title: The ovine newborn and human foetal intervertebral disc contain perlecan and aggrecan variably substituted with native 7D4 CS sulphation motif: spatiotemporal immunolocalisation and co-distribution with Notch-1 in the human foetal disc.
abstract: Composite agarose (1.2 %) polyacrylamide (0.6 %) gel electrophoresis was used to separate discrete populations of native aggrecan and perlecan in newborn to 10 year old ovine intervertebral discs (IVDs). Semi-dry immunoblotting using core-protein and glycosaminoglycan (GAG) side chain specific monoclonal antibodies in combination with chondroitin ABC lyase demonstrated intra-chain native 7-D-4 chondroitin sulphate (CS) sulphation motifs and variable proportions of non-reducing terminal Δ4,5-unsaturated uronate-N-acetylgalactosamine-4-sulphate [2B6(+)] and Δ4,5-unsaturated glucuronate-N-acetylgalactosamine-6-sulphate [3B3(+)] disaccharides. The relative abundance of 2-B-6(+) aggrecan increased with advancing age of the IVD samples while the converse was true for the 3-B-3(+) aggrecan population. Relative 7D4 levels in aggrecan and perlecan were highest in the newborn IVD and significantly lower in the older IVD and other cartilage samples. Quantitation of 7D4 proteoglycan by enzyme linked immunosorbent inhibition assay confirmed the newborn ovine nucleus pulposus (NP) and inner annulus fibrosus (AF) contained higher levels (1.2-1.32 μg 7-D-4-proteoglycan/mg tissue wet weight) than the 2 (0.35-0.42 μg/mg wet weight tissue) and 10 year old IVD samples (0.16-0.22 μg/mg tissue wet weight) with the outer AF zones consistently containing lower levels of 7-D-4 epitope in all cases (P < 0.001). Cell populations on the margins of the AF and cartilaginous vertebral rudiments in newborn ovine and human foetal IVD strongly expressed 7-D-4 CS epitope and perlecan, This was co-distributed with Notch-1 expression in human foetal IVDs consistent with the 7-D-4 CS sulphation motif representing a marker of tissue development expressed by disc progenitor cell populations.
authors.affiliation: Raymond Purves Bone and Joint Research Laboratories, Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, St. Leonards, NSW, 2065, Australia.
authors.forename: Cindy
authors.initials: C
authors.lastname: Shu
authors.forename: Clare
authors.initials: C
authors.lastname: Hughes
authors.forename: Susan M
authors.initials: SM
authors.lastname: Smith
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Smith
authors.forename: Anthony
authors.initials: A
authors.lastname: Hayes
authors.forename: Bruce
authors.initials: B
authors.lastname: Caterson
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Little
authors.forename: James
authors.initials: J
authors.lastname: Melrose
mesh_terms: D053674:Aggrecans / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002240:Carbohydrate Sequence; D002356:Cartilage / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007403:Intervertebral Disc / Q000196:embryology / Q000254:growth & development / Q000378:metabolism*; D009928:Organ Specificity; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D012756:Sheep; D013431:Sulfates / Q000378:metabolism
chemical_list: D053674:Aggrecans; D019812:Heparan Sulfate Proteoglycans; D051881:Receptor, Notch1; D013431:Sulfates; C071927:perlecan