Characterization of proteoglycans synthesized by human adult glomerular mesangial cells in culture.
1. The newly synthesized proteoglycans from human adult glomerular mesangial cells labelled in vitro for 24 h with [35S]sulphate have been characterized using biochemical and immunological techniques. 2. The following proteoglycans were identified (% of total synthesized). (i) A large chondroitin sulphate proteoglycan, CSPG-I, Mr approximately 1 x 10(6) (10.6%). This proteoglycan consisted of a protein core of Mr approximately 4 x 10(5) and glycosaminoglycan chains of Mr 2.5 x 10(4), and was present in both the cell layer and the culture medium. (ii) A major small dermatan sulphate proteoglycan, DSPG-I, Mr 3.5 x 10(5) (46%), which was mainly located in the culture medium. (iii) A second minor small dermatan sulphate, DSPG-II, Mr approximately 2 x 10(5) (9.8%). This molecule was exclusively located in the culture medium. (iv) A large heparan sulphate proteoglycan, HSPG-I, Mr 8 x 10(5) (3.3%). (v) A second large heparan sulphate proteoglycan HSPG-II, Mr approximately 6 x 10(5) (23%). HSPG-I and HSPG-II were extracted from both the culture medium and the cell layer. 3. Western blot analysis of the core proteins released by chondroitin ABC lyase treatment of DSPG-I and DSPG-II identified these dermatan sulphate proteoglycans as biglycan and decorin respectively. Both DSPG-I and DSPG-II had core proteins of Mr 45,000. 4. The cell-layer-associated forms of CSPG-I, HSPG-I and HSPG-II were accessible to limited trypsin treatment, bound to octyl-Sepharose and could be inserted into liposomes, indicating a possible cell membrane location. 5. Pulse-chase experiments indicated that the cell-layer-associated [35S]proteoglycans undergo limited metabolism to inorganic [35S]sulphate, the majority of which is accounted for by the degradation of HSPG-II and to a lesser extent DSPG-I.
Thomas
G J
GJ
Institute of Nephrology, University of Wales College of Medicine, Royal Infirmary, Cardiff, U.K.
Mason
R M
RM
Davies
M
M
D000328:Adult; D015153:Blotting, Western; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005920:Glomerular Mesangium / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011509:Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D011865:Radioisotope Dilution Technique; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes
D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes