THE BIOCHEMICALLY AND IMMUNOLOGICALLY DISTINCT CSPG OF NOTOCHORD IS APRODUCT OF THE AGGRECAN GENE

Using the monoclonal antibody S103L, which reacts specifically with an epitope in the chondroitin sulfate-rich domain of the chick cartilage chondroitin sulfate proteoglycan (CSPG) core protein, we have identif ied the predominant CSPG expressed by notochord. This large notochord CSPG is first detected immunohistochemically as early as stage 16, lon g before chondrogenesis occurs, and is expressed continuously during t he time of active neural crest migration and through the onset of scle rotomal differentiation. Because of the cross-reactivity of both notoc hord and cartilage CSPGs with the S103L antibody, extensive molecular and biochemical analysis of the two CSPGs was carried out. Striking di fferences distinguish the notochord and cartilage (aggrecan) CSPGs at the level of posttranslational modification. Notably, cartilage aggrec an carries a significant content of keratan sulfate (KS) chains, while the notochord CSPG is devoid of KS. In contrast, cartilage aggrecan l acks the HNK-1 epitope, while the notochord CSPG has a high content of HNK-1. Three different approaches were used to establish the relation ship of the two CSPGs at the molecular level. Northern blot analysis, using aggrecan probes, detected same-sized messages from notochord and cartilage RNA. Overlapping fragments, generated by RT-PCR using prime rs covering 98% of the entire coding sequence from the known cartilage structure, were of identical size in notochord and cartilage. Taking advantage of our recent studies, which demonstrated a single base chan ge in the aggrecan gene resulting in conversion of Glu to a STOP codon in exon 12 of chick aggrecan as the molecular basis of the defect nan omelia, we demonstrated that the same mutation was present in notochor d mRNA from nanomelic chicks. These results provide evidence that the chick aggrecan gene is expressed very early in development in notochor d and confirm that the core proteins expressed in chick notochord and cartilage are derived from the same gene. These findings strongly supp ort the hypothesis that the final structural characteristics of each p roteoglycan are determined not only by the core protein but also by ti ssue-specific, developmentally regulated posttranslational mechanisms, functioning within the context of the requirement for specific extrac ellular matrices. (C) 1995 Academic Press, Inc.