Subcellular co-localization and potential interaction of glucuronosyltransferases with nascent proteochondroitin sulphate at Golgi sites of chondroitin synthesis.
Microsomal membranes from chick embryo epiphyseal cartilage were fractionated by equilibrium sucrose-density-gradient centrifugation and assayed for GlcA (glucuronic acid) transferase I (the enzyme that transfers GlcA from UDP-GlcA to Gal-Gal-Xyl of proteochondroitin linkage region), for comparison with GlcA transferase II (the GlcA transferase of chondroitin polymerization). Gal(beta1-3)Galbeta1-methyl (disaccharide) and GalNAc(beta1-4)GlcA(beta1-3)GalNAc(beta1-4) GlcA(beta1-3)GalNAc(pentasaccharide) were used respectively as acceptors of [14C]GlcA from UDP-[14C]GlcA. Distributions of the two GlcA transferase activities in the sucrose-density-gradient fractions were compared with each other and with the previously reported distribution of the activities of Gal transferases (UDP-Gal to ovalbumin, and to xylose of the proteochondroitin linkage region) and GalNAc (N-acetylgalactosamine) transferase II of chondroitin polymerization. The linkage-region GlcA transferase I had a dual Golgi distribution similar to that of chondroitin-polymerizing GlcA transferase II and distinctly different from the distribution of linkage-region Gal transferases I and II, which were found exclusively in the heavier fractions. Solubilized GlcA transferase I was partly purified by sequential use of Q-Sepharose, heparin-Sepharose and wheatgerm agglutinin-agarose and was accompanied at each step by some of the GlcA transferase II activity. Both GlcA transferase I and II bound to the Q-Sepharose as though they were highly anionic. However, treatment with chondroitin ABC lyase eliminated the binding while markedly decreasing enzyme stability. The enzyme activities could not be reconstituted by adding chondroitin or chondroitin pentasaccharide to the chondroitin ABC lyase-treated enzymes. Incubation of the partly purified enzymes with both UDP-GlcA and UDP-GalNAc resulted in a 40-fold greater incorporation than with just one sugar nucleotide, indicating the presence of bound, nascent proteochondroitin serving as the acceptor for chondroitin polymerization. These results, together with the membrane co-localization, indicate that GlcA transferase I and GlcA transferase II occur closely together with nascent proteochondroitin at the site of synthesis and that this complex with the nascent proteochondroitin stabilizes both enzymes during purification.
Sugumaran
G
G
Connective Tissue Research Laboratory, Building 70, Edith Nourse Rogers Memorial Veterans Hospital, 200 Springs Road, Bedford, MA 01730, USA.
Katsman
M
M
Silbert
J E
JE
D000818:Animals; D002240:Carbohydrate Sequence; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D002807:Chondroitin / Q000096:biosynthesis*; D019765:Chondroitin ABC Lyase / Q000378:metabolism; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004795:Enzyme Stability; D014453:Glucuronosyltransferase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006132:Growth Plate / Q000196:embryology / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism / Q000494:pharmacology; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000378:metabolism
D011508:Chondroitin Sulfate Proteoglycans; D009844:Oligosaccharides; D014535:Uridine Diphosphate Glucuronic Acid; D014536:Uridine Diphosphate N-Acetylgalactosamine; D002807:Chondroitin; D014453:Glucuronosyltransferase; D019765:Chondroitin ABC Lyase