| The transfer of xylose to specific serine residues in the core protein of proteoglycans is the rate limiting and the initiating step in the proteoglycan biosynthesis pathway. This important step is catalyzed by xylosyltransferase (Xylt). Two xylosyltransferase isoforms have been identified in vertebrates, Xylt1 and Xylt2; however enzymatic activity has not been defined for Xylt2. We have cloned the full length murine Xylt2 and made use of xylosyltransferase-deficient CHO cells to express and characterize the activity of Xylt2. We found Xylt2 is capable of modifying both chondroitin sulfate and heparan sulfate proteoglycans, thereby effectively rescuing the mutant CHO cell phenotype. By generating N-terminal truncation mutants and a Xylt1-Xylt2 N-terminal hybrid, we found a potential regulator region in Xylt2 for aa 33-78, which distinguishes it from Xylt1. Our work has established that Xylt2 is an active transferase that appears to non-specifically and non-preferentially modify both chondroitin sulfate and heparan sulfate proteoglycans; and as well may impart some regulation at the initiating step of proteoglycan biosynthesis. We have also exploited the zebrafish as a model to explore the developmental functions of Xylt and address directly the roles of proteoglycans during early vertebrate development. Morpholino knockdown did not reveal a requirement for zebrafish xylt2 ; by contrast, xylt1 is required for normal development of the zebrafish embryo. Xylt1-deficient embryos have reduced proteoglycan synthesis and show defects in a variety of developing structures, including fins, somites and the nervous system, reminiscent of phenotypes associated with altered Hedgehog (Hh) signaling. In the hindbrain of Xylt1-deficient embryos we find reduced Hh signaling and concomitant alterations in dorsoventral patterning, as well as reduced proliferation. By contrast, spinal cord patterning is relatively normal, but changes in somite patterning reflect elevated Hh signaling. We conclude that proteoglycans, generated by Xylts, are necessary for proper patterning of the nervous system and somites, in part through modulation of Hh signaling, and that signaling regulation via proteoglycans varies with axial level. |
