A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for > 96% of the glycosaminoglycan chains in the adult brain proteoglycans. The 1D1, 3H1, and 3F8 proteoglycans all contain 3-sulfated HNK-1 carbohydrate epitopes as well as the novel O-glycosidic mannose-linked oligosaccharides which we have previously characterized in the chondroitin sulfate proteoglycans of brain, and these glycans show different developmental patterns in the three proteoglycans. Two other monoclonal antibodies, 5F3 and 3H7, react on immunoblots of chondroitinase-treated proteoglycans with core glycoproteins in the 350-450 and 61-105-kDa ranges, respectively. Immunocytochemical studies demonstrated that, like the total population of soluble chondroitin sulfate proteoglycans of brain whose localization we have previously examined using polyclonal antibodies, staining with all five monoclonal antibodies is generally most intense in the prospective white matter and absent from the external granule cell layer of early postnatal cerebellum, while in adult brain staining is strongest in the molecular layer and also present in the granule cell layer. However, 3F8 proteoglycan staining is most intense in the molecular layer of 7-day cerebellum, and the 5F3 and 3H7 antibodies are unusual insofar as they also stain Purkinje cells and strongly stain astrocytes in the granule cell layer. Immunoelectron microscopy using the 1D1 and 3H1 monoclonal antibodies demonstrated the extracellular location of these proteoglycans in 7-day brain, whereas predominantly intracellular (cytoplasmic) staining was seen in adult cerebellum.
