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Characterization of proteoglycans synthesized by human adult glomerular mesangial cells in culture.

ABSTRACT: 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.

SUBMITTER: Thomas GJ

PROVIDER: S-EPMC1151194 | biostudies-other | 1991 Jul

REPOSITORIES: biostudies-other

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Project description:Proteoglycans in mineralized (0.5 M-EDTA/4 M-guanidinium chloride-extractable) and non-mineralized (4 M-guanidinium chloride-extractable) matrices synthesized by a mouse osteoblastic-cell line MC3T3-E1 were characterized at different phases of mineralization in vitro. Cell cultures were labelled with [35S]sulphate and either [3H]glucosamine or 3H-labelled amino acids. At the mineralization phase a large majority of proteoglycans were extracted with 4 M-guanidinium chloride (G extract), and at least five species of labelled proteoglycans were identified; dermatan sulphate proteoglycans (DSPG), apparent Mr approx. 120,000 and 70,000), heparan sulphate proteoglycans (HSPG, apparent Mr approx. 200,000 and 120,000) and DS chains with very little core protein. DSPGs weakly bound to an octyl-Sepharose CL-4B column and HSPGs bound more tightly, whereas DS chains did not bind to the column. Amounts of labelled proteoglycans extracted with 0.5 M-EDTA/4 M-guanidinium chloride (EDTA extract) were much less than those in G extract. Although the predominant species in the EDTA extract were comparable with the DS or DSPGs in the G extract, none of them bound to octyl-Sepharose CL-4B, indicating their lack of hydrophobicity. At the nonmineralizing phase a large chondroitin sulphate proteoglycan (Mr greater than 600,000) was found in the matrix in addition to the five proteoglycan species similar to those at the mineralization phase. Although DS chains at the early phase were similar in size to those at the mineralization phase, the ratio of 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulpho-D-galactose to 2-acetamido-2-deoxy-3-O-(beta-D-gluculo-4-enepyranosyluronic acid)-6-O-sulpho-D-galactose was less than that at the mineralization phase. These results agree with those of previous studies performed in vivo and suggest that alteration in the synthesis of proteoglycans is involved in the mineralization process. They also suggest that at the osteoblastic mineralization front proteoglycans undergo partial degradation and lose their hydrophobicity.

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Characterization of proteoglycans synthesized by human adult glomerular mesangial cells in culture.

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