composition analysis indicated a different glycosaminoglycan (GAG) composition for PGs strongly associated with extracellular matrix (ECM). For CS/dermatan sulfate (DS)-GAG the content of nonsulfated, 6-O-sulfated, 2,6-O-disulfated, and 4,6-O-disulfated disaccharides were higher and for heparan sulfate (HS)-GAG, the content of 6-O-sulfated, 2-N-, 6-O-disulfated, 2-O-, 2-N-disulfated, and 2-O-, 2-N-, 6-O-trisulfated disaccharides were higher in urea extract compared with other buffer extracts. Digestions with chondroitinase ABC and hyaluronidase indicated that aggrecan, versican, neurocan, brevican, and phosphacan are retained in PNNs through binding to hyaluronan (HA). A comparison of the brain and spinal cord ECM with respect to CSPGs indicated that the PNNs in both parts of the CNS have the same composition.
authors.affiliation: Centre for Brain Repair, Cambridge University, Forvie Site, Cambridge CB2 2PY, United Kingdom.
authors.forename: Sarama Sathyaseelan
authors.initials: SS
authors.lastname: Deepa
authors.forename: Daniela
authors.initials: D
authors.lastname: Carulli
authors.forename: Clare
authors.initials: C
authors.lastname: Galtrey
authors.forename: Kate
authors.initials: K
authors.lastname: Rhodes
authors.forename: Junko
authors.initials: J
authors.lastname: Fukuda
authors.forename: Tadahisa
authors.initials: T
authors.lastname: Mikami
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Sugahara
authors.forename: James W
authors.initials: JW
authors.lastname: Fawcett
mesh_terms: D000367:Age Factors; D053674:Aggrecans; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D001923:Brain Chemistry; D058581:Brevican; D002021:Buffers; D011508:Chondroitin Sulfate Proteoglycans / Q000032:analysis / Q000302:isolation & purification; D003902:Detergents; D004187:Disaccharides / Q000032:analysis*; D016326:Extracellular Matrix Proteins / Q000032:analysis* / Q000302:isolation & purification*; D006821:Hyaluronoglucosaminidase; D037181:Lectins, C-Type / Q000032:analysis / Q000302:isolation & purification; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000302:isolation & purification; D058580:Neurocan; D009473:Neuronal Plasticity; D011509:Proteoglycans / Q000032:analysis / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5; D013116:Spinal Cord / Q000737:chemistry*; D013302:Streptomyces / Q000201:enzymology
chemical_list: C508321:Acan protein, rat; D053674:Aggrecans; C546938:BCAN protein, human; C546939:Bcan protein, rat; D058581:Brevican; D002021:Buffers; D011508:Chondroitin Sulfate Proteoglycans; D003902:Detergents; D004187:Disaccharides; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D009419:Nerve Tissue Proteins; D058580:Neurocan; D011509:Proteoglycans; C076817:NCAN protein, human; C516895:PTPRZ1 protein, human; C516897:Ptprz1 protein, rat; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5; D006821:Hyaluronoglucosaminidase