Photoaffmity analogs for the study of glycosaminoglycan biosynthetic enzymes in human myeloma cell lines

Academic Article

Abstract

  • To understand the cellular mechanisms that determine glycosaminoglycan (GAG) structure and function, we are studying several human myeloma B-cell lines known to have variable expression of the heparan sulfate (HS)-proteoglycan, syndecan-1 To begin to identify and characterize HS biosynthetic enyzymes in these cell lines, the photoaffinity analogs 5-azido-UDP-GlcUA, 5-azido-UDP-Glc and 2- or 8-azido-PAP were used Using [32P]5N3UDP-GlcUA and 32[P]5N UDP-Glc, a microsomal protein of 74 kDa has been identified as a putative heparin UDPGlcUA/UDPGIcNAc glycosyltransferase based on photoincorporation of both photoprobes Photolabeling of the 74 kDa protein was Mn2+ dependent, saturable and inhibited by UDP-sugars Cell lines which had low or no syndecan-1 expression had minimal labeling of the 74 kDa protein Partial purification of the transferase with heparin-agarose resulted in enrichment of the 74 kDa protein as indicated by enzymatic assays and photoaffinity labeling Using [32P]N3PAP, photolabeled proteins of 97 kDa and 60 kDa were observed, consistent with reported MWs of heparin sulfotransferases. A new chemical synthesis of photoaffinity analogs for UDP-GlcNAc and UDP-GalNac will also be presented.
  • Author List

  • Sunlhankar P; Dennington L; Rooke A; Sanderson R; Drake R
  • Volume

  • 10
  • Issue

  • 6