Recognition of Acceptor Proteins by UDP-D-xylose Proteoglycan Core Protein β-D-Xylosyltransferase

Abstract

The formation of chondroitin sulfate is initiated by xylosyltransferase (XT) transferring xylose from UDPxylose to consensus serine residues of proteoglycan core proteins. Our alignment of 51 amino acid sequences of chondroitin sulfate attachment sites in 19 different proteins resulted in a consensus sequence for the recognition signal of XT. The complete recognition sequence is composed of the amino acids a-a-a-a-G-S-G-a-b-a, with a ‫؍‬ E or D and b ‫؍‬ G, E, or D. This sequence was confirmed by determination of the Michaelis-Menten constants for in vitro xylosylation of different synthetic proteins and peptides using an enriched XT preparation from conditioned cell culture supernatant of human chondrocytes. The highest acceptor activity was determined by the sequence Q-E-E-E-E-G-S-G-G-G-Q, which was found in the single chondroitin sulfate attachment site of bikunin, the inhibitory active component of the human inter-␣-trypsin inhibitor.We determined the Michaelis-Menten constant (K m ) of xylosylation of the synthetic bikunin analogous peptide Q-E-E-E-G-S-G-G-G-Q-K to be 22 M, which was 9-fold decreased in comparison to deglycosylated core protein from bovine cartilage (188 M), which was previously used as acceptor for the XT activity assay. The best XT acceptors were nonglycosylated recombinant wildtype bikunin (K m ‫؍‬ 0. These results imply that the primary structure of the acceptor is not the only determinant for recognition by xylosyltransferase. Thus, protein conformation is also a main factor in determining xylosylation.

Differential Roles of TwoN-Acetylgalactosaminyltransferases, CSGalNAcT-1, and a Novel Enzyme, CSGalNAcT-2

“…Another possibility is the effect of the peptide sequence of the core protein on the enzyme recognition. A currently proposed GAG attachment motif is the Ser-Gly-Ser-Gly sequence and surrounding acidic amino acids (39,40). CSGalNAcT-2 might recognize CS binding peptide sequences of core proteins other than syndecan-4.…”

The chondroitin sulfate form of invariant chain trimerizes with conventional invariant chain and these complexes are rapidly transported from thetrans-Golgi network to the cell surface

“…Studies have suggested that xylosyltransferase is the rate-limiting enzyme in glycosaminoglycan addition [30,43]. Only a small percentage of Ii is normally modified by CS addition, potentially due to deviation of the Ii sequence from the xylosylation consensus sequence [44]. Modification of the Ii sequence to conform more closely to the consensus sequence (Figure 1) resulted in approx.…”

“…3-fold greater levels of Ii-CS. Modification of the amino acids surrounding the xylose-accepting serine residue probably changed the conformation of the protein, potentially affecting the activity of xylosyltransferase, which has been shown to be dependent on protein conformation [44]. However, it is also possible that changing the primary structure around the xylose-accepting serine residue could also affect the activity of other enzymes needed to complete the tetrasaccharide linkage region, thus increasing glycosaminoglycan addition.…”

Evidence for a glycosaminoglycan on the nudel protein important for dorsoventral patterning of the drosophila embryo

“…The mutations did not appear to affect other acidic species that were perturbed by nitrous acid treatment, suggesting that Nudel contains other sites for GAG attachment. Near the three mutated sites, Nudel contains three copies of the sequence SG, which has been shown to be sufficient for GAG attachment (Brinkmann et al, 1997).…”