In contrast to the functional role of heparan sulfate proteoglycans (HSPGs), the importance of chondroitin sulfate proteoglycans (CSPGs) in modulating signaling pathways involving hedgehog proteins, wingless-related proteins and fibroblast growth factors remains unclear. To elucidate the importance of sulfated CSPGs in signaling paradigms required for endochondral bone formation, the brachymorphic (bm) mouse was used as a model for undersulfated CSPGs. The bm mouse exhibits a postnatal chondrodysplasia caused by a mutation in the phosphoadenosine phosphosulfate (PAPS) synthetase (Papss2) gene, leading to reduced levels of PAPS and undersulfated proteoglycans. Biochemical analysis of the glycosaminoglycan (GAG) content in bm cartilage via sulfate labeling and fluorophore-assisted carbohydrate electrophoresis revealed preferential undersulfation of chondroitin chains (CS) and normal sulfation of heparan sulfate chains. In situ hybridization and immunohistochemical analysis of bm limb growth plates showed diminished Indian hedgehog (Ihh) signaling and abnormal Ihh protein distribution in the extracellular matrix. Consistent with the decrease in hedgehog signaling, BrdU incorporation exhibited a significant reduction in chondrocyte proliferation. Direct measurements of Ihh binding to defined GAG chains demonstrated that Ihh interacts with CS, particularly chondroitin-4-sulfate. Furthermore, co-immunoprecipitation experiments showed that Ihh binds to the major cartilage CSPG aggrecan via its CS chains. Overall, this study demonstrates an important function for CSPGs in modulating Ihh signaling in the developing growth plate, and highlights the importance of carbohydrate sulfation in regulating growth factor signaling.
Comparison of the amino acid sequences of three different proteoglycan core proteins reveals a 12-amino acid sequence that is about 50% homologous among these proteoglycans. In each of the proteoglycans, this sequence surrounds the serine-glycine dipeptide in which the serine is known or presumed to be substituted with a chondroitin/dermatan sulfate glycosaminoglycan chain. Peptides containing this sequence from two proteoglycans were examined for their ability to serve as acceptors for xylosyltransferase, the enzyme that begins the assembly of glycosaminoglycan chains.Those peptides corresponding to amino acid sequences known to contain glycosaminoglycan-substituted serine residues in the protein were efficient xylosyltransferase acceptors, whereas peptides from sequences with no glycosaminoglycan-substituted serine residues were not. Amino acid substitutions at four critical sites in the acceptor peptides showed that single substitutions could completely abolish acceptor activity or greatly reduce it. The results suggest that the proteoglycan recognition consensus sequence for the attachment of glycosaminoglycans to core proteins consists of acidic amino acids closely followed by the tetrapeptide Ser-Gly-Xaa.Gly, where Xaa is any amino acid. The signal appears to be contained in the primary sequence information. In this regard it resembles a number of other signals for protein processing and intracellular routing.
Sulfation is critical to the function of a wide variety of biomolecules. This common modification requires the enzymatic synthesis of an activated sulfate donor, phosphoadenosine-phosphosulfate (PAPS). In higher organisms PAPS synthesis is catalyzed by a bifunctional sulfurylase kinase (SK) polypeptide having both ATP-sulfurylase and adenosine-phosphosulfate kinase activities. We report the identification of a gene family encoding murine SK proteins with these two activities. A family member, SK2 , colocalizes with the locus for the autosomal recessive murine phenotype brachymorphism. Brachymorphic mice have normal lifespans, but abnormal hepatic detoxification, bleeding times, and postnatal growth, the latter being attributed to undersulfation of cartilage proteoglycan. A missense mutation in the SK2 coding sequence of bm mice that alters a highly conserved amino acid residue destroys adenosine-phosphosulfate kinase activity and therefore the ability of SK2 to synthesize PAPS. We conclude that a family of SK genes are responsible for sulfate activation in mammals, that a mutation in SK2 causes murine brachymorphism, and that members of this gene family have nonredundant, tissue-specific roles.
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