Amino acid determinants that drive heparan sulfate assembly in a proteoglycan.
To study how cells regulate the composition of glycosaminoglycan chains on proteoglycans, we have examined the assembly of chains on chimeric proteoglycans containing segments of betaglycan (transforming growth factor-beta Type III receptors) fused to protein A. Transient expression of the chimeras in Chinese hamster ovary cells revealed that only two glycosaminoglycan attachment sites exist. One site at Ser535 supported both chondroitin sulfate and heparan sulfate synthesis, whereas the site at Ser546 supported only chondroitin sulfate. The compositions of the chimeras were the same in CHO-K1, CHOP-C4, BHK-21, and HeLa S3 cells and in chimeras containing polyhistidine fused to the C terminus. Deletion experiments showed that the assembly of heparan sulfate chains on the chimeras required a peptide segment of < or = 16 amino acids (SPGDSS535-GWPDGYEDLE) and the first 5 amino acids were not essential. Truncation of the acidic cluster (EDLE), site-directed mutation of the acidic residues in the cluster, or deletion of the sequence between the cluster and the Ser attachment site decreased heparan sulfate assembly. Mutation of Trp537 adjacent to the site also decreased heparan sulfate assembly. More importantly, introducing tryptophan next to three different Ser-Gly dipeptides in betaglycan and syndecan-1 chimeras stimulated assembly of heparan sulfate. Thus, one type of heparan sulfate attachment site consists of a Ser-Gly dipeptide and a flanking cluster of acidic residues. An adjacent tryptophan residue can augment the proportion of heparan sulfate.
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham 35294.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002809:Chondroitin Sulfates / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011509:Proteoglycans / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D018125:Receptors, Transforming Growth Factor beta / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism
D002809:Chondroitin Sulfates; D006497:Heparan Sulfate; D011509:Proteoglycans; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C071736:betaglycan